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 equinus ATCC 33317, Enterococcus hirae strain F1129E, Streptococcus equinus strain AG46, Staphylococcus simulans strain 19, Streptococcus intermedius B196 strain G1552, Streptococcus sanguinis SK330, Streptococcus sp. C150, Streptococcus oralis subsp. oralis strain RH_1735_08, Streptococcus oralis SK313, Staphylococcus warneri strain 691, Staphylococcus stiuri strain SNUC 2430, Streptococcus gallolyticus strain AM24-4, Lactobacillus kullabergensis strain Biut2, and Streptococcus suis strain LSS83 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 equinus ATCC 33317 Cas9, Enterococcus hirae strain F1129E Cas9, Streptococcus equinus strain AG46 Cas9, Staphylococcus simulans strain 19 Cas9, Streptococcus intermedius B196 strain G1552, Streptococcus sanguinis SK330 Cas9, Streptococcus sp. C150 Cas9, Streptococcus oralis subsp. oralis strain RH_1735_08 Cas9, Streptococcus oralis SK313 Cas9, Staphylococcus warneri strain 691 Cas9, Staphylococcus sciuri strain SNUC 2430 Cas9, Streptococcus gallolyticus strain AM24-4 Cas9, Lactobacillus kullabergensis strain Biut2 Cas9, or Streptococcus suis strain LSS83 Cas9 is provided herein.
2 . The Cas9 protein of claim 1 , wherein the Streptococcus equinus ATCC 33317 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 1)
MEKSYSIGLDIGTNSVGWSVITDDYKVPAKKMRVLGNTDKKYIKK
NLLGALLFDSGETAEATRLKRTARRRYTRRKNRLRYLQEIFAKEM
AKVDESFFQRLDESFLTDDDKTFDSHPIFGNKAEEDAYHQKFPTI
YHLRKHLADSQEKADLRLIYLALAHMIKFRGHFLYDNFNDDNFDW
RNIDIQKRYEEFIETYDSTLGESYLADISVDAASILEEKVSKTER
LENLLKYYPTEKKTTFFGNLIKLILGQQAKFKAIFNLEDEISLQF
ATPTYDEDLEELLGKIDNGDSYSELFVAAQNLYNTILLASFLKTD
NKSAKAPLSTSMIERYENHKKDLAKLKDFVKKNCPDQYHDIFRDK
SKNGYAGYIDNGVSQNDFYTFIGKCLEESLKKDKGAQYFLDKIDR
DDFLRKQRTFDNGAFPYQIHLQEMHAILRRQGDYYPFLKENQDKI
EKILTFRIPYYVGPLARKDSRFAWANYRSDEAITPWNFDEVVDKE
KSAEKFITRMTLNDLYLPEEKVLPKHSLIYETFTVYNELTNIKYV
NDQGNAIHFDSELKEKIFNQLFKENRKVSKKTLIDFLNNSEGIYT
DKLVGIDEEVKYLNASLGTYHDLKKILESFMDDEINEKIIEDIIQ
TLTLFEDIEMKRQRLQKYDDIFTPKQLKELARRNYTGWGRLSYKL
INGIRNKENNKTILDYLKNGNRNFMQLINDDRLSFKQIIIDARKI
EKLDNIESVVYNLPGSPAIKKGILQSIKIVDELVKVMGHNPDNIV
IEMARENQTTNQGKNRSQQRLKRLQDSMSNFKDSSISLKDVDNSD
LQNDRLFLYYIQNGKDMYTGEELDIDHLSDYDIDHIIPQSFIKDN
SIDNRVLTSSAKNRGKSDDVPGRDVVLKMKPFWKKLYDVKLISKR
KFDNLTKSEHGGLTESDKAGFIKRQLVETRQITKYVAQILDGRFN
TKRDDNNKVIRDVKVITLKSSLVSQFRKDFGFYKVREINDYHHAH
DAYLNAVVGTAILKKYPKLAPEFVYGEYKKCDVRKLIAKSGDKSE
IGKATAKYFFYSNLMNFFKRVIRYSNGMIVVRPVIEYSKDTGEIA
WDKEKDFKTVCKVLSCPQVNIVKKVEKQSHGLDRGKPKGFYNANP
SPKPKKGSKVNLVPIKANLNPKNYGGYAGISNSYAVLVDATIEKG
AKKKLTRIQEFQGISIIDREKYEKNKVEFLKGLGYKEIYSIITLP
KYSLFELADGSRRMLASILSTNNKRGEIHKGNELVLPAKYIPLLY
HANRIHNTFETGHREYVEKHIAEFKEIAEIILEFNNKYVNAKKNS
SIIEKALESFDSFSLDEICDSFVGKLKKNNTKKNSGLFELVSLGS
ASDFEFLETKVPRYRDYTPSSLLNATLIHQSITGLYETRIDLSKL
GEE
3 . The Cas9 protein of claim 1 , wherein the Enterococcus hirae strain F1129E Cas9 has at least 80% sequence identity to
(SEQ ID NO: 2)
MTKDYTIGLDIGTNSVGWAVLTDDYQLMKRKMSVHGNTEKKKIKK
NFWGARLFDEGQTAEFRRTKRTNRRRLARRKYRLSKLQDLFAEEL
CKQDDCFFVRLEESFLVPEEKQYKPASIFPTLEEEKEYYQKYPTI
YHLRQKLVDSTEKEDLRLVYLALAHLLKYRGHFLFEGDLDTENTS
IEESFRVFLEQYSKQSDQPLIVHQPVLTILTDKLSKTKKVEEILK
YYPTEKINSFFAQCLKLIVGNQANFKRIFDLEAEVKLQFSKETYE
EDLESLLEKIGDEYLDIFLQAKKVHDAILLSEIISSTVKHTKAKL
SSGMVERYERHKADLAKFKQFVKENVPQKSTAFFKDTTKNGYAGY
IKGKTTQEEFYKFVKKELSGVVGSEPFLEKIDQETFLLKQRTYTN
GVIPHQVHLIELKAIIDQQKQHYPFLEEAGPKIIALFKFRIPYYV
GPLAKEQEASSFAWIERKTAEKIHPWNFSEVVDIEKSAMRFIQRM
TKQDTYLPTEKVLPKNSLLYQKYMIFNELTKVSYKDERGVKQYFS
GDEKQQIFKQLFQKERGKITVKKLQNFLYTHYHIENAQIFGIEKA
FNASYSTYHDFMKLAKTNQKAMQEWLEQPEMEPIFEDIVKILTIF
EDRQMIKHQLSKYQEVFGEKLLKEFARKHYTGWGRFSAKLIHGIR
DRKTNKTILDYLINDDDVPANRNRNLMQLINDEHLSFKEEIAKAT
VFSKHKSLVDVIQDLPGSPAIKKGIWQSLKIVEELIAIIGYKPKN
IVIEMARENQKTHRTKPRLKALENGLKQIGSTLLKEQPTDNKALQ
KERLYLYYLQNGRDMYTGEPLEIENLHQYEVDHIIPRSFIVDNSI
DNKVLVARKQNQKKRDDVPKKQIVNEQRIFWNQLKEAKLISPKKY
AYLTKIELTPEDKARFIQRQLVETRQITKHVANILHQSFHQEEEG
TDCDGVQIITLKATLTSQFRQTFGLYKVREINPHHHAHDAYLNGF
IANVLLKRYPKLAPEFVYGKYVKYSLARENKATAKKEFYSNILKF
FESDEPFCDENGEIYWEKSHHLPRIKKVLSSHQVNVVKKVEQQKG
GFYKETVNSKEKPDKLIERKNNWDVTKYGGFGSPVIAYAIAFVYA
KGKTQKKTRAIEGITIMEQAAFEKDPTTFLKEKGFPQVTEFIKLP
KYTLFEFDNGRRRFLASHKESQKGNPFILSDQLVTLLYHAQHYDK
ITYQESFDYVNTHLSDFSAILTEVLAFAEKYTLADKNIERIQELY
EENKYGEISMIAQSFLQLLQFNAIGAPADFKFFGVTIPRKRYTSL
TEIWDATIIYQSVTGLYETRIRMGDLWAGEQ.
4 . The Cas9 protein of claim 1 , wherein the Streptococcus equinus strain AG46 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 3)
MTNGKILGLDIGVASVGVGIIEAKTGKVIHANSRLFSAANAENNA
ERRGFRGARRLTRRKKHRVKRVRDLFEKYDISTDFRNLNLNPYEL
RVKGLSEQLTNEELFAALRTIAKRRGISYLDDAEDDSTGSSDYAK
SIDENRRLLKSMTPGQIQLERLEKYGQLRGNFTVYDENGEAHRLI
NVESTSDYKNEARKILETQSNYNKQITDEFIEDYIEILTQKRKYY
HGPGNEKSRTDYGRFRTDGTTLENIFGILIGKCSFYPEEYRASKA
SYTAQEFNFLNDLNNLKVPTETGKLSTEQKEYLVDFAKNSKTLGA
SKLLKEIAKLVDGDVKDISGYREDSKGKPDLHTFEPYRKLKFNLT
TVDIDNLSRDILDKLANILTLNTEREGIEDAINRNLPEQFTKEQI
SEIVQIRKSQSSAFNKGWHSFSAKLMNELIPELYATSEEQMTILT
RLEKFKATKKSSKNTKTIDEKEITDEIYNPVVAKSVRQTIKIINA
AVKKYGDFDKIVIEMPRDKNADDEKKFIDKKNKENKKEKDDSLKR
AAYLYNGTDKLPDDVFHGNKQLATKIRLWYQQGERCLYSGKPILI
QDLVHNSNNFEIDHILPLSLSFDDSLANKVLVYAWTNQEKGQKTP
YQVIDSMDAAWSFREMKDYVLKQKGIGKKKREYLLTTENIDKIEV
KKKFIERNLVDTRYASRVVLNSLQTALKELGKDTKVSVVRGQFTS
QLRRKWKIDKSRETYHHHAVDALIIAASSQLKLWQKHENLMFENY
GENQVVNKETGEILSISDDEYKELVFQPPYQGFVNTISSKAFEDE
ILFSYQVDSKFNRKVSDATIYSTRKAKLGKDKKEETYVLGKIKDI
YSQDGFDTFIKRYKKDKTQFLMYQKDPLTWENVIEVILRDYPTTK
KSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLKYYDKK
LGNHISITPKESKNDVVLQSLNPWRADLYFNPDTLKYELMGLKYS
DLSFEKGTGKYHISQEKYDEIKEKEGIGQNSEFKFTLYRNDLILI
KDTESGEQEIYRFLSRTMPNVKHYVELKPYDKEKFNGGQELIKSL
GEADKVGRCLKGLSKPGISIYKVRTDVLGNKFFVKKEGDKPKLDF
KNNKK
5 . The Cas9 protein of claim 1 , wherein the Staphylococcus simulans strain 19 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 4)
MNNSYILGLDIGITSVGYGIIEYETRDVIDAGVRLFKEANVENNE
GRRSKRGARRLKRRRRHRLQRVKKMLFDYKLLNEDSEISGINPYE
ARVKGLSEKLSDEEFSAALLHLAKRRGVHNVSDVEEDTGNELSTK
EQIARNNKALEDKYVAELQLERLKEQGEVRGAANRFKTSDYIKEA
KQLLKTQSDYHKIDETFIETYISLLETRRTYYEGPGEGSPFGWKD
IKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVIARD
ENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRV
TSTGKPEFTNFKIYHDIKGITSRKEILENADLLNQIAEILTIYQS
AEDIQEELAELEPKLTQEEIEQISNLTGYTGTHRLSLKAINLILD
ELWNTSDNQMTIFNRLKLVPKKVDLSQQKEIPTSLVDDFILSPVV
KRSFIQSIKVINAIIKKFGLPKDIIIELAREKNSKEAQKFINEMQ
KRNRQTNERIEKIIKETGKEKAKFLIEKIKLHDMQEGKCLYSLES
IPLEDLLNNPYHYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGN
RTPFQYLSSSDAKISYETFKKHILNLSKGKGRVSKKKKEYLLEER
DINRFSVQKDFINRNLVDTRYATRELMNLLRSYFRVNDLDVKVKS
INGGFTSFLRTKWKFKKERNQGYKHHAEDALVIANADFIFKEWKK
LDTTNKVMENQTVEEQQAENMPGIETDDEYKEIFVIPRQIQSIKD
FKDYKYSHRVDKKPNRELVNDTLYSTRKDDKGNTLIINNIKGLYD
KDNDKLKNLIKKSPEKLLMYHHDPQTYQKLKTIMEQYSNEKNPLY
KYHEETGNYLTKYSKKDNGPIIKKVKYYGKKLNAHLDITNDYSNS
QNKIVKLSLKPYRFDVYLDNGGYKFVTVKNLDVIKKEGFFKIDSN
AYEKAKSEKKIDENAVFIASFYNNDLIKIDGELYRIVGVNNDTRN
VVELNMIPITYKEYLENINDKRTPRILKTISQKTYSIEKYSTDIL
GNLYKVKSKKKPQMIMKG
6 . In some embodiments, the Streptococcus intermedius B196 strain G1552 Cas9 protein has at least 80% sequence identity to
(SEQ ID NO: 5)
MNGLVLGLDIGIASVGVGILNKETGEIIHVNSRIFPAATADSNVE
RRGFRQGRRLGRRKKHRSARLNDLFEEFGFITDFSAVPLNLNPYA
LRVKGLSEELTNEELFIALKNIIKRRGISYLDDASEDGETASNEY
GKAVEENRKLLADKTPGQIQLERFEKYGQVRGDFTVVENGENHRL
INVESTSAYKKEAERILRRQQEFNVRISDEFIEAYLTILTGKRKY
YHGPGNEKSRTDYGRFRTDGTTLDNIFGILIGKCTFYPDEYRAAK
ASYTAQEFNLLNDLNNLTVPTETKKLRPEQKRQIVEYARTAKTLG
TPTLLKYIAKLVDGSIDDIKGYRIDKSDKPEMHTFDAYRKMRTLE
LVDVDILSRETLDDLAYILTLNTESEGILEALNSKMPGTFTKEQI
DELIQFRKKNSAVFGKGWHNFSLKLMNELISELYETSEEQMTILT
RLGKQRSREISKRTKYIDEKELTDEIYNPVVAKSVRQAIKIINLA
TKKYGIFDNIVIEMARESNEDDEKKAIQNVQKANEDEKKAAMEKA
ADLYNGKKELPDSIFHGHKELATKIRLWHQQGERCLYTGKNISIH
DLIHNPHQYEIDHILPLSLSFDDGLANKVLVLATANQEKGQRTPF
QALDSMDDAWSYIEFKQYVRNSKSLSNKKKDYLLTEEDISKIEVK
QKFIERNLVDTRYSSRVVLNTLQEFYKTNDFDTKISVVRGQFTSQ
LRRKWKIEKSRDTYHHHAVDALIIAASSQLRLWKKQNNPLISYRE
GQLVDPETGEILSLTDDEYKELVFRPPYDYFVDTLKSKSFEDSIL
FSYQVDSKYNRKISDATIYGTRKAQLGKDKQEETYVLGKIKDIYS
QKGYEDFIKRYKKDTTQFLMYHKDPQTFAKVIEEILKTYPDKELN
EKGKEIPCNPFEKYRQENGPIRKYSKKGKGPEIKSLKYYDNKLGN
HIDITPVNSQNQVVLQSLKPWRTDVYFNPQTSKYELMGLKYSDLR
FEKGSGSYGISPEKYNKVKAKEGVDEDSEFKFTLYKNDLILIKDT
ETGEQQLFRYGSRNDTSKHYVELKPYEKAKFEGNQQLMNLLGTVA
KGGQCLKGINKPNLSIYKVKTDVLGNKHFIKKEGDQPQLNFKKKI
7 . The Cas9 protein of claim 1 , wherein the Streptococcus sanguinis SK330 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 6)
MENKNYSIGLDIGTNSVGWAVITDDYKVPSKKMKVFGNTDKHFIK
KNLIGALLFDEGATAEDRRLKRTARRRYTRRKNRLRYLQEIFSEE
ISKLDSSFFHRLDDSFLVPKDKRGSKYPIFATLEEEKEYHKKFPT
IYHLRKHLADSKEKTDLRLIYLALAHMIKYRGHFLYEESFDIKNN
DIQKIFNEFISIYDNTFEGSSLSGQNAQVEAIFTDKISKSAKRER
VLKLFSDEKSTSLFSEFLKLIVGNQADFKKHFDLEEKAPLQFSKD
TYDEDLENLLGQIGDGFTDLFLVAKKLYDAILLSGILTVTDPSTK
APLSASMIERYESHQKDLAALKQFIKNNLPKRYNEVFSDQSKDGY
AGYIDGKTTQEAFYKYIKNLLSKFEGADYFLDKIEREDFLRKQRT
FDNGSIPHQIHLQEMNAILRRQGEHYPFLKENREKIEKILTFRIP
YYVGPLARGNRDFAWLTRNSDQAIRPWNFEEIVDKASSAEEFINK
MTNYDLYLPEEKVLPKHSLLYETFAVYNELTKVKFIAEGLRDYQF
LDSGQKKQIVNQLFKEKRKVTEKDIIHYLHNVDGYDGIELKGIEK
QFNASLSTYHDLLKIIKDKEFMDDPKNEEILENIVHTLTIFEDRE
MIKQRLAQYDSIFDEKVIKALTRRHYTGWGKLSAKLINGICDKQT
GDTILDYLIDDGKINRNFMQLINDDGLSFKEIIQKAQVVGKTDDV
KQVVQELPGSPAIKKGILQSIKIVDELVKVMGYAPESIVIEMARE
NQTTARGKKNSQQRYKRIEDSLKNLAPGLDSNILKENPTDNIQLQ
NDRLFLYYLQNGKDMYTGKPLDIDQLSSYDIDHIIPQAFIKDDSI
DNRVLTSSKDNRGKSDNVPSLEVVQKRKAFWQQLLDSKLISERKE
NNLTKAERGGLDERDKVGFIRRQLVETRQITKHVAQILDASFNTE
VNEKNQKIRTVKIITLKSNLVSNFRKEFELYKVREINDYHHAHDA
YLNAVVAKAILKKYPKLEPEFVYGDYQKYDLKRYISRFKPSKEIE
KATEKYFFYSNLLNFFKEEVHYADGIIVKRENIEYSKDTGEIAWN
KEKDFATIKKVLSYPQVNIVKKTEIQTHGLDRGKPKGLFNSNPSP
KPSEDSKENLVPIKQGLDPRKYGGYAGISNSYAVLVKAIVEKGAK
KQQKTILEFQGISILDKINFENNKENYLLKKRYIEILSTITLPKY
SLFEFPDGTRRRLASILSTNNKRGEIHKGNELVLPGKYTTLLYHA
KNINKKLEPEHLEYVEKHRNDFAKLLECVLNFNDKYVGALKNGER
IRQAFTDWETVDIEKLCFSFIGPENSKNAGLFELTSQGSASDFEF
LGVKIPRYRDYAPSSLLKATLIHQSITGLYETRIDLSKLGED
8 . The Cas9 protein of claim 1 , wherein the Streptococcus sp. C150 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 7)
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLE
RRTNRQGRRLTRRKKHRRVRLNHLFEESGLITDFTKVSINLNPYQ
LRVKGLTAELSNEELFIALKNMVKHRGISYLDDASDDGNSSVGDY
AQIVKENSKQLETKTPGQIQLERYQKYGQLRGDFTVEEDGRKHRL
INVFPTSAYHAEALRILQTQQEFNPQITDEFINSYLEILTGKRKY
YHGPGNEKSRTDYGKYTTKKDAQGQYITLNNIFGILIGKCTFYPE
EYRAAKASYTAQEFNLLNDLNNLTVPTETKKLSEEQKYQIITYVK
NEKAMGPAKLFKYIAKLLSCDVADIKGYRIDKSDKAEIHTFEAYR
KMKTLETLDVKKMAREELDKLAYVLTLNTEREGIQEALDHEFADG
TFSQEQVDELVQFRKANSSIFGKGWHSFSVKLMMELIPELYATSE
EQMTILTRLGKQKTTSSSNKTKYIDEKQLTEEIYNPVVAKSVRQA
IKIVNAAIKKYGDFDNIVIEMARETNEDDEKKAIQKIQKANKAEK
DAAMRKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYT
GKTISIHDLINNPNQFEIDHILPLSITFDDSLANKVLVYATANQE
KGQRTPYQALDSMDDAWSFRELKAFVRESKALSNKKKEYLLTEED
ISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRTHKIDTKVSVV
RGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKN
TLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEF
EDSILFSYQVDSKFNRKISDATIYATRKAKLDKEKKEYTYTLGKI
KDIYALGTKTPSKTGFYKFLDLYKKDKSQFLMYQKDRRTWDEVIE
KILEQYRPFKEKDKNGKEVDENPFEKYRIENGPIRKYSRKGNGPE
IKSLKYYDNLLGRFVDITPSESKNPVALLSLNPWRTDVYYNTETR
KYEFLGLKYADLCFEKGGSYGISKVKYNKIREKEGIGKNSEFKFT
LYKNDLILIKDTETNRQQIFRFWSRTGKDNPKSFEKHKLELKPYE
KTRFEKGEELKVLGKVPPSSNRLQKNMQIENLSIYKVRTDVLGNQ
HIIKNEGDKPKLDF
9 . The Cas9 protein of claim 1 , wherein the Streptococcus oralis subsp. oralis strain RH_1735_08 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 8)
MNGLVLGLDIGIASVGVGILEKNSGKIIHANSRIFPAATADNNVE
RRKNRQARRLHRRKKHRGVRLQDIFEDYGLLTDFSKVSINLNPYR
LRVDGLDQQLTNEELFIALKNIVKRRGISYLDDASEDGGTVSSDY
GKAVEENRKLLAEKTPGQIQLERFEKYGQVRGDFTVVENGEKRRL
INVESTSAYRKEAERILRKQQEFNSKITDEFIEDCLKILTGKRKY
YHGPGNEKSRTDYGRFRTDGTTLDNIFGILIGKCTFYPNEYRASK
ASHTAQEFNLLNDLNNLTVPTETKKLSEEQKKVIVEYAKEAKTLG
ASTLLKYIAKMIDASVDQISGYRVDVNNKPEMHTFEVYRKMQSLE
TISVGELSRNVLDELAHILTLNTEREGIEEAINTKLKDSFSQDQV
LELVQFRKNNSSLFSRGWHNFSLKLMMELIPELYETSEEQMTILT
RLGKQKSKETSKRTKYIDEKELTEEIYNPVVAKSVRQAIKIINEA
TKKYGIFDNIVIEMARENNEEDAKKDYIKRQKANQDEKNAAMEKA
AFQYNGKKELPDNIFHGHKELATKIRLWHQQGEKCLYTGKNIPIS
DLIHNQYKYEIDHILPLSLSFDDSLSNKVLVLATANQEKGQRTPF
QALDSMDDGWSYREFKSYVKESKLLGNKKKEYLLTEEDISKIEVK
QKFIERNLVDTRYSSRVVLNALQDFYKAHKFDTTISVVRGQFTSQ
LRRKWGLEKSRETYHHHAVDALIIAASSQLRLWKKQNNPLIAYKE
GQFVDSETGEILSLSDDEYKELVFKAPYDHFVDTLRSKTFEDSIL
FSYQVDSKYNRKISDATIYATRKAKLDKDKSEETYVLGKIKDIYS
QAGYDAFIKIYNKDKSKFLMYHKDPQTFEKVIEEILRTYPSKELN
DKNKEIPCNPFEKYRQENGPIRKYSKKGNGPEIKCLKYYDNKLGN
YIDITPDGSDNQVVLQSIAPWRTDVYYNHKTGKYEFLGLKYSDLY
FEKGTGKYKISKEKYDNIKKIEGVVETSEFKFTLYKNDLILIKDV
EKGQEQLFRFLSRNNKGKHQVQLKPMNKSDFEKGEKLIDIFGTVP
NSTTQCVKGLNKSNISIFKVKTDVLGKKHIIKKEGDEPKLKF
10 . The Cas9 protein of claim 1 , wherein the Streptococcus oralis SK313 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 9)
MNGLVLGLDIGIASVGVGILEKNSGKIVHASSRIFPAATADNNVE
RRKNRQARRLHRRKKHRGARLKDLFEYYGLLTDFSKVSINLNPYR
LRVDGLDQQLTNEELFIALKNIVKRRGISYLDDASEDGGTVSSDY
GKAVEENRKLLAEQTPGQIQLERFEKYGQLRGDFTVVENSEKCRL
INVESTSAYKKEAERILRKQQEFNNQITDEFIEDYLKILTGKRKY
YHGPGNEKSRTDYGRFRTDGATLDNIFGILIGKCTFYPNEYRASK
ASYTAQEFNLLNDLNNLTVPTETKKLSEEQKKTIIEYAKSAKTLG
ASTLLKYIAKMIDASVDQIRGYRVDVNNKPEMHTFEVYRKMQSLE
TISVGELSRNILDELAHILTLNTEREGIEEAINTKLRDSFSQDQV
LELVQFRKNNSSLFSKGWHNFSLKLMMELIPELYETSEEQMTILT
RLGKQKSKETSKRTKYIDEKEVTEEIYNPVVAKSVRQAIKIINEA
TKKHGIFDNIVIEMARENNEEDAKKDYIKRQKANQDEKYAAMEKA
AFQYNGKKELPDNIFHGHKELATKIRLWHQQGEKCLYTGKSIPIS
DLIHNQYKYEIDHILPLSLSFDDSLSNKVLVLATANQEKGQRTPF
QALDSMDDAWSYREFKSYVKESKLLGNKKKEYLLTEEDISKIEVK
QKFIERNLVDTRYSSRVVLNALQDFYKNHNFDTTISVVRGQFTSQ
LRRKWGLEKSRETYHHHAVDALIIAASSQLRLWKKQNNPLIAYKE
GQFVDSQTGEIISLTDDEYKELVFKAPYDHFVDTLSSKTFEDSIL
FSYQVDSKFNRKISDATIYATRKAKLDKEKKEYTYTLGKIKDIYS
LGTKTPSKTGFYKFLDLYNKDKSQFLMFQKDRKTWDEVIEKIMEQ
YRPFKEYDKAGKLVDFNPFEKYRQENGPIRKYSKKGNGPEIKSLK
YYDILLGKHKNITPEGSRNTVALLSLNPWRTDVYYNMETKKYEFL
GLKYADLPFEEGGAYGISTETYNELREKEGIGKNSEFKFTLYKND
LILIKDTETNCQQFFRFWSRTGKDNPKSFEKHKIELKPYEKAKFE
KGEELEVLGKVPPSSNQFQKNMQIENLSIYKVKTDVLGNKHFIKK
EGDKPKLKF
11 . The Cas9 protein of claim 1 , wherein the Staphylococcus warneri strain 691 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 10)
MKEKYILGLDLGITSVGYGIINFETKKIIDAGVRLFPEANVDNNE
GRRSKRGSRRLKRRRIHRLDRVKSLLTEYNLINREQIPTSNNPYQ
IRVKGLSEILSKDELAIALLHLAKRRGIHNINVSSEDEDASNELS
TKEQINRNNKLLKNKYVCEVQLQRLKEGQIRGEKNRFKTTDILKE
IDQLLKVQKDYHNLDIDFINQYKEIVETRREYFEGPGQGSPFGWN
GDLKKWYEMLMGHCTYFPQELRSVKYAYSADLFNALNDLNNLIIQ
RNDSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGY
RITKSGTPQFTEFKLYHDLKSIVFDKSILENEAILDQIAEILTIY
QDEESIKEELNKLPEILNEQDKAEIAKLTGYNVTHRLSLKCIHLI
NEELWQTSRNQMEIFNYLNIKPNKVDLSEQNKIPKDLVDEFILSP
VVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINN
LQKKNEATRKRINEIIGQTGNQNGKRIVEKIRLHDQQEGKCLYSL
ESIPLMDLLNNPQNYEVDHIIPRSVAFDNSIHNKVLVKQIENSKK
GNRTPYQYLNSSDANLSYNQFKQHILNLSKSKDRISKKKKDYLLE
ERDINKFEVQKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKV
KTINGSFTNHLRKVWRFDKYRNHSYKHHAEDALIIANADFLFKEN
KKLQNANKILEKPTIENDTQKVTVEKEEDYNNMFETPKLVEDIKQ
YRDYKFSHRVDKKPNRQLIKDTLYSTRMKDEHNYIVQTITDIYGK
DNTNLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAK
YYEETGEYLTKYSKKNNGPIVKKIKLLGNKVGNHLDVTNKYENST
KKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIPKDL
YQELKAKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNI
IELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESIEKLTTDVLG
NLYLHTTEKAPQLIFKRGL
12 . The Cas9 protein of claim 1 , wherein the Staphylococcus sciuri strain SNUC 2430 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 11)
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNE
GRRSKRGARRLKRRRRHRLQRVKKLLFDYNLLTDHSELSGINPYE
ARVKGLSQKLSEVEFSAALLHLAKRRGVHNVNEVEEDTGNELSTK
EQISRNSKALEEKYVAELQLERLKTDGEVRGPNNRFKTSDYVKEA
KQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKD
IKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVIARD
ENEKLEYYEKFQIIENVFKQKKKPTLKQIAKELLVNEEDIKGYRV
TSIGKPEFTNFKIYHDIKGITERKEVLENAELLDQIAEILTIYQS
SEDVQEELANLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILD
ELWHTNDNQMAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVV
KRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQ
KRNRQMNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLES
IPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGN
RTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEER
DINRFSVQKDFINRNLVDTRYATRELMNLLRSYFRVNNLDVKVKS
INGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK
LDKAKKVMENQTVEEKQAESMPEIETEQEYKEIFITPHQIQHIKG
FKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYD
KDNDKLKKLMNKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLY
KYYEETGNYLTKYSKKDNGPVIKKIKYYGKNLKAHLDITDDYPNS
RNKVVKLSVKPYRFDVYLDNDIYKFVTVKNLDVIKKEDYYEVNSK
CYKEAKKLKKISDQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
RIEVNMINITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDIL
GNLYEVKSKQKPQMIMKG
13 . The Cas9 protein of claim 1 , wherein the Streptococcus gallolyticus strain AM24-4 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 12)
MTNGKILGLDIGIASVGVGIIEAKTGKVVHANSRLFSAANAENNT
ERRGFRGSRRLNRRKKHRVKRVRDLFEKHEIVTDFRNLNLSPYEL
RVKGLTEQLTNEELFAALRTISKRRGISYLDDAEDDSTGSTDYAK
SIDENRRLLKNKTPGQIQLERLEKYGQLRGNFTVYDENGEAHRLI
NVESTSDYEKEARKILETQADYNKKITAEFIDDYVEILTQKRKYY
HGPGNEKSRTDYGRFRTDGTTLENIFGILIGKCNFYPDEYRASKA
SYTAQEYNFLNDLNNLKVPTETGKLPTEQKESLVEFAKNTATLGP
SKLLKEIAKILDCKVDEIKGYREDDKGKPDLHTFEPYRKLKFNLE
SINIDDLSREVIDKLADILTLNTEREGIEDAIKRNLPNQFTEEQI
SEIIKVRKSQSTAFNKGWHSFSAKLMNELIPELYATSDEQMTILT
RLEKFKVNKKSSKNTKTIDEKEVTDEIYNPVVAKSVRQTIKIINA
AVKKYGDFDKIVIEMPRDKNADDEKKFIDKRNKENKKEKDDALKR
AAYLYNSSDKLPDEVFHGNKQLETKIRLWYQQGERCLYSGKPIPI
HDLVHNSNNFEIDHILPLSLSFDDSLANKVLVYDWANQEKGQKTP
YQVIDSMDAAWSFREMKDYVLKQKGLGKKKRDYLLTTENIDKIEV
KKKFIERNLIDTRYASRVVLNSLQSALRELCKDTKVSVIRGQFTS
QLRRKWKIDKSRETYHHHAVDALIIAASSQLKLWEKQDNLMFIDY
GNNQVVDKETGEILSVSDDEYKELVFQPPYQGFVNTISSKGFEDE
ILFSYQVDSKYNRKVSDATIYSTRKAKIGKDKKEETYVLGKIKDI
YSQNGFDTFIKKYNKDKTQFLMYQKDPLTWENVIEVILRDYPTTK
KSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLKYYDKK
LGNCIDITPEESRNKVILQSINPWRADVYFNPETLKYELMGLKYS
DLSFEKGTGKYHISQEKYDAIKEKEGIGKKSEFKFTLYRNDLILI
KDTASGEQEIYRFLSRTMPNVNHYAELKPYDKEKFDGGQELMEVF
GKVANGGQCLKSLNKSNISIYKVRTDVLGNKYFVKKEGDKPKLNF
KNNKK
14 . The Cas9 protein of claim 1 , wherein the Lactobacillus kullabergensis strain Biut2 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 13)
MKRVNEDYILGLDIGTNSCGWAVTDKKNNLLKLRGKTAIGSHLFE
EGHTAADRRGFRTTRRRLKRRKWRLRLLEEIFAEPMAKVDPGFFV
RLHQSWVSPLDKDRKKYNAIVFPTAKEDQAFYKHYATIYHLRDEL
MTQDRQFDLREIFLAIHHIVKYRGNFLQDTPVKDFEASKIEVGPI
LSHINNAFAEKIVEDQDPIELNVANAADIEDVIRGKDAEKTVYKL
DKVKKIAKLLTDSTAKEEKNVAKQIANAIMGYKTQFETILDKEID
KSDKAQWEFKLSDADADDKLDALLPDLDETDQTVVAEIEKLFSAI
TLSTIVDENKSLSQSMVEKYKKHKKDYKKLKKYINTLQDQTKAKK
LLLAYDLYVNNRHGRLLEAKKTFEDKKKKKALTKDEFYKIVKDNL
DDSDLAHEIQQEIAADNFMPKQRTNSNGVIPFQLHQIELDKIIAN
QGKYYPFLAAENPVEDHRKQAPYKLDELVRFRVPYYVGPMITADE
QEKTSGKSFAWMVRKEDGQITPWNFEQKVDRQESANKFIKRMTIK
DTYLLSEDVLPANSLLYQRFEVLNELNNIRINGSRISVDLKQQIF
NDLFEEKKTVTEKSLTSYLKQNLHLPTVEIKGLADPTKFNSSLAS
YYHLKSLHVFDKELADPQYQKDFEKIIEYSSIFEDKKIFQDKLHA
EFKWLTPEQFKAISTWRLQGWGRLSRKLLVELHDTNGQNIMEQLW
DSQKNFMQIVTEPDFKDAIAKENQNVTRANGVEEILADAYTSPAN
KKAIRQVVKVVADVVKAAGGKKPAQFAIEFTRDPDKNPQLSHIRG
TKLLKAYQETAGELVDQKLTDSLKEAMTSRKLLKDKYFLYFMQAG
RDAYTGQKINIDEVSTNYQIDHILPQSFIKDDSFDNRVLTATPLN
AEKSDDVPYKRFANNYVSDMKMTVGEMWKHWQKAGIINKHKLGNL
LLDPDRLNKFQKSGFINRQLVETSQIIKLVSVILQNKYPDAEIIT
VKAGDNSALRQRLNLYKSRDVNDYHHAIDAYLSIICGNFLYQVYP
KYRPYFVYGKYKKFSQDPDLQKEVIKHFKGFTFMWPLLQKDNSER
KAPEKIKENNSDRIVFYKHPDIFDKLRKAYNYKYMLVSRETTTEN
SGLFDVTIYPRGERDLAKTRKLIPKSNGLDPKIYGGYSGNTDAYM
VIVKIDKGKESIYKVIGVPMRALASLNRAKKQGNYKEELHQVLEP
QIMFDKNGKPKRSVKGFRIIKDHVPFKQVVLDGDKKFMLNSSTYE
INAKQLTLTPETMRIVTDNLKKGEDQDQLLVKAYDEILQKVDQYL
PLFDVNKFRNSLHLGRAKFLDLAVNDKKITLTNILNGLHDNLVTP
DLKNIGIKTPLGKLQVPSGIVLSSEAILIFQSPTGLFEKRVRIADL
15 . The Cas9 protein of claim 1 , wherein the Streptococcus suis strain LSS83 Cas9 has at least 80% sequence identity to
(SEQ ID NO: 14)
MSNGKILGLDIGIASVGVGVIDAQTGEIIHASSRIFPSANAANNA
ERRTFRGSRRLIRRKKHRIKRLDDLFNDFHINLDGEMSTDNPYVL
RVKGLSQKLTVEELYISIKNIMKRRGISYLDDAESDNEAGRSDYA
KAIERNRQLLTSKTPGEIQLERLEKYGQLRGNFTIIDEEGQSQQI
INVESTSDYVKEVEKILDCQKMYHKFISDEFCDKLIELLREKRKY
YVGPGNEKSRTDYGIYRTDGTTLENLFGILIGKCTFYPDQYRSSR
ASYTAQEFNFLNDLNNLTVPTETKKLSQEQKEFLVNYAKETSVLG
AGKILQQIAKLADCKVEDIRGYRLDNKDKPELHTFETYRAMKGLV
PLVDIGVLSREQLDILADILTLNTDFEGIREALKKQLPNVFDEKQ
VKGLASFRKSKSQLFAKGWHNLSQKIMLEVIPELYATSDEQMTIL
TRLGKFEKSSVAEYPSSINVDEITDEIYNPVVAKSIRQTIKIINA
SIKKWDEFDQIVIEMPRDRNEDEEKKRIADGQKANAKEKADSILR
AAELYCAGKVLPDYVYNGHNQLATKIRLWYQQGERCIYTGQPISI
HDLIHNQNQYEIDHILPLSLTFDDSLSNKVLVLATANQEKAQRTP
YNYLKSATSAWSYREFKDYVTKRKGIGKKKCEYLTFEEDINGFEV
RSKFIQRNLVDTRYASKVILNALQDYFKISGIQTKVSVVRGQFTS
QLRHKWGIEKTRETYHHHAVDALIIAASSQLRLWKKQESPLVVDY
QEGRQVDLETGEILELTDEQYKELVYQPPYQGFVNTISSSAFDNE
ILFSYQVDSKVNRKISDATIYATRNAQLGKDKTEGIYVLGKIKDI
YTQAGYEAFLKRYTKDKTSFLMYHKDLDTWEKVIEIILRDYREYD
EKGKEIGNPFERYRRENGYVKKYSRKGNGTAIKSLKYYDNKLGNH
IDITPENSRNAVVLQSLKPWRTDVYFNKETGKYEFLGIKYSDLSF
EKGTGEYGISQEKYDSIKIAEGVAKKSIFKFTLYKQDLLFIKDIE
NNFGKLLRFTSKNDTSKHYVELKPYDKNKFGTEEPLLPVLGNVAK
SGQCIKGLNKSNISIYKVRTDILGYRHFIKQEGEHPQLKFKK
16 . The Cas9 protein of any one of claims 1-15 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.
17 . 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, and/or a deaminase.
18 . The protein of claim 17 , wherein the Streptococcus equinus ATCC 33317 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 29)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDGSS
GSETPGTSESATPESSG PKKKRKV GTMEKSYSIGL A IGTNSVGWS
VITDDYKVPAKKMRVLGNTDKKYIKKNLLGALLFDSGETAEATRL
KRTARRRYTRRKNRLRYLQEIFAKEMAKVDESFFQRLDESFLTDD
DKTFDSHPIFGNKAEEDAYHQKFPTIYHLRKHLADSQEKADLRLI
YLALAHMIKFRGHFLYDNFNDDNFDWRNIDIQKRYEEFIETYDST
LGESYLADISVDAASILEEKVSKTERLENLLKYYPTEKKTTFFGN
LIKLILGQQAKFKAIFNLEDEISLQFATPTYDEDLEELLGKIDNG
DSYSELFVAAQNLYNTILLASFLKTDNKSAKAPLSTSMIERYENH
KKDLAKLKDFVKKNCPDQYHDIFRDKSKNGYAGYIDNGVSQNDFY
TFIGKCLEESLKKDKGAQYFLDKIDRDDFLRKQRTFDNGAFPYQI
HLQEMHAILRRQGDYYPFLKENQDKIEKILTFRIPYYVGPLARKD
SRFAWANYRSDEAITPWNFDEVVDKEKSAEKFITRMTLNDLYLPE
EKVLPKHSLIYETFTVYNELTNIKYVNDQGNAIHFDSELKEKIFN
QLFKENRKVSKKTLIDFLNNSEGIYTDKLVGIDEEVKYLNASLGT
YHDLKKILESFMDDEINEKIIEDIIQTLTLFEDIEMKRQRLQKYD
DIFTPKQLKELARRNYTGWGRLSYKLINGIRNKENNKTILDYLKN
GNRNFMQLINDDRLSFKQIIIDARKIEKLDNIESVVYNLPGSPAI
KKGILQSIKIVDELVKVMGHNPDNIVIEMARENQTTNQGKNRSQQ
RLKRLQDSMSNFKDSSISLKDVDNSDLQNDRLFLYYIQNGKDMYT
GEELDIDHLSDYDIDHIIPQSFIKDNSIDNRVLTSSAKNRGKSDD
VPGRDVVLKMKPFWKKLYDVKLISKRKFDNLTKSEHGGLTESDKA
GFIKRQLVETRQITKYVAQILDGRFNTKRDDNNKVIRDVKVITLK
SSLVSQFRKDFGFYKVREINDYHHAHDAYLNAVVGTAILKKYPKL
APEFVYGEYKKCDVRKLIAKSGDKSEIGKATAKYFFYSNLMNFFK
RVIRYSNGMIVVRPVIEYSKDTGEIAWDKEKDFKTVCKVLSCPQV
NIVKKVEKQSHGLDRGKPKGFYNANPSPKPKKGSKVNLVPIKANL
NPKNYGGYAGISNSYAVLVDATIEKGAKKKLTRIQEFQGISIIDR
EKYEKNKVEFLKGLGYKEIYSIITLPKYSLFELADGSRRMLASIL
STNNKRGEIHKGNELVLPAKYIPLLYHANRIHNTFETGHREYVEK
HIAEFKEIAEIILEFNNKYVNAKKNSSIIEKALESFDSFSLDEIC
DSFVGKLKKNNTKKNSGLFELVSLGSASDFEFLETKVPRYRDYTP
SSLLNATLIHQSITGLYETRIDLSKLGEE KRPAATKKAGQAKKKK
GS YPYDVPDYAYPYDVPDYAYPYDVPDYA .
19 . The protein of claim 17 , wherein the Enterococcus hirae strain F1129E Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 30)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MTKDYTIGL A IGTNSVGWA
VLTDDYQLMKRKMSVHGNTEKKKIKKNFWGARLFDEGQTAEFRRT
KRTNRRRLARRKYRLSKLQDLFAEELCKQDDCFFVRLEESFLVPE
EKQYKPASIFPTLEEEKEYYQKYPTIYHLRQKLVDSTEKEDLRLV
YLALAHLLKYRGHFLFEGDLDTENTSIEESFRVFLEQYSKQSDQP
LIVHQPVLTILTDKLSKTKKVEEILKYYPTEKINSFFAQCLKLIV
GNQANFKRIFDLEAEVKLQFSKETYEEDLESLLEKIGDEYLDIFL
QAKKVHDAILLSEIISSTVKHTKAKLSSGMVERYERHKADLAKFK
QFVKENVPQKSTAFFKDTTKNGYAGYIKGKTTQEEFYKFVKKELS
GVVGSEPFLEKIDQETFLLKQRTYTNGVIPHQVHLIELKAIIDQQ
KQHYPFLEEAGPKIIALFKFRIPYYVGPLAKEQEASSFAWIERKT
AEKIHPWNFSEVVDIEKSAMRFIQRMTKQDTYLPTEKVLPKNSLL
YQKYMIFNELTKVSYKDERGVKQYFSGDEKQQIFKQLFQKERGKI
TVKKLQNFLYTHYHIENAQIFGIEKAFNASYSTYHDFMKLAKTNQ
KAMQEWLEQPEMEPIFEDIVKILTIFEDRQMIKHQLSKYQEVFGE
KLLKEFARKHYTGWGRFSAKLIHGIRDRKTNKTILDYLINDDDVP
ANRNRNLMQLINDEHLSFKEEIAKATVFSKHKSLVDVIQDLPGSP
AIKKGIWQSLKIVEELIAIIGYKPKNIVIEMARENQKTHRTKPRL
KALENGLKQIGSTLLKEQPTDNKALQKERLYLYYLQNGRDMYTGE
PLEIENLHQYEVDHIIPRSFIVDNSIDNKVLVARKQNQKKRDDVP
KKQIVNEQRIFWNQLKEAKLISPKKYAYLTKIELTPEDKARFIQR
QLVETRQITKHVANILHQSFHQEEEGTDCDGVQIITLKATLTSQF
RQTFGLYKVREINPHHHAHDAYLNGFIANVLLKRYPKLAPEFVYG
KYVKYSLARENKATAKKEFYSNILKFFESDEPFCDENGEIYWEKS
HHLPRIKKVLSSHQVNVVKKVEQQKGGFYKETVNSKEKPDKLIER
KNNWDVTKYGGFGSPVIAYAIAFVYAKGKTQKKTRAIEGITIMEQ
AAFEKDPTTFLKEKGFPQVTEFIKLPKYTLFEFDNGRRRFLASHK
ESQKGNPFILSDQLVTLLYHAQHYDKITYQESFDYVNTHLSDFSA
ILTEVLAFAEKYTLADKNIERIQELYEENKYGEISMIAQSFLQLL
QFNAIGAPADFKFFGVTIPRKRYTSLTEIWDATIIYQSVTGLYET
RIRMGDLWAGEQ KRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVP
DYAYPYDVPDYA
20 . The protein of claim 17 , wherein the Streptococcus equinus strain AG46 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 31)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MTNGKILGL A IGVASVGVG
IIEAKTGKVIHANSRLFSAANAENNAERRGFRGARRLTRRKKHRV
KRVRDLFEKYDISTDFRNLNLNPYELRVKGLSEQLTNEELFAALR
TIAKRRGISYLDDAEDDSTGSSDYAKSIDENRRLLKSMTPGQIQL
ERLEKYGQLRGNFTVYDENGEAHRLINVESTSDYKNEARKILETQ
SNYNKQITDEFIEDYIEILTQKRKYYHGPGNEKSRTDYGRFRTDG
TTLENIFGILIGKCSFYPEEYRASKASYTAQEFNFLNDLNNLKVP
TETGKLSTEQKEYLVDFAKNSKTLGASKLLKEIAKLVDGDVKDIS
GYREDSKGKPDLHTFEPYRKLKFNLTTVDIDNLSRDILDKLANIL
TLNTEREGIEDAINRNLPEQFTKEQISEIVQIRKSQSSAFNKGWH
SFSAKLMNELIPELYATSEEQMTILTRLEKFKATKKSSKNTKTID
EKEITDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDK
NADDEKKFIDKKNKENKKEKDDSLKRAAYLYNGTDKLPDDVFHGN
KQLATKIRLWYQQGERCLYSGKPILIQDLVHNSNNFEIDHILPLS
LSFDDSLANKVLVYAWTNQEKGQKTPYQVIDSMDAAWSFREMKDY
VLKQKGIGKKKREYLLTTENIDKIEVKKKFIERNLVDTRYASRVV
LNSLQTALKELGKDTKVSVVRGQFTSQLRRKWKIDKSRETYHHHA
VDALIIAASSQLKLWQKHENLMFENYGENQVVNKETGEILSISDD
EYKELVFQPPYQGFVNTISSKAFEDEILFSYQVDSKFNRKVSDAT
IYSTRKAKLGKDKKEETYVLGKIKDIYSQDGFDTFIKRYKKDKTQ
FLMYQKDPLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRR
ENGLICKYSKKGKGTPIKSLKYYDKKLGNHISITPKESKNDVVLQ
SLNPWRADLYFNPDTLKYELMGLKYSDLSFEKGTGKYHISQEKYD
EIKEKEGIGQNSEFKFTLYRNDLILIKDTESGEQEIYRFLSRTMP
NVKHYVELKPYDKEKFNGGQELIKSLGEADKVGRCLKGLSKPGIS
IYKVRTDVLGNKFFVKKEGDKPKLDFKNN KKKRPAATKKAGQAKK
KK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA .
21 . The protein of claim 17 , wherein the Streptococcus intermedius B196 strain G1552 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 32)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MNGLVLGL A IGIASVGVGI
LNKETGEIIHVNSRIFPAATADSNVERRGFRQGRRLGRRKKHRSA
RLNDLFEEFGFITDFSAVPLNLNPYALRVKGLSEELTNEELFIAL
KNIIKRRGISYLDDASEDGETASNEYGKAVEENRKLLADKTPGQI
QLERFEKYGQVRGDFTVVENGENHRLINVESTSAYKKEAERILRR
QQEFNVRISDEFIEAYLTILTGKRKYYHGPGNEKSRTDYGRFRTD
GTTLDNIFGILIGKCTFYPDEYRAAKASYTAQEFNLLNDLNNLTV
PTETKKLRPEQKRQIVEYARTAKTLGTPTLLKYIAKLVDGSIDDI
KGYRIDKSDKPEMHTFDAYRKMRTLELVDVDILSRETLDDLAYIL
TLNTESEGILEALNSKMPGTFTKEQIDELIQFRKKNSAVFGKGWH
NFSLKLMNELISELYETSEEQMTILTRLGKQRSREISKRTKYIDE
KELTDEIYNPVVAKSVRQAIKIINLATKKYGIFDNIVIEMARESN
EDDEKKAIQNVQKANEDEKKAAMEKAADLYNGKKELPDSIFHGHK
ELATKIRLWHQQGERCLYTGKNISIHDLIHNPHQYEIDHILPLSL
SFDDGLANKVLVLATANQEKGQRTPFQALDSMDDAWSYIEFKQYV
RNSKSLSNKKKDYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVL
NTLQEFYKTNDFDTKISVVRGQFTSQLRRKWKIEKSRDTYHHHAV
DALIIAASSQLRLWKKQNNPLISYREGQLVDPETGEILSLTDDEY
KELVFRPPYDYFVDTLKSKSFEDSILFSYQVDSKYNRKISDATIY
GTRKAQLGKDKQEETYVLGKIKDIYSQKGYEDFIKRYKKDTTQFL
MYHKDPQTFAKVIEEILKTYPDKELNEKGKEIPCNPFEKYRQENG
PIRKYSKKGKGPEIKSLKYYDNKLGNHIDITPVNSQNQVVLQSLK
PWRTDVYFNPQTSKYELMGLKYSDLRFEKGSGSYGISPEKYNKVK
AKEGVDEDSEFKFTLYKNDLILIKDTETGEQQLFRYGSRNDTSKH
YVELKPYEKAKFEGNQQLMNLLGTVAKGGQCLKGINKPNLSIYKV
KTDVLGNKHFIKKEGDQPQLNFKKK IKRPAATKKAGQAKKKK GS Y
PYDVPDYAYPYDVPDYAYPYDVPDYA .
22 . The protein of claim 17 , wherein the Staphylococcus simulans strain 19 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 33)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MNNSYILGL A IGITSVGYG
IIEYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRL
QRVKKMLFDYKLLNEDSEISGINPYEARVKGLSEKLSDEEFSAAL
LHLAKRRGVHNVSDVEEDTGNELSTKEQIARNNKALEDKYVAELQ
LERLKEQGEVRGAANRFKTSDYIKEAKQLLKTQSDYHKIDETFIE
TYISLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEEL
RSVKYAYNADLYNALNDLNNLVIARDENEKLEYYEKFQIIENVFK
QKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNFKIYHDIKG
ITSRKEILENADLLNQIAEILTIYQSAEDIQEELAELEPKLTQEE
IEQISNLTGYTGTHRLSLKAINLILDELWNTSDNQMTIFNRLKLV
PKKVDLSQQKEIPTSLVDDFILSPVVKRSFIQSIKVINAIIKKFG
LPKDIIIELAREKNSKEAQKFINEMQKRNRQTNERIEKIIKETGK
EKAKFLIEKIKLHDMQEGKCLYSLESIPLEDLLNNPYHYEVDHII
PRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDAKISYETF
KKHILNLSKGKGRVSKKKKEYLLEERDINRFSVQKDFINRNLVDT
RYATRELMNLLRSYFRVNDLDVKVKSINGGFTSFLRTKWKFKKER
NQGYKHHAEDALVIANADFIFKEWKKLDTTNKVMENQTVEEQQAE
NMPGIETDDEYKEIFVIPRQIQSIKDFKDYKYSHRVDKKPNRELV
NDTLYSTRKDDKGNTLIINNIKGLYDKDNDKLKNLIKKSPEKLLM
YHHDPQTYQKLKTIMEQYSNEKNPLYKYHEETGNYLTKYSKKDNG
PIIKKVKYYGKKLNAHLDITNDYSNSQNKIVKLSLKPYRFDVYLD
NGGYKFVTVKNLDVIKKEGFFKIDSNAYEKAKSEKKIDENAVFIA
SFYNNDLIKIDGELYRIVGVNNDTRNVVELNMIPITYKEYLENIN
DKRTPRILKTISQKTYSIEKYSTDILGNLYKVKSKKKPQMIMKG K
RPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA
23 . The protein of claim 17 , wherein the Streptococcus sanguinis SK330 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 34)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MENKNYSIGL A IGTNSVGW
AVITDDYKVPSKKMKVFGNTDKHFIKKNLIGALLFDEGATAEDRR
LKRTARRRYTRRKNRLRYLQEIFSEEISKLDSSFFHRLDDSFLVP
KDKRGSKYPIFATLEEEKEYHKKFPTIYHLRKHLADSKEKTDLRL
IYLALAHMIKYRGHFLYEESFDIKNNDIQKIFNEFISIYDNTFEG
SSLSGQNAQVEAIFTDKISKSAKRERVLKLFSDEKSTSLFSEFLK
LIVGNQADFKKHFDLEEKAPLQFSKDTYDEDLENLLGQIGDGFTD
LFLVAKKLYDAILLSGILTVTDPSTKAPLSASMIERYESHQKDLA
ALKQFIKNNLPKRYNEVESDQSKDGYAGYIDGKTTQEAFYKYIKN
LLSKFEGADYFLDKIEREDFLRKQRTFDNGSIPHQIHLQEMNAIL
RRQGEHYPFLKENREKIEKILTFRIPYYVGPLARGNRDFAWLTRN
SDQAIRPWNFEEIVDKASSAEEFINKMTNYDLYLPEEKVLPKHSL
LYETFAVYNELTKVKFIAEGLRDYQFLDSGQKKQIVNQLFKEKRK
VTEKDIIHYLHNVDGYDGIELKGIEKQFNASLSTYHDLLKIIKDK
EFMDDPKNEEILENIVHTLTIFEDREMIKQRLAQYDSIFDEKVIK
ALTRRHYTGWGKLSAKLINGICDKQTGDTILDYLIDDGKINRNFM
QLINDDGLSFKEIIQKAQVVGKTDDVKQVVQELPGSPAIKKGILQ
SIKIVDELVKVMGYAPESIVIEMARENQTTARGKKNSQQRYKRIE
DSLKNLAPGLDSNILKENPTDNIQLQNDRLFLYYLQNGKDMYTGK
PLDIDQLSSYDIDHIIPQAFIKDDSIDNRVLTSSKDNRGKSDNVP
SLEVVQKRKAFWQQLLDSKLISERKFNNLTKAERGGLDERDKVGF
IRRQLVETRQITKHVAQILDASFNTEVNEKNQKIRTVKIITLKSN
LVSNFRKEFELYKVREINDYHHAHDAYLNAVVAKAILKKYPKLEP
EFVYGDYQKYDLKRYISRFKPSKEIEKATEKYFFYSNLLNFFKEE
VHYADGIIVKRENIEYSKDTGEIAWNKEKDFATIKKVLSYPQVNI
VKKTEIQTHGLDRGKPKGLFNSNPSPKPSEDSKENLVPIKQGLDP
RKYGGYAGISNSYAVLVKAIVEKGAKKQQKTILEFQGISILDKIN
FENNKENYLLKKRYIEILSTITLPKYSLFEFPDGTRRRLASILST
NNKRGEIHKGNELVLPGKYTTLLYHAKNINKKLEPEHLEYVEKHR
NDFAKLLECVLNFNDKYVGALKNGERIRQAFTDWETVDIEKLCFS
FIGPENSKNAGLFELTSQGSASDFEFLGVKIPRYRDYAPSSLLKA
TLIHQSITGLYETRIDLSKLGED KRPAATKKAGQAKKKK GS YPYD
VPDYAYPYDVPDYAYPYDVPDYA
24 . The protein of claim 17 , wherein the Streptococcus sp. C150 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 35)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MSDLVLGL A IGIGSVGVGI
LNKVTGEIIHKNSRIFPAAQAENNLERRTNRQGRRLTRRKKHRRV
RLNHLFEESGLITDFTKVSINLNPYQLRVKGLTAELSNEELFIAL
KNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQI
QLERYQKYGQLRGDFTVEEDGRKHRLINVFPTSAYHAEALRILQT
QQEFNPQITDEFINSYLEILTGKRKYYHGPGNEKSRTDYGKYTTK
KDAQGQYITLNNIFGILIGKCTFYPEEYRAAKASYTAQEFNLLND
LNNLTVPTETKKLSEEQKYQIITYVKNEKAMGPAKLFKYIAKLLS
CDVADIKGYRIDKSDKAEIHTFEAYRKMKTLETLDVKKMAREELD
KLAYVLTLNTEREGIQEALDHEFADGTFSQEQVDELVQFRKANSS
IFGKGWHSFSVKLMMELIPELYATSEEQMTILTRLGKQKTTSSSN
KTKYIDEKQLTEEIYNPVVAKSVRQAIKIVNAAIKKYGDFDNIVI
EMARETNEDDEKKAIQKIQKANKAEKDAAMRKAANQYNGKAELPH
SVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNPNQFEID
HILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSF
RELKAFVRESKALSNKKKEYLLTEEDISKFDVRKKFIERNLVDTR
YASRVVLNALQEHFRTHKIDTKVSVVRGQFTSQLRRHWGIEKTRD
TYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELI
SDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKIS
DATIYATRKAKLDKEKKEYTYTLGKIKDIYALGTKTPSKTGFYKF
LDLYKKDKSQFLMYQKDRRTWDEVIEKILEQYRPFKEKDKNGKEV
DENPFEKYRIENGPIRKYSRKGNGPEIKSLKYYDNLLGRFVDITP
SESKNPVALLSLNPWRTDVYYNTETRKYEFLGLKYADLCFEKGGS
YGISKVKYNKIREKEGIGKNSEFKFTLYKNDLILIKDTETNRQQI
FRFWSRTGKDNPKSFEKHKLELKPYEKTRFEKGEELKVLGKVPPS
SNRLQKNMQIENLSIYKVRTDVLGNQHIIKNEGDKPKLDF KRPAA
TKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA
25 . The protein of claim 17 , wherein the Streptococcus oralis subsp. oralis strain RH_1735_08 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 36)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MNGLVLGL A IGIASVGVGI
LEKNSGKIIHANSRIFPAATADNNVERRKNRQARRLHRRKKHRGV
RLQDIFEDYGLLTDFSKVSINLNPYRLRVDGLDQQLTNEELFIAL
KNIVKRRGISYLDDASEDGGTVSSDYGKAVEENRKLLAEKTPGQI
QLERFEKYGQVRGDFTVVENGEKRRLINVESTSAYRKEAERILRK
QQEFNSKITDEFIEDCLKILTGKRKYYHGPGNEKSRTDYGRFRTD
GTTLDNIFGILIGKCTFYPNEYRASKASHTAQEFNLLNDLNNLTV
PTETKKLSEEQKKVIVEYAKEAKTLGASTLLKYIAKMIDASVDQI
SGYRVDVNNKPEMHTFEVYRKMQSLETISVGELSRNVLDELAHIL
TLNTEREGIEEAINTKLKDSFSQDQVLELVQFRKNNSSLFSRGWH
NFSLKLMMELIPELYETSEEQMTILTRLGKQKSKETSKRTKYIDE
KELTEEIYNPVVAKSVRQAIKIINEATKKYGIFDNIVIEMARENN
EEDAKKDYIKRQKANQDEKNAAMEKAAFQYNGKKELPDNIFHGHK
ELATKIRLWHQQGEKCLYTGKNIPISDLIHNQYKYEIDHILPLSL
SFDDSLSNKVLVLATANQEKGQRTPFQALDSMDDGWSYREFKSYV
KESKLLGNKKKEYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVL
NALQDFYKAHKFDTTISVVRGQFTSQLRRKWGLEKSRETYHHHAV
DALIIAASSQLRLWKKQNNPLIAYKEGQFVDSETGEILSLSDDEY
KELVFKAPYDHFVDTLRSKTFEDSILFSYQVDSKYNRKISDATIY
ATRKAKLDKDKSEETYVLGKIKDIYSQAGYDAFIKIYNKDKSKFL
MYHKDPQTFEKVIEEILRTYPSKELNDKNKEIPCNPFEKYRQENG
PIRKYSKKGNGPEIKCLKYYDNKLGNYIDITPDGSDNQVVLQSIA
PWRTDVYYNHKTGKYEFLGLKYSDLYFEKGTGKYKISKEKYDNIK
KIEGVVETSEFKFTLYKNDLILIKDVEKGQEQLFRFLSRNNKGKH
QVQLKPMNKSDFEKGEKLIDIFGTVPNSTTQCVKGLNKSNISIFK
VKTDVLGKKHIIKKEGDEPKLKFKRPAATKKA GQAKKKK GS YPYD
VPDYAYPYDVPDYAYPYDVPDYA .
26 . The protein of claim 17 , wherein the Streptococcus oralis SK313 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 37)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MNGLVLGL A IGIASVGVGI
LEKNSGKIIHANSRIFPAATADNNVERRKNRQARRLHRRKKHRGV
RLQDIFEDYGLLTDFSKVSINLNPYRLRVDGLDQQLTNEELFIAL
KNIVKRRGISYLDDASEDGGTVSSDYGKAVEENRKLLAEKTPGQI
QLERFEKYGQVRGDFTVVENGEKRRLINVESTSAYRKEAERILRK
QQEFNSKITDEFIEDCLKILTGKRKYYHGPGNEKSRTDYGRFRTD
GTTLDNIFGILIGKCTFYPNEYRASKASHTAQEFNLLNDLNNLTV
PTETKKLSEEQKKVIVEYAKEAKTLGASTLLKYIAKMIDASVDQI
SGYRVDVNNKPEMHTFEVYRKMQSLETISVGELSRNVLDELAHIL
TLNTEREGIEEAINTKLKDSFSQDQVLELVQFRKNNSSLFSRGWH
NFSLKLMMELIPELYETSEEQMTILTRLGKQKSKETSKRTKYIDE
KELTEEIYNPVVAKSVRQAIKIINEATKKYGIFDNIVIEMARENN
EEDAKKDYIKRQKANQDEKNAAMEKAAFQYNGKKELPDNIFHGHK
ELATKIRLWHQQGEKCLYTGKNIPISDLIHNQYKYEIDHILPLSL
SFDDSLSNKVLVLATANQEKGQRTPFQALDSMDDGWSYREFKSYV
KESKLLGNKKKEYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVL
NALQDFYKAHKFDTTISVVRGQFTSQLRRKWGLEKSRETYHHHAV
DALIIAASSQLRLWKKQNNPLIAYKEGQFVDSETGEILSLSDDEY
KELVFKAPYDHFVDTLRSKTFEDSILFSYQVDSKYNRKISDATIY
ATRKAKLDKDKSEETYVLGKIKDIYSQAGYDAFIKIYNKDKSKFL
MYHKDPQTFEKVIEEILRTYPSKELNDKNKEIPCNPFEKYRQENG
PIRKYSKKGNGPEIKCLKYYDNKLGNYIDITPDGSDNQVVLQSIA
PWRTDVYYNHKTGKYEFLGLKYSDLYFEKGTGKYKISKEKYDNIK
KIEGVVETSEFKFTLYKNDLILIKDVEKGQEQLFRFLSRNNKGKH
QVQLKPMNKSDFEKGEKLIDIFGTVPNSTTQCVKGLNKSNISIFK
VKTDVLGKKHIIKKEGDEPKLKFKRPAATKKA GQAKKKK GS YPYD
VPDYAYPYDVPDYAYPYDVPDYA .
27 . The protein of claim 17 , wherein the Staphylococcus warneri strain 691 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 38)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MKEKYILGL A LGITSVGYG
IINFETKKIIDAGVRLFPEANVDNNEGRRSKRGSRRLKRRRIHRL
DRVKSLLTEYNLINREQIPTSNNPYQIRVKGLSEILSKDELAIAL
LHLAKRRGIHNINVSSEDEDASNELSTKEQINRNNKLLKNKYVCE
VQLQRLKEGQIRGEKNRFKTTDILKEIDQLLKVQKDYHNLDIDFI
NQYKEIVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQ
ELRSVKYAYSADLFNALNDLNNLIIQRNDSEKLEYHEKYHIIENV
FKQKKKPTLKQIAKEIGVNPEDIKGYRITKSGTPQFTEFKLYHDL
KSIVFDKSILENEAILDQIAEILTIYQDEESIKEELNKLPEILNE
QDKAEIAKLTGYNVTHRLSLKCIHLINEELWQTSRNQMEIFNYLN
IKPNKVDLSEQNKIPKDLVDEFILSPVVKRTFIQSINVINKVIEK
YGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIGQT
GNQNGKRIVEKIRLHDQQEGKCLYSLESIPLMDLLNNPQNYEVDH
IIPRSVAFDNSIHNKVLVKQIENSKKGNRTPYQYLNSSDANLSYN
QFKQHILNLSKSKDRISKKKKDYLLEERDINKFEVQKEFINRNLV
DTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLRKVWRFDK
YRNHSYKHHAEDALIIANADFLFKENKKLQNANKILEKPTIENDT
QKVTVEKEEDYNNMFETPKLVEDIKQYRDYKFSHRVDKKPNRQLI
KDTLYSTRMKDEHNYIVQTITDIYGKDNTNLKKQFNKNPEKFLMY
QNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLTKYSKKNNGP
IVKKIKLLGNKVGNHLDVTNKYENSTKKLVKLSIKNYRFDVYLTE
KGYKFVTIAYLNVFKKDNYYYIPKDLYQELKAKKKIKDTDQFIAS
FYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYCEINNI
KGEPRIKKTIGKKTESIEKLTTDVLGNLYLHTTEKAPQLIFKRGL
KRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA .
28 . The protein of claim 17 , wherein the Staphylococcus sciuri strain SNUC 2430 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 39)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MKRNYILGL A IGITSVGYG
IIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRL
QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEVEFSAAL
LHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQ
LERLKTDGEVRGPNNRFKTSDYVKEAKQLLKVQKAYHQLDQSFID
TYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEEL
RSVKYAYNADLYNALNDLNNLVIARDENEKLEYYEKFQIIENVFK
QKKKPTLKQIAKELLVNEEDIKGYRVTSIGKPEFTNFKIYHDIKG
ITERKEVLENAELLDQIAEILTIYQSSEDVQEELANLNSELTQEE
IEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQMAIFNRLKLV
PKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYG
LPNDIIIELAREKNSKDAQKMINEMQKRNRQMNERIEEIIRTTGK
ENAKYLIEKIKLHDMQEGKCLYSLESIPLEDLLNNPFNYEVDHII
PRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETF
KKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDT
RYATRELMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKER
NKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQTVEEKQAE
SMPEIETEQEYKEIFITPHQIQHIKGFKDYKYSHRVDKKPNRELI
NDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLMNKSPEKLLM
YHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNG
PVIKKIKYYGKNLKAHLDITDDYPNSRNKVVKLSVKPYRFDVYLD
NDIYKFVTVKNLDVIKKEDYYEVNSKCYKEAKKLKKISDQAEFIA
SFYNNDLIKINGELYRVIGVNNDLLNRIEVNMINITYREYLENMN
DKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKQKPQMIMKG K
RPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA .
29 . The protein of claim 17 , wherein the Streptococcus gallolyticus strain AM24-4 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 40)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MTNGKILGL A IGIASVGVG
IIEAKTGKVVHANSRLFSAANAENNTERRGFRGSRRLNRRKKHRV
KRVRDLFEKHEIVTDFRNLNLSPYELRVKGLTEQLTNEELFAALR
TISKRRGISYLDDAEDDSTGSTDYAKSIDENRRLLKNKTPGQIQL
ERLEKYGQLRGNFTVYDENGEAHRLINVESTSDYEKEARKILETQ
ADYNKKITAEFIDDYVEILTQKRKYYHGPGNEKSRTDYGRFRTDG
TTLENIFGILIGKCNFYPDEYRASKASYTAQEYNFLNDLNNLKVP
TETGKLPTEQKESLVEFAKNTATLGPSKLLKEIAKILDCKVDEIK
GYREDDKGKPDLHTFEPYRKLKFNLESINIDDLSREVIDKLADIL
TLNTEREGIEDAIKRNLPNQFTEEQISEIIKVRKSQSTAFNKGWH
SFSAKLMNELIPELYATSDEQMTILTRLEKFKVNKKSSKNTKTID
EKEVTDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDK
NADDEKKFIDKRNKENKKEKDDALKRAAYLYNSSDKLPDEVFHGN
KQLETKIRLWYQQGERCLYSGKPIPIHDLVHNSNNFEIDHILPLS
LSFDDSLANKVLVYDWANQEKGQKTPYQVIDSMDAAWSFREMKDY
VLKQKGLGKKKRDYLLTTENIDKIEVKKKFIERNLIDTRYASRVV
LNSLQSALRELCKDTKVSVIRGQFTSQLRRKWKIDKSRETYHHHA
VDALIIAASSQLKLWEKQDNLMFIDYGNNQVVDKETGEILSVSDD
EYKELVFQPPYQGFVNTISSKGFEDEILFSYQVDSKYNRKVSDAT
IYSTRKAKIGKDKKEETYVLGKIKDIYSQNGFDTFIKKYNKDKTQ
FLMYQKDPLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRR
ENGLICKYSKKGKGTPIKSLKYYDKKLGNCIDITPEESRNKVILQ
SINPWRADVYFNPETLKYELMGLKYSDLSFEKGTGKYHISQEKYD
AIKEKEGIGKKSEFKFTLYRNDLILIKDTASGEQEIYRFLSRTMP
NVNHYAELKPYDKEKFDGGQELMEVFGKVANGGQCLKSLNKSNIS
IYKVRTDVLGNKYFVKKEGDKPKLNFKNN KKKRPAATKKAGQAKK
KK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA
30 . The protein of claim 17 , wherein the Lactobacillus kullabergensis strain Biut2 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 41)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MKRVNEDYILGL A IGTNSC
GWAVTDKKNNLLKLRGKTAIGSHLFEEGHTAADRRGFRTTRRRLK
RRKWRLRLLEEIFAEPMAKVDPGFFVRLHQSWVSPLDKDRKKYNA
IVFPTAKEDQAFYKHYATIYHLRDELMTQDRQFDLREIFLAIHHI
VKYRGNFLQDTPVKDFEASKIEVGPILSHINNAFAEKIVEDQDPI
ELNVANAADIEDVIRGKDAEKTVYKLDKVKKIAKLLTDSTAKEEK
NVAKQIANAIMGYKTQFETILDKEIDKSDKAQWEFKLSDADADDK
LDALLPDLDETDQTVVAEIEKLESAITLSTIVDENKSLSQSMVEK
YKKHKKDYKKLKKYINTLQDQTKAKKLLLAYDLYVNNRHGRLLEA
KKTFEDKKKKKALTKDEFYKIVKDNLDDSDLAHEIQQEIAADNFM
PKQRTNSNGVIPFQLHQIELDKIIANQGKYYPFLAAENPVEDHRK
QAPYKLDELVRFRVPYYVGPMITADEQEKTSGKSFAWMVRKEDGQ
ITPWNFEQKVDRQESANKFIKRMTIKDTYLLSEDVLPANSLLYQR
FEVLNELNNIRINGSRISVDLKQQIFNDLFEEKKTVTEKSLTSYL
KQNLHLPTVEIKGLADPTKFNSSLASYYHLKSLHVFDKELADPQY
QKDFEKIIEYSSIFEDKKIFQDKLHAEFKWLTPEQFKAISTWRLQ
GWGRLSRKLLVELHDTNGQNIMEQLWDSQKNFMQIVTEPDFKDAI
AKENQNVTRANGVEEILADAYTSPANKKAIRQVVKVVADVVKAAG
GKKPAQFAIEFTRDPDKNPQLSHIRGTKLLKAYQETAGELVDQKL
TDSLKEAMTSRKLLKDKYFLYFMQAGRDAYTGQKINIDEVSTNYQ
IDHILPQSFIKDDSFDNRVLTATPLNAEKSDDVPYKRFANNYVSD
MKMTVGEMWKHWQKAGIINKHKLGNLLLDPDRLNKFQKSGFINRQ
LVETSQIIKLVSVILQNKYPDAEIITVKAGDNSALRQRLNLYKSR
DVNDYHHAIDAYLSIICGNFLYQVYPKYRPYFVYGKYKKFSQDPD
LQKEVIKHFKGFTFMWPLLQKDNSERKAPEKIKENNSDRIVFYKH
PDIFDKLRKAYNYKYMLVSRETTTENSGLFDVTIYPRGERDLAKT
RKLIPKSNGLDPKIYGGYSGNTDAYMVIVKIDKGKESIYKVIGVP
MRALASLNRAKKQGNYKEELHQVLEPQIMFDKNGKPKRSVKGFRI
IKDHVPFKQVVLDGDKKFMLNSSTYEINAKQLTLTPETMRIVTDN
LKKGEDQDQLLVKAYDEILQKVDQYLPLFDVNKFRNSLHLGRAKF
LDLAVNDKKITLTNILNGLHDNLVTPDLKNIGIKTPLGKLQVPSG
IVLSSEAILIFQSPTGLFEKRVRIADL KRPAATKKAGQAKKKK GS
YPYDVPDYAYPYDVPDYAYPYDVPDYA .
31 . The protein of claim 17 , wherein the Streptococcus suis strain LSS83 Cas9 has an amino acid sequence at least 80% identical to
(SEQ ID NO: 42)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MSNGKILGL A IGIASVGVG
VIDAQTGEIIHASSRIFPSANAANNAERRTFRGSRRLIRRKKHRI
KRLDDLFNDFHINLDGEMSTDNPYVLRVKGLSQKLTVEELYISIK
NIMKRRGISYLDDAESDNEAGRSDYAKAIERNRQLLTSKTPGEIQ
LERLEKYGQLRGNFTIIDEEGQSQQIINVESTSDYVKEVEKILDC
QKMYHKFISDEFCDKLIELLREKRKYYVGPGNEKSRTDYGIYRTD
GTTLENLFGILIGKCTFYPDQYRSSRASYTAQEFNFLNDLNNLTV
PTETKKLSQEQKEFLVNYAKETSVLGAGKILQQIAKLADCKVEDI
RGYRLDNKDKPELHTFETYRAMKGLVPLVDIGVLSREQLDILADI
LTLNTDFEGIREALKKQLPNVFDEKQVKGLASFRKSKSQLFAKGW
HNLSQKIMLEVIPELYATSDEQMTILTRLGKFEKSSVAEYPSSIN
VDEITDEIYNPVVAKSIRQTIKIINASIKKWDEFDQIVIEMPRDR
NEDEEKKRIADGQKANAKEKADSILRAAELYCAGKVLPDYVYNGH
NQLATKIRLWYQQGERCIYTGQPISIHDLIHNQNQYEIDHILPLS
LTFDDSLSNKVLVLATANQEKAQRTPYNYLKSATSAWSYREFKDY
VTKRKGIGKKKCEYLTFEEDINGFEVRSKFIQRNLVDTRYASKVI
LNALQDYFKISGIQTKVSVVRGQFTSQLRHKWGIEKTRETYHHHA
VDALIIAASSQLRLWKKQESPLVVDYQEGRQVDLETGEILELTDE
QYKELVYQPPYQGFVNTISSSAFDNEILFSYQVDSKVNRKISDAT
IYATRNAQLGKDKTEGIYVLGKIKDIYTQAGYEAFLKRYTKDKTS
FLMYHKDLDTWEKVIEIILRDYREYDEKGKEIGNPFERYRRENGY
VKKYSRKGNGTAIKSLKYYDNKLGNHIDITPENSRNAVVLQSLKP
WRTDVYFNKETGKYEFLGIKYSDLSFEKGTGEYGISQEKYDSIKI
AEGVAKKSIFKFTLYKQDLLFIKDIENNFGKLLRFTSKNDTSKHY
VELKPYDKNKFGTEEPLLPVLGNVAKSGQCIKGLNKSNISIYKVR
TDILGYRHFIKQEGEHPQLKF KKKRPAATKKAGQAKKKK GS YPYD
VPDYAYPYDVPDYAYPYDVPDYA
32 . The 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.
33 . The protein of claim 32 , wherein the mutation is an amino acid substitution.
34 . The protein of any one of the preceding claims , wherein the Cas9 protein has nickase activity.
35 . The protein of claim 32 , wherein the at least one mutation results in an inactive Cas9 (dCas9).
36 . The 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.
37 . An engineered, non-naturally occurring Cas9 fusion protein comprising a Cas9 protein having at least 80% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 and wherein the Cas9 protein is fused to a histone demethylase, a transcriptional activator, or to a deaminase.
38 . The Cas9 protein of claim 37 , wherein the Cas9 protein is fused to a cytosine deaminase or to an adenosine deaminase.
39 . The Cas9 protein of claim 38 , 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: 29)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GTMEKSYSIGL A IGTNSVGWS
VITDDYKVPAKKMRVLGNTDKKYIKKNLLGALLFDSGETAEATRL
KRTARRRYTRRKNRLRYLQEIFAKEMAKVDESFFQRLDESFLTDD
DKTFDSHPIFGNKAEEDAYHQKFPTIYHLRKHLADSQEKADLRLI
YLALAHMIKFRGHFLYDNFNDDNFDWRNIDIQKRYEEFIETYDST
LGESYLADISVDAASILEEKVSKTERLENLLKYYPTEKKTTFFGN
LIKLILGQQAKFKAIFNLEDEISLQFATPTYDEDLEELLGKIDNG
DSYSELFVAAQNLYNTILLASFLKTDNKSAKAPLSTSMIERYENH
KKDLAKLKDFVKKNCPDQYHDIFRDKSKNGYAGYIDNGVSQNDFY
TFIGKCLEESLKKDKGAQYFLDKIDRDDFLRKQRTFDNGAFPYQI
HLQEMHAILRRQGDYYPFLKENQDKIEKILTFRIPYYVGPLARKD
SRFAWANYRSDEAITPWNFDEVVDKEKSAEKFITRMTLNDLYLPE
EKVLPKHSLIYETFTVYNELTNIKYVNDQGNAIHFDSELKEKIFN
QLFKENRKVSKKTLIDFLNNSEGIYTDKLVGIDEEVKYLNASLGT
YHDLKKILESFMDDEINEKIIEDIIQTLTLFEDIEMKRQRLQKYD
DIFTPKQLKELARRNYTGWGRLSYKLINGIRNKENNKTILDYLKN
GNRNFMQLINDDRLSFKQIIIDARKIEKLDNIESVVYNLPGSPAI
KKGILQSIKIVDELVKVMGHNPDNIVIEMARENQTTNQGKNRSQQ
RLKRLQDSMSNFKDSSISLKDVDNSDLQNDRLFLYYIQNGKDMYT
GEELDIDHLSDYDIDHIIPQSFIKDNSIDNRVLTSSAKNRGKSDD
VPGRDVVLKMKPFWKKLYDVKLISKRKFDNLTKSEHGGLTESDKA
GFIKRQLVETRQITKYVAQILDGRFNTKRDDNNKVIRDVKVITLK
SSLVSQFRKDFGFYKVREINDYHHAHDAYLNAVVGTAILKKYPKL
APEFVYGEYKKCDVRKLIAKSGDKSEIGKATAKYFFYSNLMNFFK
RVIRYSNGMIVVRPVIEYSKDTGEIAWDKEKDFKTVCKVLSCPQV
NIVKKVEKQSHGLDRGKPKGFYNANPSPKPKKGSKVNLVPIKANL
NPKNYGGYAGISNSYAVLVDATIEKGAKKKLTRIQEFQGISIIDR
EKYEKNKVEFLKGLGYKEIYSIITLPKYSLFELADGSRRMLASIL
STNNKRGEIHKGNELVLPAKYIPLLYHANRIHNTFETGHREYVEK
HIAEFKEIAEIILEFNNKYVNAKKNSSIIEKALESFDSFSLDEIC
DSFVGKLKKNNTKKNSGLFELVSLGSASDFEFLETKVPRYRDYTP
SSLLNATLIHQSITGLYETRIDLSKLGEE KRPAATKKAGQAKKKK
GS YPYDVPDYAYPYDVPDYAYPYDVPDYA .
(b)
(SEQ ID NO: 30)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MTKDYTIGL A IGTNSVGWA
VLTDDYQLMKRKMSVHGNTEKKKIKKNFWGARLFDEGQTAEFRRT
KRTNRRRLARRKYRLSKLQDLFAEELCKQDDCFFVRLEESFLVPE
EKQYKPASIFPTLEEEKEYYQKYPTIYHLRQKLVDSTEKEDLRLV
YLALAHLLKYRGHELFEGDLDTENTSIEESFRVFLEQYSKQSDQP
LIVHQPVLTILTDKLSKTKKVEEILKYYPTEKINSFFAQCLKLIV
GNQANFKRIFDLEAEVKLQFSKETYEEDLESLLEKIGDEYLDIFL
QAKKVHDAILLSEIISSTVKHTKAKLSSGMVERYERHKADLAKFK
QFVKENVPQKSTAFFKDTTKNGYAGYIKGKTTQEEFYKFVKKELS
GVVGSEPFLEKIDQETFLLKQRTYTNGVIPHQVHLIELKAIIDQQ
KQHYPFLEEAGPKIIALFKFRIPYYVGPLAKEQEASSFAWIERKT
AEKIHPWNFSEVVDIEKSAMRFIQRMTKQDTYLPTEKVLPKNSLL
YQKYMIFNELTKVSYKDERGVKQYFSGDEKQQIFKQLFQKERGKI
TVKKLQNFLYTHYHIENAQIFGIEKAFNASYSTYHDFMKLAKTNQ
KAMQEWLEQPEMEPIFEDIVKILTIFEDRQMIKHQLSKYQEVFGE
KLLKEFARKHYTGWGRFSAKLIHGIRDRKTNKTILDYLINDDDVP
ANRNRNLMQLINDEHLSFKEEIAKATVFSKHKSLVDVIQDLPGSP
AIKKGIWQSLKIVEELIAIIGYKPKNIVIEMARENQKTHRTKPRL
KALENGLKQIGSTLLKEQPTDNKALQKERLYLYYLQNGRDMYTGE
PLEIENLHQYEVDHIIPRSFIVDNSIDNKVLVARKQNQKKRDDVP
KKQIVNEQRIFWNQLKEAKLISPKKYAYLTKIELTPEDKARFIQR
QLVETRQITKHVANILHQSFHQEEEGTDCDGVQIITLKATLTSQF
RQTFGLYKVREINPHHHAHDAYLNGFIANVLLKRYPKLAPEFVYG
KYVKYSLARENKATAKKEFYSNILKFFESDEPFCDENGEIYWEKS
HHLPRIKKVLSSHQVNVVKKVEQQKGGFYKETVNSKEKPDKLIER
KNNWDVTKYGGFGSPVIAYAIAFVYAKGKTQKKTRAIEGITIMEQ
AAFEKDPTTFLKEKGFPQVTEFIKLPKYTLFEFDNGRRRFLASHK
ESQKGNPFILSDQLVTLLYHAQHYDKITYQESFDYVNTHLSDFSA
ILTEVLAFAEKYTLADKNIERIQELYEENKYGEISMIAQSFLQLL
QFNAIGAPADFKFFGVTIPRKRYTSLTEIWDATIIYQSVTGLYET
RIRMGDLWAGEQ KRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVP
DYAYPYDVPDYA
(c)
(SEQ ID NO: 31)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MTNGKILGL A IGVASVGVG
IIEAKTGKVIHANSRLFSAANAENNAERRGFRGARRLTRRKKHRV
KRVRDLFEKYDISTDFRNLNLNPYELRVKGLSEQLTNEELFAALR
TIAKRRGISYLDDAEDDSTGSSDYAKSIDENRRLLKSMTPGQIQL
ERLEKYGQLRGNFTVYDENGEAHRLINVESTSDYKNEARKILETQ
SNYNKQITDEFIEDYIEILTQKRKYYHGPGNEKSRTDYGRFRTDG
TTLENIFGILIGKCSFYPEEYRASKASYTAQEFNFLNDLNNLKVP
TETGKLSTEQKEYLVDFAKNSKTLGASKLLKEIAKLVDGDVKDIS
GYREDSKGKPDLHTFEPYRKLKFNLTTVDIDNLSRDILDKLANIL
TLNTEREGIEDAINRNLPEQFTKEQISEIVQIRKSQSSAFNKGWH
SFSAKLMNELIPELYATSEEQMTILTRLEKFKATKKSSKNTKTID
EKEITDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDK
NADDEKKFIDKKNKENKKEKDDSLKRAAYLYNGTDKLPDDVFHGN
KQLATKIRLWYQQGERCLYSGKPILIQDLVHNSNNFEIDHILPLS
LSFDDSLANKVLVYAWTNQEKGQKTPYQVIDSMDAAWSFREMKDY
VLKQKGIGKKKREYLLTTENIDKIEVKKKFIERNLVDTRYASRVV
LNSLQTALKELGKDTKVSVVRGQFTSQLRRKWKIDKSRETYHHHA
VDALIIAASSQLKLWQKHENLMFENYGENQVVNKETGEILSISDD
EYKELVFQPPYQGFVNTISSKAFEDEILFSYQVDSKFNRKVSDAT
IYSTRKAKLGKDKKEETYVLGKIKDIYSQDGFDTFIKRYKKDKTQ
FLMYQKDPLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRR
ENGLICKYSKKGKGTPIKSLKYYDKKLGNHISITPKESKNDVVLQ
SLNPWRADLYFNPDTLKYELMGLKYSDLSFEKGTGKYHISQEKYD
EIKEKEGIGQNSEFKFTLYRNDLILIKDTESGEQEIYRFLSRTMP
NVKHYVELKPYDKEKFNGGQELIKSLGEADKVGRCLKGLSKPGIS
IYKVRTDVLGNKFFVKKEGDKPKLDFKNN KKKRPAATKKAGQAKK
KK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA .
(d)
(SEQ ID NO: 32)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MNGLVLGL A IGIASVGVGI
LNKETGEIIHVNSRIFPAATADSNVERRGFRQGRRLGRRKKHRSA
RLNDLFEEFGFITDESAVPLNLNPYALRVKGLSEELTNEELFIAL
KNIIKRRGISYLDDASEDGETASNEYGKAVEENRKLLADKTPGQI
QLERFEKYGQVRGDFTVVENGENHRLINVESTSAYKKEAERILRR
QQEFNVRISDEFIEAYLTILTGKRKYYHGPGNEKSRTDYGRFRTD
GTTLDNIFGILIGKCTFYPDEYRAAKASYTAQEFNLLNDLNNLTV
PTETKKLRPEQKRQIVEYARTAKTLGTPTLLKYIAKLVDGSIDDI
KGYRIDKSDKPEMHTFDAYRKMRTLELVDVDILSRETLDDLAYIL
TLNTESEGILEALNSKMPGTFTKEQIDELIQFRKKNSAVFGKGWH
NFSLKLMNELISELYETSEEQMTILTRLGKQRSREISKRTKYIDE
KELTDEIYNPVVAKSVRQAIKIINLATKKYGIFDNIVIEMARESN
EDDEKKAIQNVQKANEDEKKAAMEKAADLYNGKKELPDSIFHGHK
ELATKIRLWHQQGERCLYTGKNISIHDLIHNPHQYEIDHILPLSL
SFDDGLANKVLVLATANQEKGQRTPFQALDSMDDAWSYIEFKQYV
RNSKSLSNKKKDYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVL
NTLQEFYKTNDFDTKISVVRGQFTSQLRRKWKIEKSRDTYHHHAV
DALIIAASSQLRLWKKQNNPLISYREGQLVDPETGEILSLTDDEY
KELVFRPPYDYFVDTLKSKSFEDSILFSYQVDSKYNRKISDATIY
GTRKAQLGKDKQEETYVLGKIKDIYSQKGYEDFIKRYKKDTTQFL
MYHKDPQTFAKVIEEILKTYPDKELNEKGKEIPCNPFEKYRQENG
PIRKYSKKGKGPEIKSLKYYDNKLGNHIDITPVNSQNQVVLQSLK
PWRTDVYFNPQTSKYELMGLKYSDLRFEKGSGSYGISPEKYNKVK
AKEGVDEDSEFKFTLYKNDLILIKDTETGEQQLFRYGSRNDTSKH
YVELKPYEKAKFEGNQQLMNLLGTVAKGGQCLKGINKPNLSIYKV
KTDVLGNKHFIKKEGDQPQLNFKKKI KRPAATKKAGQAKKKK GS Y
PYDVPDYAYPYDVPDYAYPYDVPDYA .
(e)
(SEQ ID NO: 33)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MNNSYILGL A IGITSVGYG
IIEYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRL
QRVKKMLFDYKLLNEDSEISGINPYEARVKGLSEKLSDEEFSAAL
LHLAKRRGVHNVSDVEEDTGNELSTKEQIARNNKALEDKYVAELQ
LERLKEQGEVRGAANRFKTSDYIKEAKQLLKTQSDYHKIDETFIE
TYISLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEEL
RSVKYAYNADLYNALNDLNNLVIARDENEKLEYYEKFQIIENVFK
QKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNFKIYHDIKG
ITSRKEILENADLLNQIAEILTIYQSAEDIQEELAELEPKLTQEE
IEQISNLTGYTGTHRLSLKAINLILDELWNTSDNQMTIFNRLKLV
PKKVDLSQQKEIPTSLVDDFILSPVVKRSFIQSIKVINAIIKKFG
LPKDIIIELAREKNSKEAQKFINEMQKRNRQTNERIEKIIKETGK
EKAKFLIEKIKLHDMQEGKCLYSLESIPLEDLLNNPYHYEVDHII
PRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDAKISYETF
KKHILNLSKGKGRVSKKKKEYLLEERDINRFSVQKDFINRNLVDT
RYATRELMNLLRSYFRVNDLDVKVKSINGGFTSFLRTKWKFKKER
NQGYKHHAEDALVIANADFIFKEWKKLDTTNKVMENQTVEEQQAE
NMPGIETDDEYKEIFVIPRQIQSIKDFKDYKYSHRVDKKPNRELV
NDTLYSTRKDDKGNTLIINNIKGLYDKDNDKLKNLIKKSPEKLLM
YHHDPQTYQKLKTIMEQYSNEKNPLYKYHEETGNYLTKYSKKDNG
PIIKKVKYYGKKLNAHLDITNDYSNSQNKIVKLSLKPYRFDVYLD
NGGYKFVTVKNLDVIKKEGFFKIDSNAYEKAKSEKKIDENAVFIA
SFYNNDLIKIDGELYRIVGVNNDTRNVVELNMIPITYKEYLENIN
DKRTPRILKTISQKTYSIEKYSTDILGNLYKVKSKKKPQMIMKG K
RPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA .
(f)
(SEQ ID NO: 34)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MENKNYSIGL A IGTNSVGW
AVITDDYKVPSKKMKVFGNTDKHFIKKNLIGALLFDEGATAEDRR
LKRTARRRYTRRKNRLRYLQEIFSEEISKLDSSFFHRLDDSFLVP
KDKRGSKYPIFATLEEEKEYHKKFPTIYHLRKHLADSKEKTDLRL
IYLALAHMIKYRGHFLYEESFDIKNNDIQKIFNEFISIYDNTFEG
SSLSGQNAQVEAIFTDKISKSAKRERVLKLFSDEKSTSLFSEFLK
LIVGNQADFKKHFDLEEKAPLQFSKDTYDEDLENLLGQIGDGFTD
LFLVAKKLYDAILLSGILTVTDPSTKAPLSASMIERYESHQKDLA
ALKQFIKNNLPKRYNEVESDQSKDGYAGYIDGKTTQEAFYKYIKN
LLSKFEGADYFLDKIEREDFLRKQRTFDNGSIPHQIHLQEMNAIL
RRQGEHYPFLKENREKIEKILTFRIPYYVGPLARGNRDFAWLTRN
SDQAIRPWNFEEIVDKASSAEEFINKMTNYDLYLPEEKVLPKHSL
LYETFAVYNELTKVKFIAEGLRDYQFLDSGQKKQIVNQLFKEKRK
VTEKDIIHYLHNVDGYDGIELKGIEKQFNASLSTYHDLLKIIKDK
EFMDDPKNEEILENIVHTLTIFEDREMIKQRLAQYDSIFDEKVIK
ALTRRHYTGWGKLSAKLINGICDKQTGDTILDYLIDDGKINRNFM
QLINDDGLSFKEIIQKAQVVGKTDDVKQVVQELPGSPAIKKGILQ
SIKIVDELVKVMGYAPESIVIEMARENQTTARGKKNSQQRYKRIE
DSLKNLAPGLDSNILKENPTDNIQLQNDRLFLYYLQNGKDMYTGK
PLDIDQLSSYDIDHIIPQAFIKDDSIDNRVLTSSKDNRGKSDNVP
SLEVVQKRKAFWQQLLDSKLISERKFNNLTKAERGGLDERDKVGF
IRRQLVETRQITKHVAQILDASFNTEVNEKNQKIRTVKIITLKSN
LVSNFRKEFELYKVREINDYHHAHDAYLNAVVAKAILKKYPKLEP
EFVYGDYQKYDLKRYISRFKPSKEIEKATEKYFFYSNLLNFFKEE
VHYADGIIVKRENIEYSKDTGEIAWNKEKDFATIKKVLSYPQVNI
VKKTEIQTHGLDRGKPKGLFNSNPSPKPSEDSKENLVPIKQGLDP
RKYGGYAGISNSYAVLVKAIVEKGAKKQQKTILEFQGISILDKIN
FENNKENYLLKKRYIEILSTITLPKYSLFEFPDGTRRRLASILST
NNKRGEIHKGNELVLPGKYTTLLYHAKNINKKLEPEHLEYVEKHR
NDFAKLLECVLNFNDKYVGALKNGERIRQAFTDWETVDIEKLCFS
FIGPENSKNAGLFELTSQGSASDFEFLGVKIPRYRDYAPSSLLKA
TLIHQSITGLYETRIDLSKLGED KRPAATKKAGQAKKKK GS YPYD
VPDYAYPYDVPDYAYPYDVPDYA .
(g)
(SEQ ID NO: 35)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MSDLVLGL A IGIGSVGVGI
LNKVTGEIIHKNSRIFPAAQAENNLERRTNRQGRRLTRRKKHRRV
RLNHLFEESGLITDFTKVSINLNPYQLRVKGLTAELSNEELFIAL
KNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQI
QLERYQKYGQLRGDFTVEEDGRKHRLINVFPTSAYHAEALRILQT
QQEFNPQITDEFINSYLEILTGKRKYYHGPGNEKSRTDYGKYTTK
KDAQGQYITLNNIFGILIGKCTFYPEEYRAAKASYTAQEFNLLND
LNNLTVPTETKKLSEEQKYQIITYVKNEKAMGPAKLFKYIAKLLS
CDVADIKGYRIDKSDKAEIHTFEAYRKMKTLETLDVKKMAREELD
KLAYVLTLNTEREGIQEALDHEFADGTFSQEQVDELVQFRKANSS
IFGKGWHSFSVKLMMELIPELYATSEEQMTILTRLGKQKTTSSSN
KTKYIDEKQLTEEIYNPVVAKSVRQAIKIVNAAIKKYGDFDNIVI
EMARETNEDDEKKAIQKIQKANKAEKDAAMRKAANQYNGKAELPH
SVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNPNQFEID
HILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSF
RELKAFVRESKALSNKKKEYLLTEEDISKFDVRKKFIERNLVDTR
YASRVVLNALQEHFRTHKIDTKVSVVRGQFTSQLRRHWGIEKTRD
TYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELI
SDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKIS
DATIYATRKAKLDKEKKEYTYTLGKIKDIYALGTKTPSKTGFYKF
LDLYKKDKSQFLMYQKDRRTWDEVIEKILEQYRPFKEKDKNGKEV
DFNPFEKYRIENGPIRKYSRKGNGPEIKSLKYYDNLLGRFVDITP
SESKNPVALLSLNPWRTDVYYNTETRKYEFLGLKYADLCFEKGGS
YGISKVKYNKIREKEGIGKNSEFKFTLYKNDLILIKDTETNRQQI
FRFWSRTGKDNPKSFEKHKLELKPYEKTRFEKGEELKVLGKVPPS
SNRLQKNMQIENLSIYKVRTDVLGNQHIIKNEGDKPKLDF KRPAA
TKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA .
(h)
(SEQ ID NO: 36)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MNGLVLGL A IGIASVGVGI
LEKNSGKIIHANSRIFPAATADNNVERRKNRQARRLHRRKKHRGV
RLQDIFEDYGLLTDFSKVSINLNPYRLRVDGLDQQLTNEELFIAL
KNIVKRRGISYLDDASEDGGTVSSDYGKAVEENRKLLAEKTPGQI
QLERFEKYGQVRGDFTVVENGEKRRLINVESTSAYRKEAERILRK
QQEFNSKITDEFIEDCLKILTGKRKYYHGPGNEKSRTDYGRFRTD
GTTLDNIFGILIGKCTFYPNEYRASKASHTAQEFNLLNDLNNLTV
PTETKKLSEEQKKVIVEYAKEAKTLGASTLLKYIAKMIDASVDQI
SGYRVDVNNKPEMHTFEVYRKMQSLETISVGELSRNVLDELAHIL
TLNTEREGIEEAINTKLKDSFSQDQVLELVQFRKNNSSLFSRGWH
NFSLKLMMELIPELYETSEEQMTILTRLGKQKSKETSKRTKYIDE
KELTEEIYNPVVAKSVRQAIKIINEATKKYGIFDNIVIEMARENN
EEDAKKDYIKRQKANQDEKNAAMEKAAFQYNGKKELPDNIFHGHK
ELATKIRLWHQQGEKCLYTGKNIPISDLIHNQYKYEIDHILPLSL
SFDDSLSNKVLVLATANQEKGQRTPFQALDSMDDGWSYREFKSYV
KESKLLGNKKKEYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVL
NALQDFYKAHKFDTTISVVRGQFTSQLRRKWGLEKSRETYHHHAV
DALIIAASSQLRLWKKQNNPLIAYKEGQFVDSETGEILSLSDDEY
KELVFKAPYDHFVDTLRSKTFEDSILFSYQVDSKYNRKISDATIY
ATRKAKLDKDKSEETYVLGKIKDIYSQAGYDAFIKIYNKDKSKFL
MYHKDPQTFEKVIEEILRTYPSKELNDKNKEIPCNPFEKYRQENG
PIRKYSKKGNGPEIKCLKYYDNKLGNYIDITPDGSDNQVVLQSIA
PWRTDVYYNHKTGKYEFLGLKYSDLYFEKGTGKYKISKEKYDNIK
KIEGVVETSEFKFTLYKNDLILIKDVEKGQEQLFRFLSRNNKGKH
QVQLKPMNKSDFEKGEKLIDIFGTVPNSTTQCVKGLNKSNISIFK
VKTDVLGKKHIIKKEGDEPKLKFKRPAATKKA GQAKKKK GS YPYD
VPDYAYPYDVPDYAYPYDVPDYA .
(i)
(SEQ ID NO: 37)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MNGLVLGL A IGIASVGVGI
LEKNSGKIIHANSRIFPAATADNNVERRKNRQARRLHRRKKHRGV
RLQDIFEDYGLLTDFSKVSINLNPYRLRVDGLDQQLTNEELFIAL
KNIVKRRGISYLDDASEDGGTVSSDYGKAVEENRKLLAEKTPGQI
QLERFEKYGQVRGDFTVVENGEKRRLINVESTSAYRKEAERILRK
QQEFNSKITDEFIEDCLKILTGKRKYYHGPGNEKSRTDYGRFRTD
GTTLDNIFGILIGKCTFYPNEYRASKASHTAQEFNLLNDLNNLTV
PTETKKLSEEQKKVIVEYAKEAKTLGASTLLKYIAKMIDASVDQI
SGYRVDVNNKPEMHTFEVYRKMQSLETISVGELSRNVLDELAHIL
TLNTEREGIEEAINTKLKDSFSQDQVLELVQFRKNNSSLFSRGWH
NFSLKLMMELIPELYETSEEQMTILTRLGKQKSKETSKRTKYIDE
KELTEEIYNPVVAKSVRQAIKIINEATKKYGIFDNIVIEMARENN
EEDAKKDYIKRQKANQDEKNAAMEKAAFQYNGKKELPDNIFHGHK
ELATKIRLWHQQGEKCLYTGKNIPISDLIHNQYKYEIDHILPLSL
SFDDSLSNKVLVLATANQEKGQRTPFQALDSMDDGWSYREFKSYV
KESKLLGNKKKEYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVL
NALQDFYKAHKFDTTISVVRGQFTSQLRRKWGLEKSRETYHHHAV
DALIIAASSQLRLWKKQNNPLIAYKEGQFVDSETGEILSLSDDEY
KELVFKAPYDHFVDTLRSKTFEDSILFSYQVDSKYNRKISDATIY
ATRKAKLDKDKSEETYVLGKIKDIYSQAGYDAFIKIYNKDKSKFL
MYHKDPQTFEKVIEEILRTYPSKELNDKNKEIPCNPFEKYRQENG
PIRKYSKKGNGPEIKCLKYYDNKLGNYIDITPDGSDNQVVLQSIA
PWRTDVYYNHKTGKYEFLGLKYSDLYFEKGTGKYKISKEKYDNIK
KIEGVVETSEFKFTLYKNDLILIKDVEKGQEQLFRFLSRNNKGKH
QVQLKPMNKSDFEKGEKLIDIFGTVPNSTTQCVKGLNKSNISIFK
VKTDVLGKKHIIKKEGDEPKLKFKRPAATKKA GQAKKKK GS YPYD
VPDYAYPYDVPDYAYPYDVPDYA .
(j)
(SEQ ID NO: 38)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRAR
DEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGL
VMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGA
AGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNA
QKKAQSSTD GSSGSETPGTSESATPESSG PKKKRKV GT MKEKYIL
GL A LGITSVGYGIINFETKKIIDAGVRLFPEANVDNNEGRRSKRG
SRRLKRRRIHRLDRVKSLLTEYNLINREQIPTSNNPYQIRVKGLS
EILSKDELAIALLHLAKRRGIHNINVSSEDEDASNELSTKEQINR
NNKLLKNKYVCEVQLQRLKEGQIRGEKNRFKTTDILKEIDQLLKV
QKDYHNLDIDFINQYKEIVETRREYFEGPGQGSPFGWNGDLKKWY
EMLMGHCTYFPQELRSVKYAYSADLFNALNDLNNLIIQRNDSEKL
EYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGYRITKSGT
PQFTEFKLYHDLKSIVFDKSILENEAILDQIAEILTIYQDEESIK
EELNKLPEILNEQDKAEIAKLTGYNVTHRLSLKCIHLINEELWQT
SRNQMEIFNYLNIKPNKVDLSEQNKIPKDLVDEFILSPVVKRTFI
QSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEA
TRKRINEIIGQTGNQNGKRIVEKIRLHDQQEGKCLYSLESIPLMD
LLNNPQNYEVDHIIPRSVAFDNSIHNKVLVKQIENSKKGNRTPYQ
YLNSSDANLSYNQFKQHILNLSKSKDRISKKKKDYLLEERDINKF
EVQKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKVKTINGSF
TNHLRKVWRFDKYRNHSYKHHAEDALIIANADFLFKENKKLQNAN
KILEKPTIENDTQKVTVEKEEDYNNMFETPKLVEDIKQYRDYKFS
HRVDKKPNRQLIKDTLYSTRMKDEHNYIVQTITDIYGKDNTNLKK
QFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGE
YLTKYSKKNNGPIVKKIKLLGNKVGNHLDVTNKYENSTKKLVKLS
IKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIPKDLYQELKAK
KKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYD
IKYKDYCEINNIKGEPRIKKTIGKKTESIEKLTTDVLGNLYLHTT
EKAPQLIFKRGL KRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVP
DYAYPYDVPDYA .
(k)
(SEQ ID NO: 39)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MKRNYILGL A IGITSVGYG
IIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRL
QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEVEFSAAL
LHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQ
LERLKTDGEVRGPNNRFKTSDYVKEAKQLLKVQKAYHQLDQSFID
TYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEEL
RSVKYAYNADLYNALNDLNNLVIARDENEKLEYYEKFQIIENVFK
QKKKPTLKQIAKELLVNEEDIKGYRVTSIGKPEFTNFKIYHDIKG
ITERKEVLENAELLDQIAEILTIYQSSEDVQEELANLNSELTQEE
IEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQMAIFNRLKLV
PKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYG
LPNDIIIELAREKNSKDAQKMINEMQKRNRQMNERIEEIIRTTGK
ENAKYLIEKIKLHDMQEGKCLYSLESIPLEDLLNNPFNYEVDHII
PRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETF
KKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDT
RYATRELMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKER
NKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQTVEEKQAE
SMPEIETEQEYKEIFITPHQIQHIKGFKDYKYSHRVDKKPNRELI
NDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLMNKSPEKLLM
YHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNG
PVIKKIKYYGKNLKAHLDITDDYPNSRNKVVKLSVKPYRFDVYLD
NDIYKFVTVKNLDVIKKEDYYEVNSKCYKEAKKLKKISDQAEFIA
SFYNNDLIKINGELYRVIGVNNDLLNRIEVNMINITYREYLENMN
DKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKQKPQMIMKGK
RPAATKKA GQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA .
(l)
(SEQ ID NO: 40)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MTNGKILGL A IGIASVGVG
IIEAKTGKVVHANSRLFSAANAENNTERRGFRGSRRLNRRKKHRV
KRVRDLFEKHEIVTDFRNLNLSPYELRVKGLTEQLTNEELFAALR
TISKRRGISYLDDAEDDSTGSTDYAKSIDENRRLLKNKTPGQIQL
ERLEKYGQLRGNFTVYDENGEAHRLINVESTSDYEKEARKILETQ
ADYNKKITAEFIDDYVEILTQKRKYYHGPGNEKSRTDYGRFRTDG
TTLENIFGILIGKCNFYPDEYRASKASYTAQEYNFLNDLNNLKVP
TETGKLPTEQKESLVEFAKNTATLGPSKLLKEIAKILDCKVDEIK
GYREDDKGKPDLHTFEPYRKLKFNLESINIDDLSREVIDKLADIL
TLNTEREGIEDAIKRNLPNQFTEEQISEIIKVRKSQSTAFNKGWH
SFSAKLMNELIPELYATSDEQMTILTRLEKFKVNKKSSKNTKTID
EKEVTDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDK
NADDEKKFIDKRNKENKKEKDDALKRAAYLYNSSDKLPDEVFHGN
KQLETKIRLWYQQGERCLYSGKPIPIHDLVHNSNNFEIDHILPLS
LSFDDSLANKVLVYDWANQEKGQKTPYQVIDSMDAAWSFREMKDY
VLKQKGLGKKKRDYLLTTENIDKIEVKKKFIERNLIDTRYASRVV
LNSLQSALRELCKDTKVSVIRGQFTSQLRRKWKIDKSRETYHHHA
VDALIIAASSQLKLWEKQDNLMFIDYGNNQVVDKETGEILSVSDD
EYKELVFQPPYQGFVNTISSKGFEDEILFSYQVDSKYNRKVSDAT
IYSTRKAKIGKDKKEETYVLGKIKDIYSQNGFDTFIKKYNKDKTQ
FLMYQKDPLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRR
ENGLICKYSKKGKGTPIKSLKYYDKKLGNCIDITPEESRNKVILQ
SINPWRADVYFNPETLKYELMGLKYSDLSFEKGTGKYHISQEKYD
AIKEKEGIGKKSEFKFTLYRNDLILIKDTASGEQEIYRFLSRTMP
NVNHYAELKPYDKEKFDGGQELMEVFGKVANGGQCLKSLNKSNIS
IYKVRTDVLGNKYFVKKEGDKPKLNFKNN KKKRPAATKKAGQAKK
KK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA .
(m)
(SEQ ID NO: 41)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MKRVNEDYILGL A IGTNSC
GWAVTDKKNNLLKLRGKTAIGSHLFEEGHTAADRRGFRTTRRRLK
RRKWRLRLLEEIFAEPMAKVDPGFFVRLHQSWVSPLDKDRKKYNA
IVFPTAKEDQAFYKHYATIYHLRDELMTQDRQFDLREIFLAIHHI
VKYRGNFLQDTPVKDFEASKIEVGPILSHINNAFAEKIVEDQDPI
ELNVANAADIEDVIRGKDAEKTVYKLDKVKKIAKLLTDSTAKEEK
NVAKQIANAIMGYKTQFETILDKEIDKSDKAQWEFKLSDADADDK
LDALLPDLDETDQTVVAEIEKLESAITLSTIVDENKSLSQSMVEK
YKKHKKDYKKLKKYINTLQDQTKAKKLLLAYDLYVNNRHGRLLEA
KKTFEDKKKKKALTKDEFYKIVKDNLDDSDLAHEIQQEIAADNFM
PKQRTNSNGVIPFQLHQIELDKIIANQGKYYPFLAAENPVEDHRK
QAPYKLDELVRFRVPYYVGPMITADEQEKTSGKSFAWMVRKEDGQ
ITPWNFEQKVDRQESANKFIKRMTIKDTYLLSEDVLPANSLLYQR
FEVLNELNNIRINGSRISVDLKQQIFNDLFEEKKTVTEKSLTSYL
KQNLHLPTVEIKGLADPTKFNSSLASYYHLKSLHVFDKELADPQY
QKDFEKIIEYSSIFEDKKIFQDKLHAEFKWLTPEQFKAISTWRLQ
GWGRLSRKLLVELHDTNGQNIMEQLWDSQKNFMQIVTEPDFKDAI
AKENQNVTRANGVEEILADAYTSPANKKAIRQVVKVVADVVKAAG
GKKPAQFAIEFTRDPDKNPQLSHIRGTKLLKAYQETAGELVDQKL
TDSLKEAMTSRKLLKDKYFLYFMQAGRDAYTGQKINIDEVSTNYQ
IDHILPQSFIKDDSFDNRVLTATPLNAEKSDDVPYKRFANNYVSD
MKMTVGEMWKHWQKAGIINKHKLGNLLLDPDRLNKFQKSGFINRQ
LVETSQIIKLVSVILQNKYPDAEIITVKAGDNSALRQRLNLYKSR
DVNDYHHAIDAYLSIICGNFLYQVYPKYRPYFVYGKYKKFSQDPD
LQKEVIKHFKGFTFMWPLLQKDNSERKAPEKIKENNSDRIVFYKH
PDIFDKLRKAYNYKYMLVSRETTTENSGLFDVTIYPRGERDLAKT
RKLIPKSNGLDPKIYGGYSGNTDAYMVIVKIDKGKESIYKVIGVP
MRALASLNRAKKQGNYKEELHQVLEPQIMFDKNGKPKRSVKGFRI
IKDHVPFKQVVLDGDKKFMLNSSTYEINAKQLTLTPETMRIVTDN
LKKGEDQDQLLVKAYDEILQKVDQYLPLFDVNKFRNSLHLGRAKF
LDLAVNDKKITLTNILNGLHDNLVTPDLKNIGIKTPLGKLQVPSG
IVLSSEAILIFQSPTGLFEKRVRIADL KRPAATKKAGQAKKKK GS
YPYDVPDYAYPYDVPDYAYPYDVPDYA .
(n)
(SEQ ID NO: 42)
M PAAKRVKLD GS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLV
LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL
YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG
MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD GSS
GSETPGTSESATPESSG PKKKRKV GT MSNGKILGL A IGIASVGVG
VIDAQTGEIIHASSRIFPSANAANNAERRTFRGSRRLIRRKKHRI
KRLDDLFNDFHINLDGEMSTDNPYVLRVKGLSQKLTVEELYISIK
NIMKRRGISYLDDAESDNEAGRSDYAKAIERNRQLLTSKTPGEIQ
LERLEKYGQLRGNFTIIDEEGQSQQIINVESTSDYVKEVEKILDC
QKMYHKFISDEFCDKLIELLREKRKYYVGPGNEKSRTDYGIYRTD
GTTLENLFGILIGKCTFYPDQYRSSRASYTAQEFNFLNDLNNLTV
PTETKKLSQEQKEFLVNYAKETSVLGAGKILQQIAKLADCKVEDI
RGYRLDNKDKPELHTFETYRAMKGLVPLVDIGVLSREQLDILADI
LTLNTDFEGIREALKKQLPNVFDEKQVKGLASFRKSKSQLFAKGW
HNLSQKIMLEVIPELYATSDEQMTILTRLGKFEKSSVAEYPSSIN
VDEITDEIYNPVVAKSIRQTIKIINASIKKWDEFDQIVIEMPRDR
NEDEEKKRIADGQKANAKEKADSILRAAELYCAGKVLPDYVYNGH
NQLATKIRLWYQQGERCIYTGQPISIHDLIHNQNQYEIDHILPLS
LTEDDSLSNKVLVLATANQEKAQRTPYNYLKSATSAWSYREFKDY
VTKRKGIGKKKCEYLTFEEDINGFEVRSKFIQRNLVDTRYASKVI
LNALQDYFKISGIQTKVSVVRGQFTSQLRHKWGIEKTRETYHHHA
VDALIIAASSQLRLWKKQESPLVVDYQEGRQVDLETGEILELTDE
QYKELVYQPPYQGFVNTISSSAFDNEILFSYQVDSKVNRKISDAT
IYATRNAQLGKDKTEGIYVLGKIKDIYTQAGYEAFLKRYTKDKTS
FLMYHKDLDTWEKVIEIILRDYREYDEKGKEIGNPFERYRRENGY
VKKYSRKGNGTAIKSLKYYDNKLGNHIDITPENSRNAVVLQSLKP
WRTDVYFNKETGKYEFLGIKYSDLSFEKGTGEYGISQEKYDSIKI
AEGVAKKSIFKFTLYKQDLLFIKDIENNFGKLLRFTSKNDTSKHY
VELKPYDKNKFGTEEPLLPVLGNVAKSGQCIKGLNKSNISIYKVR
TDILGYRHFIKQEGEHPQLKF KKKRPAATKKAGQAKKKK GS YPYD
VPDYAYPYDVPDYAYPYDVPDYA .
40 . The Cas9 protein of claim 38 , wherein the Cas9 protein is fused to a cytosine deaminase.
41 . The Cas9 protein of claim 2 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NRGNR-3′.
42 . The Cas9 protein of claim 3 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NRG-3′, wherein H is adenine, cytosine, or thymine.
43 . The Cas9 protein of claim 4, 11 or 12 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NNGR-3′, wherein H is adenine, cytosine or thymine, and R is adenine or guanine.
44 . The Cas9 protein of claim 5 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NNGRRT-3′.
45 . The Cas9 protein of claim 6 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NNAAAA-3′.
46 . The Cas9 protein of claim 7 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NGGNG-3′.
47 . The Cas9 protein of claim 8 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NNGNRG-3′.
48 . The Cas9 protein of claim 9 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NNAAAC-3′.
49 . The Cas9 protein of claim 10 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NNRAAG-3′.
50 . The Cas9 protein of claim 13 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NNAYAA-3′.
51 . The Cas9 protein of claim 13 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NNGAAA-3′.
52 . The Cas9 protein of claim 13 , wherein the Cas9 protein recognizes a PAM sequence comprising 5′-NNAAA-3′. (H=A, C or T; R=A or G).
53 . A nucleic acid encoding the Cas9 protein of any one of the preceding claims .
54 . The nucleic acid of claim 53 , wherein the nucleic acid is codon-optimized for expression in mammalian cells.
55 . The nucleic acid of claim 54 , wherein the nucleic acid is codon-optimized for expression in human cells.
56 . A eukaryotic cell comprising the Cas9 protein of any one of claims 1-52 .
57 . The eukaryotic cell of claim 55 , wherein the cell is a human cell.
58 . A method of cleaving a target nucleic acid in a eukaryotic cell comprising:
contacting the cell with a Cas9 of any one of claims 1-52 , 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.
59 . 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 1-52 , 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.
60 . 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 1-52 , 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.
61 . A method of modifying a target nucleic acid in a eukaryotic cell comprising:
contacting the cell with a Cas9 of any one of claims 1-52 , 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.
62 . The method of claim 60 or 61 , wherein the Cas9 protein is an inactive Cas9 (dCas9).
63 . The method of claim 62 , wherein the dCas9 is fused to a deaminase.
64 . The method of any one of claims 58-63 , wherein the RNA guide comprises a crRNA and a tracrRNA.
65 . The method of any one of claims 58-64 , wherein the RNA guide comprises a sgRNA.
66 . The method of claim 65 , wherein the sgRNA for use with Seq2Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGUACUCUGUAAUUUUGAAAAAAAUUACAGAAUCUACUAAAACAAGG CAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGACGAGAUUUUUUU-3′ (SEQ ID NO: 43).
67 . The method of claim 65 , wherein the sgRNA for use with EhiCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGAGCUAUGUUGGAAACAACAUAGCGAGUUAAAAUAAGGCAUUGUCC GUUAUCAGCUUUUAAAGCAAGCACUGUCUCGGUGCUUUUUU-3′ (SEQ ID NO:
44).
68 . The method of claim 65 , wherein the sgRNA for use with SeqCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAGCCUACAAAGAUAAG GCUUCAUGCCGAAUUCAAGCACCCCAUGUUUACAUGGGGUGCUUUU-3′ (SEQ ID NO: 45).
69 . The method of claim 65 , wherein the sgRNA for use with SsiCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGUACUCUGUAAUUUUGAAAAAAAUUACAGAAUCUACUAAAACAAGG CAAAAUGCCGUGUUUAUCUCGUCAACUUGUUUGGCGAGAUUUUUUUU-3′ (SEQ ID NO: 46).
70 . The method of claim 65 , wherein the sgRNA for use with SinCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAG GCUUCAUGCCGAAAUCAACACCCUGUCUAUGACGGGGUGUUUU-3′ (SEQ ID NO: 47).
71 . The method of claim 65 , wherein the sgRNA for use with SsaCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGAGCUGUGUUGUGAAAACAACACAGCAAGUUAAAAUAAGGCUUUGU 15 CCGUACACAACUUGAAAAAGUGCGCACCGAUUCGGUGCUUUUUU-3′ (SEQ ID NO: 48).
72 . The method of claim 65 , wherein the sgRNA for use with Ssc2Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAG GCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUGUUUU-3′ (SEQ ID NO: 49).
73 . The method of claim 65 , wherein the sgRNA for use with Sor2Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAG GCUUCAUGCCGAAUUCAACACCCUGUCAUUUAUGGCGGGGUGUUUU-3′ (SEQ ID NO: 50).
74 . The method of claim 65 , wherein the sgRNA for use with SorCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGUAGAAGCUACAAAGAUAAG GCUUCAUGCCGAAUUCAACACCCUGUCAUUUAUGGCGGGGUGUUUCGUUUU-3′ (SEQ ID NO: 51).
75 . The method of claim 65 , wherein the sgRNA for use with SwaCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGUACUCUGUAAUUUUGAAAAAAAUUACAGAAUCUACUGAAACAAGA CUAUAUGUCGUGUUUAUCCCACUAAUUUAUUAGUGGGAUUUUUU-3′ (SEQ ID NO: 52).
76 . The method of claim 65 , wherein the sgRNA for use with SscCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGUACUCUGUAAUUUUGAAAAAAAUUACAGAAUCUACUAAAACAAGG CAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGACGAGAUUUUUUU-3′ (SEQ ID NO: 53).
77 . The method of claim 65 , wherein the sgRNA for use with SgaCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCUGAGCCUACAAAGAUAAG GCUUUAUGCCGAAUUCAAGCACCCCAUGUUUUGACAUGGGGUGCUUUU-3′ (SEQ ID NO: 54).
78 . The method of claim 65 , wherein the sgRNA for use with LkuCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGAUGAUUGUUAGAUCGAAAGAUCUAACAACCAGAUUUUAAAAUCAA ACAAUGUAUCUUUGAUACUAAGUUUCAACGCGGUAUUAUUACCGUCCUGCCUC AGCUCUAUAGCGGAGGUUUUUU-3′ (SEQ ID NO: 55).
79 . The method of claim 65 , wherein the sgRNA for use with SsuCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAGCCUACAAAGAUAAG GCUUUAUGCCGAAAUCAAGCACCCCGUUUCGUACGGGGUGCUUUU-3′ (SEQ ID NO: 56).
80 . The method of claim 64 , wherein the crRNA comprises a guide sequence of between about 16 and 26 nucleotides long.
81 . The method of claim 64 , wherein the crRNA comprises a guide sequence between 18 and 24 nucleotides long.
82 . The method of claim 80 or 81 , wherein the break in the target nucleic acid is a single-stranded or double-stranded break.
83 . The method of claim 82 , wherein the break in the target nucleic acid is a single-stranded break.
84 . The method of claim 60 or 61 , 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.
85 . The method of any one of claims 58-84 , wherein the target nucleic acid is 5′ to a protospacer adjacent motif (PAM) sequence.
86 . The method of any one of claims 58-85 , 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.
87 . The method of claim 86 , wherein the eukaryotic cell is a human cell.
88 . The method of claim 86 , wherein the promoter sequence is a eukaryotic or viral promoter.
89 . 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 CRISPR-associated (Cas) protein having at least 80% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, 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.
90 . 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 CRISPR-associated (Cas) protein having at least 80% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14;
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.
91 . The system of claim 90 , wherein the Cas9 protein is an inactive Cas9 (dCas9).
92 . The system of claim of any one of claims 90-91 , wherein the RNA guide comprises a crRNA and a tracrRNA.
93 . The system of any one of claims 90-92 , wherein the RNA guide comprises an sgRNA.
94 . The system of claim 93 , wherein the sgRNA for use with Seq2Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGUACUCUGUAAUUUUGAAAAAAAUUACAGAAUCUACUAAAACAAGG CAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGACGAGAUUUUUUU-3′ (SEQ ID NO: 43).
95 . The system of claim 93 , the sgRNA for use with EhiCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGAGCUAUGUUGGAAACAACAUAGCGAGUUAAAAUAAGGCAUUGUCC GUUAUCAGCUUUUAAAGCAAGCACUGUCUCGGUGCUUUUUU-3′ (SEQ ID NO: 44).
96 . The system of claim 93 , wherein the sgRNA for use with SeqCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAGCCUACAAAGAUAAG GCUUCAUGCCGAAUUCAAGCACCCCAUGUUUACAUGGGGUGCUUUU-3′ (SEQ ID NO: 45).
97 . The system of claim 93 , wherein the sgRNA for use with SsiCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGUACUCUGUAAUUUUGAAAAAAAUUACAGAAUCUACUAAAACAAGG CAAAAUGCCGUGUUUAUCUCGUCAACUUGUUUGGCGAGAUUUUUUUU-3′ (SEQ ID NO: 46).
98 . The system of claim 93 , wherein the sgRNA for use with SinCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAG GCUUCAUGCCGAAAUCAACACCCUGUCUAUGACGGGGUGUUUU-3′ (SEQ ID NO: 47).
99 . The system of claim 93 , wherein the sgRNA for use with SsaCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGAGCUGUGUUGUGAAAACAACACAGCAAGUUAAAAUAAGGCUUUGU 20 CCGUACACAACUUGAAAAAGUGCGCACCGAUUCGGUGCUUUUUU-3′ (SEQ ID NO: 48).
100 . The system of claim 93 , wherein the sgRNA for use with Sor2Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAG GCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUGUUUU-3′ (SEQ ID NO: 49).
101 . The system of claim 93 , wherein the sgRNA for use with Ssc2Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAG GCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUGUUUU-3′ (SEQ ID NO: 50).
102 . The system of claim 93 , wherein the sgRNA for use with Sor2Cas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAG 5 GCUUCAUGCCGAAUUCAACACCCUGUCAUUUAUGGCGGGGUGUUUU-3′ (SEQ ID NO: 51).
103 . The system of claim 93 , wherein the sgRNA for use with SwaCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGUACUCUGUAAUUUUGAAAAAAAUUACAGAAUCUACUGAAACAAGA CUAUAUGUCGUGUUUAUCCCACUAAUUUAUUAGUGGGAUUUUUU-3′ (SEQ ID NO: 52).
104 . The system of claim 93 , wherein the sgRNA for use with SscCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGUACUCUGUAAUUUUGAAAAAAAUUACAGAAUCUACUAAAACAAGG CAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGACGAGAUUUUUUU-3′ (SEQ ID NO: 53).
105 . The system of claim 93 , wherein the sgRNA for use with SgaCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCUGAGCCUACAAAGAUAAG GCUUUAUGCCGAAUUCAAGCACCCCAUGUUUUGACAUGGGGUGCUUUU-3′ (SEQ ID NO: 54).
106 . The system of claim 93 , wherein the sgRNA for use with LkuCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUAGAUGAUUGUUAGAUCGAAAGAUCUAACAACCAGAUUUUAAAAUCAA ACAAUGUAUCUUUGAUACUAAGUUUCAACGCGGUAUUAUUACCGUCCUGCCUC AGCUCUAUAGCGGAGGUUUUUU-3′ (SEQ ID NO: 55).
107 . The system of claim 93 , wherein the sgRNA for use with SsuCas9 comprises a scaffold comprising a sequence having at least about 80% identity to 5′-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAGCCUACAAAGAUAAG GCUUUAUGCCGAAAUCAAGCACCCCGUUUCGUACGGGGUGCUUUU-3′ (SEQ ID NO: 56).
108 . The system of any one of claims 90-107 , wherein the nucleic acid encoding 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.
109 . The system of claim 108 , wherein the eukaryotic cell is a human cell.
110 . The system of claim 108 , wherein the promoter sequence is a eukaryotic promoter sequence.
111 . A nucleic acid encoding the system of any one of claims 90-110 .
112 . A vector comprising the system of any one of claims 90-110 .
113 . The vector of claim 112 , wherein the vector is a plasmid vector or a viral vector.
114 . The vector of claim 113 , wherein the viral vector is an adeno associated virus (AAV) vector or a lentiviral vector.
115 . The vector of claim 114 , wherein the viral vector is an AAV vector.
116 . The vector of claim 115 , wherein more than one AAV vector is used for packaging the system of claims 90-110 .
118 . 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 90-111 .
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.
119 . The method of claim 118 , wherein the guide RNA is complementary to about 18-24 nucleotides.
120 . The method of claim 118 , wherein the guide RNA is complementary to 20 nucleotides.
121 . A base editor comprising the fusion protein of any one of claims 37-40 .
122 . The base editor of claim 118 comprising an adenosine deaminase domain or a cytidine deaminase domain.
123 . A method of editing a nucleobase of a polynucleotide, the method comprising contacting the polynucleotide with the base editor of claim 118 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.
124 . A method of editing a nucleobase of a polynucleotide, the method comprising contacting the polynucleotide with the base editor of claim 118 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.
125 . The method of claim 123 or 124 , wherein the editing results in less than 50% indel formation in the target polynucleotide sequence.
126 . The method of any one of claims 123-125 , wherein the editing generates a point mutation.