                         SEQUENCE LISTING

<110>  Keygene N.V.
 
<120>  Methods of targeted genetic alteration in plant cells

<130>  P6069740PCT

<150>  NL 2018381
<151>  2017-02-15

<160>  38    

<170>  PatentIn version 3.5

<210>  1
<211>  5082
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  DNA sequence encoding the rAPOBEC-Cas9-UGI fusion protein

<400>  1
atgggtagca gccatcatca tcaccatcat atgagcagcg aaacaggtcc ggttgcagtt       60

gatccgaccc tgcgtcgtcg tattgaaccg catgaatttg aagttttttt tgatccgcgt      120

gagctgcgta aagaaacctg tctgctgtat gaaattaact ggggtggtcg tcatagcatt      180

tggcgtcata ccagccagaa taccaataaa catgtggaag tgaacttcat cgagaaattt      240

accaccgaac gttatttttg tccgaatacc cgttgtagca ttacctggtt tctgagctgg      300

tcaccgtgtg gtgaatgtag ccgtgcaatt accgaatttc tgagccgtta tccgcatgtt      360

accctgttta tctatattgc ccgtctgtat catcatgcag atccgcgtaa tcgtcagggt      420

ctgcgtgatc tgattagcag cggtgttacc attcagatta tgaccgaaca agaaagcggt      480

tattgctggc gtaattttgt gaattatagc ccgagcaatg aagcacattg gcctcgctat      540

ccgcatctgt gggttcgtct gtatgttctg gaactgtatt gtattattct gggtctgcct      600

ccgtgtctga atattctgcg tcgtaaacag ccgcagctga ccttttttac cattgcactg      660

cagagctgtc attatcagcg tctgccaccg catattctgt gggcaacagg tctgaaaagc      720

ggtagcgaaa caccgggtac aagcgaaagc gcaacaccgg aaagcgacaa aaaatacagc      780

attggtctgg acattggtac caacagcgtg ggctgggccg tgatcaccga cgagtacaag      840

gtgcccagca agaagttcaa ggtgctgggc aacaccgacc gccacagcat caagaagaac      900

ctgatcggcg ccctgctgtt cgacagcggc gagaccgccg aggccacccg cctgaagcgc      960

accgcccgcc gccgctacac ccgccgcaag aaccgcatct gctacctgca ggagatcttc     1020

agcaacgaga tggccaaggt ggacgacagc ttcttccacc gcctggagga gagcttcctg     1080

gtggaggagg acaagaagca cgagcgccac cccatcttcg gcaacatcgt ggacgaggtg     1140

gcctaccacg agaagtaccc caccatctac cacctgcgca agaagctggt ggacagcacc     1200

gacaaggccg acctgcgcct gatctacctg gccctggccc acatgatcaa gttccgcggc     1260

cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc     1320

cagctggtgc agacctacaa ccagctgttc gaggagaacc ccatcaacgc cagcggcgtg     1380

gacgccaagg ccatcctgag cgcccgcctg agcaagagcc gccgcctgga gaacctgatc     1440

gcccagctgc ccggcgagaa gaagaacggc ctgttcggca acctgatcgc cctgagcctg     1500

ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggacgccaa gctgcagctg     1560

agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac     1620

gccgacctgt tcctggccgc caagaacctg agcgacgcca tcctgctgag cgacatcctg     1680

cgcgtgaaca ccgagatcac caaggccccc ctgagcgcca gcatgatcaa gcgctacgac     1740

gagcaccacc aggacctgac cctgctgaag gccctggtgc gccagcagct gcccgagaag     1800

tacaaggaga tcttcttcga ccagagcaag aacggctacg ccggctacat cgacggcggc     1860

gccagccagg aggagttcta caagttcatc aagcccatcc tggagaagat ggacggcacc     1920

gaggagctgc tggtgaagct gaaccgcgag gacctgctgc gcaagcagcg caccttcgac     1980

aacggcagca tcccccacca gatccacctg ggcgagctgc acgccatcct gcgccgccag     2040

gaggacttct accccttcct gaaggacaac cgcgagaaga tcgagaagat cctgaccttc     2100

cgcatcccct actacgtggg ccccctggcc cgcggcaaca gccgcttcgc ctggatgacc     2160

cgcaagagcg aggagaccat caccccctgg aacttcgagg aggtggtgga caagggcgcc     2220

agcgcccaga gcttcatcga gcgcatgacc aacttcgaca agaacctgcc caacgagaag     2280

gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtacaacga gctgaccaag     2340

gtgaagtacg tgaccgaggg catgcgcaag cccgccttcc tgagcggcga gcagaagaag     2400

gccatcgtgg acctgctgtt caagaccaac cgcaaggtga ccgtgaagca gctgaaggag     2460

gactacttca agaagatcga gtgcttcgac agcgtggaga tcagcggcgt ggaggaccgc     2520

ttcaacgcca gcctgggcac ctaccacgac ctgctgaaga tcatcaagga caaggacttc     2580

ctggacaacg aggagaacga ggacatcctg gaggacatcg tgctgaccct gaccctgttc     2640

gaggaccgcg agatgatcga ggagcgcctg aagacctacg cccacctgtt cgacgacaag     2700

gtgatgaagc agctgaagcg ccgccgctac accggctggg gccgcctgag ccgcaagctt     2760

atcaacggca tccgcgacaa gcagagcggc aagaccatcc tggacttcct gaagagcgac     2820

ggcttcgcca accgcaactt catgcagctg atccacgacg acagcctgac cttcaaggag     2880

gacatccaga aggcccaggt gagcggccag ggcgacagcc tgcacgagca catcgccaac     2940

ctggccggca gccccgccat caagaagggc atcctgcaga ccgtgaaggt ggtggacgag     3000

ctggtgaagg tgatgggccg ccacaagccc gagaacatcg tgatcgagat ggcccgcgag     3060

aaccagacca cccagaaggg ccagaagaac agccgcgagc gcatgaagcg catcgaggag     3120

ggcatcaagg agctgggcag ccagatcctg aaggagcacc ccgtggagaa cacccagctg     3180

cagaacgaga agctgtacct gtactacctg cagaacggcc gcgacatgta cgtggaccag     3240

gagctggaca tcaaccgcct gagcgactac gacgtggacc acatcgtgcc ccagagcttc     3300

ctgaaggacg acagcatcga caacaaggtg ctgacccgca gcgacaagaa ccgcggcaag     3360

agcgacaacg tgcccagcga ggaggtggtg aagaagatga agaactactg gcgccagctg     3420

ctgaacgcca agctgatcac ccagcgcaag ttcgacaacc tgaccaaggc cgagcgcggc     3480

ggcctgagcg agctggacaa ggccggcttc atcaagcgcc agctggtgga gacccgccag     3540

atcaccaagc acgtggccca gatcctggac agccgcatga acaccaagta cgacgagaac     3600

gacaagctga tccgcgaggt gaaggtgatc accctgaaga gcaagctggt gagcgacttc     3660

cgcaaggact tccagttcta caaggtgcgc gagatcaaca actaccacca cgcccacgac     3720

gcctacctga acgccgtggt gggcaccgcc ctgatcaaga agtaccccaa gctggagagc     3780

gagttcgtgt acggcgacta caaggtgtac gacgtgcgca agatgatcgc caagagcgag     3840

caggagatcg gcaaggccac cgccaagtac ttcttctaca gcaacatcat gaacttcttc     3900

aagaccgaga tcaccctggc caacggcgag atccgcaagc gccccctgat cgagaccaac     3960

ggcgagaccg gcgagatcgt gtgggacaag ggccgcgact tcgccaccgt gcgcaaggtg     4020

ctgagcatgc cccaggtgaa catcgtgaag aagaccgagg tgcagaccgg cggcttcagc     4080

aaggagagca tcctgcccaa gcgcaacagc gacaagctga tcgcccgcaa gaaggactgg     4140

gaccccaaga agtacggcgg cttcgacagc cccaccgtgg cctacagcgt gctggtggtg     4200

gccaaggtgg agaagggcaa gagcaagaag ctgaagagcg tgaaggagct gctgggcatc     4260

accatcatgg agcgcagcag cttcgagaag aaccccatcg acttcctgga ggccaagggc     4320

tacaaggagg tgaagaagga cctgatcatc aagctgccca agtacagcct gttcgagctg     4380

gagaacggcc gcaagcgcat gctggccagc gccggcgagc tgcagaaggg caacgagctg     4440

gccctgccca gcaagtacgt gaacttcctg tacctggcca gccactacga gaagctgaag     4500

ggcagccccg aggacaacga gcagaagcag ctgttcgtgg agcagcacaa gcactacctg     4560

gacgagatca tcgagcagat cagcgagttc agcaagcgcg tgatcctggc cgacgccaac     4620

ctggacaagg tgctgagcgc ctacaacaag caccgcgaca agcccatccg cgagcaggcc     4680

gagaacatca tccacctgtt caccctgacc aacctgggcg cccccgccgc cttcaagtac     4740

ttcgacacca ccatcgaccg caagcgctac accagcacca aggaggtgct ggacgccacc     4800

ctgatccacc agagcatcac cggtctgtat gaaacccgta ttgatctgag ccagctgggt     4860

ggtgatagcg gtggtagcac caatctgagc gatatcattg aaaaagaaac cggcaaacag     4920

ctggtgattc aagaaagcat tctgatgctg cctgaagaag tggaagaagt tattggtaat     4980

aaaccggaaa gcgatattct ggttcatacc gcatatgatg aaagcaccga tgaaaatgtt     5040

atgctgagcg gtggttcccc gaaaaaaaaa cgtaaagttt aa                        5082


<210>  2
<211>  1693
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  rAPOBEC-Cas9-UGI fusion protein

<400>  2

Met Gly Ser Ser His His His His His His Met Ser Ser Glu Thr Gly 
1               5                   10                  15      


Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro His Glu 
            20                  25                  30          


Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu 
        35                  40                  45              


Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg His Thr 
    50                  55                  60                  


Ser Gln Asn Thr Asn Lys His Val Glu Val Asn Phe Ile Glu Lys Phe 
65                  70                  75                  80  


Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp 
                85                  90                  95      


Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu 
            100                 105                 110         


Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg 
        115                 120                 125             


Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu 
    130                 135                 140                 


Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser Gly 
145                 150                 155                 160 


Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu Ala His 
                165                 170                 175     


Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu Glu Leu 
            180                 185                 190         


Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg 
        195                 200                 205             


Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser Cys His 
    210                 215                 220                 


Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu Lys Ser 
225                 230                 235                 240 


Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Asp 
                245                 250                 255     


Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp 
            260                 265                 270         


Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val 
        275                 280                 285             


Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala 
    290                 295                 300                 


Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg 
305                 310                 315                 320 


Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu 
                325                 330                 335     


Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe 
            340                 345                 350         


His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu 
        355                 360                 365             


Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu 
    370                 375                 380                 


Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr 
385                 390                 395                 400 


Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile 
                405                 410                 415     


Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn 
            420                 425                 430         


Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln 
        435                 440                 445             


Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala 
    450                 455                 460                 


Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile 
465                 470                 475                 480 


Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile 
                485                 490                 495     


Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu 
            500                 505                 510         


Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp 
        515                 520                 525             


Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe 
    530                 535                 540                 


Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu 
545                 550                 555                 560 


Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile 
                565                 570                 575     


Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu 
            580                 585                 590         


Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln 
        595                 600                 605             


Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu 
    610                 615                 620                 


Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr 
625                 630                 635                 640 


Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln 
                645                 650                 655     


Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu 
            660                 665                 670         


Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys 
        675                 680                 685             


Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr 
    690                 695                 700                 


Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr 
705                 710                 715                 720 


Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val 
                725                 730                 735     


Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe 
            740                 745                 750         


Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu 
        755                 760                 765             


Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val 
    770                 775                 780                 


Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys 
785                 790                 795                 800 


Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys 
                805                 810                 815     


Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val 
            820                 825                 830         


Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr 
        835                 840                 845             


His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu 
    850                 855                 860                 


Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe 
865                 870                 875                 880 


Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu 
                885                 890                 895     


Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly 
            900                 905                 910         


Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln 
        915                 920                 925             


Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn 
    930                 935                 940                 


Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu 
945                 950                 955                 960 


Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu 
                965                 970                 975     


His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu 
            980                 985                 990         


Gln Thr Val Lys Val Val Asp Glu  Leu Val Lys Val Met  Gly Arg His 
        995                 1000                 1005             


Lys Pro  Glu Asn Ile Val Ile  Glu Met Ala Arg Glu  Asn Gln Thr 
    1010                 1015                 1020             


Thr Gln  Lys Gly Gln Lys Asn  Ser Arg Glu Arg Met  Lys Arg Ile 
    1025                 1030                 1035             


Glu Glu  Gly Ile Lys Glu Leu  Gly Ser Gln Ile Leu  Lys Glu His 
    1040                 1045                 1050             


Pro Val  Glu Asn Thr Gln Leu  Gln Asn Glu Lys Leu  Tyr Leu Tyr 
    1055                 1060                 1065             


Tyr Leu  Gln Asn Gly Arg Asp  Met Tyr Val Asp Gln  Glu Leu Asp 
    1070                 1075                 1080             


Ile Asn  Arg Leu Ser Asp Tyr  Asp Val Asp His Ile  Val Pro Gln 
    1085                 1090                 1095             


Ser Phe  Leu Lys Asp Asp Ser  Ile Asp Asn Lys Val  Leu Thr Arg 
    1100                 1105                 1110             


Ser Asp  Lys Asn Arg Gly Lys  Ser Asp Asn Val Pro  Ser Glu Glu 
    1115                 1120                 1125             


Val Val  Lys Lys Met Lys Asn  Tyr Trp Arg Gln Leu  Leu Asn Ala 
    1130                 1135                 1140             


Lys Leu  Ile Thr Gln Arg Lys  Phe Asp Asn Leu Thr  Lys Ala Glu 
    1145                 1150                 1155             


Arg Gly  Gly Leu Ser Glu Leu  Asp Lys Ala Gly Phe  Ile Lys Arg 
    1160                 1165                 1170             


Gln Leu  Val Glu Thr Arg Gln  Ile Thr Lys His Val  Ala Gln Ile 
    1175                 1180                 1185             


Leu Asp  Ser Arg Met Asn Thr  Lys Tyr Asp Glu Asn  Asp Lys Leu 
    1190                 1195                 1200             


Ile Arg  Glu Val Lys Val Ile  Thr Leu Lys Ser Lys  Leu Val Ser 
    1205                 1210                 1215             


Asp Phe  Arg Lys Asp Phe Gln  Phe Tyr Lys Val Arg  Glu Ile Asn 
    1220                 1225                 1230             


Asn Tyr  His His Ala His Asp  Ala Tyr Leu Asn Ala  Val Val Gly 
    1235                 1240                 1245             


Thr Ala  Leu Ile Lys Lys Tyr  Pro Lys Leu Glu Ser  Glu Phe Val 
    1250                 1255                 1260             


Tyr Gly  Asp Tyr Lys Val Tyr  Asp Val Arg Lys Met  Ile Ala Lys 
    1265                 1270                 1275             


Ser Glu  Gln Glu Ile Gly Lys  Ala Thr Ala Lys Tyr  Phe Phe Tyr 
    1280                 1285                 1290             


Ser Asn  Ile Met Asn Phe Phe  Lys Thr Glu Ile Thr  Leu Ala Asn 
    1295                 1300                 1305             


Gly Glu  Ile Arg Lys Arg Pro  Leu Ile Glu Thr Asn  Gly Glu Thr 
    1310                 1315                 1320             


Gly Glu  Ile Val Trp Asp Lys  Gly Arg Asp Phe Ala  Thr Val Arg 
    1325                 1330                 1335             


Lys Val  Leu Ser Met Pro Gln  Val Asn Ile Val Lys  Lys Thr Glu 
    1340                 1345                 1350             


Val Gln  Thr Gly Gly Phe Ser  Lys Glu Ser Ile Leu  Pro Lys Arg 
    1355                 1360                 1365             


Asn Ser  Asp Lys Leu Ile Ala  Arg Lys Lys Asp Trp  Asp Pro Lys 
    1370                 1375                 1380             


Lys Tyr  Gly Gly Phe Asp Ser  Pro Thr Val Ala Tyr  Ser Val Leu 
    1385                 1390                 1395             


Val Val  Ala Lys Val Glu Lys  Gly Lys Ser Lys Lys  Leu Lys Ser 
    1400                 1405                 1410             


Val Lys  Glu Leu Leu Gly Ile  Thr Ile Met Glu Arg  Ser Ser Phe 
    1415                 1420                 1425             


Glu Lys  Asn Pro Ile Asp Phe  Leu Glu Ala Lys Gly  Tyr Lys Glu 
    1430                 1435                 1440             


Val Lys  Lys Asp Leu Ile Ile  Lys Leu Pro Lys Tyr  Ser Leu Phe 
    1445                 1450                 1455             


Glu Leu  Glu Asn Gly Arg Lys  Arg Met Leu Ala Ser  Ala Gly Glu 
    1460                 1465                 1470             


Leu Gln  Lys Gly Asn Glu Leu  Ala Leu Pro Ser Lys  Tyr Val Asn 
    1475                 1480                 1485             


Phe Leu  Tyr Leu Ala Ser His  Tyr Glu Lys Leu Lys  Gly Ser Pro 
    1490                 1495                 1500             


Glu Asp  Asn Glu Gln Lys Gln  Leu Phe Val Glu Gln  His Lys His 
    1505                 1510                 1515             


Tyr Leu  Asp Glu Ile Ile Glu  Gln Ile Ser Glu Phe  Ser Lys Arg 
    1520                 1525                 1530             


Val Ile  Leu Ala Asp Ala Asn  Leu Asp Lys Val Leu  Ser Ala Tyr 
    1535                 1540                 1545             


Asn Lys  His Arg Asp Lys Pro  Ile Arg Glu Gln Ala  Glu Asn Ile 
    1550                 1555                 1560             


Ile His  Leu Phe Thr Leu Thr  Asn Leu Gly Ala Pro  Ala Ala Phe 
    1565                 1570                 1575             


Lys Tyr  Phe Asp Thr Thr Ile  Asp Arg Lys Arg Tyr  Thr Ser Thr 
    1580                 1585                 1590             


Lys Glu  Val Leu Asp Ala Thr  Leu Ile His Gln Ser  Ile Thr Gly 
    1595                 1600                 1605             


Leu Tyr  Glu Thr Arg Ile Asp  Leu Ser Gln Leu Gly  Gly Asp Ser 
    1610                 1615                 1620             


Gly Gly  Ser Thr Asn Leu Ser  Asp Ile Ile Glu Lys  Glu Thr Gly 
    1625                 1630                 1635             


Lys Gln  Leu Val Ile Gln Glu  Ser Ile Leu Met Leu  Pro Glu Glu 
    1640                 1645                 1650             


Val Glu  Glu Val Ile Gly Asn  Lys Pro Glu Ser Asp  Ile Leu Val 
    1655                 1660                 1665             


His Thr  Ala Tyr Asp Glu Ser  Thr Asp Glu Asn Val  Met Leu Ser 
    1670                 1675                 1680             


Gly Gly  Ser Pro Lys Lys Lys  Arg Lys Val 
    1685                 1690             


<210>  3
<211>  5328
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  DNA sequence encoding the TadA-Cas9 D10A-NLS fusion protein

<400>  3
atgtccgagg tcgagttctc tcatgagtac tggatgaggc acgctctcac tcttgctaaa       60

agagcttggg acgagagaga ggttccagtt ggagctgttt tggtgcacaa caaccgtgtg      120

attggcgaag gatggaacag gccaattgga aggcatgatc caactgctca cgctgagatt      180

atggccctta gacaaggtgg actcgtgatg cagaactaca ggcttatcga cgccactctc      240

tacgtgacac ttgagccatg tgttatgtgc gctggtgcca tgattcactc caggattgga      300

agggttgtgt tcggagctag agatgctaaa actggcgctg ccggatctct catggatgtg      360

cttcatcatc ctgggatgaa ccacagggtt gagatcactg agggaatcct tgctgatgag      420

tgcgctgctc tcctgtctga ttttttcagg atgaggcgtc aagagatcaa ggcccagaag      480

aaggctcagt cctctactga ttctggcgga tcttctggtg ggtcatctgg atctgaaacc      540

cctggaactt ctgagtccgc tactccagaa tcatccggtg gatctagtgg tggttctagc      600

gaggttgagt tcagccacga atactggatg cgtcacgcac ttactttggc taagagggct      660

cgtgatgaga gggaagttcc tgttggtgca gtgctcgtgc ttaacaacag agtgatcgga      720

gaaggctgga atcgtgctat cggacttcat gatcctaccg cacatgcaga gatcatggct      780

ttgaggcaag gtgggcttgt catgcaaaat taccgtctga tcgacgctac cttgtacgtc      840

acattcgagc cttgcgtgat gtgtgctggg gctatgatcc attctaggat cggtagagtg      900

gtgttcggtg tgaggaatgc taagacaggt gctgctggct cacttatgga tgtgttgcat      960

taccccggca tgaaccaccg tgtggaaatt acagagggca tcttggcaga tgagtgtgcc     1020

gctcttttgt gctacttctt caggatgcca cgtcaggtgt tcaacgctca aaagaaggcc     1080

caatccagca ccgattctgg tggtagtagt ggtggatctt ccggatcaga gactcctggt     1140

actagtgagt ctgctacccc tgaaagtagc ggaggttcaa gtggtgggtc cgacaagaag     1200

tactctatcg gattggctat cgggaccaac tctgttggat gggctgtgat cactgacgag     1260

tacaaggtgc cctccaagaa gttcaaggtt ctcggaaaca ccgacaggca ctccatcaag     1320

aagaacctca tcggggctct gcttttcgat tcaggtgaaa ctgctgaggc caccaggctt     1380

aagagaactg ctagaagaag gtacacccgt aggaagaaca ggatctgcta cctccaagag     1440

atcttctcca acgagatggc taaggtggac gactcattct tccacaggct cgaagagtcc     1500

ttcttggtgg aagaggataa gaagcacgag aggcacccaa tcttcgggaa cattgtggat     1560

gaagtggcct accacgagaa gtacccaacc atctaccacc tgaggaagaa gctcgttgac     1620

tccaccgata aggctgacct gaggcttatc taccttgctc tcgctcacat gatcaagttc     1680

cgtgggcact tccttatcga aggggatctg aacccagaca actccgatgt ggacaagctg     1740

ttcattcagc tcgtgcagac ctacaaccag ctgttcgaag agaacccaat caacgcttct     1800

ggtgtggacg ctaaggctat cctttctgcc aggctttcca agtccagaag gcttgagaac     1860

ctgattgctc agcttcctgg ggagaagaag aacggacttt tcgggaacct gatcgccctc     1920

tctcttggac ttactcccaa cttcaagtcc aacttcgacc tcgctgagga tgccaagctt     1980

cagctctcta aggataccta cgatgacgac ctcgacaacc tccttgctca gattggagat     2040

cagtacgccg accttttcct cgccgctaaa aacctctctg acgccatcct cctgtccgat     2100

attcttaggg tgaacaccga gatcaccaag gcaccacttt ccgcctctat gatcaagcgt     2160

tacgatgagc accaccagga cctcactttg cttaaggctc ttgttaggca gcagctccca     2220

gagaagtaca aagagatttt cttcgaccag tccaagaacg ggtacgccgg ttatattgat     2280

ggtggggctt ctcaagaaga gttctacaag ttcatcaagc ccatcttgga aaagatggac     2340

gggaccgaag agttgctcgt gaagcttaac cgtgaggacc ttcttaggaa gcagcgaact     2400

ttcgacaacg gctctattcc tcaccagatc caccttggag agctgcacgc tattcttcgt     2460

aggcaagagg acttctaccc attcctcaag gacaaccgtg agaagatcga gaagattctc     2520

accttcagga tcccttacta cgtgggacca cttgctaggg gaaattctag gttcgcttgg     2580

atgacccgta agagcgaaga gactatcact ccatggaact tcgaagaggt ggtggacaaa     2640

ggtgctagcg ctcagtcttt catcgagagg atgactaact tcgacaagaa cctgccaaac     2700

gagaaggtgc tcccaaagca ctctctgctc tacgagtact tcaccgtgta caacgagctg     2760

accaaggtca agtatgtgac cgagggaatg cgtaagccag ctttccttag tggtgagcag     2820

aaaaaggcca tcgtggacct cttgttcaag accaatagaa aggtgaccgt gaagcagctc     2880

aaagaggact acttcaaaaa gatcgagtgc ttcgactccg tcgagatctc tggtgttgag     2940

gataggttca acgcctcctt gggaacttac cacgacctcc tcaagatcat caaggataag     3000

gatttcttgg acaacgagga aaacgaggac atcttggagg acatcgtgct cacccttacc     3060

ttgttcgagg atcgagagat gatcgaggaa cgactcaaga cctacgctca cctgttcgat     3120

gacaaggtca tgaagcagtt gaagaggcgt aggtacactg gatggggacg tttgtcccgt     3180

aagctcatta acggaatcag ggacaagcag tccggcaaga ctatcctcga cttcctcaag     3240

tctgatgggt tcgccaaccg taacttcatg cagctcatcc acgacgacag cctgaccttt     3300

aaagaggaca tccaaaaggc ccaggtgtcc ggtcaaggcg attctcttca tgagcacatt     3360

gctaacctcg ctgggtcacc agctatcaag aagggaattc tccagactgt gaaggtcgtg     3420

gacgagttgg ttaaggtgat gggtagacac aagcccgaga acatcgtgat tgagatggct     3480

cgtgagaacc agactactca gaaggggcag aagaactcca gggaacgtat gaagaggatc     3540

gaagagggga tcaaagagct ggggtcccag attcttaaag agcacccagt tgagaacacc     3600

cagctccaga atgagaagct ctacctctac tacctgcaga acggcaggga tatgtacgtg     3660

gaccaagagc tggatatcaa caggctctcc gactacgatg ttgaccacat tgtgccccag     3720

tctttcttga aggacgactc catcgacaac aaggtgctca ccaggtctga taagaaccgt     3780

gggaagtctg acaacgtgcc atctgaagag gtcgtgaaga agatgaagaa ctactggcgt     3840

cagctcctca acgccaagct tattactcag aggaagttcg acaacttgac caaggctgag     3900

cgtggtggac tttccgaact tgataaggcc ggattcatca agaggcagct cgtggaaact     3960

aggcagatca ctaagcacgt ggcccagatc ttggactcta ggatgaacac caagtacgac     4020

gagaacgaca agctcatccg tgaggtgaag gtcatcaccc tcaagagcaa gctggtgtcc     4080

gatttcagaa aggacttcca attctacaag gtgagagaga tcaacaacta ccatcacgct     4140

cacgacgctt accttaacgc tgttgttgga accgctctca tcaaaaagta ccccaagctc     4200

gagtccgagt tcgtgtacgg tgattacaag gtgtacgacg tgcgtaagat gatcgccaag     4260

tcagagcaag agatcggtaa ggctaccgcc aagtatttct tctactccaa catcatgaat     4320

ttcttcaaga ctgagatcac cctcgccaac ggggagatta gaaagaggcc acttatcgag     4380

actaacggcg agactggtga aatcgtgtgg gataagggaa gagacttcgc cactgtgcgt     4440

aaggtgttgt ctatgccaca ggtgaacatc gtcaagaaaa ccgaggttca gaccggcggg     4500

ttctccaaag aatctatcct tccaaagagg aactccgaca agctgatcgc taggaagaag     4560

gattgggacc caaaaaagta cggtgggttc gattctccaa ccgtggctta ctctgttctt     4620

gttgtggcca aggttgagaa ggggaagtct aagaaactca agtccgtgaa agagctgctc     4680

gggatcacta tcatggaaag gtccagcttc gagaagaatc caatcgattt cctcgaggcc     4740

aagggctaca aagaggtgaa gaaggacctt atcatcaagc tccccaagta cagcctcttc     4800

gagttggaga acggacgtaa gaggatgctt gcttctgctg gggaacttca gaagggaaac     4860

gaactcgctc tgccctctaa gtacgtgaac ttcctgtacc tcgcttccca ctacgagaag     4920

cttaagggat ctccagagga taacgagcaa aagcagcttt tcgtcgagca gcacaagcac     4980

tacctcgacg agattatcga gcagatctcc gagttctcca agcgtgtgat tctcgctgat     5040

gccaacttgg acaaggtgtt gagcgcttac aacaagcacc gtgacaagcc aattagagag     5100

caggctgaga acatcatcca cctgttcact ctcaccaacc ttggtgctcc agctgccttt     5160

aagtacttcg ataccaccat cgaccgtaag aggtacacct ccaccaaaga ggttttggac     5220

gctaccctta tccaccagtc catcactgga ctttacgaga ctaggatcga cctctcacag     5280

ctcggtggtg actctggtgg atcaccaaag aagaagagga aggtctaa                  5328


<210>  4
<211>  1775
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  TadA-Cas9 D10A-NLS fusion protein

<400>  4

Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 
1               5                   10                  15      


Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 
            20                  25                  30          


Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 
        35                  40                  45              


Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 
    50                  55                  60                  


Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 
65                  70                  75                  80  


Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 
                85                  90                  95      


Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly 
            100                 105                 110         


Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 
        115                 120                 125             


Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 
    130                 135                 140                 


Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys 
145                 150                 155                 160 


Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser 
                165                 170                 175     


Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser 
            180                 185                 190         


Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr 
        195                 200                 205             


Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg 
    210                 215                 220                 


Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly 
225                 230                 235                 240 


Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala 
                245                 250                 255     


Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg 
            260                 265                 270         


Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys 
        275                 280                 285             


Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val 
    290                 295                 300                 


Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His 
305                 310                 315                 320 


Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala 
                325                 330                 335     


Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln 
            340                 345                 350         


Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly 
        355                 360                 365             


Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser 
    370                 375                 380                 


Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys 
385                 390                 395                 400 


Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val 
                405                 410                 415     


Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly 
            420                 425                 430         


Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu 
        435                 440                 445             


Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala 
    450                 455                 460                 


Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu 
465                 470                 475                 480 


Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg 
                485                 490                 495     


Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His 
            500                 505                 510         


Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr 
        515                 520                 525             


Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys 
    530                 535                 540                 


Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe 
545                 550                 555                 560 


Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp 
                565                 570                 575     


Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe 
            580                 585                 590         


Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu 
        595                 600                 605             


Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln 
    610                 615                 620                 


Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu 
625                 630                 635                 640 


Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu 
                645                 650                 655     


Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp 
            660                 665                 670         


Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala 
        675                 680                 685             


Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val 
    690                 695                 700                 


Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg 
705                 710                 715                 720 


Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg 
                725                 730                 735     


Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys 
            740                 745                 750         


Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe 
        755                 760                 765             


Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu 
    770                 775                 780                 


Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr 
785                 790                 795                 800 


Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His 
                805                 810                 815     


Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn 
            820                 825                 830         


Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val 
        835                 840                 845             


Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys 
    850                 855                 860                 


Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys 
865                 870                 875                 880 


Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys 
                885                 890                 895     


Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu 
            900                 905                 910         


Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu 
        915                 920                 925             


Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile 
    930                 935                 940                 


Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu 
945                 950                 955                 960 


Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile 
                965                 970                 975     


Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp 
            980                 985                 990         


Leu Leu Lys Ile Ile Lys Asp Lys  Asp Phe Leu Asp Asn  Glu Glu Asn 
        995                 1000                 1005             


Glu Asp  Ile Leu Glu Asp Ile  Val Leu Thr Leu Thr  Leu Phe Glu 
    1010                 1015                 1020             


Asp Arg  Glu Met Ile Glu Glu  Arg Leu Lys Thr Tyr  Ala His Leu 
    1025                 1030                 1035             


Phe Asp  Asp Lys Val Met Lys  Gln Leu Lys Arg Arg  Arg Tyr Thr 
    1040                 1045                 1050             


Gly Trp  Gly Arg Leu Ser Arg  Lys Leu Ile Asn Gly  Ile Arg Asp 
    1055                 1060                 1065             


Lys Gln  Ser Gly Lys Thr Ile  Leu Asp Phe Leu Lys  Ser Asp Gly 
    1070                 1075                 1080             


Phe Ala  Asn Arg Asn Phe Met  Gln Leu Ile His Asp  Asp Ser Leu 
    1085                 1090                 1095             


Thr Phe  Lys Glu Asp Ile Gln  Lys Ala Gln Val Ser  Gly Gln Gly 
    1100                 1105                 1110             


Asp Ser  Leu His Glu His Ile  Ala Asn Leu Ala Gly  Ser Pro Ala 
    1115                 1120                 1125             


Ile Lys  Lys Gly Ile Leu Gln  Thr Val Lys Val Val  Asp Glu Leu 
    1130                 1135                 1140             


Val Lys  Val Met Gly Arg His  Lys Pro Glu Asn Ile  Val Ile Glu 
    1145                 1150                 1155             


Met Ala  Arg Glu Asn Gln Thr  Thr Gln Lys Gly Gln  Lys Asn Ser 
    1160                 1165                 1170             


Arg Glu  Arg Met Lys Arg Ile  Glu Glu Gly Ile Lys  Glu Leu Gly 
    1175                 1180                 1185             


Ser Gln  Ile Leu Lys Glu His  Pro Val Glu Asn Thr  Gln Leu Gln 
    1190                 1195                 1200             


Asn Glu  Lys Leu Tyr Leu Tyr  Tyr Leu Gln Asn Gly  Arg Asp Met 
    1205                 1210                 1215             


Tyr Val  Asp Gln Glu Leu Asp  Ile Asn Arg Leu Ser  Asp Tyr Asp 
    1220                 1225                 1230             


Val Asp  His Ile Val Pro Gln  Ser Phe Leu Lys Asp  Asp Ser Ile 
    1235                 1240                 1245             


Asp Asn  Lys Val Leu Thr Arg  Ser Asp Lys Asn Arg  Gly Lys Ser 
    1250                 1255                 1260             


Asp Asn  Val Pro Ser Glu Glu  Val Val Lys Lys Met  Lys Asn Tyr 
    1265                 1270                 1275             


Trp Arg  Gln Leu Leu Asn Ala  Lys Leu Ile Thr Gln  Arg Lys Phe 
    1280                 1285                 1290             


Asp Asn  Leu Thr Lys Ala Glu  Arg Gly Gly Leu Ser  Glu Leu Asp 
    1295                 1300                 1305             


Lys Ala  Gly Phe Ile Lys Arg  Gln Leu Val Glu Thr  Arg Gln Ile 
    1310                 1315                 1320             


Thr Lys  His Val Ala Gln Ile  Leu Asp Ser Arg Met  Asn Thr Lys 
    1325                 1330                 1335             


Tyr Asp  Glu Asn Asp Lys Leu  Ile Arg Glu Val Lys  Val Ile Thr 
    1340                 1345                 1350             


Leu Lys  Ser Lys Leu Val Ser  Asp Phe Arg Lys Asp  Phe Gln Phe 
    1355                 1360                 1365             


Tyr Lys  Val Arg Glu Ile Asn  Asn Tyr His His Ala  His Asp Ala 
    1370                 1375                 1380             


Tyr Leu  Asn Ala Val Val Gly  Thr Ala Leu Ile Lys  Lys Tyr Pro 
    1385                 1390                 1395             


Lys Leu  Glu Ser Glu Phe Val  Tyr Gly Asp Tyr Lys  Val Tyr Asp 
    1400                 1405                 1410             


Val Arg  Lys Met Ile Ala Lys  Ser Glu Gln Glu Ile  Gly Lys Ala 
    1415                 1420                 1425             


Thr Ala  Lys Tyr Phe Phe Tyr  Ser Asn Ile Met Asn  Phe Phe Lys 
    1430                 1435                 1440             


Thr Glu  Ile Thr Leu Ala Asn  Gly Glu Ile Arg Lys  Arg Pro Leu 
    1445                 1450                 1455             


Ile Glu  Thr Asn Gly Glu Thr  Gly Glu Ile Val Trp  Asp Lys Gly 
    1460                 1465                 1470             


Arg Asp  Phe Ala Thr Val Arg  Lys Val Leu Ser Met  Pro Gln Val 
    1475                 1480                 1485             


Asn Ile  Val Lys Lys Thr Glu  Val Gln Thr Gly Gly  Phe Ser Lys 
    1490                 1495                 1500             


Glu Ser  Ile Leu Pro Lys Arg  Asn Ser Asp Lys Leu  Ile Ala Arg 
    1505                 1510                 1515             


Lys Lys  Asp Trp Asp Pro Lys  Lys Tyr Gly Gly Phe  Asp Ser Pro 
    1520                 1525                 1530             


Thr Val  Ala Tyr Ser Val Leu  Val Val Ala Lys Val  Glu Lys Gly 
    1535                 1540                 1545             


Lys Ser  Lys Lys Leu Lys Ser  Val Lys Glu Leu Leu  Gly Ile Thr 
    1550                 1555                 1560             


Ile Met  Glu Arg Ser Ser Phe  Glu Lys Asn Pro Ile  Asp Phe Leu 
    1565                 1570                 1575             


Glu Ala  Lys Gly Tyr Lys Glu  Val Lys Lys Asp Leu  Ile Ile Lys 
    1580                 1585                 1590             


Leu Pro  Lys Tyr Ser Leu Phe  Glu Leu Glu Asn Gly  Arg Lys Arg 
    1595                 1600                 1605             


Met Leu  Ala Ser Ala Gly Glu  Leu Gln Lys Gly Asn  Glu Leu Ala 
    1610                 1615                 1620             


Leu Pro  Ser Lys Tyr Val Asn  Phe Leu Tyr Leu Ala  Ser His Tyr 
    1625                 1630                 1635             


Glu Lys  Leu Lys Gly Ser Pro  Glu Asp Asn Glu Gln  Lys Gln Leu 
    1640                 1645                 1650             


Phe Val  Glu Gln His Lys His  Tyr Leu Asp Glu Ile  Ile Glu Gln 
    1655                 1660                 1665             


Ile Ser  Glu Phe Ser Lys Arg  Val Ile Leu Ala Asp  Ala Asn Leu 
    1670                 1675                 1680             


Asp Lys  Val Leu Ser Ala Tyr  Asn Lys His Arg Asp  Lys Pro Ile 
    1685                 1690                 1695             


Arg Glu  Gln Ala Glu Asn Ile  Ile His Leu Phe Thr  Leu Thr Asn 
    1700                 1705                 1710             


Leu Gly  Ala Pro Ala Ala Phe  Lys Tyr Phe Asp Thr  Thr Ile Asp 
    1715                 1720                 1725             


Arg Lys  Arg Tyr Thr Ser Thr  Lys Glu Val Leu Asp  Ala Thr Leu 
    1730                 1735                 1740             


Ile His  Gln Ser Ile Thr Gly  Leu Tyr Glu Thr Arg  Ile Asp Leu 
    1745                 1750                 1755             


Ser Gln  Leu Gly Gly Asp Ser  Gly Gly Ser Pro Lys  Lys Lys Arg 
    1760                 1765                 1770             


Lys Val  
    1775 


<210>  5
<211>  183
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  LIN5 sgRNA1

<400>  5
ggagtgatca aaagtcccac atcgatcagg tgatatatag cagcttagtt tatataatga       60

tagagtcgac atagcgattg cctgacgatg aaattaagaa gttttagagc tagaaatagc      120

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt      180

ttt                                                                    183


<210>  6
<211>  183
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  LIN5 sgRNA2

<400>  6
ggagtgatca aaagtcccac atcgatcagg tgatatatag cagcttagtt tatataatga       60

tagagtcgac atagcgattg ttcatcgtca ggtaatacat gttttagagc tagaaatagc      120

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt      180

ttt                                                                    183


<210>  7
<211>  183
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  ALS2 sgRNA1

<400>  7
ggagtgatca aaagtcccac atcgatcagg tgatatatag cagcttagtt tatataatga       60

tagagtcgac atagcgattg caagtgccga ggaggatgat gttttagagc tagaaatagc      120

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt      180

ttt                                                                    183


<210>  8
<211>  183
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  ALS2 sgRNA2

<400>  8
ggagtgatca aaagtcccac atcgatcagg tgatatatag cagcttagtt tatataatga       60

tagagtcgac atagcgattg catcctcctc ggcacttgac gttttagagc tagaaatagc      120

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt      180

ttt                                                                    183


<210>  9
<211>  183
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  ALS2 sgRNA3

<400>  9
ggagtgatca aaagtcccac atcgatcagg tgatatatag cagcttagtt tatataatga       60

tagagtcgac atagcgattg ttaccggtca agtgccgagg gttttagagc tagaaatagc      120

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt      180

ttt                                                                    183


<210>  10
<211>  183
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  ALS2 S640 sgRNA1

<400>  10
ggagtgatca aaagtcccac atcgatcagg tgatatatag cagcttagtt tatataatga       60

tagagtcgac atagcgattg gcaccgccac tgggaatcat gttttagagc tagaaatagc      120

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt      180

ttt                                                                    183


<210>  11
<211>  183
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  ALS2 S640 sgRNA2

<400>  11
ggagtgatca aaagtcccac atcgatcagg tgatatatag cagcttagtt tatataatga       60

tagagtcgac atagcgattg tctttgaaag caccgccact gttttagagc tagaaatagc      120

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt      180

ttt                                                                    183


<210>  12
<211>  183
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  ALS2 S640 sgRNA3

<400>  12
ggagtgatca aaagtcccac atcgatcagg tgatatatag cagcttagtt tatataatga       60

tagagtcgac atagcgattg ctaccgatga ttcccagtgg gttttagagc tagaaatagc      120

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt      180

ttt                                                                    183


<210>  13
<211>  23
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  LIN5 target site

<400>  13
cctgacgatg aaattaagaa agg                                               23


<210>  14
<211>  23
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  LIN5 target site

<400>  14
cctgacgatg aaattaagaa agg                                               23


<210>  15
<211>  23
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  LIN5 target site

<400>  15
ccgatgtatt acctgacgat gaa                                               23


<210>  16
<211>  23
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  LIN5 target site

<400>  16
cctgacgatg aaattaagaa agg                                               23


<210>  17
<211>  23
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  LIN5 target site

<400>  17
cctgacgatg aaattaagaa agg                                               23


<210>  18
<211>  20
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  WT target sequence

<400>  18
atgtattacc tgacgatgaa                                                   20


<210>  19
<211>  20
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  line 1 target sequence

<400>  19
atgtattayc tgaygatgaa                                                   20


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

<220>
<223>  line 2 target sequence

<400>  20
atgtattacy tgaygatgaa                                                   20


<210>  21
<211>  20
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  line 3 target sequence

<400>  21
atgtattayy tgaygatgaa                                                   20


<210>  22
<211>  20
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  line 4 target sequence

<400>  22
atgtattacc tgacratraa                                                   20


<210>  23
<211>  20
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  line 5 target sequence

<400>  23
atgtattacc tracratraa                                                   20


<210>  24
<211>  20
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  line 6 target sequence

<400>  24
atgtattacc taacaataaa                                                   20


<210>  25
<211>  39
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  target site

<400>  25
tgctattacc ggtcaagtgc cgaggaggat gattggtac                              39


<210>  26
<211>  39
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  target site

<400>  26
acgataatgg ccagttcacg gctcctccta ctaaccatg                              39


<210>  27
<211>  39
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  target site

<400>  27
tgttctaccg atgattccca gtggcggtgc tttcaaaga                              39


<210>  28
<211>  39
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  target site

<400>  28
acaagatggc tactaagggt caccgccacg aaagtttct                              39


<210>  29
<211>  39
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  P184 ref

<400>  29
tgctattacc ggtcaagtgc cgaggaggat gattggtac                              39


<210>  30
<211>  39
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  S640 ref

<400>  30
tgttctaccg atgattccca gtggcggtgc tttcaaaga                              39


<210>  31
<211>  39
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  S640 ref

<400>  31
tgttctaccg atgattccca gtggcggtgc tttcaaaga                              39


<210>  32
<211>  11
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  linker

<400>  32

Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser 
1               5                   10      


<210>  33
<211>  16
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  linker

<400>  33

Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 
1               5                   10                  15      


<210>  34
<211>  17
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  linker

<400>  34

Lys Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 
1               5                   10                  15      


Ser 
    


<210>  35
<211>  21
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  forward primer

<400>  35
cactattggc atgtatcaca c                                                 21


<210>  36
<211>  22
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  reversed primer

<400>  36
gtgatgctga gatcccttta ac                                                22


<210>  37
<211>  6
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  His-tag

<400>  37

His His His His His His 
1               5       


<210>  38
<211>  5
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  linker

<400>  38

Glu Ala Ala Ala Lys 
1               5   


