NOVEL CRISPR ENZYMES, METHODS, SYSTEMS AND USES THEREOF
The present invention provides novel systems, methods and compositions for making and using recombinantly engineered novel Cas9 enzymes optimized for human cells, for nucleic acid targeting and manipulation. The present invention is based on the discovery of novel Cas9 enzymes from Streptococcus constellatus, Sharpen spp. isolate RUG017, Veillonella parvula, Ezakiella peruensis, Lactobacillus fermentum strain AF15-40LB strain and Peptoniphilus sp. Marseille-P3761 bacteria that were codon-optimized and recombinantly produced for use in human cells. In some embodiments, novel Cas9 enzymes can be used for base editing. In some embodiments, the novel engineered Cas9 enzymes are used to treat human diseases.
1 . An engineered, non-naturally occurring Cas9 protein modified from Streptococcus constellatus Cas9, Sharpea Cas9, Veillonella parvula Cas9, Ezakiella peruensis Cas9, Lactobacillus fermentum strain AF15-40LB Cas9, or Peptoniphilus sp. Marseille-P3761 Cas9.
2 . The Cas9 protein of claim 1 , wherein the Streptococcus constellatus Cas9 has at least 80% sequence identity to
(SEQ ID NO: 1)
MGKPYSIGLDIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKQSIKKNLLGALLEDSGETAEAT
RLKRTARRRYTRRKNRLRYLQEIFTGEMNKVDENFFQRLDDSELVDEDKRGEHHPIFGNIAA
EVKYHDDFPTIYHLRRHLADTSKKADLRLVYLALAHMIKERGHELYEGDLKAENTDVQALFK
DFVEEYDKTIEESHLSEITVDALSILTEKVSKSSRLENLIAHYPTEKKNTLFGNLIALSLDL
HPNFKTNFQLSEDAKLQFSKDTYEEDLEGFLGEVGDEYADLFASAKNLYDAILLSGILTVDD
NSTKAPLSASMVKRYEEHQKDLKKLKDFIKVNAPDQYNAIFKDKNKKGYASYIESGVKQDEF
YKYLKGILLKINGSGDFLDKIDREDFLRKQRTFDNGSIPHQIHLQEMHAILRRQGEHYPFLK
ENQDKIEKILTFRIPYYVGPLARKGSRFAWAEYKADEKITPWNEDDILDKEKSAEKFITRMT
LNDLYLPEEKVLPKHSPLYEAFTVYNELTKVKYVNEQGEAKFFDTNMKQEIFDHVFKENRKV
TKDKLLNYLNKEFEEFRIVNLTGLDKENKAFNSSLGTYHDLRKILDKSFLDDKANEKTIEDI
IQTLTLFEDREMIRQRLQKYSDIFTKAQLKKLERRHYTGWGRLSYKLINGIRNKENKKTILD
YLIDDGYANRNFMQLINDDALSFKEEIARAQIIDDVDDIANVVHDLPGSPAIKKGILQSVKI
VDELVKVMGHNPANIIIEMARENQTTDKGRRNSQQRLKLLQDSLKNLDNPVNIKNVENQQLQ
NDRLFLYYIQNGKDMYTGETLDINNLSQYDIDHIIPQAFIKDNSLDNRVLTRSDKNRGKSDD
VPSIEVVHEMKSFWSKLLSVKLITQRKEDNLTKAERGGLTEEDKAGFIKRQLVETRQITKHV
AQILDERENTEFDGNKRRIRNVKIITLKSNLVSNFRKEFELYKVREINDYHHAHDAYLNAVV
GNALLLKYPQLEPEFVYGEYPKYNSYRSRKSATEKFLFYSNILRFFKKEDIQTNEDGEIAWN
KEKHIKILRKVLSYPQVNIVKKTEEQTGGFSKESILPKGESDKLIPRKTKNSYWDPKKYGGF
DSPVVAYSILVFADVEKGKSKKLRKVQDMVGITIMEKKRFEKNPVDFLEQRGYRNVRLEKII
KLPKYSLFELENKRRRLLASAKELQKGNELVIPQRFTTLLYHSYRIEKDYEPEHREYVEKHK
DEFKELLEYISVFSRKYVLADNNLTKIEMLFSKNKDAEVSSLAKSFISLLTFTAFGAPAAEN
FFGENIDRKRYTSVTECLNATLIHQSITGLYETRIDLSKLGED.
3 . The Cas9 protein of claim 1 , wherein the Sharpea Cas9 has at least 80% sequence identity to
(SEQ ID NO: 4)
MAKNKDIRYSIGLDIGTNSVGWAVMDEHYELLKKGNHHMWGSRLFDAAEPAATRRASRSIRR
RYNKRRERIRLLRDLLGDMVMEVDPTFFIRLLNVSFLDEEDKQKNLGNDYKDNYNLFIEKDE
NDKTYYDKYPTIYHLRKELCENKEKADPRLIYLALHHIVKYRGNFLKEGQSFAKVYEDIEEK
LDNTLKKFMSLNDLDNLFVDNDINSMITVLSKIYQRSKKADDLLKIMNPTKEERAAYKEFTK
ALVGLKFNVSKMILAQEVKKDDKDIELDESNVDYDSTVDGLQAELGEYIEFIEMLHSINSWV
ELQDILGNNSTISAAMVERYEEHKNDLRVLKKVIREELPDKYNEVFREDNPKLHNYLGYIKY
PKNTPVEEFYEYIKRLLAKVDTGEAREILERIDLEKFMLKQNSRINGSIPYQMQKDEMIQII
DNQSVYYPQLKENREKLISILEFRIPYYFGPLNTHSEFAWIKKFEDKQKERILPWNYDQIVD
IDATAEGFIERMQNTGTYFPDKPVMAKNSLTVSKFEVLNELNKIRINGKLIPVETKKELLSD
LFMKNKTITDKKLKDWLVTHQYYDTNEELKIEGYQKDLQFSTSLAPWIDFTKIFGEINASNY
QLIEKIIYDISIFEDKKILKRRLKKVYQLDDLLVDKILKLNYTGWSRLSEKLLTGIKSKNSK
ETILSILENSNMNLMEIINDESLGFKQIIEESNKKDIEGPFRYDEVKKLAGSPAIKRGIWQA
LLVVQEITKFMKHEPSHIYIEFAREEQEKVRTESRIAKLQKIYKDLNLQTKEDQLVYESLKK
EDAKKKIDTDALYLYYLQMGKSMYSGKPLDIDKLSTYHIDHILPRSLIKDDSLDNRVLVLPK
ENEWKLDSETVPFEIRNKMMGFWQKLHENGLMSNKKFFSLIRTDENEKDKKRFINRQLVETR
QIIKNVAVIINDHYTNTNVVTVRAELSHQFRERYKIYKNRDLNDLHHAHDAYIACILGQFIH
QNFGNMDVNMIYGQYKKNYKKDVQEHNNYGFILNSMNHIHENDDNSVIWDPSYIGKIKSCFC
YKDVYVTKKLEQNDAKLEDLTILPSDKNSENGVTKAKIPVNKYRKDVNKYGGFSGDAPIMLA
IEADKGKKHVRQVIAFPLRLKNYNDEERIKFIEKEKNLKNVKILTEVKKNQLILINHQYFFI
TGTNELVNATQLKLSAKNTKNLFNLVDANKHNKLESIDDANFNEVIQELICKLQEPIYSRYN
SIGKEFEDSYEKINAVTKQDKLYIIEYLIAIMSAKATQGYIKPELAREIGTNGKNKGRIKSF
TIDLNKTTFISTSVTGLESKKYKL.
4 . The Cas9 protein of claim 1 , wherein the Veillonella parvula Cas9 has at least 80% sequence identity to
(SEQ ID NO: 8)
MSIINFQRRGLMETQASNQLISSHLKGYPIKDYFVGLDIGTSSVGWAVTNKAYELLKERSHK
MWGSRLFDEGESAVARRGERSMRRRLERRKLRLKLLEELFADAMAQVDPTFFMRLRESKYHY
EDKTTGHSSKHILFIDKNYNDQDYFKEYPTVYHLRSELMKSGTDDIRKLFLAVHHILKYRGN
FLYEGATFDSNASTLDDVIKQALENITENCEDCNSAISSIGQILMEAGKTKSDKAKAIEHLV
DTYIATDTVDTSSKTQKDQVKEDKKRLKAFANLVLGLNASLIDLFGSVEELEEDLKKLQITG
DTYDDKRDELAKAWSDEIYIIDDCKSVYDAIILLSIKEPGLTISESKVKAFNKHKDDLAILK
SLLKSDRSIYNTMFKVDEKGLHNYVHYIKQGRTEETSCNREDFYKYTKKIVEGLSDSKDKEY
ILSQIELQILLPLQRIKDNGVIPYQLHLEELKAILAKCGPKFPFLNEVADGESVAEKLIKML
EFRIPYYVGPLNTHHNVDNGGFAWAVRKASGRVTPWNEDDKIDREKSAAAFIKNLINKCTYL
LGEDVLPKSSLLYSEFMLLNELNNVRIDGKPLEKVVKEHLIEAVFKQDHKKMTKNRIEQFLK
DNGYISETHKHEITGLDGEIKNDLASYRDMVRILGDGEDRSMAEEIITDITIFGESKKMLRE
TLRKKFASCLDDEAIKKLTKLRYRDWGRLSQKLLNGIEGCDKAGDGTPETIIILMRNESYNL
MELLGDKFSFMERIQEINAKLTEGQIVNPHDIIDDLALSPAVKRAVWQALRIVDEVAHIKKA
LPARIFVEVTRSNKNEKKKKDSRQKRLSDLYAAIKKDDVLLNGLNNEIFGELKSSLAKYDDA
ALRSKKLYLYYTQMGRCAYTGEIIELSLLNTDNYDIDHIYPRSLTKDDSFDNLVLCKRTANA
QKSDAYPISEEIQKTQKPFWTFLKQQGLISERKYERLTRITPLTADDLSGFIARQLVETNQS
VKAATTLLRRLYPGVDVVFVKAENVTDERHDNNFIKVRSLNHHHHAKDAYLNIVVGNVYHER
FTRNFRAFFKKNGANRTYNLAKMENYDVNCTNAKDGKAWDVKTSMDTVKKMMDSNDVRVTKR
LLEQTGALADATIYKATVAGKAKDGAYIGMKTKSSVFADVSKYGGMTKIKNAYSIIVQYTGK
KGEVIKEIVPLPIYLTNRNTTDQDLINYVASIIPQAKDISIIYGKLCINQLVKVNGFYYYLG
GKTNSKFCIDNAIQVIVSNEWIPYLKVLEKENNMRKDNKDLKANVVSTRALDNKHTIEVRIV
EEKNIEFFDYLVSKLKMPIYQKMKGNKAAELSEKGYGLFKKMSLEEQSIHLIELLNLLTNQK
TTFEVKPLGITASRSTVGSKISNQDEFKVINESITGLYSNEVTIV.
5 . The Cas9 protein of claim 1 , wherein the Ezakiella peruensis Cas9 has at least 80% sequence identity to
(SEQ ID NO: 14)
MTKVKDYYIGLDIGTSSVGWAVTDEAYNVLKFNSKKMWGVRLEDDAKTAEERRGQRGARRRL
DRKKERLSLLQDFFAEEVAKVDPNFFLRLDNSDLYMEDKDQKLKSKYTLFNDKDFKDKNFHK
KYPTIHHLLMDLIEDDSKKDIRLVYLACHYLLKNRGHFIFEGQKFDTKSSFENSLNELKVHL
NDEYGLDLEFDNENLINILTDPKLNKTAKKKELKSVIGDTKFLKAVSAIMIGSSQKLVDLFE
NPEDFDDSAIKSVDFSTTSFDDKYSDYELALGDKIALVNILKEIYDSSILENLLKEADKSKD
GNKYISNAFVKKYNKHGQDLKEFKRLVRQYHKSAYFDIFRSEKVNDNYVSYTKSSISNNKRV
KANKFTDQEAFYKFAKKHLETIKYKINKVNGSKADLELIDGMLRDMEFKNEMPKIKSSDNGV
IPYQLKLMELNKILENQSKHHEFLNVSDEYGSVCDKIASIMEFRIPYYVGPLNPNSKYAWIK
KQKDSEITPWNFKDVVDLDSSREEFIDSLIGRCTYLKDEKVLPKASLLYNEYMVLNELNNLK
LNDLPITEEMKKKIFDQLFKTRKKVTLKAVANLLKKEFNINGEILLSGTDGDFKQGLNSYND
FKAIVGDKVDSDDYRDKIEEIIKLIVLYGDDKSYLQKKIKAGYGKYFTDSEIKKMAGLNYKD
WGRLSKKLLTGLEGANKITGERGSIIHEMREYNLNLMELMSASFTFTEEIQKLNPVDDRKLS
YEMVDELYLSPSVKRMLWQSLRIVDEIKNIMGTDSKKIFIEMARGKEEVKARKESRKNQLLK
FYKDGKKAFISEIGEERYSYLLSEIEGEEENKERWDNLYLYYTQLGRCMYSLEPIDISELSS
KNIYDQDHIYPKSKIYDDSIENRVLVKKDLNSKKGNSYPIPDEILNKNCYAYWKILYDKGLI
GQKKYTRLTRRTGFTDDELVQFISRQIVETRQATKETANLLKTICKNSEIVYSKAENASRER
QEFDIVKCRAVNDLHHMHDAYINIIVGNVYNTKFTKDPMNFVKKQEKARSYNLENMEKYDVK
RGGYTAWIADDEKGTVKNASIKRIRKELEGTNYRFTRMNYIESGALFNATLQRKNKGSRPLK
DKGPKSSIEKYGGYTNINKACFAVLDIKSKNKIERKLMPVEREIYAKQKNDKKLSDEIFSKY
LKDRFGIEDYRVVYPVVKMRTLLKIDGSYYFITGGSDKTLELRSALQLILPKKNEWAIKQID
KSSENDYLTIERIQDLTEELVYNTFDIIVNKFKTSVEKKSFLNLFQDDKIENIDFKEKSMDF
KEKCKTLLMLVKAIRASGVRQDLKSIDLKSDYGRLSSKINNIGNYQEFKIINQSITGLFENE
VDLLKL.
6 . The Cas9 protein of claim 1 , wherein the Lactobacillus fermentum strain AF15-40LB Cas9 has at least 80% sequence identity to
(SEQ ID NO: 84)
MKEYHIGLDIGTSSIGWAVTDSQFKLMRIKGKTAIGVRLFEEGKTAAERRTFRTTRRRLKRR
KWRLHYLDEIFAPHLQEVDENFLRRLKQSNIHPEDPAKNQAFIGKLLFPDLLKKNERGYPTL
IKMRDELPVEQRAHYPVTNIYKLREAMINEDRQFDLREVYLAVHHIVKYRGHELNNASVDKF
KVGRIDFDKSFNVLNEAYEELQNGEGSFTIEPSKVEKIGQLLLDTKMRKLDRQKAVAKLLEV
KVADKEETKRNKQIATAMSKLVLGYKADFATVAMANGNEWKIDLSSETSEDEIEKFREELSD
AQNDILTEITSLESQIMLNEIVPNGMSISESMMDRYWTHERQLAEVKEYLATQPASARKEFD
QVYNKYIGQAPKEKGFDLEKGLKKILSKKENWKEIDELLKAGDELPKQRTSANGVIPHQMHQ
QELDRIIEKQAKYYPWLATENPATGERDRHQAKYELDQLVSFRIPYYVGPLVTPEVQKATSG
AKFAWAKRKEDGEITPWNLWDKIDRAESAEAFIKRMTVKDTYLLNEDVLPANSLLYQKYNVL
NELNNVRVNGRRLSVGIKQDIYTELFKKKKTVKAGDVASLVMAKTRGVNKPSVEGLSDPKKF
NSNLATYLDLKSIVGDKVDDNRYQMDLENIIEWRSVFEDGEIFADKLTEVEWLTDEQRSALV
KKRYKGWGRLSKKLLTGIVDENGQRIIDLMWNTDQNFMQIVNQPVFKEQIDQLNQKAITNDG
MTLRERVESVLDDAYTSPQNKKAIWQVVRVVEDIVKAVGNAPKSISIEFARNEGNKGEITRS
RRTQLQKLFEDQAHELVKDTSLTEELEKAPDLSDRYYFYFTQGGKDMYTGDPINFDEISTKY
DIDHILPQSFVKDDSLDNRVLVSRAENNKKSDRVPAKLYAAKMKPYWNQLLKQGLITQRKFE
NLTMDVDQTIKYRSLGFVKRQLVETRQVIKLTANILGSMYQEAGTDIIETRAGLTKQLREEF
DLPKVREVNDYHHAVDAYLTTFAGQYLNRRYPKLRSFFVYGEYMKFKHGSDLKLRNENFFHE
LMEGDKSQGKVVDQQTGELITTRDEVADYFDWVINLKVMLISNETYEETGKYFDASHESSSL
YLKNQNKKSKLVVPLKNKLQPEYYGAYTGITQGYMVILKLLDKKGGFGVYRIPRYAADILNK
CHDEVAYRNKIAEIISSDPRAPKSFEVVVPRVLKGTFLVDGEEKFILSSYRYKVNATQLILP
VSDIKLIQDNFKALKKLNVEMQTKKLIEIYDNILRQVDKYYKLYDINKFRAKLHDGRSKFVE
LDDFGQDASKEKVIIKILRGLHFGSDLQNLKEIGFGTTPLGQFQVSEAGIRLSNTAFIIFKS
PTGLENRKLYLKNL.
7 . The Cas9 protein of claim 1 , wherein the Peptoniphilus sp. Marseille P3761 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 86)
MEKKTNYTIGLDIGTDSVGWAVVKDDLELVKKRMKVLGNTETNYIKKNLWGSLLFESGQTAK
DRRLKRVARRRYERRRNRLTELQKIFAPAIDEVDENFFFRLNESFLVPEDKAFSKNPIFGTL
GEDKTYYKTYPTIYHLRQHLADSEEKADVRLIYLALAHMIKYRGHFLIEGKLDTEHIAINEN
LEQFFESYNALFSEEPIELRKEELIAIENILREKNSRTVKEKRITSFLKDIGRANKQSPMMA
FITLIVGKKAKFKAAFNLEEEISLNLTDDSYDENLEILLNTIGSDFADLFDHAQRVYNAVEL
AGILSGDVKNTHAKLSAQMVAMYERHKEQLKEYKSFIKANLPDQYDMTFVAPKDAQKKDLKG
YAGYIDGNMSQDSFYKFVKDQLKEVPGSEKFLDSIEKEDELRKQRSFYNGVIPNQVHLAEME
AILDRQENYYPWLKENREKIISLLTFRIPYYVGPLADGQSEFAWLERKSDEKIKPWNFSDVV
DLDRSAEKFIEQLIGRDTYLPDEYVLPKKSLIYQKYMVFNELTKIAYLDERQKRMNLSSVEK
KEIFETLFKKRSKVTEKQLVKFFENYLQIDNPTIFGIEDAFNADYSTYVELAKVPGMKSMMD
DPDNEDLMEEIVKILTVFEDRKMRRKQLEKYKERLSPEQIKELAKKHYTGWGRLSKKLLVGI
RDKETQKTILDYLVEDDNHSGGRQHLNRNLMQLINDDRLSFKKTIAELQMIDPSADLYAQVQ
EIAGSPAIKKGILLGLKIVDEIIRVMGEKPENIVIEMARENQTTARGKALSKRREAKIKEGL
AALGSSLLKENLPGNADLSQRKIYLYYTQNGKDIYLDEPLDEDRLSQYDEDHIIPQSFTVDN
SLDNLVLTNSSQNRGNKKDDVPSLEVVNRQLAYWRSLKDAGLMTQRKFDNLTKAMRGGLTDK
DRERFIQRQLVETRQITKNVAKLLDMRLNDKKDEAGNKIRETNIVLLKSAMASEFRKMERLY
KVRELNDYHHAHDAYLNAAIAINLLALYPYMADDEVYGEFRYKKKPQAEKATYEKLRQWNLI
KRFGEKQLFTPDHEDCWNKERDIKTIKKVMGYRQVNVVKKAEERTGMLFKETINGKTNKGSR
IPIKKDLDPSKYGGYIEEKMAYYAVISYEDKKKKPGKTIVGISIMDKKEFEYDSISYLGKLG
FSNPVVQIILKNYSLIAYPDGRRRYITGATKTTKGKVELQKANQIAMEQDLVNFIYHLKNYD
EISHPESYAFVQSHTDYFDRLEDSIEHYTRRFLDAETNINRLRRIYEEEKKKDPVDIEALVA
SFIELLKLTSAGAPADFIFMGEAISRRRYNSMTGLEDGQVIYQSLTGLYETRMRFED.
8 . The Cas9 protein of any one of claims 2 - 7 comprising an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 1, 4, 8, 14, 84 or 86.
9 . The Cas9 protein of any one of the preceding claims, further comprising a nuclear localization sequence (NLS) and/or a FLAG, HIS or HA tag.
10 . The Cas9 protein of claim 9 , wherein the Streptococcus constellatus Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 2)
MPKKKRKVGGKPYSIGLDIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKQSIKKNLLGALLFD
SGETAEATRLKRTARRRYTRRKNRLRYLQEIFTGEMNKVDENFFQRLDDSELVDEDKRGEHH
PIFGNIAAEVKYHDDFPTIYHLRRHLADTSKKADLRLVYLALAHMIKERGHFLYEGDLKAEN
TDVQALFKDFVEEYDKTIEESHLSEITVDALSILTEKVSKSSRLENLIAHYPTEKKNTLFGN
LIALSLDLHPNFKTNFQLSEDAKLQFSKDTYEEDLEGFLGEVGDEYADLFASAKNLYDAILL
SGILTVDDNSTKAPLSASMVKRYEEHQKDLKKLKDFIKVNAPDQYNAIFKDKNKKGYASYIE
SGVKQDEFYKYLKGILLKINGSGDFLDKIDREDFLRKQRTEDNGSIPHQIHLQEMHAILRRQ
GEHYPFLKENQDKIEKILTFRIPYYVGPLARKGSRFAWAEYKADEKITPWNEDDILDKEKSA
EKFITRMTLNDLYLPEEKVLPKHSPLYEAFTVYNELTKVKYVNEQGEAKFEDTNMKQEIFDH
VFKENRKVTKDKLLNYLNKEFEEFRIVNLTGLDKENKAFNSSLGTYHDLRKILDKSFLDDKA
NEKTIEDIIQTLTLFEDREMIRQRLQKYSDIFTKAQLKKLERRHYTGWGRLSYKLINGIRNK
ENKKTILDYLIDDGYANRNFMQLINDDALSFKEEIARAQIIDDVDDIANVVHDLPGSPAIKK
GILQSVKIVDELVKVMGHNPANIIIEMARENQTTDKGRRNSQQRLKLLQDSLKNLDNPVNIK
NVENQQLQNDRLFLYYIQNGKDMYTGETLDINNLSQYDIDHIIPQAFIKDNSLDNRVLTRSD
KNRGKSDDVPSIEVVHEMKSFWSKLLSVKLITQRKFDNLTKAERGGLTEEDKAGFIKRQLVE
TRQITKHVAQILDERENTEFDGNKRRIRNVKIITLKSNLVSNFRKEFELYKVREINDYHHAH
DAYLNAVVGNALLLKYPQLEPEFVYGEYPKYNSYRSRKSATEKFLFYSNILRFFKKEDIQTN
EDGEIAWNKEKHIKILRKVLSYPQVNIVKKTEEQTGGFSKESILPKGESDKLIPRKTKNSYW
DPKKYGGFDSPVVAYSILVFADVEKGKSKKLRKVQDMVGITIMEKKRFEKNPVDFLEQRGYR
NVRLEKIIKLPKYSLFELENKRRRLLASAKELQKGNELVIPQRFTTLLYHSYRIEKDYEPEH
REYVEKHKDEFKELLEYISVESRKYVLADNNLTKIEMLESKNKDAEVSSLAKSFISLLTFTA
FGAPAAFNFFGENIDRKRYTSVTECLNATLIHQSITGLYETRIDLSKLGEDGKRPAATKKAG
QAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA.
10 b. The Cas9 protein of claim 9 , wherein the Streptococcus constellatus Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 95)
MPKKKRKVGMGKPYSIGLDIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKQSIKKNLLGALLF
DSGETAEATRLKRTARRRYTRRKNRLRYLQEIFTGEMNKVDENFFQRLDDSFLVDEDKRGEH
HPIFGNIAAEVKYHDDFPTIYHLRRHLADTSKKADLRLVYLALAHMIKERGHFLYEGDLKAE
NTDVQALFKDFVEEYDKTIEESHLSEITVDALSILTEKVSKSSRLENLIAHYPTEKKNTLFG
NLIALSLDLHPNEKTNFQLSEDAKLQFSKDTYEEDLEGELGEVGDEYADLFASAKNLYDAIL
LSGILTVDDNSTKAPLSASMVKRYEEHQKDLKKLKDFIKVNAPDQYNAIFKDKNKKGYASYI
ESGVKQDEFYKYLKGILLKINGSGDFLDKIDREDFLRKQRTFDNGSIPHQIHLQEMHAILRR
QGEHYPFLKENQDKIEKILTFRIPYYVGPLARKGSRFAWAEYKADEKITPWNEDDILDKEKS
AEKFITRMTLNDLYLPEEKVLPKHSPLYEAFTVYNELTKVKYVNEQGEAKFFDTNMKQEIFD
HVFKENRKVTKDKLLNYLNKEFEEFRIVNLTGLDKENKAFNSSLGTYHDLRKILDKSFLDDK
ANEKTIEDIIQTLTLFEDREMIRQRLQKYSDIFTKAQLKKLERRHYTGWGRLSYKLINGIRN
KENKKTILDYLIDDGYANRNFMQLINDDALSFKEEIARAQIIDDVDDIANVVHDLPGSPAIK
KGILQSVKIVDELVKVMGHNPANIIIEMARENQTTDKGRRNSQQRLKLLQDSLKNLDNPVNI
KNVENQQLQNDRLFLYYIQNGKDMYTGETLDINNLSQYDIDHIIPQAFIKDNSLDNRVLTRS
DKNRGKSDDVPSIEVVHEMKSFWSKLLSVKLITQRKEDNLTKAERGGLTEEDKAGFIKRQLV
ETRQITKHVAQILDERENTEFDGNKRRIRNVKIITLKSNLVSNFRKEFELYKVREINDYHHA
HDAYLNAVVGNALLLKYPQLEPEFVYGEYPKYNSYRSRKSATEKFLFYSNILRFFKKEDIQT
NEDGEIAWNKEKHIKILRKVLSYPQVNIVKKTEEQTGGFSKESILPKGESDKLIPRKTKNSY
WDPKKYGGFMQPVVAYSILVFADVEKGKSKKLRKVQDMVGITIMEKKRFEKNPVDFLEQRGY
RNVRLEKIIKLPKYSLFELENKRRRLLASAKFLQKGNELVIPQRFTTLLYHSYRIEKDYEPE
HREYVEKHKDEFKELLEYISVESRKYVLADNNLTKIEMLESKNKDAEVSSLAKSFISLLTFT
AFGAPRAFNFFGENIARKEYRSVTECLNATLIHQSITGLYETRIDLSKLGEDGEGADKRTAD
GSEFESPKKKRKV.
10 c. The Cas9 protein of claim 9 , wherein the Streptococcus constellatus Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 96)
MPKKKRKVGMGKPYSIGLDIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKQSIKKNLLGALLE
DSGETAEATRLKRTARRRYTRRKNRLRYLQEIFTGEMNKVDENFFQRLDDSELVDEDKRGEH
HPIFGNIAAEVKYHDDFPTIYHLRRHLADTSKKADLRLVYLALAHMIKFRGHELYEGDLKAE
NTDVQALFKDEVEEYDKTIEESHLSEITVDALSILTEKVSKSSRLENLIAHYPTEKKNTLFG
NLIALSLDLHPNFKTNFQLSEDAKLQFSKDTYEEDLEGFLGEVGDEYADLFASAKNLYDAIL
LSGILTVDDNSTKAPLSASMVKRYEEHQKDLKKLKDFIKVNAPDQYNAIFKDKNKKGYASYI
ESGVKQDEFYKYLKGILLKINGSGDELDKIDREDELRKQRTEDNGIIPHQIHLQEMHAILRR
QGEHYPFLKENQDKIEKILTFRIPYYVGPLARKGSRFAWAEYKADEKITPWNEDDILDKEKS
AEKFITRMTLNDLYLPEEKVLPKHSPLYEAFTVYNELTKVKYVNEQGEAKFFDTNMKQEIFD
HVFKENRKVTKDKLLNYLNKEFEEFRIVNLTGLDKENKAFNSSLGTYHDLRKILDKSFLDDK
ANEKTIEDIIQTLTLFEDREMIRQRLQKYSDIFTKAQLKKLERLHYTGWGRLSYKLINGIRN
KENKKTILDYLIDDGYANRNEMQLINDDALSFKEEIARAQIIDDVDDIANVVHDLPGSPAIK
KGILQSVKIVDELVKVMGHNPANIIIEMARENQTTDKGRRNSQQRLKLLQDSLKNLDNPVNI
KNVENQQLQNDRLFLYYIQNGKDMYTGETLDINNLSQYDIDHIIPQAFIKDNSLDNRVLTRS
DKNRGKSDDVPSIEVVHEMKSFWSKLLSVKLITQRKEDNLTKAERGGLTEEDKAGFIKRQLV
ETRQITKHVAQILDERENTEFDGNKRRIRNVKIITLKSNLVSNFRKEFELYKVREINDYHHA
HDAYLNAVVGNALLLKYPQLEPEFVYGEYPKYNSYRSRKSATEKFLFYSNILRFFKKEDIQT
NEDGEIAWNKEKHIKILRKVLSYPQVNIVKKTEEQTGGFSKESILPKGESDKLIPRKTKNSY
WDPKKYGGFMQPVVAYSILVFADVEKGKSKKLRKVQDMVGITIMEKKRFEKNPVDFLEQRGY
RNVRLEKIIKLPKYSLFELENKRRRLLASAKFLQKGNELVIPQRFTTLLYHSYRIEKDYEPE
HREYVEKHKDEFKELLEYISVFSRKYVLADNNLTKIEMLFSKNKDAEVSSLAKSFISLLTFT
AFGAPRAFNFFGENIARKEYRSVTECLNATLIHQSITGLYETRIDLSKLGEDGEGADKRTAD
GSEFESPKKKRKV.
11 . The Cas9 protein of claim 9 , wherein the Sharpea Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 5)
MPKKKRKVGAKNKDIRYSIGLDIGTNSVGWAVMDEHYELLKKGNHHMWGSRLFDAAEPAATR
RASRSIRRRYNKRRERIRLLRDLLGDMVMEVDPTFFIRLLNVSFLDEEDKQKNLGNDYKDNY
NLFIEKDENDKTYYDKYPTIYHLRKELCENKEKADPRLIYLALHHIVKYRGNELKEGQSFAK
VYEDIEEKLDNTLKKEMSLNDLDNLFVDNDINSMITVLSKIYQRSKKADDLLKIMNPTKEER
AAYKEFTKALVGLKFNVSKMILAQEVKKDDKDIELDFSNVDYDSTVDGLQAELGEYIEFIEM
LHSINSWVELQDILGNNSTISAAMVERYEEHKNDLRVLKKVIREELPDKYNEVFREDNPKLH
NYLGYIKYPKNTPVEEFYEYIKRLLAKVDTGEAREILERIDLEKEMLKQNSRINGSIPYQMQ
KDEMIQIIDNQSVYYPQLKENREKLISILEFRIPYYFGPLNTHSEFAWIKKFEDKQKERILP
WNYDQIVDIDATAEGFIERMQNTGTYFPDKPVMAKNSLTVSKFEVLNELNKIRINGKLIPVE
TKKELLSDLFMKNKTITDKKLKDWLVTHQYYDTNEELKIEGYQKDLQFSTSLAPWIDFTKIF
GEINASNYQLIEKIIYDISIFEDKKILKRRLKKVYQLDDLLVDKILKLNYTGWSRLSEKLLT
GIKSKNSKETILSILENSNMNLMEIINDESLGFKQIIEESNKKDIEGPFRYDEVKKLAGSPA
IKRGIWQALLVVQEITKFMKHEPSHIYIEFAREEQEKVRTESRIAKLQKIYKDLNLQTKEDQ
LVYESLKKEDAKKKIDTDALYLYYLQMGKSMYSGKPLDIDKLSTYHIDHILPRSLIKDDSLD
NRVLVLPKENEWKLDSETVPFEIRNKMMGFWQKLHENGLMSNKKFFSLIRTDENEKDKKRFI
NRQLVETRQIIKNVAVIINDHYTNTNVVTVRAELSHQFRERYKIYKNRDLNDLHHAHDAYIA
CILGQFIHQNFGNMDVNMIYGQYKKNYKKDVQEHNNYGFILNSMNHIHENDDNSVIWDPSYI
GKIKSCFCYKDVYVTKKLEQNDAKLEDLTILPSDKNSENGVTKAKIPVNKYRKDVNKYGGFS
GDAPIMLAIEADKGKKHVRQVIAFPLRLKNYNDEERIKFIEKEKNLKNVKILTEVKKNQLIL
INHQYFFITGTNELVNATQLKLSAKNTKNLFNLVDANKHNKLESIDDANFNEVIQELICKLQ
EPIYSRYNSIGKEFEDSYEKINAVTKQDKLYIIEYLIAIMSAKATQGYIKPELAREIGTNGK
NKGRIKSFTIDLNKTTFISTSVTGLESKKYKLGKRPAATKKAGQAKKKKGSYPYDVPDYAYP
YDVPDYAYPYDVPDYA.
12 . The Cas9 protein of claim 9 , wherein the Veillonella parvula Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 9)
MPKKKRKVGSIINFQRRGLMETQASNQLISSHLKGYPIKDYFVGLDIGTSSVGWAVINKAYE
LLKFRSHKMWGSRLFDEGESAVARRGFRSMRRRLERRKLRLKLLEELFADAMAQVDPTFFMR
LRESKYHYEDKTTGHSSKHILFIDKNYNDQDYFKEYPTVYHLRSELMKSGTDDIRKLFLAVH
HILKYRGNFLYEGATFDSNASTLDDVIKQALENITENCFDCNSAISSIGQILMEAGKTKSDK
AKAIEHLVDTYIATDTVDTSSKTQKDQVKEDKKRLKAFANLVLGLNASLIDLFGSVEELEED
LKKLQITGDTYDDKRDELAKAWSDEIYIIDDCKSVYDAIILLSIKEPGLTISESKVKAFNKH
KDDLAILKSLLKSDRSIYNTMFKVDEKGLHNYVHYIKQGRTEETSCNREDFYKYTKKIVEGL
SDSKDKEYILSQIELQILLPLQRIKDNGVIPYQLHLEELKAILAKCGPKFPFLNEVADGESV
AEKLIKMLEFRIPYYVGPLNTHHNVDNGGFAWAVRKASGRVTPWNEDDKIDREKSAAAFIKN
LTNKCTYLLGEDVLPKSSLLYSEFMLLNELNNVRIDGKPLEKVVKEHLIEAVFKQDHKKMTK
NRIEQFLKDNGYISETHKHEITGLDGEIKNDLASYRDMVRILGDGEDRSMAEEIITDITIFG
ESKKMLRETLRKKFASCLDDEAIKKLTKLRYRDWGRLSQKLLNGIEGCDKAGDGTPETIIIL
MRNFSYNLMELLGDKFSFMERIQEINAKLTEGQIVNPHDIIDDLALSPAVKRAVWQALRIVD
EVAHIKKALPARIFVEVTRSNKNEKKKKDSRQKRLSDLYAAIKKDDVLLNGLNNEIFGELKS
SLAKYDDAALRSKKLYLYYTQMGRCAYTGEIIELSLLNTDNYDIDHIYPRSLTKDDSFDNLV
LCKRTANAQKSDAYPISEEIQKTQKPFWTELKQQGLISERKYERLTRITPLTADDLSGFIAR
QLVETNQSVKAATTLLRRLYPGVDVVFVKAENVTDERHDNNFIKVRSLNHHHHAKDAYLNIV
VGNVYHERFTRNFRAFFKKNGANRTYNLAKMFNYDVNCTNAKDGKAWDVKTSMDTVKKMMDS
NDVRVTKRLLEQTGALADATIYKATVAGKAKDGAYIGMKTKSSVFADVSKYGGMTKIKNAYS
IIVQYTGKKGEVIKEIVPLPIYLTNRNTTDQDLINYVASIIPQAKDISIIYGKLCINQLVKV
NGFYYYLGGKTNSKFCIDNAIQVIVSNEWIPYLKVLEKENNMRKDNKDLKANVVSTRALDNK
HTIEVRIVEEKNIEFFDYLVSKLKMPIYQKMKGNKAAELSEKGYGLFKKMSLEEQSIHLIEL
LNLLTNQKTTFEVKPLGITASRSTVGSKISNQDEFKVINESITGLYSNEVTIVGKRPAATKK
AGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA.
13 . The Cas9 protein of claim 9 , wherein the Ezakiella peruensis Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 15)
MPKKKRKVGTKVKDYYIGLDIGTSSVGWAVTDEAYNVLKENSKKMWGVRLEDDAKTAEERRG
QRGARRRLDRKKERLSLLQDFFAEEVAKVDPNFFLRLDNSDLYMEDKDQKLKSKYTLENDKD
FKDKNFHKKYPTIHHLLMDLIEDDSKKDIRLVYLACHYLLKNRGHFIFEGQKEDTKSSFENS
LNELKVHLNDEYGLDLEFDNENLINILTDPKLNKTAKKKELKSVIGDTKFLKAVSAIMIGSS
QKLVDLFENPEDEDDSAIKSVDESTTSFDDKYSDYELALGDKIALVNILKEIYDSSILENLL
KEADKSKDGNKYISNAFVKKYNKHGQDLKEFKRLVRQYHKSAYFDIFRSEKVNDNYVSYTKS
SISNNKRVKANKFTDQEAFYKFAKKHLETIKYKINKVNGSKADLELIDGMLRDMEFKNFMPK
IKSSDNGVIPYQLKLMELNKILENQSKHHEFLNVSDEYGSVCDKIASIMEFRIPYYVGPLNP
NSKYAWIKKQKDSEITPWNFKDVVDLDSSREEFIDSLIGRCTYLKDEKVLPKASLLYNEYMV
LNELNNLKLNDLPITEEMKKKIFDQLFKTRKKVTLKAVANLLKKEFNINGEILLSGTDGDEK
QGLNSYNDFKAIVGDKVDSDDYRDKIEEIIKLIVLYGDDKSYLQKKIKAGYGKYFTDSEIKK
MAGLNYKDWGRLSKKLLTGLEGANKITGERGSIIHFMREYNLNLMELMSASFTFTEEIQKLN
PVDDRKLSYEMVDELYLSPSVKRMLWQSLRIVDEIKNIMGTDSKKIFIEMARGKEEVKARKE
SRKNQLLKFYKDGKKAFISEIGEERYSYLLSEIEGEEENKFRWDNLYLYYTQLGRCMYSLEP
IDISELSSKNIYDQDHIYPKSKIYDDSIENRVLVKKDLNSKKGNSYPIPDEILNKNCYAYWK
ILYDKGLIGQKKYTRLTRRTGFTDDELVQFISRQIVETRQATKETANLLKTICKNSEIVYSK
AENASRFRQEFDIVKCRAVNDLHHMHDAYINIIVGNVYNTKFTKDPMNFVKKQEKARSYNLE
NMFKYDVKRGGYTAWIADDEKGTVKNASIKRIRKELEGTNYRFTRMNYIESGALFNATLQRK
NKGSRPLKDKGPKSSIEKYGGYTNINKACFAVLDIKSKNKIERKLMPVEREIYAKQKNDKKL
SDEIFSKYLKDRFGIEDYRVVYPVVKMRTLLKIDGSYYFITGGSDKTLELRSALQLILPKKN
EWAIKQIDKSSENDYLTIERIQDLTEELVYNTFDIIVNKFKTSVEKKSFLNLFQDDKIENID
FKFKSMDFKEKCKTLLMLVKAIRASGVRQDLKSIDLKSDYGRLSSKINNIGNYQEFKIINQS
ITGLFENEVDLLKLGKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA
(D12A mutant in bold).
14 . The Cas9 protein of claim 9 , wherein the Lactobacillus fermentum strain AF15-40LB Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 85)
MPKKKRKVGKEYHIGLDIGTSSIGWAVTDSQFKLMRIKGKTAIGVRLFEEGKTAAERRTFRT
TRRRLKRRKWRLHYLDEIFAPHLQEVDENFLRRLKQSNIHPEDPAKNQAFIGKLLFPDLLKK
NERGYPTLIKMRDELPVEQRAHYPVTNIYKLREAMINEDRQFDLREVYLAVHHIVKYRGHEL
NNASVDKFKVGRIDFDKSFNVLNEAYEELQNGEGSFTIEPSKVEKIGQLLLDTKMRKLDRQK
AVAKLLEVKVADKEETKRNKQIATAMSKLVLGYKADFATVAMANGNEWKIDLSSETSEDEIE
KFREELSDAQNDILTEITSLESQIMLNEIVPNGMSISESMMDRYWTHERQLAEVKEYLATQP
ASARKEFDQVYNKYIGQAPKEKGFDLEKGLKKILSKKENWKEIDELLKAGDELPKQRTSANG
VIPHQMHQQELDRIIEKQAKYYPWLATENPATGERDRHQAKYELDQLVSFRIPYYVGPLVTP
EVQKATSGAKFAWAKRKEDGEITPWNLWDKIDRAESAEAFIKRMTVKDTYLLNEDVLPANSL
LYQKYNVLNELNNVRVNGRRLSVGIKQDIYTELFKKKKTVKAGDVASLVMAKTRGVNKPSVE
GLSDPKKENSNLATYLDLKSIVGDKVDDNRYQMDLENIIEWRSVFEDGEIFADKLTEVEWLT
DEQRSALVKKRYKGWGRLSKKLLTGIVDENGQRIIDLMWNTDQNFMQIVNQPVFKEQIDQLN
QKAITNDGMTLRERVESVLDDAYTSPQNKKAIWQVVRVVEDIVKAVGNAPKSISIEFARNEG
NKGEITRSRRTQLQKLFEDQAHELVKDTSLTEELEKAPDLSDRYYFYFTQGGKDMYTGDPIN
FDEISTKYDIDHILPQSFVKDDSLDNRVLVSRAENNKKSDRVPAKLYAAKMKPYWNQLLKQG
LITQRKFENLTMDVDQTIKYRSLGFVKRQLVETRQVIKLTANILGSMYQEAGTDIIETRAGL
TKQLREEFDLPKVREVNDYHHAVDAYLTTFAGQYLNRRYPKLRSFFVYGEYMKFKHGSDLKL
RNFNFFHELMEGDKSQGKVVDQQTGELITTRDEVADYFDWVINLKVMLISNETYEETGKYFD
ASHESSSLYLKNQNKKSKLVVPLKNKLQPEYYGAYTGITQGYMVILKLLDKKGGFGVYRIPR
YAADILNKCHDEVAYRNKIAEIISSDPRAPKSFEVVVPRVLKGTFLVDGEEKFILSSYRYKV
NATQLILPVSDIKLIQDNFKALKKLNVEMQTKKLIEIYDNILRQVDKYYKLYDINKFRAKLH
DGRSKFVELDDFGQDASKEKVIIKILRGLHFGSDLQNLKEIGFGTTPLGQFQVSEAGIRLSN
TAFIIFKSPTGLFNRKLYLKNLGKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPY
DVPDYA.
15 . The Cas9 protein of claim 9 , wherein the Peptoniphilus sp. Marseille-P3761 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 87)
MPKKKRKVGEKKTNYTIGLDIGTDSVGWAVVKDDLELVKKRMKVLGNTETNYIKKNLWGSLL
FESGQTAKDRRLKRVARRRYERRRNRLTELQKIFAPAIDEVDENFFERLNESFLVPEDKAFS
KNPIFGTLGEDKTYYKTYPTIYHLRQHLADSEEKADVRLIYLALAHMIKYRGHFLIEGKLDT
EHIAINENLEQFFESYNALFSEEPIELRKEELIAIENILREKNSRTVKEKRITSFLKDIGRA
NKQSPMMAFITLIVGKKAKFKAAFNLEEEISLNLTDDSYDENLEILLNTIGSDFADLEDHAQ
RVYNAVELAGILSGDVKNTHAKLSAQMVAMYERHKEQLKEYKSFIKANLPDQYDMTFVAPKD
AQKKDLKGYAGYIDGNMSQDSFYKFVKDQLKEVPGSEKELDSIEKEDELRKQRSFYNGVIPN
QVHLAEMEAILDRQENYYPWLKENREKIISLLTFRIPYYVGPLADGQSEFAWLERKSDEKIK
PWNFSDVVDLDRSAEKFIEQLIGRDTYLPDEYVLPKKSLIYQKYMVFNELTKIAYLDERQKR
MNLSSVEKKEIFETLFKKRSKVTEKQLVKFFENYLQIDNPTIFGIEDAFNADYSTYVELAKV
PGMKSMMDDPDNEDLMEEIVKILTVFEDRKMRRKQLEKYKERLSPEQIKELAKKHYTGWGRL
SKKLLVGIRDKETQKTILDYLVEDDNHSGGRQHLNRNLMQLINDDRLSFKKTIAELQMIDPS
ADLYAQVQEIAGSPAIKKGILLGLKIVDEIIRVMGEKPENIVIEMARENQTTARGKALSKRR
EAKIKEGLAALGSSLLKENLPGNADLSQRKIYLYYTQNGKDIYLDEPLDEDRLSQYDEDHII
PQSFTVDNSLDNLVLTNSSQNRGNKKDDVPSLEVVNRQLAYWRSLKDAGLMTQRKEDNLTKA
MRGGLTDKDRERFIQRQLVETRQITKNVAKLLDMRLNDKKDEAGNKIRETNIVLLKSAMASE
FRKMFRLYKVRELNDYHHAHDAYLNAAIAINLLALYPYMADDEVYGEFRYKKKPQAEKATYE
KLRQWNLIKRFGEKQLFTPDHEDCWNKERDIKTIKKVMGYRQVNVVKKAEERTGMLFKETIN
GKTNKGSRIPIKKDLDPSKYGGYIEEKMAYYAVISYEDKKKKPGKTIVGISIMDKKEFEYDS
ISYLGKLGFSNPVVQIILKNYSLIAYPDGRRRYITGATKTTKGKVELQKANQIAMEQDLVNE
IYHLKNYDEISHPESYAFVQSHTDYFDRLEDSIEHYTRRELDAETNINRLRRIYEEEKKKDP
VDIEALVASFIELLKLTSAGAPADFIFMGEAISRRRYNSMTGLEDGQVIYQSLTGLYETRMR
FEDGKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA.
16 . The Cas9 protein of any one of the preceding claims, wherein the amino acid sequence of the Cas9 protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 mutations in SEQ ID NO: 1, 4, 8, 14, 84 or 86.
17 . The Cas9 protein of claim 16 , wherein the mutation is an amino acid substitution.
18 . The Cas9 protein of any one of the preceding claims, wherein the Cas9 protein has nickase activity.
18 b. The Cas9 protein of claim 18 , wherein the nickase mutation at an amino acid positions corresponds to one or more amino acids 10, 12, 17, 762, 840, 854, 863, 982, 983, 984, 986, 987 of wild type SpCas9.
19 . The Cas9 protein of claim 16 , wherein the at least one mutation results in an inactive Cas9 (dCas9).
20 . The Cas9 protein of any one of the preceding claims, wherein the Cas9 protein comprises at least one amino acid mutation in PAM Interacting, HNH and/or RuvC domain.
20 b. The Cas9 protein of claim 20 , wherein the mutation at an amino acid position corresponds to amino acid 14 in the RuvC domain of SirCas9.
20 c. The Cas9 protein of claim 20 , wherein the mutation at an amino acid position corresponds to amino acid 12 in the RuvC domain of EpeCas9.
20 d. The Cas9 protein of claim 20 , wherein the mutation at an amino acid position corresponds to amino acid 9 in the RuvC domain of LfeCas9.
20 e. The Cas9 protein of claim 20 , wherein the mutation at an amino acid position corresponds to amino acid 12 in the RuvC domain of PmaCas9.
20 f. The Cas9 protein of claim 20 , wherein the Cas9 protein is a hyper-accurate Cas9.
20 g. The Cas9 protein of claim 20 , wherein the Cas9 protein comprises mutations corresponding to N692A, M694A, Q695A and/or H698A with reference to SpyCas9 (SEQ ID NO: 173).
20 h. The Cas9 protein of claim 20 , wherein the Cas9 protein is a high-fidelity Cas9.
20 i. The Cas9 protein of claim 20 , wherein the Cas9 protein comprises mutations corresponding to N467A, R661A, Q695A and/or Q926A with reference to SpyCas9 (SEQ ID NO: 173).
20 j. The Cas9 protein of claim 20 , wherein the Cas9 protein is a SuperFi-Cas9.
20 k. The Cas9 protein of claim 20 , wherein Y1016, R1019, Y1010, Y1013, K1031, Q1027 and/or V1018 residues corresponding to SpyCas9 are mutated to aspartic acid.
21 . An engineered, non-naturally occurring Cas9 fusion protein comprising a Cas9 protein having at least 80% identity to SEQ ID NOs: 1, 4, 8, 14, 84 or 86 and wherein the Cas9 protein is fused to a histone demethylase, a transcriptional activator, or to a deaminase.
21 b. The engineered, non-naturally occurring Cas9 fusion protein of claim 21 further comprising a nuclear localization sequence (NLS) and/or a FLAG, HIS or HA tag.
21 c. The engineered, non-naturally occurring Cas9 fusion protein of claim 22 having at least 80% identity to SEQ ID NOs: 2, 5, 9, 15, 85, 87, 95 or 96.
22 . The Cas9 protein of claim 21 , wherein the Cas9 protein is fused to a cytosine deaminase or to an adenosine deaminase.
23 . The Cas9 protein of claim 22 , wherein the Cas9 protein is fused to a adenosine deaminase and has an amino acid sequence at least 80% identical to
(a)
(SEQ ID NO: 20)
MPAAKRVKLDGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH
DPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAA
GSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVENAQKKAQSSTDGSSGSETPG
TSESATPESSGPKKKRKVGGKPYSIGLAIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKQSIK
KNLLGALLFDSGETAEATRLKRTARRRYTRRKNRLRYLQEIFTGEMNKVDENFFQRLDDSEL
VDEDKRGEHHPIFGNIAAEVKYHDDFPTIYHLRRHLADTSKKADLRLVYLALAHMIKFRGHF
LYEGDLKAENTDVQALFKDFVEEYDKTIEESHLSEITVDALSILTEKVSKSSRLENLIAHYP
TEKKNTLFGNLIALSLDLHPNEKTNFQLSEDAKLQFSKDTYEEDLEGELGEVGDEYADLFAS
AKNLYDAILLSGILTVDDNSTKAPLSASMVKRYEEHQKDLKKLKDFIKVNAPDQYNAIFKDK
NKKGYASYIESGVKQDEFYKYLKGILLKINGSGDFLDKIDREDFLRKQRTFDNGSIPHQIHL
QEMHAILRRQGEHYPFLKENQDKIEKILTFRIPYYVGPLARKGSRFAWAEYKADEKITPWNF
DDILDKEKSAEKFITRMTLNDLYLPEEKVLPKHSPLYEAFTVYNELTKVKYVNEQGEAKFFD
TNMKQEIFDHVFKENRKVTKDKLLNYLNKEFEEFRIVNLTGLDKENKAFNSSLGTYHDLRKI
LDKSFLDDKANEKTIEDIIQTLTLFEDREMIRQRLQKYSDIFTKAQLKKLERRHYTGWGRLS
YKLINGIRNKENKKTILDYLIDDGYANRNFMQLINDDALSFKEEIARAQIIDDVDDIANVVH
DLPGSPAIKKGILQSVKIVDELVKVMGHNPANIIIEMARENQTTDKGRRNSQQRLKLLQDSL
KNLDNPVNIKNVENQQLQNDRLFLYYIQNGKDMYTGETLDINNLSQYDIDHIIPQAFIKDNS
LDNRVLTRSDKNRGKSDDVPSIEVVHEMKSFWSKLLSVKLITQRKEDNLTKAERGGLTEEDK
AGFIKRQLVETRQITKHVAQILDERENTEFDGNKRRIRNVKIITLKSNLVSNERKEFELYKV
REINDYHHAHDAYLNAVVGNALLLKYPQLEPEFVYGEYPKYNSYRSRKSATEKFLFYSNILR
FFKKEDIQTNEDGEIAWNKEKHIKILRKVLSYPQVNIVKKTEEQTGGFSKESILPKGESDKL
IPRKTKNSYWDPKKYGGFDSPVVAYSILVFADVEKGKSKKLRKVQDMVGITIMEKKRFEKNP
VDFLEQRGYRNVRLEKIIKLPKYSLFELENKRRRLLASAKELQKGNELVIPQRFTTLLYHSY
RIEKDYEPEHREYVEKHKDEFKELLEYISVFSRKYVLADNNLTKIEMLFSKNKDAEVSSLAK
SFISLLTFTAFGAPAAFNFFGENIDRKRYTSVTECLNATLIHQSITGLYETRIDLSKLGEDG
KRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA;
(b)
(SEQ ID NO: 6)
MPAAKRVKLDGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH
DPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAA
GSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVENAQKKAQSSTDGSSGSETPG
TSESATPESSGPKKKRKVGAKNKDIRYSIGLAIGTNSVGWAVMDEHYELLKKGNHHMWGSRL
FDAAEPAATRRASRSIRRRYNKRRERIRLLRDLLGDMVMEVDPTFFIRLLNVSFLDEEDKQK
NLGNDYKDNYNLFIEKDENDKTYYDKYPTIYHLRKELCENKEKADPRLIYLALHHIVKYRGN
FLKEGQSFAKVYEDIEEKLDNTLKKFMSLNDLDNLFVDNDINSMITVLSKIYQRSKKADDLL
KIMNPTKEERAAYKEFTKALVGLKFNVSKMILAQEVKKDDKDIELDFSNVDYDSTVDGLQAE
LGEYIEFIEMLHSINSWVELQDILGNNSTISAAMVERYEEHKNDLRVLKKVIREELPDKYNE
VFREDNPKLHNYLGYIKYPKNTPVEEFYEYIKRLLAKVDTGEAREILERIDLEKFMLKQNSR
TNGSIPYQMQKDEMIQIIDNQSVYYPQLKENREKLISILEFRIPYYFGPLNTHSEFAWIKKE
EDKQKERILPWNYDQIVDIDATAEGFIERMQNTGTYFPDKPVMAKNSLTVSKFEVLNELNKI
RINGKLIPVETKKELLSDLFMKNKTITDKKLKDWLVTHQYYDTNEELKIEGYQKDLQFSTSL
APWIDFTKIFGEINASNYQLIEKIIYDISIFEDKKILKRRLKKVYQLDDLLVDKILKLNYTG
WSRLSEKLLTGIKSKNSKETILSILENSNMNLMEIINDESLGFKQIIEESNKKDIEGPFRYD
EVKKLAGSPAIKRGIWQALLVVQEITKFMKHEPSHIYIEFAREEQEKVRTESRIAKLQKIYK
DLNLQTKEDQLVYESLKKEDAKKKIDTDALYLYYLQMGKSMYSGKPLDIDKLSTYHIDHILP
RSLIKDDSLDNRVLVLPKENEWKLDSETVPFEIRNKMMGFWQKLHENGLMSNKKFFSLIRTD
FNEKDKKRFINRQLVETRQIIKNVAVIINDHYTNTNVVTVRAELSHQFRERYKIYKNRDLND
LHHAHDAYIACILGQFIHQNFGNMDVNMIYGQYKKNYKKDVQEHNNYGFILNSMNHIHENDD
NSVIWDPSYIGKIKSCFCYKDVYVTKKLEQNDAKLEDLTILPSDKNSENGVTKAKIPVNKYR
KDVNKYGGFSGDAPIMLAIEADKGKKHVRQVIAFPLRLKNYNDEERIKFIEKEKNLKNVKIL
TEVKKNQLILINHQYFFITGTNELVNATQLKLSAKNTKNLFNLVDANKHNKLESIDDANENE
VIQELICKLQEPIYSRYNSIGKEFEDSYEKINAVTKQDKLYIIEYLIAIMSAKATQGYIKPE
LAREIGTNGKNKGRIKSFTIDLNKTTFISTSVTGLFSKKYKLGKRPAATKKAGQAKKKKGSY
PYDVPDYAYPYDVPDYAYPYDVPDYA;
(c)
(SEQ ID NO: 10)
MPAAKRVKLDGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH
DPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAA
GSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPG
TSESATPESSGPKKKRKVGSIINFQRRGLMETQASNQLISSHLKGYPIKDYFVGLAIGTSSV
GWAVTNKAYELLKFRSHKMWGSRLFDEGESAVARRGFRSMRRRLERRKLRLKLLEELFADAM
AQVDPTFFMRLRESKYHYEDKTTGHSSKHILFIDKNYNDQDYFKEYPTVYHLRSELMKSGTD
DIRKLFLAVHHILKYRGNFLYEGATFDSNASTLDDVIKQALENITENCFDCNSAISSIGQIL
MEAGKTKSDKAKAIEHLVDTYIATDTVDTSSKTQKDQVKEDKKRLKAFANLVLGLNASLIDL
FGSVEELEEDLKKLQITGDTYDDKRDELAKAWSDEIYIIDDCKSVYDAIILLSIKEPGLTIS
ESKVKAFNKHKDDLAILKSLLKSDRSIYNTMFKVDEKGLHNYVHYIKQGRTEETSCNREDFY
KYTKKIVEGLSDSKDKEYILSQIELQILLPLQRIKDNGVIPYQLHLEELKAILAKCGPKFPF
LNEVADGFSVAEKLIKMLEFRIPYYVGPLNTHHNVDNGGFAWAVRKASGRVTPWNEDDKIDR
EKSAAAFIKNLTNKCTYLLGEDVLPKSSLLYSEFMLLNELNNVRIDGKPLEKVVKEHLIEAV
FKQDHKKMTKNRIEQFLKDNGYISETHKHEITGLDGEIKNDLASYRDMVRILGDGEDRSMAE
EIITDITIFGESKKMLRETLRKKFASCLDDEAIKKLTKLRYRDWGRLSQKLLNGIEGCDKAG
DGTPETIIILMRNFSYNLMELLGDKFSFMERIQEINAKLTEGQIVNPHDIIDDLALSPAVKR
AVWQALRIVDEVAHIKKALPARIFVEVTRSNKNEKKKKDSRQKRLSDLYAAIKKDDVLLNGL
NNEIFGELKSSLAKYDDAALRSKKLYLYYTQMGRCAYTGEIIELSLLNTDNYDIDHIYPRSL
TKDDSFDNLVLCKRTANAQKSDAYPISEEIQKTQKPFWTFLKQQGLISERKYERLTRITPLT
ADDLSGFIARQLVETNQSVKAATTLLRRLYPGVDVVFVKAENVTDERHDNNFIKVRSLNHHH
HAKDAYLNIVVGNVYHERFTRNFRAFFKKNGANRTYNLAKMENYDVNCTNAKDGKAWDVKTS
MDTVKKMMDSNDVRVTKRLLEQTGALADATIYKATVAGKAKDGAYIGMKTKSSVFADVSKYG
GMTKIKNAYSIIVQYTGKKGEVIKEIVPLPIYLTNRNTTDQDLINYVASIIPQAKDISIIYG
KLCINQLVKVNGFYYYLGGKTNSKFCIDNAIQVIVSNEWIPYLKVLEKENNMRKDNKDLKAN
VVSTRALDNKHTIEVRIVEEKNIEFFDYLVSKLKMPIYQKMKGNKAAELSEKGYGLFKKMSL
EEQSIHLIELLNLLTNQKTTFEVKPLGITASRSTVGSKISNQDEFKVINESITGLYSNEVTI
VGKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA;
(d)
(SEQ ID NO: 11)
MPKKKRKVSIINFQRRGLMETQASNQLISSHLKGYPIKDYFVGLAIGTSSVGWAVINKAYEL
LKFRSHKMWGSRLFDEGESAVARRGFRSMRRRLERRKLRLKLLEELFADAMAQVDPTFFMRL
RESKYHYEDKTTGHSSKHILFIDKNYNDQDYFKEYPTVYHLRSELMKSGTDDIRKLFLAVHH
ILKYRGNFLYEGATFDSNASTLDDVIKQALENITENCEDCNSAISSIGQILMEAGKTKSDKA
KAIEHLVDTYIATDTVDTSSKTQKDQVKEDKKRLKAFANLVLGLNASLIDLFGSVEELEEDL
KKLQITGDTYDDKRDELAKAWSDEIYIIDDCKSVYDAIILLSIKEPGLTISESKVKAFNKHK
DDLAILKSLLKSDRSIYNTMFKVDEKGLHNYVHYIKQGRTEETSCNREDFYKYTKKIVEGLS
DSKDKEYILSQIELQILLPLQRIKDNGVIPYQLHLEELKAILAKCGPKFPFLNEVADGESVA
EKLIKMLEFRIPYYVGPLNTHHNVDNGGFAWAVRKASGRVTPWNEDDKIDREKSAAAFIKNL
TNKCTYLLGEDVLPKSSLLYSEFMLLNELNNVRIDGKPLEKVVKEHLIEAVFKQDHKKMTKN
RIEQFLKDNGYISETHKHEITGLDGEIKNDLASYRDMVRILGDGFDRSMAEEIITDITIFGE
SKKMLRETLRKKFASCLDDEAIKKLTKLRYRDWGRLSQKLLNGIEGCDKAGDGTPETIIILM
RNFSYNLMELLGDKFSFMERIQEINAKLTEGQIVNPHDIIDDLALSPAVKRAVWQALRIVDE
VAHIKKALPARIFVEVTRSNKNEKKKKDSRQKRLSDLYAAIKKDDVLLNGLNNEIFGELKSS
LAKYDDAALRSKKLYLYYTQMGRCAYTGEIIELSLLNTDNYDIDHIYPRSLTKDDSFDNLVL
CKRTANAQKSDAYPISEEIQKTQKPFWTFLKQQGLISERKYERLTRITPLTADDLSGFIARQ
LVETNQSVKAATTLLRRLYPGVDVVFVKAENVTDFRHDNNFIKVRSLNHHHHAKDAYLNIVV
GNVYHERFTRNFRAFFKKNGANRTYNLAKMENYDVNCTNAKDGKAWDVKTSMDTVKKMMDSN
DVRVTKRLLEQTGALADATIYKATVAGKAKDGAYIGMKTKSSVFADVSKYGGMTKIKNAYSI
IVQYTGKKGEVIKEIVPLPIYLTNRNTTDQDLINYVASIIPQAKDISIIYGKLCINQLVKVN
GFYYYLGGKTNSKFCIDNAIQVIVSNEWIPYLKVLEKENNMRKDNKDLKANVVSTRALDNKH
TIEVRIVEEKNIEFFDYLVSKLKMPIYQKMKGNKAAELSEKGYGLFKKMSLEEQSIHLIELL
NLLTNQKTTFEVKPLGITASRSTVGSKISNQDEFKVINESITGLYSNEVTIVKRPAATKKAG
QAKKKKSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNR
VIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGR
VVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVENAQKKAQ
SSTDPAAKRVKLDGSYPYDVPDYAYPYDVPDYAYPYDVPDYA;
(e)
(SEQ ID NO: 16)
MPAAKRVKLDGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH
DPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAA
GSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVENAQKKAQSSTDGSSGSETPG
TSESATPESSGPKKKRKVGTKVKDYYIGLAIGTSSVGWAVTDEAYNVLKFNSKKMWGVRLFD
DAKTAEERRGQRGARRRLDRKKERLSLLQDFFAEEVAKVDPNFFLRLDNSDLYMEDKDQKLK
SKYTLFNDKDFKDKNFHKKYPTIHHLLMDLIEDDSKKDIRLVYLACHYLLKNRGHFIFEGQK
FDTKSSFENSLNELKVHLNDEYGLDLEFDNENLINILTDPKLNKTAKKKELKSVIGDTKFLK
AVSAIMIGSSQKLVDLFENPEDFDDSAIKSVDFSTTSFDDKYSDYELALGDKIALVNILKEI
YDSSILENLLKEADKSKDGNKYISNAFVKKYNKHGQDLKEFKRLVRQYHKSAYEDIFRSEKV
NDNYVSYTKSSISNNKRVKANKFTDQEAFYKFAKKHLETIKYKINKVNGSKADLELIDGMLR
DMEFKNFMPKIKSSDNGVIPYQLKLMELNKILENQSKHHEFLNVSDEYGSVCDKIASIMEFR
IPYYVGPLNPNSKYAWIKKQKDSEITPWNFKDVVDLDSSREEFIDSLIGRCTYLKDEKVLPK
ASLLYNEYMVLNELNNLKLNDLPITEEMKKKIFDQLFKTRKKVTLKAVANLLKKEFNINGEI
LLSGTDGDFKQGLNSYNDFKAIVGDKVDSDDYRDKIEEIIKLIVLYGDDKSYLQKKIKAGYG
KYFTDSEIKKMAGLNYKDWGRLSKKLLTGLEGANKITGERGSIIHEMREYNLNLMELMSASF
TFTEEIQKLNPVDDRKLSYEMVDELYLSPSVKRMLWQSLRIVDEIKNIMGTDSKKIFIEMAR
GKEEVKARKESRKNQLLKFYKDGKKAFISEIGEERYSYLLSEIEGEEENKERWDNLYLYYTQ
LGRCMYSLEPIDISELSSKNIYDQDHIYPKSKIYDDSIENRVLVKKDLNSKKGNSYPIPDEI
LNKNCYAYWKILYDKGLIGQKKYTRLTRRTGFTDDELVQFISRQIVETRQATKETANLLKTI
CKNSEIVYSKAENASRFRQEFDIVKCRAVNDLHHMHDAYINIIVGNVYNTKFTKDPMNFVKK
QEKARSYNLENMFKYDVKRGGYTAWIADDEKGTVKNASIKRIRKELEGTNYRFTRMNYIESG
ALFNATLQRKNKGSRPLKDKGPKSSIEKYGGYTNINKACFAVLDIKSKNKIERKLMPVEREI
YAKQKNDKKLSDEIFSKYLKDRFGIEDYRVVYPVVKMRTLLKIDGSYYFITGGSDKTLELRS
ALQLILPKKNEWAIKQIDKSSENDYLTIERIQDLTEELVYNTFDIIVNKFKTSVEKKSFLNL
FQDDKIENIDFKFKSMDFKEKCKTLLMLVKAIRASGVRQDLKSIDLKSDYGRLSSKINNIGN
YQEFKIINQSITGLFENEVDLLKLGKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAY
PYDVPDYA;
(f)
(SEQ ID NO: 17)
MPKKKRKVTKVKDYYIGLAIGTSSVGWAVTDEAYNVLKENSKKMWGVRLEDDAKTAEERRGQ
RGARRRLDRKKERLSLLQDFFAEEVAKVDPNFFLRLDNSDLYMEDKDQKLKSKYTLFNDKDF
KDKNFHKKYPTIHHLLMDLIEDDSKKDIRLVYLACHYLLKNRGHFIFEGQKEDTKSSFENSL
NELKVHLNDEYGLDLEFDNENLINILTDPKLNKTAKKKELKSVIGDTKFLKAVSAIMIGSSQ
KLVDLFENPEDFDDSAIKSVDFSTTSFDDKYSDYELALGDKIALVNILKEIYDSSILENLLK
EADKSKDGNKYISNAFVKKYNKHGQDLKEFKRLVRQYHKSAYFDIFRSEKVNDNYVSYTKSS
ISNNKRVKANKFTDQEAFYKFAKKHLETIKYKINKVNGSKADLELIDGMLRDMEFKNEMPKI
KSSDNGVIPYQLKLMELNKILENQSKHHEFLNVSDEYGSVCDKIASIMEFRIPYYVGPLNPN
SKYAWIKKQKDSEITPWNFKDVVDLDSSREEFIDSLIGRCTYLKDEKVLPKASLLYNEYMVL
NELNNLKLNDLPITEEMKKKIFDQLFKTRKKVTLKAVANLLKKEFNINGEILLSGTDGDFKQ
GLNSYNDFKAIVGDKVDSDDYRDKIEEIIKLIVLYGDDKSYLQKKIKAGYGKYFTDSEIKKM
AGLNYKDWGRLSKKLLTGLEGANKITGERGSIIHFMREYNLNLMELMSASFTFTEEIQKLNP
VDDRKLSYEMVDELYLSPSVKRMLWQSLRIVDEIKNIMGTDSKKIFIEMARGKEEVKARKES
RKNQLLKFYKDGKKAFISEIGEERYSYLLSEIEGEEENKFRWDNLYLYYTQLGRCMYSLEPI
DISELSSKNIYDQDHIYPKSKIYDDSIENRVLVKKDLNSKKGNSYPIPDEILNKNCYAYWKI
LYDKGLIGQKKYTRLTRRTGFTDDELVQFISRQIVETRQATKETANLLKTICKNSEIVYSKA
ENASRFRQEFDIVKCRAVNDLHHMHDAYINIIVGNVYNTKFTKDPMNFVKKQEKARSYNLEN
MFKYDVKRGGYTAWIADDEKGTVKNASIKRIRKELEGTNYRFTRMNYIESGALENATLQRKN
KGSRPLKDKGPKSSIEKYGGYTNINKACFAVLDIKSKNKIERKLMPVEREIYAKQKNDKKLS
DEIFSKYLKDRFGIEDYRVVYPVVKMRTLLKIDGSYYFITGGSDKTLELRSALQLILPKKNE
WAIKQIDKSSENDYLTIERIQDLTEELVYNTFDIIVNKFKTSVFKKSFLNLFQDDKIENIDE
KFKSMDFKEKCKTLLMLVKAIRASGVRQDLKSIDLKSDYGRLSSKINNIGNYQEFKIINQSI
TGLFENEVDLLKLKRPAATKKAGQAKKKKSGSETPGTSESATPESSGSEVEFSHEYWMRHAL
TLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDA
TLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADE
CAALLCRFFRMPRRVFNAQKKAQSSTDPAAKRVKLDGSYPYDVPDYAYPYDVPDYAYPYDVP
DYA;
(g)
(SEQ ID NO: 88)
MPAAKRVKLDGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH
DPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAA
GSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPG
TSESATPESSGPKKKRKVGKEYHIGLAIGTSSIGWAVTDSQFKLMRIKGKTAIGVRLFEEGK
TAAERRTFRTTRRRLKRRKWRLHYLDEIFAPHLQEVDENFLRRLKQSNIHPEDPAKNQAFIG
KLLFPDLLKKNERGYPTLIKMRDELPVEQRAHYPVTNIYKLREAMINEDRQFDLREVYLAVH
HIVKYRGHFLNNASVDKFKVGRIDFDKSFNVLNEAYEELQNGEGSFTIEPSKVEKIGQLLLD
TKMRKLDRQKAVAKLLEVKVADKEETKRNKQIATAMSKLVLGYKADFATVAMANGNEWKIDL
SSETSEDEIEKFREELSDAQNDILTEITSLESQIMLNEIVPNGMSISESMMDRYWTHERQLA
EVKEYLATQPASARKEFDQVYNKYIGQAPKEKGFDLEKGLKKILSKKENWKEIDELLKAGDF
LPKQRTSANGVIPHQMHQQELDRIIEKQAKYYPWLATENPATGERDRHQAKYELDQLVSFRI
PYYVGPLVTPEVQKATSGAKFAWAKRKEDGEITPWNLWDKIDRAESAEAFIKRMTVKDTYLL
NEDVLPANSLLYQKYNVLNELNNVRVNGRRLSVGIKQDIYTELFKKKKTVKAGDVASLVMAK
TRGVNKPSVEGLSDPKKFNSNLATYLDLKSIVGDKVDDNRYQMDLENIIEWRSVFEDGEIFA
DKLTEVEWLTDEQRSALVKKRYKGWGRLSKKLLTGIVDENGQRIIDLMWNTDQNFMQIVNQP
VFKEQIDQLNQKAITNDGMTLRERVESVLDDAYTSPQNKKAIWQVVRVVEDIVKAVGNAPKS
ISIEFARNEGNKGEITRSRRTQLQKLFEDQAHELVKDTSLTEELEKAPDLSDRYYFYFTQGG
KDMYTGDPINFDEISTKYDIDHILPQSFVKDDSLDNRVLVSRAENNKKSDRVPAKLYAAKMK
PYWNQLLKQGLITQRKFENLTMDVDQTIKYRSLGFVKRQLVETRQVIKLTANILGSMYQEAG
TDIIETRAGLTKQLREEFDLPKVREVNDYHHAVDAYLTTFAGQYLNRRYPKLRSFFVYGEYM
KFKHGSDLKLRNFNFFHELMEGDKSQGKVVDQQTGELITTRDEVADYFDWVINLKVMLISNE
TYEETGKYFDASHESSSLYLKNQNKKSKLVVPLKNKLQPEYYGAYTGITQGYMVILKLLDKK
GGFGVYRIPRYAADILNKCHDEVAYRNKIAEIISSDPRAPKSFEVVVPRVLKGTFLVDGEEK
FILSSYRYKVNATQLILPVSDIKLIQDNFKALKKLNVEMQTKKLIEIYDNILRQVDKYYKLY
DINKFRAKLHDGRSKFVELDDFGQDASKEKVIIKILRGLHFGSDLQNLKEIGFGTTPLGQFQ
VSEAGIRLSNTAFIIFKSPTGLFNRKLYLKNLGKRPAATKKAGQAKKKKGSYPYDVPDYAYP
YDVPDYAYPYDVPDYA;
(h)
(SEQ ID NO: 89)
MPKKKRKVGKEYHIGLAIGTSSIGWAVTDSQFKLMRIKGKTAIGVRLFEEGKTAAERRTFRT
TRRRLKRRKWRLHYLDEIFAPHLQEVDENFLRRLKQSNIHPEDPAKNQAFIGKLLFPDLLKK
NERGYPTLIKMRDELPVEQRAHYPVTNIYKLREAMINEDRQFDLREVYLAVHHIVKYRGHEL
NNASVDKFKVGRIDFDKSFNVLNEAYEELQNGEGSFTIEPSKVEKIGQLLLDTKMRKLDRQK
AVAKLLEVKVADKEETKRNKQIATAMSKLVLGYKADFATVAMANGNEWKIDLSSETSEDEIE
KFREELSDAQNDILTEITSLESQIMLNEIVPNGMSISESMMDRYWTHERQLAEVKEYLATQP
ASARKEFDQVYNKYIGQAPKEKGFDLEKGLKKILSKKENWKEIDELLKAGDFLPKQRTSANG
VIPHQMHQQELDRIIEKQAKYYPWLATENPATGERDRHQAKYELDQLVSFRIPYYVGPLVTP
EVQKATSGAKFAWAKRKEDGEITPWNLWDKIDRAESAEAFIKRMTVKDTYLLNEDVLPANSL
LYQKYNVLNELNNVRVNGRRLSVGIKQDIYTELFKKKKTVKAGDVASLVMAKTRGVNKPSVE
GLSDPKKENSNLATYLDLKSIVGDKVDDNRYQMDLENIIEWRSVFEDGEIFADKLTEVEWLT
DEQRSALVKKRYKGWGRLSKKLLTGIVDENGQRIIDLMWNTDQNFMQIVNQPVFKEQIDQLN
QKAITNDGMTLRERVESVLDDAYTSPQNKKAIWQVVRVVEDIVKAVGNAPKSISIEFARNEG
NKGEITRSRRTQLQKLFEDQAHELVKDTSLTEELEKAPDLSDRYYFYFTQGGKDMYTGDPIN
FDEISTKYDIDHILPQSFVKDDSLDNRVLVSRAENNKKSDRVPAKLYAAKMKPYWNQLLKQG
LITQRKFENLTMDVDQTIKYRSLGFVKRQLVETRQVIKLTANILGSMYQEAGTDIIETRAGL
TKQLREEFDLPKVREVNDYHHAVDAYLTTFAGQYLNRRYPKLRSFFVYGEYMKFKHGSDLKL
RNFNFFHELMEGDKSQGKVVDQQTGELITTRDEVADYFDWVINLKVMLISNETYEETGKYED
ASHESSSLYLKNQNKKSKLVVPLKNKLQPEYYGAYTGITQGYMVILKLLDKKGGFGVYRIPR
YAADILNKCHDEVAYRNKIAEIISSDPRAPKSFEVVVPRVLKGTFLVDGEEKFILSSYRYKV
NATQLILPVSDIKLIQDNFKALKKLNVEMQTKKLIEIYDNILRQVDKYYKLYDINKFRAKLH
DGRSKFVELDDFGQDASKEKVIIKILRGLHFGSDLQNLKEIGFGTTPLGQFQVSEAGIRLSN
TAFIIFKSPTGLENRKLYLKNLKRPAATKKAGQAKKKKSGSETPGTSESATPESSGSEVEFS
HEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLV
MQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVE
ITEGILADECAALLCRFFRMPRRVENAQKKAQSSTDPAAKRVKLDGSYPYDVPDYAYPYDVP
DYAYPYDVPDYA;
or
(i)
(SEQ ID NO: 98)
MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA
LRQGGLVMQNYRLYDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHP
GMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSES
ATPESSGGSSGGSGKPYSIGLAIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKQSIKKNLLGA
LLFDSGETAEATRLKRTARRRYTRRKNRLRYLQEIFTGEMNKVDENFFQRLDDSFLVDEDKR
GEHHPIFGNIAAEVKYHDDFPTIYHLRRHLADTSKKADLRLVYLALAHMIKERGHFLYEGDL
KAENTDVQALFKDEVEEYDKTIEESHLSEITVDALSILTEKVSKSSRLENLIAHYPTEKKNT
LFGNLIALSLDLHPNEKTNFQLSEDAKLQFSKDTYEEDLEGFLGEVGDEYADLFASAKNLYD
AILLSGILTVDDNSTKAPLSASMVKRYEEHQKDLKKLKDFIKVNAPDQYNAIFKDKNKKGYA
SYIESGVKQDEFYKYLKGILLKINGSGDFLDKIDREDFLRKQRTFDNGSIPHQIHLQEMHAI
LRRQGEHYPFLKENQDKIEKILTFRIPYYVGPLARKGSRFAWAEYKADEKITPWNEDDILDK
EKSAEKFITRMTLNDLYLPEEKVLPKHSPLYEAFTVYNELTKVKYVNEQGEAKFFDTNMKQE
IFDHVFKENRKVTKDKLLNYLNKEFEEFRIVNLTGLDKENKAFNSSLGTYHDLRKILDKSFL
DDKANEKTIEDIIQTLTLFEDREMIRQRLQKYSDIFTKAQLKKLERRHYTGWGRLSYKLING
IRNKENKKTILDYLIDDGYANRNFMQLINDDALSFKEEIARAQIIDDVDDIANVVHDLPGSP
AIKKGILQSVKIVDELVKVMGHNPANIIIEMARENQTTDKGRRNSQQRLKLLQDSLKNLDNP
VNIKNVENQQLQNDRLFLYYIQNGKDMYTGETLDINNLSQYDIDHIIPQAFIKDNSLDNRVL
TRSDKNRGKSDDVPSIEVVHEMKSFWSKLLSVKLITQRKEDNLTKAERGGLTEEDKAGFIKR
QLVETRQITKHVAQILDERENTEFDGNKRRIRNVKIITLKSNLVSNFRKEFELYKVREINDY
HHAHDAYLNAVVGNALLLKYPQLEPEFVYGEYPKYNSYRSRKSATEKFLFYSNILRFFKKED
IQTNEDGEIAWNKEKHIKILRKVLSYPQVNIVKKTEEQTGGFSKESILPKGESDKLIPRKTK
NSYWDPKKYGGFMQPVVAYSILVFADVEKGKSKKLRKVQDMVGITIMEKKRFEKNPVDFLEQ
RGYRNVRLEKIIKLPKYSLFELENKRRRLLASAKFLQKGNELVIPQRFTTLLYHSYRIEKDY
EPEHREYVEKHKDEFKELLEYISVESRKYVLADNNLTKIEMLESKNKDAEVSSLAKSFISLL
TFTAFGAPRAFNFFGENIARKEYRSVTECLNATLIHQSITGLYETRIDLSKLGEDGEGADKR
TADGSEFESPKKKRKV;
(j)
(SEQ ID NO: 99)
MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA
LRQGGLVMQNYRLYDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHP
GMNHRVEITEGILADECAALLCRFFRMPRRVENAQKKAQSSTDSGGSSGGSSGSETPGTSES
ATPESSGGSSGGSGKPYSIGLAIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKQSIKKNLLGA
LLFDSGETAEATRLKRTARRRYTRRKNRLRYLQEIFTGEMNKVDENFFQRLDDSFLVDEDKR
GEHHPIFGNIAAEVKYHDDFPTIYHLRRHLADTSKKADLRLVYLALAHMIKFRGHELYEGDL
KAENTDVQALFKDFVEEYDKTIEESHLSEITVDALSILTEKVSKSSRLENLIAHYPTEKKNT
LFGNLIALSLDLHPNFKTNFQLSEDAKLQFSKDTYEEDLEGFLGEVGDEYADLFASAKNLYD
AILLSGILTVDDNSTKAPLSASMVKRYEEHQKDLKKLKDFIKVNAPDQYNAIFKDKNKKGYA
SYIESGVKQDEFYKYLKGILLKINGSGDFLDKIDREDELRKQRTEDNGIIPHQIHLQEMHAI
LRRQGEHYPFLKENQDKIEKILTFRIPYYVGPLARKGSRFAWAEYKADEKITPWNEDDILDK
EKSAEKFITRMTLNDLYLPEEKVLPKHSPLYEAFTVYNELTKVKYVNEQGEAKFEDTNMKQE
IFDHVFKENRKVTKDKLLNYLNKEFEEFRIVNLTGLDKENKAFNSSLGTYHDLRKILDKSFL
DDKANEKTIEDIIQTLTLFEDREMIRQRLQKYSDIFTKAQLKKLERLHYTGWGRLSYKLING
IRNKENKKTILDYLIDDGYANRNFMQLINDDALSFKEEIARAQIIDDVDDIANVVHDLPGSP
AIKKGILQSVKIVDELVKVMGHNPANIIIEMARENQTTDKGRRNSQQRLKLLQDSLKNLDNP
VNIKNVENQQLQNDRLFLYYIQNGKDMYTGETLDINNLSQYDIDHIIPQAFIKDNSLDNRVL
TRSDKNRGKSDDVPSIEVVHEMKSFWSKLLSVKLITQRKEDNLTKAERGGLTEEDKAGFIKR
QLVETRQITKHVAQILDERENTEFDGNKRRIRNVKIITLKSNLVSNFRKEFELYKVREINDY
HHAHDAYLNAVVGNALLLKYPQLEPEFVYGEYPKYNSYRSRKSATEKFLFYSNILRFFKKED
IQTNEDGEIAWNKEKHIKILRKVLSYPQVNIVKKTEEQTGGFSKESILPKGESDKLIPRKTK
NSYWDPKKYGGFMQPVVAYSILVFADVEKGKSKKLRKVQDMVGITIMEKKRFEKNPVDFLEQ
RGYRNVRLEKIIKLPKYSLFELENKRRRLLASAKFLQKGNELVIPQRFTTLLYHSYRIEKDY
EPEHREYVEKHKDEFKELLEYISVFSRKYVLADNNLTKIEMLESKNKDAEVSSLAKSFISLL
TFTAFGAPRAFNFFGENIARKEYRSVTECLNATLIHQSITGLYETRIDLSKLGEDGEGADKR
TADGSEFESPKKKRKV.
24 . The Cas9 protein of claim 22 , wherein the Cas9 protein is fused to a cytosine deaminase and has an amino acid sequence at least 80% identical to
(a)
(SEQ ID NO: 21)
MPAAKRVKLDTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRS
SGKNTTNHVEVNFIKKFTSERRFHSSISCSITWELSWSPCWECSQAIREFLSQHPGVTLVIY
VARLFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMML
YALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWRLKS
GGSSGGSSGSETPGTSESATPESSGGSSGGSPKKKRKVGGKPYSIGLAIGTNSVGWAVVTDD
YKVPAKKMKVLGNTDKQSIKKNLLGALLFDSGETAEATRLKRTARRRYTRRKNRLRYLQEIF
TGEMNKVDENFFQRLDDSFLVDEDKRGEHHPIFGNIAAEVKYHDDFPTIYHLRRHLADTSKK
ADLRLVYLALAHMIKFRGHFLYEGDLKAENTDVQALFKDEVEEYDKTIEESHLSEITVDALS
ILTEKVSKSSRLENLIAHYPTEKKNTLFGNLIALSLDLHPNFKTNFQLSEDAKLQFSKDTYE
EDLEGFLGEVGDEYADLFASAKNLYDAILLSGILTVDDNSTKAPLSASMVKRYEEHQKDLKK
LKDFIKVNAPDQYNAIFKDKNKKGYASYIESGVKQDEFYKYLKGILLKINGSGDFLDKIDRE
DFLRKQRTFDNGSIPHOIHLQEMHAILRRQGEHYPFLKENQDKIEKILTFRIPYYVGPLARK
GSRFAWAEYKADEKITPWNEDDILDKEKSAEKFITRMTLNDLYLPEEKVLPKHSPLYEAFTV
YNELTKVKYVNEQGEAKFFDTNMKQEIFDHVFKENRKVTKDKLLNYLNKEFEEFRIVNLTGL
DKENKAFNSSLGTYHDLRKILDKSFLDDKANEKTIEDIIQTLTLFEDREMIRQRLOKYSDIF
TKAQLKKLERRHYTGWGRLSYKLINGIRNKENKKTILDYLIDDGYANRNFMQLINDDALSEK
EEIARAQIIDDVDDIANVVHDLPGSPAIKKGILQSVKIVDELVKVMGHNPANIIIEMARENQ
TTDKGRRNSQQRLKLLQDSLKNLDNPVNIKNVENQQLQNDRLFLYYIQNGKDMYTGETLDIN
NLSQYDIDHIIPQAFIKDNSLDNRVLTRSDKNRGKSDDVPSIEVVHEMKSFWSKLLSVKLIT
QRKFDNLTKAERGGLTEEDKAGFIKRQLVETRQITKHVAQILDERENTEFDGNKRRIRNVKI
ITLKSNLVSNFRKEFELYKVREINDYHHAHDAYLNAVVGNALLLKYPQLEPEFVYGEYPKYN
SYRSRKSATEKFLFYSNILRFFKKEDIQTNEDGEIAWNKEKHIKILRKVLSYPQVNIVKKTE
EQTGGFSKESILPKGESDKLIPRKTKNSYWDPKKYGGFDSPVVAYSILVFADVEKGKSKKLR
KVQDMVGITIMEKKRFEKNPVDFLEQRGYRNVRLEKIIKLPKYSLFELENKRRRLLASAKEL
QKGNELVIPQRFTTLLYHSYRIEKDYEPEHREYVEKHKDEFKELLEYISVESRKYVLADNNL
TKIEMLFSKNKDAEVSSLAKSFISLLTFTAFGAPAAFNFFGENIDRKRYTSVTECLNATLIH
QSITGLYETRIDLSKLGEDGKRPAATKKAGQAKKKKGSSGGSGGSGGSTNLSDIIEKETGKQ
LVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNG
ENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLYPYDVPDYAYPYDVPDYAYPYDVP
DYA;
(b)
(SEQ ID NO: 12)
MPAAKRVKLDTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRS
SGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIY
VARLFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMML
YALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWRLKS
GGSSGGSSGSETPGTSESATPESSGGSSGGSPKKKRKVGSIINFQRRGLMETQASNQLISSH
LKGYPIKDYFVGLAIGTSSVGWAVTNKAYELLKFRSHKMWGSRLFDEGESAVARRGERSMRR
RLERRKLRLKLLEELFADAMAQVDPTFFMRLRESKYHYEDKTTGHSSKHILFIDKNYNDQDY
FKEYPTVYHLRSELMKSGTDDIRKLFLAVHHILKYRGNFLYEGATFDSNASTLDDVIKQALE
NITFNCFDCNSAISSIGQILMEAGKTKSDKAKAIEHLVDTYIATDTVDTSSKTQKDQVKEDK
KRLKAFANLVLGLNASLIDLFGSVEELEEDLKKLQITGDTYDDKRDELAKAWSDEIYIIDDC
KSVYDAIILLSIKEPGLTISESKVKAFNKHKDDLAILKSLLKSDRSIYNTMFKVDEKGLHNY
VHYIKQGRTEETSCNREDFYKYTKKIVEGLSDSKDKEYILSQIELQILLPLQRIKDNGVIPY
QLHLEELKAILAKCGPKFPFLNEVADGESVAEKLIKMLEFRIPYYVGPLNTHHNVDNGGFAW
AVRKASGRVTPWNFDDKIDREKSAAAFIKNLTNKCTYLLGEDVLPKSSLLYSEFMLLNELNN
VRIDGKPLEKVVKEHLIEAVFKQDHKKMTKNRIEQFLKDNGYISETHKHEITGLDGEIKNDL
ASYRDMVRILGDGFDRSMAEEIITDITIFGESKKMLRETLRKKFASCLDDEAIKKLTKLRYR
DWGRLSQKLLNGIEGCDKAGDGTPETIIILMRNFSYNLMELLGDKFSFMERIQEINAKLTEG
QIVNPHDIIDDLALSPAVKRAVWQALRIVDEVAHIKKALPARIFVEVTRSNKNEKKKKDSRQ
KRLSDLYAAIKKDDVLLNGLNNEIFGELKSSLAKYDDAALRSKKLYLYYTQMGRCAYTGEII
ELSLLNTDNYDIDHIYPRSLTKDDSFDNLVLCKRTANAQKSDAYPISEEIQKTQKPFWTFLK
QQGLISERKYERLTRITPLTADDLSGFIARQLVETNQSVKAATTLLRRLYPGVDVVFVKAEN
VTDFRHDNNFIKVRSLNHHHHAKDAYLNIVVGNVYHERFTRNFRAFFKKNGANRTYNLAKME
NYDVNCTNAKDGKAWDVKTSMDTVKKMMDSNDVRVTKRLLEQTGALADATIYKATVAGKAKD
GAYIGMKTKSSVFADVSKYGGMTKIKNAYSIIVQYTGKKGEVIKEIVPLPIYLTNRNTTDQD
LINYVASIIPQAKDISIIYGKLCINQLVKVNGFYYYLGGKTNSKFCIDNAIQVIVSNEWIPY
LKVLEKFNNMRKDNKDLKANVVSTRALDNKHTIEVRIVEEKNIEFFDYLVSKLKMPIYQKMK
GNKAAELSEKGYGLFKKMSLEEQSIHLIELLNLLTNQKTTFEVKPLGITASRSTVGSKISNQ
DEFKVINESITGLYSNEVTIVGKRPAATKKAGQAKKKKGSSGGSGGSGGSTNLSDIIEKETG
KQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDS
NGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVH
TAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLYPYDVPDYAYPYDVPDYAY;
(c)
(SEQ ID NO: 18)
MPAAKRVKLDTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRS
SGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIY
VARLFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMML
YALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWRLKS
GGSSGGSSGSETPGTSESATPESSGGSSGGSPKKKRKVGTKVKDYYIGLAIGTSSVGWAVTD
EAYNVLKFNSKKMWGVRLFDDAKTAEERRGQRGARRRLDRKKERLSLLQDFFAEEVAKVDPN
FFLRLDNSDLYMEDKDQKLKSKYTLENDKDEKDKNFHKKYPTIHHLLMDLIEDDSKKDIRLV
YLACHYLLKNRGHFIFEGQKFDTKSSFENSLNELKVHLNDEYGLDLEFDNENLINILTDPKL
NKTAKKKELKSVIGDTKFLKAVSAIMIGSSQKLVDLFENPEDEDDSAIKSVDESTTSFDDKY
SDYELALGDKIALVNILKEIYDSSILENLLKEADKSKDGNKYISNAFVKKYNKHGQDLKEFK
RLVRQYHKSAYFDIFRSEKVNDNYVSYTKSSISNNKRVKANKFTDQEAFYKFAKKHLETIKY
KINKVNGSKADLELIDGMLRDMEFKNFMPKIKSSDNGVIPYQLKLMELNKILENQSKHHEFL
NVSDEYGSVCDKIASIMEFRIPYYVGPLNPNSKYAWIKKQKDSEITPWNFKDVVDLDSSREE
FIDSLIGRCTYLKDEKVLPKASLLYNEYMVLNELNNLKLNDLPITEEMKKKIFDQLFKTRKK
VTLKAVANLLKKEFNINGEILLSGTDGDFKQGLNSYNDFKAIVGDKVDSDDYRDKIEEIIKL
IVLYGDDKSYLQKKIKAGYGKYFTDSEIKKMAGLNYKDWGRLSKKLLTGLEGANKITGERGS
IIHFMREYNLNLMELMSASFTFTEEIQKLNPVDDRKLSYEMVDELYLSPSVKRMLWQSLRIV
DEIKNIMGTDSKKIFIEMARGKEEVKARKESRKNQLLKFYKDGKKAFISEIGEERYSYLLSE
IEGEEENKFRWDNLYLYYTQLGRCMYSLEPIDISELSSKNIYDQDHIYPKSKIYDDSIENRV
LVKKDLNSKKGNSYPIPDEILNKNCYAYWKILYDKGLIGQKKYTRLTRRTGFTDDELVQFIS
RQIVETRQATKETANLLKTICKNSEIVYSKAENASRFRQEFDIVKCRAVNDLHHMHDAYINI
IVGNVYNTKFTKDPMNFVKKQEKARSYNLENMEKYDVKRGGYTAWIADDEKGTVKNASIKRI
RKELEGTNYRFTRMNYIESGALFNATLQRKNKGSRPLKDKGPKSSIEKYGGYTNINKACFAV
LDIKSKNKIERKLMPVEREIYAKQKNDKKLSDEIFSKYLKDREGIEDYRVVYPVVKMRTLLK
IDGSYYFITGGSDKTLELRSALQLILPKKNEWAIKQIDKSSENDYLTIERIQDLTEELVYNT
FDIIVNKFKTSVFKKSFLNLFQDDKIENIDFKFKSMDFKEKCKTLLMLVKAIRASGVRQDLK
SIDLKSDYGRLSSKINNIGNYQEFKIINQSITGLFENEVDLLKLGKRPAATKKAGQAKKKKG
SSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTD
ENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQES
ILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKM
LYPYDVPDYAYPYDVPDYAY;
(d)
(SEQ ID NO: 90)
MPAAKRVKLDTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRS
SGKNTTNHVEVNFIKKFTSERRFHSSISCSITWELSWSPCWECSQAIREFLSQHPGVTLVIY
VARLFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMML
YALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWRLKS
GGSSGGSSGSETPGTSESATPESSGGSSGGSPKKKRKVGKEYHIGLAIGTSSIGWAVTDSQF
KLMRIKGKTAIGVRLFEEGKTAAERRTFRTTRRRLKRRKWRLHYLDEIFAPHLQEVDENFLR
RLKQSNIHPEDPAKNQAFIGKLLFPDLLKKNERGYPTLIKMRDELPVEQRAHYPVINIYKLR
EAMINEDRQFDLREVYLAVHHIVKYRGHFLNNASVDKFKVGRIDEDKSENVLNEAYEELQNG
EGSFTIEPSKVEKIGQLLLDTKMRKLDRQKAVAKLLEVKVADKEETKRNKQIATAMSKLVLG
YKADFATVAMANGNEWKIDLSSETSEDEIEKFREELSDAQNDILTEITSLESQIMLNEIVPN
GMSISESMMDRYWTHERQLAEVKEYLATQPASARKEFDQVYNKYIGQAPKEKGEDLEKGLKK
ILSKKENWKEIDELLKAGDELPKQRTSANGVIPHQMHQQELDRIIEKQAKYYPWLATENPAT
GERDRHQAKYELDQLVSFRIPYYVGPLVTPEVQKATSGAKFAWAKRKEDGEITPWNLWDKID
RAESAEAFIKRMTVKDTYLLNEDVLPANSLLYQKYNVLNELNNVRVNGRRLSVGIKQDIYTE
LFKKKKTVKAGDVASLVMAKTRGVNKPSVEGLSDPKKENSNLATYLDLKSIVGDKVDDNRYQ
MDLENIIEWRSVFEDGEIFADKLTEVEWLTDEQRSALVKKRYKGWGRLSKKLLTGIVDENGQ
RIIDLMWNTDQNFMQIVNQPVFKEQIDQLNQKAITNDGMTLRERVESVLDDAYTSPQNKKAI
WQVVRVVEDIVKAVGNAPKSISIEFARNEGNKGEITRSRRTQLQKLFEDQAHELVKDTSLTE
ELEKAPDLSDRYYFYFTQGGKDMYTGDPINFDEISTKYDIDHILPQSFVKDDSLDNRVLVSR
AENNKKSDRVPAKLYAAKMKPYWNQLLKQGLITQRKFENLTMDVDQTIKYRSLGFVKRQLVE
TRQVIKLTANILGSMYQEAGTDIIETRAGLTKQLREEFDLPKVREVNDYHHAVDAYLTTFAG
QYLNRRYPKLRSFFVYGEYMKFKHGSDLKLRNENFFHELMEGDKSQGKVVDQQTGELITTRD
EVADYFDWVINLKVMLISNETYEETGKYFDASHESSSLYLKNQNKKSKLVVPLKNKLQPEYY
GAYTGITQGYMVILKLLDKKGGFGVYRIPRYAADILNKCHDEVAYRNKIAEIISSDPRAPKS
FEVVVPRVLKGTFLVDGEEKFILSSYRYKVNATQLILPVSDIKLIQDNFKALKKLNVEMQTK
KLIEIYDNILRQVDKYYKLYDINKFRAKLHDGRSKFVELDDFGQDASKEKVIIKILRGLHFG
SDLQNLKEIGFGTTPLGQFQVSEAGIRLSNTAFIIFKSPTGLENRKLYLKNLGKRPAATKKA
GQAKKKKGSSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVH
TAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETG
KQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDS
NGENKIKMLYPYDVPDYAYPYDVPDYAY;
(e)
(SEQ ID NO: 93)
MPAAKRVKLDTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRS
SGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIY
VARLFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMML
YALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWRLKS
GGSSGGSSGSETPGTSESATPESSGGSSGGSPKKKRKVGEKKTNYTIGLAIGTDSVGWAVVK
DDLELVKKRMKVLGNTETNYIKKNLWGSLLFESGQTAKDRRLKRVARRRYERRRNRLTELQK
IFAPAIDEVDENFFFRLNESFLVPEDKAFSKNPIFGTLGEDKTYYKTYPTIYHLRQHLADSE
EKADVRLIYLALAHMIKYRGHFLIEGKLDTEHIAINENLEQFFESYNALFSEEPIELRKEEL
IAIENILREKNSRTVKEKRITSFLKDIGRANKQSPMMAFITLIVGKKAKFKAAFNLEEEISL
NLTDDSYDENLEILLNTIGSDFADLEDHAQRVYNAVELAGILSGDVKNTHAKLSAQMVAMYE
RHKEQLKEYKSFIKANLPDQYDMTFVAPKDAQKKDLKGYAGYIDGNMSQDSFYKFVKDQLKE
VPGSEKFLDSIEKEDFLRKQRSFYNGVIPNQVHLAEMEAILDRQENYYPWLKENREKIISLL
TFRIPYYVGPLADGQSEFAWLERKSDEKIKPWNFSDVVDLDRSAEKFIEQLIGRDTYLPDEY
VLPKKSLIYQKYMVFNELTKIAYLDERQKRMNLSSVEKKEIFETLFKKRSKVTEKQLVKFFE
NYLQIDNPTIFGIEDAFNADYSTYVELAKVPGMKSMMDDPDNEDLMEEIVKILTVFEDRKMR
RKQLEKYKERLSPEQIKELAKKHYTGWGRLSKKLLVGIRDKETQKTILDYLVEDDNHSGGRQ
HLNRNLMQLINDDRLSFKKTIAELQMIDPSADLYAQVQEIAGSPAIKKGILLGLKIVDEIIR
VMGEKPENIVIEMARENQTTARGKALSKRREAKIKEGLAALGSSLLKENLPGNADLSQRKIY
LYYTQNGKDIYLDEPLDFDRLSQYDEDHIIPQSFTVDNSLDNLVLTNSSQNRGNKKDDVPSL
EVVNRQLAYWRSLKDAGLMTQRKFDNLTKAMRGGLTDKDRERFIQRQLVETRQITKNVAKLL
DMRLNDKKDEAGNKIRETNIVLLKSAMASEFRKMFRLYKVRELNDYHHAHDAYLNAAIAINL
LALYPYMADDEVYGEFRYKKKPQAEKATYEKLRQWNLIKRFGEKQLFTPDHEDCWNKERDIK
TIKKVMGYRQVNVVKKAEERTGMLFKETINGKTNKGSRIPIKKDLDPSKYGGYIEEKMAYYA
VISYEDKKKKPGKTIVGISIMDKKEFEYDSISYLGKLGFSNPVVQIILKNYSLIAYPDGRRR
YITGATKTTKGKVELQKANQIAMEQDLVNFIYHLKNYDEISHPESYAFVQSHTDYFDRLEDS
IEHYTRRFLDAETNINRLRRIYEEEKKKDPVDIEALVASFIELLKLTSAGAPADFIFMGEAI
SRRRYNSMTGLEDGQVIYQSLTGLYETRMRFEDGKRPAATKKAGQAKKKKGSSGGSGGSGGS
TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAP
EYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEV
IGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLYPYDVPDYAY
PYDVPDYAY;
(f)
(SEQ ID NO: 94)
MPAAKRVKLDTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDE
NVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESI
LMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML
SGGSGGSGGSPKKKRKVEKKTNYTIGLAIGTDSVGWAVVKDDLELVKKRMKVLGNTETNYIK
KNLWGSLLFESGQTAKDRRLKRVARRRYERRRNRLTELQKIFAPAIDEVDENFFERLNESEL
VPEDKAFSKNPIFGTLGEDKTYYKTYPTIYHLRQHLADSEEKADVRLIYLALAHMIKYRGHF
LIEGKLDTEHIAINENLEQFFESYNALFSEEPIELRKEELIAIENILREKNSRTVKEKRITS
FLKDIGRANKQSPMMAFITLIVGKKAKFKAAFNLEEEISLNLTDDSYDENLEILLNTIGSDE
ADLFDHAQRVYNAVELAGILSGDVKNTHAKLSAQMVAMYERHKEQLKEYKSFIKANLPDQYD
MTFVAPKDAQKKDLKGYAGYIDGNMSQDSFYKFVKDQLKEVPGSEKFLDSIEKEDELRKQRS
FYNGVIPNQVHLAEMEAILDRQENYYPWLKENREKIISLLTFRIPYYVGPLADGQSEFAWLE
RKSDEKIKPWNFSDVVDLDRSAEKFIEQLIGRDTYLPDEYVLPKKSLIYQKYMVENELTKIA
YLDERQKRMNLSSVEKKEIFETLFKKRSKVTEKQLVKFFENYLQIDNPTIFGIEDAFNADYS
TYVELAKVPGMKSMMDDPDNEDLMEEIVKILTVFEDRKMRRKQLEKYKERLSPEQIKELAKK
HYTGWGRLSKKLLVGIRDKETQKTILDYLVEDDNHSGGRQHLNRNLMQLINDDRLSFKKTIA
ELQMIDPSADLYAQVQEIAGSPAIKKGILLGLKIVDEIIRVMGEKPENIVIEMARENQTTAR
GKALSKRREAKIKEGLAALGSSLLKENLPGNADLSQRKIYLYYTQNGKDIYLDEPLDEDRLS
QYDEDHIIPQSFTVDNSLDNLVLTNSSQNRGNKKDDVPSLEVVNRQLAYWRSLKDAGLMTQR
KFDNLTKAMRGGLTDKDRERFIQRQLVETRQITKNVAKLLDMRLNDKKDEAGNKIRETNIVL
LKSAMASEFRKMERLYKVRELNDYHHAHDAYLNAAIAINLLALYPYMADDFVYGEFRYKKKP
QAEKATYEKLRQWNLIKRFGEKQLFTPDHEDCWNKERDIKTIKKVMGYRQVNVVKKAEERTG
MLFKETINGKTNKGSRIPIKKDLDPSKYGGYIEEKMAYYAVISYEDKKKKPGKTIVGISIMD
KKEFEYDSISYLGKLGFSNPVVQIILKNYSLIAYPDGRRRYITGATKTTKGKVELQKANQIA
MEQDLVNFIYHLKNYDEISHPESYAFVQSHTDYFDRLEDSIEHYTRRFLDAETNINRLRRIY
EEEKKKDPVDIEALVASFIELLKLTSAGAPADFIFMGEAISRRRYNSMTGLEDGQVIYQSLT
GLYETRMRFEDKRPAATKKAGQAKKKKGSSGGSSGGSSGSETPGTSESATPESSGGSSGGST
SEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEV
NFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVARLFWHMDQR
NRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILS
LPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWRYPYDVPDYAYPYDV
PDYAYPYDVPDYA.
25 . The Cas9 protein of claim 2 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NGG-3′.
26 . The Cas9 protein of claim 3 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NAGHC-3′, wherein H is adenine, cytosine, or thymine.
27 . The Cas9 protein of claim 4 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NRHRRH-3′, wherein H is adenine, cytosine or thymine, and R is adenine or guanine.
28 . The Cas9 protein of claim 5 or claim 7 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NGG-3′.
29 . The Cas9 protein of claim 6 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NNAAA-3′.
29 b. The Cas9 protein of claim 10 b or 10 c, wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NGG-3′.
30 . A nucleic acid encoding the Cas9 protein of any one of the preceding claims.
31 . The nucleic acid of claim 30 , wherein the nucleic acid is codon-optimized for expression in mammalian cells.
32 . The nucleic acid of claim 31 , wherein the nucleic acid is codon-optimized for expression in human cells.
33 . A eukaryotic cell comprising the Cas9 protein of any one of claim 29 .
34 . The eukaryotic cell of claim 33 , wherein the cell is a human cell.
35 . A method of cleaving a target nucleic acid in a eukaryotic cell comprising:
contacting the cell with a Cas9 of any one of claims 1 - 29 , and an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and
wherein the Cas9 protein is capable of binding to the RNA guide and of causing a break in the target nucleic acid sequence complementary to the RNA guide.
36 . A method of altering expression of a target nucleic acid in a eukaryotic cell comprising:
contacting the cell with a Cas9 of any one of claims 1 - 29 , and an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and
wherein the Cas9 protein is capable of binding to the RNA guide and of causing a break in the target nucleic acid sequence complementary to the RNA guide.
37 . A method of altering expression of a target nucleic acid in a eukaryotic cell comprising:
contacting the cell with a Cas9 of any one of claims 1 - 29 , and an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and
wherein the Cas9 protein is capable of binding to the RNA guide and editing the target nucleic acid sequence complementary to the RNA guide.
38 . A method of modifying a target nucleic acid in a eukaryotic cell comprising:
contacting the cell with a Cas9 of any one of claims 1 - 29 , and an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and
wherein the Cas9 protein is capable of binding to the RNA guide and editing the target nucleic acid sequence complementary to the RNA guide.
39 . The method of claim 37 or 38 , wherein the Cas9 protein is an inactive Cas9 (dCas9).
40 . The method of claim 39 , wherein the dCas9 is fused to a deaminase.
41 . The method of any one of claims 35 - 40 , wherein the RNA guide comprises a crRNA and a tracrRNA.
42 . The method of any one of claims 35 - 39 , wherein the RNA guide comprises a sgRNA.
43 . The method of claim 42 , wherein the sgRNA for use with Streptococcus constellatus Cas 9 comprises a scaffold comprising a sequence having at least about 80% identity to
(SEQ ID NO: 3)
5′-GUUUUAGAGCUGUGCUGUUUAAACAACACAGCAAGUUAAAAUAAGGC
UUUGUCCGUACUCAAGCUUGCAAAAGCGUGCACCGAUUCGGUGCU-3′
44 . The method of claim 42 , wherein the sgRNA for use with Sharpea Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to
(SEQ ID NO: 7)
5′-GUUUUAGAGUUGUGUUAUUGAAAAAUAACACAACGAGUUAAAAUAAA
GCUUAUGCUUAAAUGCCAGCUUUGCUGGUGUCAUUUAGAUGACUUUACUA
AGGUUGCUUCGGCAACCUUUUU-3′
45 . The method of claim 42 , wherein the sgRNA for use with Veillonella parvula Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to
(SEQ ID NO: 13)
5′-GUUUGAGAGUAGUGUGAAAACAUUACGAGUUCAAAUACAAAUUAAUU
UACAAUGCCUUCGGGCUGCCCGACGUAGGGCACCUACUCUCAAUUCUUCG
GAAUUGAGUU-3′
46 . The method of claim 42 , wherein the sgRNA for use with Ezakiella peruensis Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to
(SEQ ID NO: 19)
5′-GUUUGAGAGUUAUGUAAUUGAAAAAUUACAUGACGAGUUCAAAUAAA
AAUUUAUUCAAACCGCCUAUUUAUAGGCCGCAGAUGUUCUGCAUUAUGCU
UGCUAUUGCAAGCUU-3′
47 . The method of claim 42 , wherein the sgRNA for use with Lactobacillus fermentum strain AF15-40LB Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to
(SEQ ID NO: 95)
5′-GUCUUGGAUGAGUGUGAAAACACUCAUAGUCAAGAUCAAACGAGUGG
UUUUCCACGAGUUAUUACUUUUGAGGUCUUAUAUGGCCCAUACAUAAAAA
GGAGUCGGAAUUUCCGGCUCCUUUUCUU-3′
48 . The method of claim 42 , wherein the sgRNA for use with Peptoniphilus sp. Marseille-P 3761 Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to
(SEQ ID NO: 96)
5′-GUUUUAGAGCCAUGUAGAAAUACAUUGCAAGUUAAAAUAAGGCUUUG
UCCGUAAUCAACUUGAAAAAGUGGCGCUGUUUCGGCGCUUU-3′
49 . The method of claim 41 , wherein the crRNA comprises a guide sequence of between about 16 and 26 nucleotides long.
50 . The method of claim 49 , wherein the crRNA comprises a guide sequence between 18 and 24 nucleotides long.
51 . The method of claim 35 or 36 , wherein the break in the target nucleic acid is a single-stranded or double-stranded break.
52 . The method of claim 51 , wherein the break in the target nucleic acid is a single-stranded break.
53 . The method of claim 34 or 35 , wherein the Cas9 protein is a nuclease that cleaves both strands of the target nucleic acid sequence, or is a nickase that cleaves one strand of the target nucleic acid sequence.
54 . The method of any one of claims 34 - 53 , wherein the target nucleic acid is 5′ to a protospacer adjacent motif (PAM) sequence.
55 . The method of any one of claims 34 - 54 , wherein the Cas9 is operably linked to a promoter sequence for expression in a eukaryotic cell, and wherein the guide RNA is operably linked to a promoter sequence for expression in a eukaryotic cell.
56 . The method of claim 55 , wherein the eukaryotic cell is a human cell.
57 . The method of claim 55 , wherein the promoter sequence is a eukaryotic or viral promoter.
58 . An engineered, non-naturally occurring CRISPR-Cas system comprising:
an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid; and
a codon-optimized CRISPR-associated (Cas) protein having at least 80% sequence identity to SEQ ID NOs: 1, 4, 8, 14, 84 or 86, and wherein the Cas protein is capable of binding to the RNA guide and of causing a break in the target nucleic acid sequence complementary to the RNA guide.
58 b. The engineered, non-naturally occurring CRISPR-Cas system of claim 58 where the codon-optimized CRISPR-associated (Cas) protein further comprising a nuclear localization sequence (NLS) and/or a FLAG, HIS or HA tag.
58 c. The engineered, non-naturally occurring CRISPR-Cas system of claim 59 where the codon-optimized CRISPR-associated (Cas) protein having at least 80% sequence identity to SEQ ID NOs: 2, 5, 9, 15, 85, 87, 95 or 96, and wherein the Cas protein is capable of binding to the RNA guide and of causing a break in the target nucleic acid sequence complementary to the RNA guide.
59 . An engineered, non-naturally occurring CRISPR-Cas system comprising:
an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid; and
a codon-optimized CRISPR-associated (Cas) protein having at least 80% sequence identity to SEQ ID NOs: 1, 4, 8, 14, 84 or 86;
wherein the Cas protein is fused to a deaminase, and wherein the Cas protein fusion is capable of binding to the RNA guide and of editing the target nucleic acid sequence complementary to the RNA guide.
59 b. The engineered, non-naturally occurring CRISPR-Cas system of claim 59 where the codon-optimized CRISPR-associated (Cas) protein further comprising a nuclear localization sequence (NLS) and/or a FLAG, HIS or HA tag.
59 c. The engineered, non-naturally occurring CRISPR-Cas system of claim 59 b where the codon-optimized CRISPR-associated (Cas) protein having at least 80% sequence identity to SEQ ID NOs: 2, 5, 9, 15, 85, 87, 95 or 96, wherein the Cas protein is fused to a deaminase, and wherein the Cas protein fusion is capable of binding to the RNA guide and of editing the target nucleic acid sequence complementary to the RNA guide.
60 . The system of claim 59 , wherein the Cas9 protein is an inactive Cas9 (dCas9).
61 . The system of claim any one of claims 58 - 60 , wherein the RNA guide comprises a crRNA and a tracrRNA.
62 . The system of any one of claims 58 - 60 , wherein the RNA guide comprises an sgRNA.
63 . The system of claim 62 , wherein the sgRNA for use with Streptococcus constellatus Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to
(SEQ ID NO: 3)
5′-GUUUUAGAGCUGUGCUGUUUAAACAACACAGCAAGUUAAAAUAAGGC
UUUGUCCGUACUCAAGCUUGCAAAAGCGUGCACCGAUUCGGUGCU-3′
64 . The system of claim 62 , the sgRNA for use with Sharpea Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to
(SEQ ID NO: 7)
5′-GUUUUAGAGUUGUGUUAUUGAAAAAUAACACAACGAGUUAAAAUAAA
GCUUAUGCUUAAAUGCCAGCUUUGCUGGUGUCAUUUAGAUGACUUUACUA
AGGUUGCUUCGGCAACCUUUUU-3′
65 . The system of claim 62 , wherein the sgRNA for use with Veillonella parvula Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to
(SEQ ID NO: 13)
5′-GUUUGAGAGUAGUGUGAAAACAUUACGAGUUCAAAUACAAAUUAAUU
UACAAUGCCUUCGGGCUGCCCGACGUAGGGCACCUACUCUCAAUUCUUCG
GAAUUGAGUU-3′
66 . The system of claim 62 , wherein the sgRNA for use with Ezakiella peruensis Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to
(SEQ ID NO: 19)
5′-GUUUGAGAGUUAUGUAAUUGAAAAAUUACAUGACGAGUUCAAAUAAA
AAUUUAUUCAAACCGCCUAUUUAUAGGCCGCAGAUGUUCUGCAUUAUGCU
UGCUAUUGCAAGCUU-3′
67 . The system of claim 62 , wherein the sgRNA for use with Lactobacillus fermentum strain AF15-40LB Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to
(SEQ ID NO: 95)
5′-GUCUUGGAUGAGUGUGAAAACACUCAUAGUCAAGAUCAAACGAGUGG
UUUUCCACGAGUUAUUACUUUUGAGGUCUUAUAUGGCCCAUACAUAAAAA
GGAGUCGGAAUUUCCGGCUCCUUUUCUU-3′
68 . The system of claim 62 , wherein the sgRNA for use with Peptoniphilus sp. Marseille-P3761 Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to
(SEQ ID NO: 96)
5′-GUUUUAGAGCCAUGUAGAAAUACAUUGCAAGUUAAAAUAAGGCUUUG
UCCGUAAUCAACUUGAAAAAGUGGCGCUGUUUCGGCGCUUU-3′
69 . The system of any one of claims 58 - 68 , wherein the Cas protein is operably linked to a promoter sequence for expression in a eukaryotic cell, and wherein the guide RNA is operably linked to a promoter sequence for expression in a eukaryotic cell.
70 . The system of claim 69 , wherein the eukaryotic cell is a human cell.
71 . The system of claim 70 , wherein the promoter sequence is a eukaryotic promoter sequence.
72 . A nucleic acid encoding the system of any one of claims 58 - 71 .
73 . A vector comprising the system of any one of claims 58 - 72 .
74 . The vector of claim 73 , wherein the vector is a plasmid vector or a viral vector.
75 . The vector of claim 74 , wherein the viral vector is an adeno associated virus (AAV) vector or a lentiviral vector.
76 . The vector of claim 75 , wherein the viral vector is an AAV vector.
77 . The vector of claim 76 , wherein more than one AAV vector is used for packaging the system of claims 59 - 71 .
78 . A method of treating a disorder or a disease in a subject in need thereof, the method comprising administering to the subject a system of any one of claims 58 - 71 ,
wherein the guide RNA is complementary to at least 10 nucleotides of a target nucleic acid associated with the condition or disease;
wherein the Cas protein associates with the guide RNA;
wherein the guide RNA binds to the target nucleic acid;
wherein the Cas protein causes a break in the target nucleic acid, optionally wherein the Cas9 is an inactive Cas9 (dCas9) fused to a deaminase and results in one or more base edits in the target nucleic acid, thereby treating the disorder or disease.
79 . The method of claim 78 , wherein the guide RNA is complementary to about 18-24 nucleotides.
80 . The method of claim 79 , wherein the guide RNA is complementary to 20 nucleotides.
81 . A base editor comprising the fusion protein of any one of claims 16 - 19 .
82 . The base editor of claim 81 comprising an adenosine deaminase domain or a cytidine deaminase domain.
82 b. The base editor of claim 81 comprising an adenosine deaminase domain and a cytidine deaminase domain.
83 . A method of editing a nucleobase of a polynucleotide, the method comprising contacting the polynucleotide with the base editor of claim 81 in complex with one or more guide RNAs, wherein the base editor comprises an adenosine deaminase domain and wherein the one or more guide RNAs target the base editor to effect an A•T to G•C alteration in the polynucleotide.
84 . A method of editing a nucleobase of a polynucleotide, the method comprising contacting the polynucleotide with the base editor of claim 81 in complex with one or more guide RNAs, wherein the base editor comprises a cytidine deaminase domain, and wherein the one or more guide RNAs target the base editor to effect an C•G to T•A alteration in the polynucleotide.
85 . The method of claim 83 or 84 , wherein the editing results in less than 50% indel formation in the target polynucleotide sequence.
86 . The method of any one of claims 83 - 85 , wherein the editing generates a point mutation.