﻿               SEQUENCE LISTING

<110> Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e.V.

<120> CAS9 variants with enhanced specificity

<130> AC1374 PCT S3

<140> 
<141> 

<160> 78

<170> BiSSAP 1.3.6

<210> 1
<211> 1368
<212> PRT
<213> Streptococcus pyogenes


<220> 
<223> Wild-type SpCas9

<400> 1
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 
1               5                   10                  15      
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 
            20                  25                  30          
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 
        35                  40                  45              
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 
    50                  55                  60                  
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 
65                  70                  75                  80  
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 
                85                  90                  95      
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 
            100                 105                 110         
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 
        115                 120                 125             
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 
    130                 135                 140                 
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 
145                 150                 155                 160 
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 
                165                 170                 175     
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 
            180                 185                 190         
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 
        195                 200                 205             
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 
    210                 215                 220                 
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 
225                 230                 235                 240 
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 
                245                 250                 255     
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 
            260                 265                 270         
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 
        275                 280                 285             
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 
    290                 295                 300                 
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 
305                 310                 315                 320 
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 
                325                 330                 335     
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 
            340                 345                 350         
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 
        355                 360                 365             
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 
    370                 375                 380                 
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 
385                 390                 395                 400 
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 
                405                 410                 415     
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 
            420                 425                 430         
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 
        435                 440                 445             
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 
    450                 455                 460                 
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 
465                 470                 475                 480 
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 
                485                 490                 495     
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 
            500                 505                 510         
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 
        515                 520                 525             
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 
    530                 535                 540                 
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 
545                 550                 555                 560 
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 
                565                 570                 575     
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 
            580                 585                 590         
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 
        595                 600                 605             
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 
    610                 615                 620                 
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 
625                 630                 635                 640 
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 
                645                 650                 655     
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 
            660                 665                 670         
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 
        675                 680                 685             
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 
    690                 695                 700                 
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 
705                 710                 715                 720 
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 
                725                 730                 735     
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 
            740                 745                 750         
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 
        755                 760                 765             
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 
    770                 775                 780                 
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 
785                 790                 795                 800 
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 
                805                 810                 815     
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 
            820                 825                 830         
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 
        835                 840                 845             
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 
    850                 855                 860                 
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 
865                 870                 875                 880 
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 
                885                 890                 895     
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 
            900                 905                 910         
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 
        915                 920                 925             
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 
    930                 935                 940                 
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 
945                 950                 955                 960 
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 
                965                 970                 975     
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 
            980                 985                 990         
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 
        995                 1000                1005            
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys 
    1010                1015                1020                
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser 
1025                1030                1035                1040
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu 
                1045                1050                1055    
Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile 
            1060                1065                1070        
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser 
        1075                1080                1085            
Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly 
    1090                1095                1100                
Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile 
1105                1110                1115                1120
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser 
                1125                1130                1135    
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly 
            1140                1145                1150        
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 
        1155                1160                1165            
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala 
    1170                1175                1180                
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys 
1185                1190                1195                1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser 
                1205                1210                1215    
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr 
            1220                1225                1230        
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 
        1235                1240                1245            
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His 
    1250                1255                1260                
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val 
1265                1270                1275                1280
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys 
                1285                1290                1295    
His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu 
            1300                1305                1310        
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp 
        1315                1320                1325            
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 
    1330                1335                1340                
Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile 
1345                1350                1355                1360
Asp Leu Ser Gln Leu Gly Gly Asp 
                1365            

<210> 2
<211> 1368
<212> PRT
<213> Artificial Sequence


<220> 
<223> Wild type SpCas9 amino acid sequence wherein the arginine (R) at
      position 63 and the glutamine (Q) at position 768 are each
      replaced by alanine (A)

<400> 2
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 
1               5                   10                  15      
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 
            20                  25                  30          
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 
        35                  40                  45              
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Ala Leu 
    50                  55                  60                  
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 
65                  70                  75                  80  
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 
                85                  90                  95      
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 
            100                 105                 110         
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 
        115                 120                 125             
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 
    130                 135                 140                 
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 
145                 150                 155                 160 
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 
                165                 170                 175     
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 
            180                 185                 190         
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 
        195                 200                 205             
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 
    210                 215                 220                 
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 
225                 230                 235                 240 
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 
                245                 250                 255     
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 
            260                 265                 270         
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 
        275                 280                 285             
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 
    290                 295                 300                 
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 
305                 310                 315                 320 
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 
                325                 330                 335     
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 
            340                 345                 350         
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 
        355                 360                 365             
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 
    370                 375                 380                 
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 
385                 390                 395                 400 
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 
                405                 410                 415     
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 
            420                 425                 430         
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 
        435                 440                 445             
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 
    450                 455                 460                 
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 
465                 470                 475                 480 
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 
                485                 490                 495     
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 
            500                 505                 510         
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 
        515                 520                 525             
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 
    530                 535                 540                 
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 
545                 550                 555                 560 
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 
                565                 570                 575     
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 
            580                 585                 590         
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 
        595                 600                 605             
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 
    610                 615                 620                 
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 
625                 630                 635                 640 
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 
                645                 650                 655     
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 
            660                 665                 670         
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 
        675                 680                 685             
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 
    690                 695                 700                 
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 
705                 710                 715                 720 
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 
                725                 730                 735     
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 
            740                 745                 750         
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Ala 
        755                 760                 765             
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 
    770                 775                 780                 
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 
785                 790                 795                 800 
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 
                805                 810                 815     
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 
            820                 825                 830         
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 
        835                 840                 845             
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 
    850                 855                 860                 
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 
865                 870                 875                 880 
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 
                885                 890                 895     
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 
            900                 905                 910         
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 
        915                 920                 925             
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 
    930                 935                 940                 
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 
945                 950                 955                 960 
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 
                965                 970                 975     
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 
            980                 985                 990         
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 
        995                 1000                1005            
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys 
    1010                1015                1020                
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser 
1025                1030                1035                1040
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu 
                1045                1050                1055    
Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile 
            1060                1065                1070        
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser 
        1075                1080                1085            
Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly 
    1090                1095                1100                
Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile 
1105                1110                1115                1120
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser 
                1125                1130                1135    
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly 
            1140                1145                1150        
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 
        1155                1160                1165            
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala 
    1170                1175                1180                
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys 
1185                1190                1195                1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser 
                1205                1210                1215    
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr 
            1220                1225                1230        
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 
        1235                1240                1245            
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His 
    1250                1255                1260                
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val 
1265                1270                1275                1280
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys 
                1285                1290                1295    
His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu 
            1300                1305                1310        
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp 
        1315                1320                1325            
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 
    1330                1335                1340                
Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile 
1345                1350                1355                1360
Asp Leu Ser Gln Leu Gly Gly Asp 
                1365            

<210> 3
<211> 1368
<212> PRT
<213> Artificial Sequence


<220> 
<223> Wild type SpCas9 amino acid sequence wherein the arginine (R) at
      position 66 and the glutamine (Q) at position 768 are each
      replaced by alanine (A)

<400> 3
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 
1               5                   10                  15      
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 
            20                  25                  30          
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 
        35                  40                  45              
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 
    50                  55                  60                  
Lys Ala Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 
65                  70                  75                  80  
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 
                85                  90                  95      
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 
            100                 105                 110         
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 
        115                 120                 125             
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 
    130                 135                 140                 
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 
145                 150                 155                 160 
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 
                165                 170                 175     
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 
            180                 185                 190         
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 
        195                 200                 205             
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 
    210                 215                 220                 
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 
225                 230                 235                 240 
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 
                245                 250                 255     
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 
            260                 265                 270         
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 
        275                 280                 285             
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 
    290                 295                 300                 
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 
305                 310                 315                 320 
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 
                325                 330                 335     
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 
            340                 345                 350         
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 
        355                 360                 365             
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 
    370                 375                 380                 
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 
385                 390                 395                 400 
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 
                405                 410                 415     
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 
            420                 425                 430         
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 
        435                 440                 445             
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 
    450                 455                 460                 
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 
465                 470                 475                 480 
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 
                485                 490                 495     
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 
            500                 505                 510         
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 
        515                 520                 525             
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 
    530                 535                 540                 
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 
545                 550                 555                 560 
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 
                565                 570                 575     
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 
            580                 585                 590         
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 
        595                 600                 605             
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 
    610                 615                 620                 
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 
625                 630                 635                 640 
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 
                645                 650                 655     
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 
            660                 665                 670         
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 
        675                 680                 685             
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 
    690                 695                 700                 
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 
705                 710                 715                 720 
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 
                725                 730                 735     
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 
            740                 745                 750         
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Ala 
        755                 760                 765             
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 
    770                 775                 780                 
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 
785                 790                 795                 800 
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 
                805                 810                 815     
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 
            820                 825                 830         
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 
        835                 840                 845             
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 
    850                 855                 860                 
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 
865                 870                 875                 880 
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 
                885                 890                 895     
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 
            900                 905                 910         
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 
        915                 920                 925             
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 
    930                 935                 940                 
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 
945                 950                 955                 960 
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 
                965                 970                 975     
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 
            980                 985                 990         
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 
        995                 1000                1005            
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys 
    1010                1015                1020                
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser 
1025                1030                1035                1040
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu 
                1045                1050                1055    
Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile 
            1060                1065                1070        
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser 
        1075                1080                1085            
Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly 
    1090                1095                1100                
Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile 
1105                1110                1115                1120
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser 
                1125                1130                1135    
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly 
            1140                1145                1150        
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 
        1155                1160                1165            
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala 
    1170                1175                1180                
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys 
1185                1190                1195                1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser 
                1205                1210                1215    
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr 
            1220                1225                1230        
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 
        1235                1240                1245            
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His 
    1250                1255                1260                
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val 
1265                1270                1275                1280
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys 
                1285                1290                1295    
His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu 
            1300                1305                1310        
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp 
        1315                1320                1325            
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 
    1330                1335                1340                
Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile 
1345                1350                1355                1360
Asp Leu Ser Gln Leu Gly Gly Asp 
                1365            

<210> 4
<211> 4179
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9 (including N-terminal His-tag),
      recombinant expressed in E.coli, purified and used for in vitro
      experiments

<400> 4
atgggccatc atcatcatca tcatcatcat catcacagca gcggccatat cgaaggtcgt       60

catagcgtcg acatggataa gaaatactca ataggcttag atatcggcac aaatagcgtc      120

ggatgggcgg tgatcactga tgaatataag gttccgtcta aaaagttcaa ggttctggga      180

aatacagacc gccacagtat caaaaaaaat cttatagggg ctcttttatt tgacagtgga      240

gagacagcgg aagcgactcg tctcaaacgg acagctcgta gaaggtatac acgtcggaag      300

aatcgtattt gttatctaca ggagattttt tcaaatgaga tggcgaaagt agatgatagt      360

ttctttcatc gacttgaaga gtcttttttg gtggaagaag acaagaagca tgaacgtcat      420

cctatttttg gaaatatagt agatgaagtt gcttatcatg agaaatatcc aactatctat      480

catctgcgaa aaaaattggt agattctact gataaagcgg atttgcgctt aatctatttg      540

gccttagcgc atatgattaa gtttcgtggt cattttttga ttgagggaga tttaaatcct      600

gataatagtg atgtggacaa actatttatc cagttggtac aaacctacaa tcaattattt      660

gaagaaaacc ctattaacgc aagtggagta gatgctaaag cgattctttc tgcacgattg      720

agtaaatcaa gacgattaga aaatctcatt gctcagctcc ccggtgagaa gaaaaatggc      780

ttatttggga atctcattgc tttgtcattg ggtttgaccc ctaattttaa atcaaatttt      840

gatttggcag aagatgctaa attacagctt tcaaaagata cttacgatga tgatttagat      900

aatttattgg cgcaaattgg agatcaatat gctgatttgt ttttggcagc taagaattta      960

tcagatgcta ttttactttc agatatccta agagtaaata ctgaaataac taaggctccc     1020

ctatcagctt caatgattaa acgctacgat gaacatcatc aagacttgac tcttttaaaa     1080

gctttagttc gacaacaact tccagaaaag tataaagaaa tcttttttga tcaatcaaaa     1140

aacggatatg caggttatat tgatggggga gctagccaag aagaatttta taaatttatc     1200

aaaccaattt tagaaaaaat ggatggtact gaggaattat tggtgaaact aaatcgtgaa     1260

gatttgctgc gcaagcaacg gacctttgac aacggctcta ttccccatca aattcacttg     1320

ggtgagctgc atgctatttt gagaagacaa gaagactttt atccattttt aaaagacaat     1380

cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt attatgttgg tccattggcg     1440

cgtggcaata gtcgttttgc atggatgact cggaagtctg aagaaacaat taccccatgg     1500

aattttgaag aagttgtcga taaaggtgct tcagctcaat catttattga acgcatgaca     1560

aactttgata aaaatcttcc aaatgaaaaa gtactaccaa aacatagttt gctttatgag     1620

tattttacgg tttataacga attgacaaag gtcaaatatg ttactgaagg aatgcgaaaa     1680

ccagcatttc tttcaggtga acagaagaaa gccattgttg atttactctt caaaacaaat     1740

cgaaaagtaa ccgttaagca attaaaagaa gattatttca aaaaaataga atgttttgat     1800

agtgttgaaa tttcaggagt tgaagataga tttaatgctt cattaggtac ctaccatgat     1860

ttgctaaaaa ttattaaaga taaagatttt ttggataatg aagaaaatga agatatctta     1920

gaggatattg ttttaacatt gaccttattt gaagataggg agatgattga ggaaagactt     1980

aaaacatatg ctcacctctt tgatgataag gtgatgaaac agcttaaacg tcgccgttat     2040

actggttggg gacgtttgtc tcgaaaattg attaatggta ttagggataa gcaatctggc     2100

aaaacaatat tagatttttt gaaatcagat ggttttgcca atcgcaattt tatgcagctg     2160

atccatgatg atagtttgac atttaaagaa gacattcaaa aagcacaagt gtctggacaa     2220

ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat taaaaaaggt     2280

attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg gcataagcca     2340

gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa ctcaaaaggg ccagaaaaat     2400

tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag tcagattctt     2460

aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct ctattatctc     2520

caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt aagtgattat     2580

gatgtcgatc acattgttcc acaaagtttc cttaaagacg attcaataga caataaggtc     2640

ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga agaagtagtc     2700

aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac tcaacgtaag     2760

tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa agctggtttt     2820

atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca aattttggat     2880

agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt taaagtgatt     2940

accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta taaagtacgt     3000

gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt tggaactgct     3060

ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta taaagtttat     3120

gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac cgcaaaatat     3180

ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc aaatggagag     3240

attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt ctgggataaa     3300

gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag     3360

aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa aagaaattcg     3420

gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg ttttgatagt     3480

ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag     3540

ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc ctttgaaaaa     3600

aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga cttaatcatt     3660

aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat gctggctagt     3720

gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt gaatttttta     3780

tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga acaaaaacaa     3840

ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat cagtgaattt     3900

tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc atataacaaa     3960

catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt tacgttgacg     4020

aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat     4080

acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat     4140

gaaacacgca ttgatttgag tcagctagga ggtgactga                            4179


<210> 5
<211> 4179
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9_R63A (including N-terminal
      His-tag), recombinant expressed in E.coli, purified and used for
      in vitro experiments

<400> 5
atgggccatc atcatcatca tcatcatcat catcacagca gcggccatat cgaaggtcgt       60

catagcgtcg acatggataa gaaatactca ataggcttag atatcggcac aaatagcgtc      120

ggatgggcgg tgatcactga tgaatataag gttccgtcta aaaagttcaa ggttctggga      180

aatacagacc gccacagtat caaaaaaaat cttatagggg ctcttttatt tgacagtgga      240

gagacagcgg aagcgactgc tctcaaacgg acagctcgta gaaggtatac acgtcggaag      300

aatcgtattt gttatctaca ggagattttt tcaaatgaga tggcgaaagt agatgatagt      360

ttctttcatc gacttgaaga gtcttttttg gtggaagaag acaagaagca tgaacgtcat      420

cctatttttg gaaatatagt agatgaagtt gcttatcatg agaaatatcc aactatctat      480

catcttcgaa aaaaattggt agattctact gataaagcgg atttgcgctt aatctatttg      540

gccttagcgc atatgattaa gtttcgtggt cattttttga ttgagggaga tttaaatcct      600

gataatagtg atgtggacaa actatttatc cagttggtac aaacctacaa tcaattattt      660

gaagaaaacc ctattaacgc aagtggagta gatgctaaag cgattctttc tgcacgattg      720

agtaaatcaa gacgattaga aaatctcatt gctcagctcc ccggtgagaa gaaaaatggc      780

ttatttggga atctcattgc tttgtcattg ggtttgaccc ctaattttaa atcaaatttt      840

gatttggcag aagatgctaa attacagctt tcaaaagata cttacgatga tgatttagat      900

aatttattgg cgcaaattgg agatcaatat gctgatttgt ttttggcagc taagaattta      960

tcagatgcta ttttactttc agatatccta agagtaaata ctgaaataac taaggctccc     1020

ctatcagctt caatgattaa acgctacgat gaacatcatc aagacttgac tcttttaaaa     1080

gctttagttc gacaacaact tccagaaaag tataaagaaa tcttttttga tcaatcaaaa     1140

aacggatatg caggttatat tgatggggga gctagccaag aagaatttta taaatttatc     1200

aaaccaattt tagaaaaaat ggatggtact gaggaattat tggtgaaact aaatcgtgaa     1260

gatttgctgc gcaagcaacg gacctttgac aacggctcta ttccccatca aattcacttg     1320

ggtgagctgc atgctatttt gagaagacaa gaagactttt atccattttt aaaagacaat     1380

cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt attatgttgg tccattggcg     1440

cgtggcaata gtcgttttgc atggatgact cggaagtctg aagaaacaat taccccatgg     1500

aattttgaag aagttgtcga taaaggtgct tcagctcaat catttattga acgcatgaca     1560

aactttgata aaaatcttcc aaatgaaaaa gtactaccaa aacatagttt gctttatgag     1620

tattttacgg tttataacga attgacaaag gtcaaatatg ttactgaagg aatgcgaaaa     1680

ccagcatttc tttcaggtga acagaagaaa gccattgttg atttactctt caaaacaaat     1740

cgaaaagtaa ccgttaagca attaaaagaa gattatttca aaaaaataga atgttttgat     1800

agtgttgaaa tttcaggagt tgaagataga tttaatgctt cattaggtac ctaccatgat     1860

ttgctaaaaa ttattaaaga taaagatttt ttggataatg aagaaaatga agatatctta     1920

gaggatattg ttttaacatt gaccttattt gaagataggg agatgattga ggaaagactt     1980

aaaacatatg ctcacctctt tgatgataag gtgatgaaac agcttaaacg tcgccgttat     2040

actggttggg gacgtttgtc tcgaaaattg attaatggta ttagggataa gcaatctggc     2100

aaaacaatat tagatttttt gaaatcagat ggttttgcca atcgcaattt tatgcagctg     2160

atccatgatg atagtttgac atttaaagaa gacattcaaa aagcacaagt gtctggacaa     2220

ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat taaaaaaggt     2280

attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg gcataagcca     2340

gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa ctcaaaaggg ccagaaaaat     2400

tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag tcagattctt     2460

aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct ctattatctc     2520

caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt aagtgattat     2580

gatgtcgatc acattgttcc acaaagtttc cttaaagacg attcaataga caataaggtc     2640

ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga agaagtagtc     2700

aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac tcaacgtaag     2760

tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa agctggtttt     2820

atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca aattttggat     2880

agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt taaagtgatt     2940

accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta taaagtacgt     3000

gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt tggaactgct     3060

ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta taaagtttat     3120

gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac cgcaaaatat     3180

ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc aaatggagag     3240

attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt ctgggataaa     3300

gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag     3360

aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa aagaaattcg     3420

gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg ttttgatagt     3480

ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag     3540

ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc ctttgaaaaa     3600

aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga cttaatcatt     3660

aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat gctggctagt     3720

gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt gaatttttta     3780

tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga acaaaaacaa     3840

ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat cagtgaattt     3900

tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc atataacaaa     3960

catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt tacgttgacg     4020

aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat     4080

acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat     4140

gaaacacgca ttgatttgag tcagctagga ggtgactga                            4179


<210> 6
<211> 4179
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9_R66A (including N-terminal
      His-tag), recombinant expressed in E.coli, purified and used for
      in vitro experiments

<400> 6
atgggccatc atcatcatca tcatcatcat catcacagca gcggccatat cgaaggtcgt       60

catagcgtcg acatggataa gaaatactca ataggcttag atatcggcac aaatagcgtc      120

ggatgggcgg tgatcactga tgaatataag gttccgtcta aaaagttcaa ggttctggga      180

aatacagacc gccacagtat caaaaaaaat cttatagggg ctcttttatt tgacagtgga      240

gagacagcgg aagcgactcg tctcaaagcg acagctcgta gaaggtatac acgtcggaag      300

aatcgtattt gttatctaca ggagattttt tcaaatgaga tggcgaaagt agatgatagt      360

ttctttcatc gacttgaaga gtcttttttg gtggaagaag acaagaagca tgaacgtcat      420

cctatttttg gaaatatagt agatgaagtt gcttatcatg agaaatatcc aactatctat      480

catcttcgaa aaaaattggt agattctact gataaagcgg atttgcgctt aatctatttg      540

gccttagcgc atatgattaa gtttcgtggt cattttttga ttgagggaga tttaaatcct      600

gataatagtg atgtggacaa actatttatc cagttggtac aaacctacaa tcaattattt      660

gaagaaaacc ctattaacgc aagtggagta gatgctaaag cgattctttc tgcacgattg      720

agtaaatcaa gacgattaga aaatctcatt gctcagctcc ccggtgagaa gaaaaatggc      780

ttatttggga atctcattgc tttgtcattg ggtttgaccc ctaattttaa atcaaatttt      840

gatttggcag aagatgctaa attacagctt tcaaaagata cttacgatga tgatttagat      900

aatttattgg cgcaaattgg agatcaatat gctgatttgt ttttggcagc taagaattta      960

tcagatgcta ttttactttc agatatccta agagtaaata ctgaaataac taaggctccc     1020

ctatcagctt caatgattaa acgctacgat gaacatcatc aagacttgac tcttttaaaa     1080

gctttagttc gacaacaact tccagaaaag tataaagaaa tcttttttga tcaatcaaaa     1140

aacggatatg caggttatat tgatggggga gctagccaag aagaatttta taaatttatc     1200

aaaccaattt tagaaaaaat ggatggtact gaggaattat tggtgaaact aaatcgtgaa     1260

gatttgctgc gcaagcaacg gacctttgac aacggctcta ttccccatca aattcacttg     1320

ggtgagctgc atgctatttt gagaagacaa gaagactttt atccattttt aaaagacaat     1380

cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt attatgttgg tccattggcg     1440

cgtggcaata gtcgttttgc atggatgact cggaagtctg aagaaacaat taccccatgg     1500

aattttgaag aagttgtcga taaaggtgct tcagctcaat catttattga acgcatgaca     1560

aactttgata aaaatcttcc aaatgaaaaa gtactaccaa aacatagttt gctttatgag     1620

tattttacgg tttataacga attgacaaag gtcaaatatg ttactgaagg aatgcgaaaa     1680

ccagcatttc tttcaggtga acagaagaaa gccattgttg atttactctt caaaacaaat     1740

cgaaaagtaa ccgttaagca attaaaagaa gattatttca aaaaaataga atgttttgat     1800

agtgttgaaa tttcaggagt tgaagataga tttaatgctt cattaggtac ctaccatgat     1860

ttgctaaaaa ttattaaaga taaagatttt ttggataatg aagaaaatga agatatctta     1920

gaggatattg ttttaacatt gaccttattt gaagataggg agatgattga ggaaagactt     1980

aaaacatatg ctcacctctt tgatgataag gtgatgaaac agcttaaacg tcgccgttat     2040

actggttggg gacgtttgtc tcgaaaattg attaatggta ttagggataa gcaatctggc     2100

aaaacaatat tagatttttt gaaatcagat ggttttgcca atcgcaattt tatgcagctg     2160

atccatgatg atagtttgac atttaaagaa gacattcaaa aagcacaagt gtctggacaa     2220

ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat taaaaaaggt     2280

attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg gcataagcca     2340

gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa ctcaaaaggg ccagaaaaat     2400

tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag tcagattctt     2460

aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct ctattatctc     2520

caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt aagtgattat     2580

gatgtcgatc acattgttcc acaaagtttc cttaaagacg attcaataga caataaggtc     2640

ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga agaagtagtc     2700

aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac tcaacgtaag     2760

tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa agctggtttt     2820

atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca aattttggat     2880

agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt taaagtgatt     2940

accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta taaagtacgt     3000

gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt tggaactgct     3060

ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta taaagtttat     3120

gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac cgcaaaatat     3180

ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc aaatggagag     3240

attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt ctgggataaa     3300

gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag     3360

aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa aagaaattcg     3420

gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg ttttgatagt     3480

ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag     3540

ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc ctttgaaaaa     3600

aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga cttaatcatt     3660

aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat gctggctagt     3720

gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt gaatttttta     3780

tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga acaaaaacaa     3840

ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat cagtgaattt     3900

tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc atataacaaa     3960

catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt tacgttgacg     4020

aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat     4080

acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat     4140

gaaacacgca ttgatttgag tcagctagga ggtgactga                            4179


<210> 7
<211> 4179
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9_R70A (including N-terminal
      His-tag), recombinant expressed in E.coli, purified and used for
      in vitro experiments

<400> 7
atgggccatc atcatcatca tcatcatcat catcacagca gcggccatat cgaaggtcgt       60

catagcgtcg acatggataa gaaatactca ataggcttag atatcggcac aaatagcgtc      120

ggatgggcgg tgatcactga tgaatataag gttccgtcta aaaagttcaa ggttctggga      180

aatacagacc gccacagtat caaaaaaaat cttatagggg ctcttttatt tgacagtgga      240

gagacagcgg aagcgactcg tctcaaacgg acagctcgtg caaggtatac acgtcggaag      300

aatcgtattt gttatctaca ggagattttt tcaaatgaga tggcgaaagt agatgatagt      360

ttctttcatc gacttgaaga gtcttttttg gtggaagaag acaagaagca tgaacgtcat      420

cctatttttg gaaatatagt agatgaagtt gcttatcatg agaaatatcc aactatctat      480

catcttcgaa aaaaattggt agattctact gataaagcgg atttgcgctt aatctatttg      540

gccttagcgc atatgattaa gtttcgtggt cattttttga ttgagggaga tttaaatcct      600

gataatagtg atgtggacaa actatttatc cagttggtac aaacctacaa tcaattattt      660

gaagaaaacc ctattaacgc aagtggagta gatgctaaag cgattctttc tgcacgattg      720

agtaaatcaa gacgattaga aaatctcatt gctcagctcc ccggtgagaa gaaaaatggc      780

ttatttggga atctcattgc tttgtcattg ggtttgaccc ctaattttaa atcaaatttt      840

gatttggcag aagatgctaa attacagctt tcaaaagata cttacgatga tgatttagat      900

aatttattgg cgcaaattgg agatcaatat gctgatttgt ttttggcagc taagaattta      960

tcagatgcta ttttactttc agatatccta agagtaaata ctgaaataac taaggctccc     1020

ctatcagctt caatgattaa acgctacgat gaacatcatc aagacttgac tcttttaaaa     1080

gctttagttc gacaacaact tccagaaaag tataaagaaa tcttttttga tcaatcaaaa     1140

aacggatatg caggttatat tgatggggga gctagccaag aagaatttta taaatttatc     1200

aaaccaattt tagaaaaaat ggatggtact gaggaattat tggtgaaact aaatcgtgaa     1260

gatttgctgc gcaagcaacg gacctttgac aacggctcta ttccccatca aattcacttg     1320

ggtgagctgc atgctatttt gagaagacaa gaagactttt atccattttt aaaagacaat     1380

cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt attatgttgg tccattggcg     1440

cgtggcaata gtcgttttgc atggatgact cggaagtctg aagaaacaat taccccatgg     1500

aattttgaag aagttgtcga taaaggtgct tcagctcaat catttattga acgcatgaca     1560

aactttgata aaaatcttcc aaatgaaaaa gtactaccaa aacatagttt gctttatgag     1620

tattttacgg tttataacga attgacaaag gtcaaatatg ttactgaagg aatgcgaaaa     1680

ccagcatttc tttcaggtga acagaagaaa gccattgttg atttactctt caaaacaaat     1740

cgaaaagtaa ccgttaagca attaaaagaa gattatttca aaaaaataga atgttttgat     1800

agtgttgaaa tttcaggagt tgaagataga tttaatgctt cattaggtac ctaccatgat     1860

ttgctaaaaa ttattaaaga taaagatttt ttggataatg aagaaaatga agatatctta     1920

gaggatattg ttttaacatt gaccttattt gaagataggg agatgattga ggaaagactt     1980

aaaacatatg ctcacctctt tgatgataag gtgatgaaac agcttaaacg tcgccgttat     2040

actggttggg gacgtttgtc tcgaaaattg attaatggta ttagggataa gcaatctggc     2100

aaaacaatat tagatttttt gaaatcagat ggttttgcca atcgcaattt tatgcagctg     2160

atccatgatg atagtttgac atttaaagaa gacattcaaa aagcacaagt gtctggacaa     2220

ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat taaaaaaggt     2280

attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg gcataagcca     2340

gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa ctcaaaaggg ccagaaaaat     2400

tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag tcagattctt     2460

aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct ctattatctc     2520

caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt aagtgattat     2580

gatgtcgatc acattgttcc acaaagtttc cttaaagacg attcaataga caataaggtc     2640

ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga agaagtagtc     2700

aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac tcaacgtaag     2760

tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa agctggtttt     2820

atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca aattttggat     2880

agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt taaagtgatt     2940

accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta taaagtacgt     3000

gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt tggaactgct     3060

ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta taaagtttat     3120

gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac cgcaaaatat     3180

ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc aaatggagag     3240

attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt ctgggataaa     3300

gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag     3360

aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa aagaaattcg     3420

gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg ttttgatagt     3480

ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag     3540

ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc ctttgaaaaa     3600

aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga cttaatcatt     3660

aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat gctggctagt     3720

gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt gaatttttta     3780

tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga acaaaaacaa     3840

ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat cagtgaattt     3900

tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc atataacaaa     3960

catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt tacgttgacg     4020

aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat     4080

acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat     4140

gaaacacgca ttgatttgag tcagctagga ggtgactga                            4179


<210> 8
<211> 4179
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9_R63A/Q768A (including N-terminal
      His-tag), recombinant expressed in E.coli, purified and used for
      in vitro experiments

<400> 8
atgggccatc atcatcatca tcatcatcat catcacagca gcggccatat cgaaggtcgt       60

catagcgtcg acatggataa gaaatactca ataggcttag atatcggcac aaatagcgtc      120

ggatgggcgg tgatcactga tgaatataag gttccgtcta aaaagttcaa ggttctggga      180

aatacagacc gccacagtat caaaaaaaat cttatagggg ctcttttatt tgacagtgga      240

gagacagcgg aagcgactgc tctcaaacgg acagctcgta gaaggtatac acgtcggaag      300

aatcgtattt gttatctaca ggagattttt tcaaatgaga tggcgaaagt agatgatagt      360

ttctttcatc gacttgaaga gtcttttttg gtggaagaag acaagaagca tgaacgtcat      420

cctatttttg gaaatatagt agatgaagtt gcttatcatg agaaatatcc aactatctat      480

catcttcgaa aaaaattggt agattctact gataaagcgg atttgcgctt aatctatttg      540

gccttagcgc atatgattaa gtttcgtggt cattttttga ttgagggaga tttaaatcct      600

gataatagtg atgtggacaa actatttatc cagttggtac aaacctacaa tcaattattt      660

gaagaaaacc ctattaacgc aagtggagta gatgctaaag cgattctttc tgcacgattg      720

agtaaatcaa gacgattaga aaatctcatt gctcagctcc ccggtgagaa gaaaaatggc      780

ttatttggga atctcattgc tttgtcattg ggtttgaccc ctaattttaa atcaaatttt      840

gatttggcag aagatgctaa attacagctt tcaaaagata cttacgatga tgatttagat      900

aatttattgg cgcaaattgg agatcaatat gctgatttgt ttttggcagc taagaattta      960

tcagatgcta ttttactttc agatatccta agagtaaata ctgaaataac taaggctccc     1020

ctatcagctt caatgattaa acgctacgat gaacatcatc aagacttgac tcttttaaaa     1080

gctttagttc gacaacaact tccagaaaag tataaagaaa tcttttttga tcaatcaaaa     1140

aacggatatg caggttatat tgatggggga gctagccaag aagaatttta taaatttatc     1200

aaaccaattt tagaaaaaat ggatggtact gaggaattat tggtgaaact aaatcgtgaa     1260

gatttgctgc gcaagcaacg gacctttgac aacggctcta ttccccatca aattcacttg     1320

ggtgagctgc atgctatttt gagaagacaa gaagactttt atccattttt aaaagacaat     1380

cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt attatgttgg tccattggcg     1440

cgtggcaata gtcgttttgc atggatgact cggaagtctg aagaaacaat taccccatgg     1500

aattttgaag aagttgtcga taaaggtgct tcagctcaat catttattga acgcatgaca     1560

aactttgata aaaatcttcc aaatgaaaaa gtactaccaa aacatagttt gctttatgag     1620

tattttacgg tttataacga attgacaaag gtcaaatatg ttactgaagg aatgcgaaaa     1680

ccagcatttc tttcaggtga acagaagaaa gccattgttg atttactctt caaaacaaat     1740

cgaaaagtaa ccgttaagca attaaaagaa gattatttca aaaaaataga atgttttgat     1800

agtgttgaaa tttcaggagt tgaagataga tttaatgctt cattaggtac ctaccatgat     1860

ttgctaaaaa ttattaaaga taaagatttt ttggataatg aagaaaatga agatatctta     1920

gaggatattg ttttaacatt gaccttattt gaagataggg agatgattga ggaaagactt     1980

aaaacatatg ctcacctctt tgatgataag gtgatgaaac agcttaaacg tcgccgttat     2040

actggttggg gacgtttgtc tcgaaaattg attaatggta ttagggataa gcaatctggc     2100

aaaacaatat tagatttttt gaaatcagat ggttttgcca atcgcaattt tatgcagctg     2160

atccatgatg atagtttgac atttaaagaa gacattcaaa aagcacaagt gtctggacaa     2220

ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat taaaaaaggt     2280

attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg gcataagcca     2340

gaaaatatcg ttattgaaat ggcacgtgaa aatgcgacaa ctcaaaaggg ccagaaaaat     2400

tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag tcagattctt     2460

aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct ctattatctc     2520

caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt aagtgattat     2580

gatgtcgatc acattgttcc acaaagtttc cttaaagacg attcaataga caataaggtc     2640

ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga agaagtagtc     2700

aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac tcaacgtaag     2760

tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa agctggtttt     2820

atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca aattttggat     2880

agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt taaagtgatt     2940

accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta taaagtacgt     3000

gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt tggaactgct     3060

ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta taaagtttat     3120

gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac cgcaaaatat     3180

ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc aaatggagag     3240

attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt ctgggataaa     3300

gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag     3360

aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa aagaaattcg     3420

gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg ttttgatagt     3480

ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag     3540

ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc ctttgaaaaa     3600

aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga cttaatcatt     3660

aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat gctggctagt     3720

gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt gaatttttta     3780

tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga acaaaaacaa     3840

ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat cagtgaattt     3900

tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc atataacaaa     3960

catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt tacgttgacg     4020

aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat     4080

acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat     4140

gaaacacgca ttgatttgag tcagctagga ggtgactga                            4179


<210> 9
<211> 4179
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9 Q768A (including N-terminal
      His-tag), recombinant expressed in E.coli, purified and used for
      in vitro experiments

<400> 9
atgggccatc atcatcatca tcatcatcat catcacagca gcggccatat cgaaggtcgt       60

catagcgtcg acatggataa gaaatactca ataggcttag atatcggcac aaatagcgtc      120

ggatgggcgg tgatcactga tgaatataag gttccgtcta aaaagttcaa ggttctggga      180

aatacagacc gccacagtat caaaaaaaat cttatagggg ctcttttatt tgacagtgga      240

gagacagcgg aagcgactcg tctcaaacgg acagctcgta gaaggtatac acgtcggaag      300

aatcgtattt gttatctaca ggagattttt tcaaatgaga tggcgaaagt agatgatagt      360

ttctttcatc gacttgaaga gtcttttttg gtggaagaag acaagaagca tgaacgtcat      420

cctatttttg gaaatatagt agatgaagtt gcttatcatg agaaatatcc aactatctat      480

catctgcgaa aaaaattggt agattctact gataaagcgg atttgcgctt aatctatttg      540

gccttagcgc atatgattaa gtttcgtggt cattttttga ttgagggaga tttaaatcct      600

gataatagtg atgtggacaa actatttatc cagttggtac aaacctacaa tcaattattt      660

gaagaaaacc ctattaacgc aagtggagta gatgctaaag cgattctttc tgcacgattg      720

agtaaatcaa gacgattaga aaatctcatt gctcagctcc ccggtgagaa gaaaaatggc      780

ttatttggga atctcattgc tttgtcattg ggtttgaccc ctaattttaa atcaaatttt      840

gatttggcag aagatgctaa attacagctt tcaaaagata cttacgatga tgatttagat      900

aatttattgg cgcaaattgg agatcaatat gctgatttgt ttttggcagc taagaattta      960

tcagatgcta ttttactttc agatatccta agagtaaata ctgaaataac taaggctccc     1020

ctatcagctt caatgattaa acgctacgat gaacatcatc aagacttgac tcttttaaaa     1080

gctttagttc gacaacaact tccagaaaag tataaagaaa tcttttttga tcaatcaaaa     1140

aacggatatg caggttatat tgatggggga gctagccaag aagaatttta taaatttatc     1200

aaaccaattt tagaaaaaat ggatggtact gaggaattat tggtgaaact aaatcgtgaa     1260

gatttgctgc gcaagcaacg gacctttgac aacggctcta ttccccatca aattcacttg     1320

ggtgagctgc atgctatttt gagaagacaa gaagactttt atccattttt aaaagacaat     1380

cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt attatgttgg tccattggcg     1440

cgtggcaata gtcgttttgc atggatgact cggaagtctg aagaaacaat taccccatgg     1500

aattttgaag aagttgtcga taaaggtgct tcagctcaat catttattga acgcatgaca     1560

aactttgata aaaatcttcc aaatgaaaaa gtactaccaa aacatagttt gctttatgag     1620

tattttacgg tttataacga attgacaaag gtcaaatatg ttactgaagg aatgcgaaaa     1680

ccagcatttc tttcaggtga acagaagaaa gccattgttg atttactctt caaaacaaat     1740

cgaaaagtaa ccgttaagca attaaaagaa gattatttca aaaaaataga atgttttgat     1800

agtgttgaaa tttcaggagt tgaagataga tttaatgctt cattaggtac ctaccatgat     1860

ttgctaaaaa ttattaaaga taaagatttt ttggataatg aagaaaatga agatatctta     1920

gaggatattg ttttaacatt gaccttattt gaagataggg agatgattga ggaaagactt     1980

aaaacatatg ctcacctctt tgatgataag gtgatgaaac agcttaaacg tcgccgttat     2040

actggttggg gacgtttgtc tcgaaaattg attaatggta ttagggataa gcaatctggc     2100

aaaacaatat tagatttttt gaaatcagat ggttttgcca atcgcaattt tatgcagctg     2160

atccatgatg atagtttgac atttaaagaa gacattcaaa aagcacaagt gtctggacaa     2220

ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat taaaaaaggt     2280

attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg gcataagcca     2340

gaaaatatcg ttattgaaat ggcacgtgaa aatgcgacaa ctcaaaaggg ccagaaaaat     2400

tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag tcagattctt     2460

aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct ctattatctc     2520

caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt aagtgattat     2580

gatgtcgatc acattgttcc acaaagtttc cttaaagacg attcaataga caataaggtc     2640

ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga agaagtagtc     2700

aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac tcaacgtaag     2760

tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa agctggtttt     2820

atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca aattttggat     2880

agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt taaagtgatt     2940

accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta taaagtacgt     3000

gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt tggaactgct     3060

ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta taaagtttat     3120

gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac cgcaaaatat     3180

ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc aaatggagag     3240

attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt ctgggataaa     3300

gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag     3360

aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa aagaaattcg     3420

gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg ttttgatagt     3480

ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag     3540

ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc ctttgaaaaa     3600

aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga cttaatcatt     3660

aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat gctggctagt     3720

gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt gaatttttta     3780

tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga acaaaaacaa     3840

ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat cagtgaattt     3900

tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc atataacaaa     3960

catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt tacgttgacg     4020

aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat     4080

acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat     4140

gaaacacgca ttgatttgag tcagctagga ggtgactga                            4179


<210> 10
<211> 4179
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9_R66A/Q768A (including N-terminal
      His-tag), recombinant expressed in E.coli, purified and used for
      in vitro experiments

<400> 10
atgggccatc atcatcatca tcatcatcat catcacagca gcggccatat cgaaggtcgt       60

catagcgtcg acatggataa gaaatactca ataggcttag atatcggcac aaatagcgtc      120

ggatgggcgg tgatcactga tgaatataag gttccgtcta aaaagttcaa ggttctggga      180

aatacagacc gccacagtat caaaaaaaat cttatagggg ctcttttatt tgacagtgga      240

gagacagcgg aagcgactcg tctcaaagcg acagctcgta gaaggtatac acgtcggaag      300

aatcgtattt gttatctaca ggagattttt tcaaatgaga tggcgaaagt agatgatagt      360

ttctttcatc gacttgaaga gtcttttttg gtggaagaag acaagaagca tgaacgtcat      420

cctatttttg gaaatatagt agatgaagtt gcttatcatg agaaatatcc aactatctat      480

catcttcgaa aaaaattggt agattctact gataaagcgg atttgcgctt aatctatttg      540

gccttagcgc atatgattaa gtttcgtggt cattttttga ttgagggaga tttaaatcct      600

gataatagtg atgtggacaa actatttatc cagttggtac aaacctacaa tcaattattt      660

gaagaaaacc ctattaacgc aagtggagta gatgctaaag cgattctttc tgcacgattg      720

agtaaatcaa gacgattaga aaatctcatt gctcagctcc ccggtgagaa gaaaaatggc      780

ttatttggga atctcattgc tttgtcattg ggtttgaccc ctaattttaa atcaaatttt      840

gatttggcag aagatgctaa attacagctt tcaaaagata cttacgatga tgatttagat      900

aatttattgg cgcaaattgg agatcaatat gctgatttgt ttttggcagc taagaattta      960

tcagatgcta ttttactttc agatatccta agagtaaata ctgaaataac taaggctccc     1020

ctatcagctt caatgattaa acgctacgat gaacatcatc aagacttgac tcttttaaaa     1080

gctttagttc gacaacaact tccagaaaag tataaagaaa tcttttttga tcaatcaaaa     1140

aacggatatg caggttatat tgatggggga gctagccaag aagaatttta taaatttatc     1200

aaaccaattt tagaaaaaat ggatggtact gaggaattat tggtgaaact aaatcgtgaa     1260

gatttgctgc gcaagcaacg gacctttgac aacggctcta ttccccatca aattcacttg     1320

ggtgagctgc atgctatttt gagaagacaa gaagactttt atccattttt aaaagacaat     1380

cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt attatgttgg tccattggcg     1440

cgtggcaata gtcgttttgc atggatgact cggaagtctg aagaaacaat taccccatgg     1500

aattttgaag aagttgtcga taaaggtgct tcagctcaat catttattga acgcatgaca     1560

aactttgata aaaatcttcc aaatgaaaaa gtactaccaa aacatagttt gctttatgag     1620

tattttacgg tttataacga attgacaaag gtcaaatatg ttactgaagg aatgcgaaaa     1680

ccagcatttc tttcaggtga acagaagaaa gccattgttg atttactctt caaaacaaat     1740

cgaaaagtaa ccgttaagca attaaaagaa gattatttca aaaaaataga atgttttgat     1800

agtgttgaaa tttcaggagt tgaagataga tttaatgctt cattaggtac ctaccatgat     1860

ttgctaaaaa ttattaaaga taaagatttt ttggataatg aagaaaatga agatatctta     1920

gaggatattg ttttaacatt gaccttattt gaagataggg agatgattga ggaaagactt     1980

aaaacatatg ctcacctctt tgatgataag gtgatgaaac agcttaaacg tcgccgttat     2040

actggttggg gacgtttgtc tcgaaaattg attaatggta ttagggataa gcaatctggc     2100

aaaacaatat tagatttttt gaaatcagat ggttttgcca atcgcaattt tatgcagctg     2160

atccatgatg atagtttgac atttaaagaa gacattcaaa aagcacaagt gtctggacaa     2220

ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat taaaaaaggt     2280

attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg gcataagcca     2340

gaaaatatcg ttattgaaat ggcacgtgaa aatgcgacaa ctcaaaaggg ccagaaaaat     2400

tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag tcagattctt     2460

aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct ctattatctc     2520

caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt aagtgattat     2580

gatgtcgatc acattgttcc acaaagtttc cttaaagacg attcaataga caataaggtc     2640

ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga agaagtagtc     2700

aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac tcaacgtaag     2760

tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa agctggtttt     2820

atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca aattttggat     2880

agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt taaagtgatt     2940

accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta taaagtacgt     3000

gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt tggaactgct     3060

ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta taaagtttat     3120

gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac cgcaaaatat     3180

ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc aaatggagag     3240

attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt ctgggataaa     3300

gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag     3360

aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa aagaaattcg     3420

gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg ttttgatagt     3480

ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag     3540

ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc ctttgaaaaa     3600

aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga cttaatcatt     3660

aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat gctggctagt     3720

gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt gaatttttta     3780

tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga acaaaaacaa     3840

ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat cagtgaattt     3900

tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc atataacaaa     3960

catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt tacgttgacg     4020

aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat     4080

acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat     4140

gaaacacgca ttgatttgag tcagctagga ggtgactga                            4179


<210> 11
<211> 4167
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9 (including N-terminal His-tag),
      used for in vivo experiments in E.coli, note that only the coding
      sequence for the His-tag differs, the CDS for Cas9 is the same as
      for the recombinant purified ones

<400> 11
atgaaacatc accatcacca tcacaacact agtcatatcg aaggtcgtca tagcgtcgac       60

atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg      120

atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc      180

cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa      240

gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt      300

tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga      360

cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga      420

aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa      480

aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat      540

atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat      600

gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct      660

attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga      720

cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat      780

ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa      840

gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg      900

caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt      960

ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca     1020

atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga     1080

caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca     1140

ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta     1200

gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc     1260

aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat     1320

gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt     1380

gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt     1440

cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa     1500

gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa     1560

aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt     1620

tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt     1680

tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc     1740

gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt     1800

tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt     1860

attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt     1920

ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct     1980

cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga     2040

cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta     2100

gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat     2160

agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta     2220

catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact     2280

gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt     2340

attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt     2400

atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct     2460

gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga     2520

gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac     2580

attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct     2640

gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa     2700

aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta     2760

acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa     2820

ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat     2880

actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct     2940

aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat     3000

taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa     3060

tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa     3120

atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct     3180

aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc     3240

cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt     3300

gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta     3360

cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt     3420

gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct     3480

tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt     3540

aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac     3600

tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa     3660

tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta     3720

caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt     3780

cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag     3840

cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt     3900

attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa     3960

ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct     4020

cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa     4080

gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt     4140

gatttgagtc agctaggagg tgactga                                         4167


<210> 12
<211> 4167
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9_R63A (including N-terminal
      His-tag), used for in vivo experiments in E.coli, note that only
      the coding sequence for the His-tag differs, the CDS for Cas9 is
      the same as for the recombinant purified ones

<400> 12
atgaaacatc accatcacca tcacaacact agtcatatcg aaggtcgtca tagcgtcgac       60

atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg      120

atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc      180

cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa      240

gcgactgctc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt      300

tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga      360

cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga      420

aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tcttcgaaaa      480

aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat      540

atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat      600

gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct      660

attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga      720

cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat      780

ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa      840

gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg      900

caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt      960

ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca     1020

atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga     1080

caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca     1140

ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta     1200

gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc     1260

aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat     1320

gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt     1380

gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt     1440

cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa     1500

gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa     1560

aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt     1620

tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt     1680

tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc     1740

gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt     1800

tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt     1860

attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt     1920

ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct     1980

cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga     2040

cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta     2100

gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat     2160

agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta     2220

catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact     2280

gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt     2340

attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt     2400

atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct     2460

gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga     2520

gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac     2580

attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct     2640

gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa     2700

aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta     2760

acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa     2820

ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat     2880

actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct     2940

aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat     3000

taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa     3060

tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa     3120

atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct     3180

aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc     3240

cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt     3300

gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta     3360

cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt     3420

gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct     3480

tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt     3540

aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac     3600

tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa     3660

tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta     3720

caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt     3780

cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag     3840

cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt     3900

attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa     3960

ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct     4020

cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa     4080

gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt     4140

gatttgagtc agctaggagg tgactga                                         4167


<210> 13
<211> 4167
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9_R66A (including N-terminal
      His-tag), used for in vivo experiments in E.coli, note that only
      the coding sequence for the His-tag differs, the CDS for Cas9 is
      the same as for the recombinant purified ones

<400> 13
atgaaacatc accatcacca tcacaacact agtcatatcg aaggtcgtca tagcgtcgac       60

atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg      120

atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc      180

cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa      240

gcgactcgtc tcaaagcgac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt      300

tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga      360

cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga      420

aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tcttcgaaaa      480

aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat      540

atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat      600

gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct      660

attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga      720

cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat      780

ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa      840

gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg      900

caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt      960

ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca     1020

atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga     1080

caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca     1140

ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta     1200

gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc     1260

aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat     1320

gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt     1380

gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt     1440

cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa     1500

gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa     1560

aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt     1620

tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt     1680

tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc     1740

gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt     1800

tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt     1860

attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt     1920

ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct     1980

cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga     2040

cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta     2100

gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat     2160

agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta     2220

catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact     2280

gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt     2340

attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt     2400

atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct     2460

gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga     2520

gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac     2580

attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct     2640

gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa     2700

aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta     2760

acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa     2820

ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat     2880

actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct     2940

aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat     3000

taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa     3060

tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa     3120

atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct     3180

aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc     3240

cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt     3300

gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta     3360

cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt     3420

gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct     3480

tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt     3540

aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac     3600

tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa     3660

tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta     3720

caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt     3780

cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag     3840

cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt     3900

attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa     3960

ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct     4020

cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa     4080

gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt     4140

gatttgagtc agctaggagg tgactga                                         4167


<210> 14
<211> 4167
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9_R70A (including N-terminal
      His-tag), used for in vivo experiments in E.coli, note that only
      the coding sequence for the His-tag differs, the CDS for Cas9 is
      the same as for the recombinant purified ones

<400> 14
atgaaacatc accatcacca tcacaacact agtcatatcg aaggtcgtca tagcgtcgac       60

atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg      120

atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc      180

cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa      240

gcgactcgtc tcaaacggac agctcgtgca aggtatacac gtcggaagaa tcgtatttgt      300

tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga      360

cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga      420

aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tcttcgaaaa      480

aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat      540

atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat      600

gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct      660

attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga      720

cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat      780

ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa      840

gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg      900

caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt      960

ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca     1020

atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga     1080

caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca     1140

ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta     1200

gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc     1260

aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat     1320

gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt     1380

gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt     1440

cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa     1500

gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa     1560

aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt     1620

tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt     1680

tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc     1740

gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt     1800

tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt     1860

attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt     1920

ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct     1980

cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga     2040

cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta     2100

gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat     2160

agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta     2220

catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact     2280

gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt     2340

attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt     2400

atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct     2460

gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga     2520

gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac     2580

attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct     2640

gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa     2700

aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta     2760

acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa     2820

ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat     2880

actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct     2940

aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat     3000

taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa     3060

tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa     3120

atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct     3180

aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc     3240

cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt     3300

gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta     3360

cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt     3420

gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct     3480

tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt     3540

aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac     3600

tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa     3660

tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta     3720

caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt     3780

cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag     3840

cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt     3900

attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa     3960

ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct     4020

cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa     4080

gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt     4140

gatttgagtc agctaggagg tgactga                                         4167


<210> 15
<211> 4167
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9 Q768A (including N-terminal
      His-tag), used for in vivo experiments in E.coli, note that only
      the coding sequence for the His-tag differs, the CDS for Cas9 is
      the same as for the recombinant purified ones

<400> 15
atgaaacatc accatcacca tcacaacact agtcatatcg aaggtcgtca tagcgtcgac       60

atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg      120

atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc      180

cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa      240

gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt      300

tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga      360

cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga      420

aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa      480

aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat      540

atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat      600

gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct      660

attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga      720

cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat      780

ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa      840

gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg      900

caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt      960

ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca     1020

atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga     1080

caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca     1140

ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta     1200

gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc     1260

aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat     1320

gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt     1380

gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt     1440

cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa     1500

gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa     1560

aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt     1620

tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt     1680

tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc     1740

gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt     1800

tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt     1860

attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt     1920

ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct     1980

cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga     2040

cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta     2100

gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat     2160

agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta     2220

catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact     2280

gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt     2340

attgaaatgg cacgtgaaaa tgcgacaact caaaagggcc agaaaaattc gcgagagcgt     2400

atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct     2460

gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga     2520

gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac     2580

attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct     2640

gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa     2700

aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta     2760

acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa     2820

ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat     2880

actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct     2940

aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat     3000

taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa     3060

tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa     3120

atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct     3180

aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc     3240

cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt     3300

gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta     3360

cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt     3420

gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct     3480

tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt     3540

aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac     3600

tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa     3660

tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta     3720

caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt     3780

cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag     3840

cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt     3900

attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa     3960

ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct     4020

cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa     4080

gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt     4140

gatttgagtc agctaggagg tgactga                                         4167


<210> 16
<211> 4167
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9_R63A/Q768A (including N-terminal
      His-tag), used for in vivo experiments in E.coli, note that only
      the coding sequence for the His-tag differs, the CDS for Cas9 is
      the same as for the recombinant purified ones

<400> 16
atgaaacatc accatcacca tcacaacact agtcatatcg aaggtcgtca tagcgtcgac       60

atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg      120

atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc      180

cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa      240

gcgactgctc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt      300

tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga      360

cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga      420

aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tcttcgaaaa      480

aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat      540

atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat      600

gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct      660

attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga      720

cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat      780

ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa      840

gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg      900

caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt      960

ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca     1020

atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga     1080

caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca     1140

ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta     1200

gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc     1260

aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat     1320

gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt     1380

gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt     1440

cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa     1500

gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa     1560

aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt     1620

tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt     1680

tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc     1740

gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt     1800

tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt     1860

attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt     1920

ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct     1980

cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga     2040

cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta     2100

gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat     2160

agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta     2220

catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact     2280

gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt     2340

attgaaatgg cacgtgaaaa tgcgacaact caaaagggcc agaaaaattc gcgagagcgt     2400

atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct     2460

gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga     2520

gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac     2580

attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct     2640

gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa     2700

aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta     2760

acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa     2820

ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat     2880

actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct     2940

aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat     3000

taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa     3060

tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa     3120

atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct     3180

aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc     3240

cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt     3300

gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta     3360

cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt     3420

gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct     3480

tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt     3540

aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac     3600

tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa     3660

tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta     3720

caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt     3780

cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag     3840

cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt     3900

attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa     3960

ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct     4020

cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa     4080

gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt     4140

gatttgagtc agctaggagg tgactga                                         4167


<210> 17
<211> 4167
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9_R66A/Q768A (including N-terminal
      His-tag), used for in vivo experiments in E.coli, note that only
      the coding sequence for the His-tag differs, the CDS for Cas9 is
      the same as for the recombinant purified ones

<400> 17
atgaaacatc accatcacca tcacaacact agtcatatcg aaggtcgtca tagcgtcgac       60

atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg      120

atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc      180

cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa      240

gcgactcgtc tcaaagcgac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt      300

tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga      360

cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga      420

aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tcttcgaaaa      480

aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat      540

atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat      600

gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct      660

attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga      720

cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat      780

ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa      840

gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg      900

caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt      960

ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca     1020

atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga     1080

caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca     1140

ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta     1200

gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc     1260

aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat     1320

gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt     1380

gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt     1440

cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa     1500

gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa     1560

aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt     1620

tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt     1680

tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc     1740

gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt     1800

tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt     1860

attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt     1920

ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct     1980

cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga     2040

cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta     2100

gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat     2160

agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta     2220

catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact     2280

gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt     2340

attgaaatgg cacgtgaaaa tgcgacaact caaaagggcc agaaaaattc gcgagagcgt     2400

atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct     2460

gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga     2520

gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac     2580

attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct     2640

gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa     2700

aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta     2760

acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa     2820

ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat     2880

actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct     2940

aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat     3000

taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa     3060

tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa     3120

atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct     3180

aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc     3240

cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt     3300

gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta     3360

cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt     3420

gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct     3480

tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt     3540

aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac     3600

tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa     3660

tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta     3720

caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt     3780

cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag     3840

cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt     3900

attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa     3960

ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct     4020

cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa     4080

gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt     4140

gatttgagtc agctaggagg tgactga                                         4167


<210> 18
<211> 4176
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9 (including NLS and C-terminal
      Flag-Tag), used for in vivo experiments in HaCat and MCF7, note
      that the sequence is Codon optimized for expression in these cell
      lines

<400> 18
atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg       60

atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg      120

cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag      180

gccaccgccc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc      240

tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga      300

ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc      360

aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag      420

aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac      480

atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac      540

gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc      600

atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga      660

cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac      720

ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag      780

gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc      840

cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc      900

ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct      960

atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg     1020

cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc     1080

ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg     1140

gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg     1200

aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac     1260

gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc     1320

gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc     1380

agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa     1440

gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag     1500

aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg     1560

tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg     1620

agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc     1680

gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc     1740

tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt     1800

atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg     1860

ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc     1920

cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc     1980

aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg     2040

gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac     2100

agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg     2160

cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca     2220

gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg     2280

atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga     2340

atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc     2400

gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg     2460

gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat     2520

atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc     2580

gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag     2640

aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg     2700

accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag     2760

ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac     2820

actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc     2880

aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac     2940

taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag     3000

taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag     3060

atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc     3120

aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg     3180

cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt     3240

gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg     3300

cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc     3360

gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc     3420

tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg     3480

aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac     3540

tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag     3600

tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg     3660

cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc     3720

cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa     3780

cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg     3840

atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag     3900

cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc     3960

cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa     4020

gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc     4080

gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc tggacaggct     4140

aagaagaaga aagattacaa agacgatgac gataag                               4176


<210> 19
<211> 4176
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9_R63A/Q768A (including NLS and
      C-terminal Flag-Tag), used for in vivo experiments in HaCat and
      MCF7, note that the sequence is Codon optimized for expression in
      these cell lines

<400> 19
atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg       60

atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg      120

cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag      180

gccaccgccc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc      240

tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga      300

ctcgaggagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc      360

aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag      420

aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac      480

atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac      540

gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc      600

atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga      660

cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac      720

ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag      780

gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc      840

cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc      900

ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct      960

atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg     1020

cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc     1080

ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg     1140

gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg     1200

aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac     1260

gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc     1320

gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc     1380

agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa     1440

gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag     1500

aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg     1560

tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg     1620

agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc     1680

gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc     1740

tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt     1800

atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg     1860

ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc     1920

cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc     1980

aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg     2040

gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac     2100

agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg     2160

cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca     2220

gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg     2280

atcgaaatgg ccagagagaa cgccaccacc cagaagggac agaagaacag ccgcgagaga     2340

atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc     2400

gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg     2460

gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat     2520

atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc     2580

gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag     2640

aactactggc gccagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg     2700

accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag     2760

ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac     2820

actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc     2880

aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac     2940

taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag     3000

taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag     3060

atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc     3120

aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg     3180

cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt     3240

gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg     3300

cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc     3360

gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc     3420

tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg     3480

aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac     3540

tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag     3600

tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg     3660

cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc     3720

cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa     3780

cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg     3840

atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag     3900

cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc     3960

cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa     4020

gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc     4080

gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc tggacaggct     4140

aagaagaaga aagattacaa agacgatgac gataag                               4176


<210> 20
<211> 4176
<212> DNA
<213> Artificial Sequence


<220> 
<223> Sequence for S. pyogenes Cas9_R66A/Q768A (including NLS and
      C-terminal Flag-Tag), used for in vivo experiments in HaCat and
      MCF7, note that the sequence is Codon optimized for expression in
      these cell lines

<400> 20
atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg       60

atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg      120

cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag      180

gccacccggc tgaaggcaac cgccagaaga agatacacca gacggaagaa ccggatctgc      240

tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga      300

ctcgaggagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc      360

aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag      420

aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac      480

atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac      540

gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc      600

atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga      660

cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac      720

ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag      780

gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc      840

cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc      900

ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct      960

atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg     1020

cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc     1080

ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg     1140

gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg     1200

aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac     1260

gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc     1320

gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc     1380

agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa     1440

gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag     1500

aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg     1560

tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg     1620

agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc     1680

gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc     1740

tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt     1800

atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg     1860

ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc     1920

cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc     1980

aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg     2040

gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac     2100

agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg     2160

cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca     2220

gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg     2280

atcgaaatgg ccagagagaa cgccaccacc cagaagggac agaagaacag ccgcgagaga     2340

atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc     2400

gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg     2460

gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat     2520

atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc     2580

gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag     2640

aactactggc gccagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg     2700

accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag     2760

ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac     2820

actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc     2880

aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac     2940

taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag     3000

taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag     3060

atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc     3120

aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg     3180

cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt     3240

gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg     3300

cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc     3360

gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc     3420

tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg     3480

aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac     3540

tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag     3600

tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg     3660

cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc     3720

cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa     3780

cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg     3840

atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag     3900

cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc     3960

cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa     4020

gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc     4080

gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc tggacaggct     4140

aagaagaaga aagattacaa agacgatgac gataag                               4176


<210> 21
<211> 77
<212> RNA
<213> Artificial Sequence


<220> 
<223> tracrRNA for in vitro experiments

<400> 21
aaaacagcau agcaaguuaa aauaaggcua guccguuauc aacuugaaaa aguggcaccg     60

agagucggug cuuuuuu                                                    77


<210> 22
<211> 42
<212> RNA
<213> Artificial Sequence


<220> 
<223> crRNA for in vitro experiments targeting speM protospacer

<400> 22
auaacucaau uuguaaaaaa guuuuagagc uaugcuguuu ug                        42


<210> 23
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed

<400> 23
ugcaccuuga agcgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 24
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 1 (counting from PAM)

<400> 24
ugcaccuuga agcgcaugau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 25
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 2 (counting from PAM)

<400> 25
ugcaccuuga agcgcaugua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 26
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 3 (counting from PAM)

<400> 26
ugcaccuuga agcgcaucaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 27
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 4 (counting from PAM)

<400> 27
ugcaccuuga agcgcaagaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 28
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 5 (counting from PAM)

<400> 28
ugcaccuuga agcgcuugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 29
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 6 (counting from PAM)

<400> 29
ugcaccuuga agcggaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 30
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 7 (counting from PAM)

<400> 30
ugcaccuuga agcccaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 31
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 8 (counting from PAM)

<400> 31
ugcaccuuga agggcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 32
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 9 (counting from PAM)

<400> 32
ugcaccuuga accgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 33
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 10 (counting from PAM)

<400> 33
ugcaccuuga ugcgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 34
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 11 (counting from PAM)

<400> 34
ugcaccuugu agcgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 35
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 12 (counting from PAM)

<400> 35
ugcaccuuca agcgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 36
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 13 (counting from PAM)

<400> 36
ugcaccuaga agcgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 37
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 14 (counting from PAM)

<400> 37
ugcaccauga agcgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 38
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 15 (counting from PAM)

<400> 38
ugcacguuga agcgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 39
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 16 (counting from PAM)

<400> 39
ugcagcuuga agcgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 40
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 17 (counting from PAM)

<400> 40
ugcuccuuga agcgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 41
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 18 (counting from PAM)

<400> 41
uggaccuuga agcgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 42
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 19 (counting from PAM)

<400> 42
uccaccuuga agcgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 43
<211> 96
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgRNA for in vivo E. coli assays targeting 5â CDS of DsRed,
      mismatch at position 20 (counting from PAM)

<400> 43
agcaccuuga agcgcaugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc     60

cguuaucaac uugaaaaagu ggcaccgagu cggugc                               96


<210> 44
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments (PAM)

<400> 44
ttatatgaac ataactcaat ttgtaaaaaa tgg                                  33


<210> 45
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 1 (counted from PAM)

<400> 45
ttatatgaac ataactcaat ttgtaaaaat tgg                                  33


<210> 46
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 2 (counted from PAM)

<400> 46
ttatatgaac ataactcaat ttgtaaaata tgg                                  33


<210> 47
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 3 (counted from PAM)

<400> 47
ttatatgaac ataactcaat ttgtaaataa tgg                                  33


<210> 48
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 4 (counted from PAM)

<400> 48
ttatatgaac ataactcaat ttgtaataaa tgg                                  33


<210> 49
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 5 (counted from PAM)

<400> 49
ttatatgaac ataactcaat ttgtataaaa tgg                                  33


<210> 50
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 6 (counted from PAM)

<400> 50
ttatatgaac ataactcaat ttgttaaaaa tgg                                  33


<210> 51
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 7 (counted from PAM)

<400> 51
ttatatgaac ataactcaat ttgaaaaaaa tgg                                  33


<210> 52
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 8 (counted from PAM)

<400> 52
ttatatgaac ataactcaat ttctaaaaaa tgg                                  33


<210> 53
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 9 (counted from PAM)

<400> 53
ttatatgaac ataactcaat tagtaaaaaa tgg                                  33


<210> 54
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 10 (counted from PAM)

<400> 54
ttatatgaac ataactcaat atgtaaaaaa tgg                                  33


<210> 55
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 11 (counted from PAM)

<400> 55
ttatatgaac ataactcaaa ttgtaaaaaa tgg                                  33


<210> 56
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 12 (counted from PAM)

<400> 56
ttatatgaac ataactcatt ttgtaaaaaa tgg                                  33


<210> 57
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 13 (counted from PAM)

<400> 57
ttatatgaac ataactctat ttgtaaaaaa tgg                                  33


<210> 58
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 14 (counted from PAM)

<400> 58
ttatatgaac ataactgaat ttgtaaaaaa tgg                                  33


<210> 59
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 15 (counted from PAM)

<400> 59
ttatatgaac ataacacaat ttgtaaaaaa tgg                                  33


<210> 60
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 16 (counted from PAM)

<400> 60
ttatatgaac ataagtcaat ttgtaaaaaa tgg                                  33


<210> 61
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 17 (counted from PAM)

<400> 61
ttatatgaac atatctcaat ttgtaaaaaa tgg                                  33


<210> 62
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 18 (counted from PAM)

<400> 62
ttatatgaac attactcaat ttgtaaaaaa tgg                                  33


<210> 63
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 19 (counted from PAM)

<400> 63
ttatatgaac aaaactcaat ttgtaaaaaa tgg                                  33


<210> 64
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> speM protospacer used for in vitro experiments, mismatch to crRNA
      at position 20 (counted from PAM)

<400> 64
ttatatgaac ttaactcaat ttgtaaaaaa tgg                                  33


<210> 65
<211> 23
<212> DNA
<213> Artificial Sequence


<220> 
<223> DSRed target sequence used in in vivo E.coli experiements (PAM )

<400> 65
tgcaccttga agcgcatgaa ggg                                            23


<210> 66
<211> 20
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgEpCAM-1

<400> 66
gatcctgact gcgatgagag                                                20


<210> 67
<211> 20
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgEpCAM-2

<400> 67
gatcacaacg cgttatcaac                                                20


<210> 68
<211> 20
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgEpCAM-3

<400> 68
taatgttatc actattgatc                                                20


<210> 69
<211> 20
<212> RNA
<213> Artificial Sequence


<220> 
<223> sgEpCAM-4

<400> 69
caagcagaag cccgaacgcg                                                20


<210> 70
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> non-target strand

<400> 70
tgaacataac tcaatttgta aaaaatcccg cag                                  33


<210> 71
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> target strand

<400> 71
ctgcgccatt ttttacaaat tgagttatgt tca                                  33


<210> 72
<211> 28
<212> RNA
<213> Artificial Sequence


<220> 
<223> crRNA strand

<400> 72
auaacucaau uuguaaaaaa guuuuaga                                       28


<210> 73
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> no bubble

<400> 73
tgaacataac tcaatttgta aaaaatggta ctc                                  33


<210> 74
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> no bubble

<400> 74
gagtaccatt ttttacaaat tgagttatgt tca                                  33


<210> 75
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> 5 nt bubble

<400> 75
tgaacataac tcaatttgta ttttttggta ctc                                  33


<210> 76
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> 5 nt bubble

<400> 76
gagtaccatt ttttacaaat tgagttatgt tca                                  33


<210> 77
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> 20 nt bubble

<400> 77
tgaacagcca gaccgggtgc ccccctggta ctc                                  33


<210> 78
<211> 33
<212> DNA
<213> Artificial Sequence


<220> 
<223> 20 nt bubble

<400> 78
gagtaccatt ttttacaaat tgagttatgt tca                                  33


