New CRISPR-Cas12i system and its applications

Abstract

Provided herein are Cas12i polypeptides, fusion proteins comprising such Cas12i polypeptides, CRISPR-Cas12i systems comprising such Cas12i polypeptides or fusion proteins, and methods of using the same.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to Chinese Patent Application No. 202111289092.6, proposed on November 2, 2021, entitled "NOVEL CRISPR-CAS12I SYSTEMS," Chinese Patent Application No. 202210081981.1, proposed on January 24, 2022, entitled "NOVEL CRISPR-CAS12I SYSTEMS," and PCT Patent Application No. PCT / CN2022 / 089074, proposed on April 25, 2022, entitled "NOVEL CRISPR-CAS12I SYSTEMS," the entire contents of which (including any sequence listings and drawings) are incorporated herein by reference in their entirety. Use of electronic sequence listing

[0002] The contents of the electronic sequence listing ("xxx.xml", xxx bytes in size and created on xxx) are incorporated herein by reference in their entirety. If the sequence is an RNA sequence, T in the sequence should be considered as U. [Technical field]

[0003] The present disclosure relates generally to Cas12i polypeptides, fusion proteins comprising such Cas12i polypeptides, CRISPR-Cas12i systems comprising such Cas12i polypeptides or fusion proteins, and methods of use thereof. [Background technology]

[0004] Clustered regularly interspaced short repeats-Cas (CRISPR-Cas) systems, including type II Cas9 and type V Cas12 systems, play a role in adaptive immunity against viruses in prokaryotes, have already been developed as genome editing tools. 1-3Compared with the type II system, the type V system, which includes VA to VK, shows more functional diversity. 4,5 Here, compared to SpCas9 and Cas12a, Cas12i has a relatively small size (1033-1093aa) and a preference for the 5'-TTN protospacer adjacent motif (PAM). 4,6,7 A feature of Cas12i is that it can autonomously process precursor crRNA (pre-crRNA) to form short mature crRNA. Cas12i mediates the cleavage of dsDNA with a single RuvC domain by first cleaving the non-target strand and then cleaving the target strand. 8-10 These unique features of Cas12i can realize multiplexed high-fidelity genome editing. However, previous Cas12i (Cas12i1 and Cas12i2) showed low editing efficiency, which limited their effectiveness in therapeutic gene editing. Therefore, it is necessary to develop a more efficient CRISPR-Cas12i system for practical application.

[0005] Citation or identification of any document in this application is not an admission that such document is obtained as prior art to the present disclosure. Summary of the Invention

[0006] To address the limitations of previous Cas12i, applicants screened 10 Cas12i and found that one xCas12i (also referred to herein as "SiCas12i") had robust and high activity in HEK293T cells. Engineering xCas12i with arginine substitutions in the PAM-interacting (PI) domain, REC domain and RuvC domain produces a mutant high-fidelity Cas12Max (hfCas12Max) with significantly elevated editing activity and minimal off-target cleavage efficiency. Applicants also evaluated the base editing efficiency of xCas12i-based base editors, thus expanding the genome editing toolbox. Applicants further demonstrated that hfCas12Max can be an effective genome editing tool ex vivo and in vivo via ribonucleoproteins (RNPs) and lipid nanoliposomes (LNPs), respectively, demonstrating its excellent potential for therapeutic genome editing applications.

[0007] According to some aspects, the present disclosure provides a Cas12i polypeptide, the Cas12i polypeptide comprising: (1) As shown in any one of SEQ ID NO: 1-3, 6 and 10; (2) SEQ ID NO: Contains any one of the amino acid sequences of 1-3, 6, and 10; or (3) An amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to any one of the amino acid sequences of SEQ ID NOs: 1-3, 6, and 10.

[0008] According to some aspects, the disclosure provides a Cas12i polypeptide, the Cas12i polypeptide comprising an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%) but less than 100% sequence identity to the amino acid sequence of a reference Cas12i polypeptide of any one of SEQ ID NOs: 1-3, 6 and 10; Optionally, wherein the Cas12i polypeptide has a function of the reference Cas12i polypeptide (e.g., a modified function that is increased or decreased compared to the function of the reference Cas12i polypeptide). (for example, (a) the ability to form a complex with a guide RNA that is capable of forming a complex with the reference Cas12i polypeptide; and / or (b) Spacer sequence-specific dsDNA cleavage activity.

[0009] In some embodiments, when both are used in combination with the same guide RNA, SEQ ID Compared to any one of the reference Cas12i polypeptides of NOs:1-3, 6, and 10, the Cas12i polypeptide has increased spacer sequence-specific dsDNA and / or ssDNA cleavage activity, for example, by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or more.

[0010] In some embodiments, the Cas12i polypeptide has reduced spacer sequence-specific dsDNA and / or ssDNA cleavage activity, e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, reduced, compared to the reference Cas12i polypeptide of any one of SEQ ID NOs: 1-3, 6 and 10 when both are used in combination with the same guide RNA.

[0011] In some embodiments, the Cas12i polypeptide is a non-Cas12i polypeptide that has essentially no spacer sequence-specific dsDNA and / or ssDNA cleavage activity, e.g., at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the spacer sequence-specific dsDNA and / or ssDNA cleavage activity of the reference Cas12i polypeptide of any one of SEQ ID NOs:1-3, 6 and 10.

[0012] In some embodiments, the Cas12i polypeptide comprises a substitution selected from D650A, D700A, E875A, and D1049A of SEQ ID NO:1, or a combination thereof.

[0013] In some embodiments, the Cas12i polypeptide is a Cas12i nickase having spacer sequence-specific ssDNA cleavage activity.

[0014] In some embodiments, the Cas12i polypeptide is a Cas12i nickase that has spacer sequence-specific ssDNA cleavage activity against the target strand of a target dsDNA.

[0015] In some embodiments, the Cas12i polypeptide has spacer sequence-specific ssDNA cleavage activity against a target strand of a target dsDNA and essentially no spacer sequence-specific dsDNA cleavage activity, e.g., a Cas12i nickase having at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the spacer sequence-specific dsDNA cleavage activity of the reference Cas12i polypeptide of any one of SEQ ID NOs:1-3, 6 and 10.

[0016] In some embodiments, the Cas12i polypeptide comprises a substitution selected from a mutant of SEQ ID NO:1 in Tables 11-14 or a combination thereof.

[0017] In some embodiments, the Cas12i polypeptide is not any of SEQ ID NOs: 1-3, 6 and 10.

[0018] In some embodiments, the Cas12i polypeptide has reduced spacer sequence-independent (off-target) dsDNA and / or ssDNA cleavage activity, e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, reduced, compared to the reference Cas12i polypeptide of any one of SEQ ID NOs: 1-3, 6 and 10 when both are used in combination with the same guide RNA.

[0019] In some embodiments, the Cas12i polypeptide comprises one or more mutations (e.g., insertions, deletions, or substitutions) in one or more amino acids that correspond to one or more amino acids in the amino acid sequence of the reference Cas12i polypeptide of any one of SEQ ID NOs: 1-3, 6, and 10.

[0020] In some embodiments, the one or more mutations are within the PI domain, the REC-I domain, and / or the RuvC-II domain corresponding to the reference Cas12i polypeptide of any one of SEQ ID NOs: 1-3, 6, and 10.

[0021] In some embodiments, the one or more mutations are in the PI domain at positions 173-291, the REC-I domain at positions 427-473, and / or the RuvC-II domain at positions 800-1082 of the reference Cas12i polypeptide of SEQ ID NO:1.

[0022] In some embodiments, the Cas12i polypeptide comprises one or more mutations (e.g., insertions, deletions, or substitutions) in one or more amino acids, the one or more amino acids being identical to or different from the amino acid sequence of the reference Cas12i polypeptide of any one of SEQ ID NOs: 1-3, 6, and 10. Any one of positions from position 1 to the end of any one of the reference Cas12i polypeptides of SEQ ID NOs: 1-3, 6, and 10, e.g., 1080, e.g., positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112 3, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 10 3, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 , 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 , 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196 , 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227 , 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、773、774、775、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、791、792、793、794、795、796、797、798、799、800、801、802、803、804、805、806、807、808、809、810、811、812、813、814、815、816、817、818、819、820、821、822、823、824、825、826、827、828、829、830、831、832、833、834、835、836、837、838、839、840、841、842、843、844、845、846、847、848、849、850、851、852、853、854、855、856、857、858、859、860、861、862、863、864、865、866、867、868、869、870、871、872、873、874、875、876、877、878、879、880、881、882、883、884、885、886、887、888、889、890、891、892、893、894、895、896、897、898、899、900、901、902、903、904、905、906、907、908、909、910、911、912、913、914、915、916、917、918、919、920、921、922、923、924、925、926、927、928、929、930、931、932、933、934、935、936、937、938、939、940、941、942、943、944、945、946、947、948、949、950、951、952、953、954、955、956、957、958、959、960、961、962、963、964、965、966、967、968、969、970、971、972、973、974、975、976、977、978、979、980、981、982、983、984、985、986、987、988、989、990、991、992、993、994、995、996、997、998、999、1000、1001、1002、1003、1004、1005、1006、1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1 047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080.

[0023] In some embodiments, the Cas12i polypeptide comprises one or more mutations (e.g., insertions, deletions, or substitutions) in one or more amino acids, the one or more amino acids being similar to or different from the amino acid sequence of the reference Cas12i polypeptide of SEQ ID NO:1. Any one of positions 1 to 1080, e.g. positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 , 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 1 77, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 20 8, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 , 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、773、774、775、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、791、792、793、794、795、796、797、798、799、800、801、802、803、804、805、806、807、808、809、810、811、812、813、814、815、816、817、818、819、820、821、822、823、824、825、826、827、828、829、830、831、832、833、834、835、836、837、838、839、840、841、842、843、844、845、846、847、848、849、850、851、852、853、854、855、856、857、858、859、860、861、862、863、864、865、866、867、868、869、870、871、872、873、874、875、876、877、878、879、880、881、882、883、884、885、886、887、888、889、890、891、892、893、894、895、896、897、898、899、900、901、902、903、904、905、906、907、908、909、910、911、912、913、914、915、916、917、918、919、920、921、922、923、924、925、926、927、928、929、930、931、932、933、934、935、936、937、938、939、940、941、942、943、944、945、946、947、948、949、950、951、952、953、954、955、956、957、958、959、960、961、962、963、964、965、966、967、968、969、970、971、972、973、974、975、976、977、978、979、980、981、982、983、984、985、986、987、988、989、990、991、992、993、994、995、996、997、998、999、1000、1001、1002、1003、1004、1005、1006、1007、1008、1009、1010、1011、1012、1013、1014、1015、1016、1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1 052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080.

[0024] In some embodiments, the Cas12i polypeptide comprises one or more mutations (e.g., insertions, deletions, or substitutions) in one or more amino acids, the one or more amino acids being similar to or different from the amino acid sequence of the reference Cas12i polypeptide of SEQ ID NO:1. K109、N110、Y111、L112、M113、S114、N115、I116、D117、S118、D119、F121、V122、W123、V124、D125、C126、127、K128、F129、A130、K131、D132、F133、A134、Y135、Q136、M137、E138、L139、G140、F141、H142、E143、F144、T145、V146、L147、A148、E149、T150、L151、L152、A153、N154、S155、I156、L157、V158、L159、N160、E161、S162、T163、K164、A165、N166、W167、A168、W169、G170、T171、V172、S173、A174、L175、Y176、G177、G178、G179、D180、K181、E182、D183、S184、T185、L186、K187、S188、K189、I190、L191、L192、A193、F194、V195、D196、A197、L198、N199、N200、H201、E202、L203、K204、T205、K206、E208、I209、L210、N211、Q212、V213、C214、E215、S216、L217、K218、Y219、Q220、S221、Y222、Q223、D224、M225、Y226、V227、D228、F229、S231、V232、V233、D234、E235、N236、G237、N238、K239、K240、S241、P242、N243、G244、S245、M246、P247、I248、V249、T250、K251、F252、E253、T254、D255、D256、L257、I258、S259、D260、N261、Q262、K264、A265、M266、I267、S268、N269、F270、T271、K272、N273、A274、A275、A276、K277、A278、A279、K280、K281、P282、I283、P284、Y285、L286、D287、288、L289、K290、E291、M293、V294、S295、L296、C297、D298、Y300、N301、V302、Y303、A304、W305、A306、A307、A308、I309、T310、N311、S312、N313、A314、<h2 style=";text-align:left;direction:ltr">D315, V316, T317, A318, N320, T321, L324, T325, F326, I327, G328, E329, Q330, N331, S332, K335, E336, L337, S338, V339, L340, Q341, T342, T343, T344 N345, E346, K347, A348, K349, D350, I351, L352, N353, K354, N356, D357, N358, L359, I360, Q361, E362, V363, Y365, T366, P367, A368, K370, H371, L372 G373, D375, L376, A377, N378, L379, F380, D381, T382, L383, K384, E385, K386, D387, I388, N389, N390, I391, E392, N393, E394, E395, E396, K397, Q398 N399、V400、I401、N402、D403、C404、I405、E406、Q407、Y408、V409、D410、D4 11、C412、L415、N416、N418、P419、I420、A421、A422、L423、L424、K425、H426、 I427、S428、Y430、Y431、E432、D433、F434、S435、A436、K437、N438、F439、L4 40、D441、G442、A443、K444、L445、N446、V447、L448、T449、E450、V451、V452、 N453、Q455、K456、A457、H458、P459、T460、I461、W462、S463、E464、I800、S8 01、L802、K803、M804、I805、S806、D807、F808、K809、G810、V811、V812、Q813、 S814、Y815、F816、S817、V818、S819、G820、C821、V822、D823、D824、A825、S8 26、K827、K828、A829、H830、D831、S832、M833、L834、F835、T836、F837、M838、 C839、A840、A841、E842、E843、K844、T846、N847、K848、E850、E851、K852、T8 53、N854、A856、A857、S858、F859、I860、L861、Q862、K863、A864、Y865、L866、<h2 style=";text-align:left;direction:ltr">H867、G868、C869、K870、M871、I872、V873、C874、E875、D876、D877、L878、P8 79、V880、A881、D882、G883、K884、T885、G886、K887、A888、Q889、N890、A891、 D892, M894, D895, W896, C897, A898, A900, L901, A902, K903, K904, V905, N906, D907, G908, C909, V910, A911, M912, S913, I914, C915, Y916, A918, P920 A921, Y922, M923, S924, S925, H926, Q927, D928, P929, F930, V931, H932, M933, Q934, D935, K936, K937, T938, S939, V940, L941, P943, F945, M946, E947 V948, N949, K950, D951, S952, I953, D955, Y956, H957, V958, A959, G960, L961, L965, N966, S967, K968, S969, D970, A971, G972, T973, S974, V975, Y976 Y977, Q979, A980, A981, L982, H983, F984, C985, E986, A987, L988, G989, V990, S991, P992, E993, L994, V995, K996, N997, K998, K999, T1000, H1001, A10 02、A1003、E1004、L1005、G1006、M1009、G1010、S1011、A1012、M1013、L1014 、M1015、P1016、W1017、G1019、G1020、V1022、Y1023、I1024、A1025、S1026、K1 027、K1028、L1029、T1030、S1031、D1032、A1033、K1034、S1035、V1036、K103 7、Y1038、C1039、G1040、E1041、D1042、M1043、W1044、Q1045、Y1046、H1047、A 1048、D1049、E1050、I1051、A1052、A1053、V1054、N1055、I1056、A1057、M10 58、Y1059、E1060、V1061、C1062、C1063、Q1064、T1065、G1066、A1067、F1068、Corresponding to one or more amino acids at one or more of positions G1069, K1070, K1071, Q1072, K1073, K1074, S1075, D1076, E1077, L1078, P1079 and G1080.

[0025] In some embodiments, the Cas12i polypeptide comprises one or more mutations (e.g., insertions, deletions, or substitutions) in one or more amino acids, the one or more amino acids being similar to or different from the amino acid sequence of the reference Cas12i polypeptide of SEQ ID NO:1. <h2 style=";text-align:left;direction:ltr">S118、D119、F121、W123、Q136、E138、E143、V146、S155、V158、E161、S162、 T163、A165、N166、G178、D180、T185、K189、A193、D196、N199、N200、E202、L 203, S221, V233, E235, N236, S241, N243, S245, K251, D255, L257, N273, D287, S295, V302, S332, E336, S338, V339, E362, D375, A377, N378, D381, T3 82、E385、D387、N390、E395、E396、Q398、N399、V400、D403、E406、Q407、V4 09、D411、C412、N416、N418、L440、L448、V451、Q455、E464、S806、S817、V81 8、S819、S832、M833、F835、T836、F837、C839、A840、E842、E843、K844、T846 、N847、K848、N854、A856、S858、Q862、K863、Y865、L866、G868、K870、M871、 D876、D877、V880、G883、K884、G886、K887、A888、A891、D892、M894、A900、 K903、K904、N906、V910、M912、S913、C915、Y916、A918、M923、S925、H926、Q 927、V931、M933、Q934、D935、K936、K937、T938、S939、V940、F945、M946、V 948、N949、K950、D951、S952、D955、Y956、A959、G960、N966、S967、K968、S9 69, D970, A971, G972, S974, V975, Y976, Q979, A980, L982, H983, C985, E986, A987, G989, V990, S991, P992, E993, L994, V995, K996, N997, K998, K99 9、T1000、H1001、A1002、A1003、E1004、G1006、G1010、A1012、M1013、L1014 、W1017、V1022、K1028、D1032、K1034、K1037、C1039、G1040、Q1045、H1047、Corresponding to one or more amino acids at one or more positions of C1063 and G1069.

[0026] In some embodiments, the Cas12i polypeptide comprises one or more mutations (e.g., insertions, deletions, or substitutions) in one or more amino acids, the one or more amino acids being similar to or different from the amino acid sequence of the reference Cas12i polypeptide of SEQ ID NO:1. It corresponds to one or more amino acids at one or more of positions N243, E336, V880, G883, D892 and M923.

[0027] In some embodiments, the one or more mutations are substituted with R.

[0028] In some embodiments, the Cas12i polypeptide further comprises one or more mutations (e.g., insertions, deletions, or substitutions) in one or more amino acids, wherein the one or more amino acids are identical to or different from the amino acid sequence of the reference Cas12i polypeptide of SEQ ID NO:1. It corresponds to one or more amino acids at one or more of positions V880, G883, D892 and M923.

[0029] In some embodiments, the one or more mutations are substituted with R.

[0030] In some embodiments, the Cas12i polypeptide comprises one or more mutations (e.g., insertions, deletions, or substitutions) in one or more amino acids, the one or more amino acids being similar to or different from the amino acid sequence of the reference Cas12i polypeptide of SEQ ID NO:1. K109, L112, D125, 127, F144, L147, A148, L151, L157, V195, Y226, F252, I258, M293, W30 5, A308, I309, S312, A314, D315, V316, A318, L324, I327, A348, L352, Y365, L372, L376, L 379, L383, I405, L424, I427, A436, F439, A443, V447, A457, H458, P459, T460, S463, S814, F859, A864, H867, Y977, S1031, A1053 and F1068.

[0031] In some embodiments, the one or more mutations are substituted with R.

[0032] In some embodiments, the substitution at N243 is R, A, V, L, I, M, F, W, S, T, C, Y, N, Q, E, K or H.

[0033] In some embodiments, the mutation is a substitution.

[0034] In some embodiments, the substitution is made at a non-polar amino acid residue (e.g., glycine (Gly / G), alanine (Ala / A), valine (Val / V), cysteine ​​(Cys / C), proline (Pro / P), leucine (Leu / L), isoleucine (Ile / I), methionine (Met / M), tryptophan (Trp / W), phenylalanine (Phe / F)), a polar amino acid residue (e.g., serine (Ser / S), threonine (Thr / T), tyrosine (Tyr / Y), asparagine (Asn / N), glutamine (Gln / Q)), a positively charged amino acid residue (e.g., lysine (Lys / K), arginine (Arg / R), histidine (His / H)), or a negatively charged amino acid residue (e.g., aspartic acid (Asp / D), glutamic acid (Glue / E)).

[0035] In some embodiments, the substitution is with a positively charged amino acid residue, such as arginine (R).

[0036] In some embodiments, the substitution is with a non-polar amino acid residue, such as alanine (A).

[0037] In some embodiments, the Cas12i polypeptide comprises a substitution corresponding to any one of the mutations in Table 6 or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO:1.

[0038] In some embodiments, the Cas12i polypeptide comprises a substitution corresponding to any one of the mutations in Table 6 or a combination thereof, when both are used in combination with the same guide RNA, SEQ ID NO: and wherein the Cas12i polypeptide has increased spacer sequence-specific dsDNA cleavage activity compared to said reference Cas12i polypeptide of SEQ ID NO:1, e.g., increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or more, and wherein said amino acid positions are relative to SEQ ID NO:1.

[0039] In some embodiments, the Cas12i polypeptide is an xCas12i-N243R mutant.

[0040] In some embodiments, the Cas12i polypeptide comprises a substitution corresponding to any one of the mutations in Table 8 or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO:1.

[0041] In some embodiments, the Cas12i polypeptide is an xCas12i-N243R+E336R+D892R mutant.

[0042] In some embodiments, the Cas12i polypeptide is an xCas12i-N243R+E336R+G883R mutant.

[0043] According to one aspect, the present disclosure provides a Cas12i polypeptide, the Cas12i polypeptide comprising: (1) As shown in the amino acid sequence of the xCas12i-N243R mutant, (2) comprising the amino acid sequence of the xCas12i-N243R mutant, or (3) An amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to the amino acid sequence of the xCas12i-N243R mutant.

[0044] According to one aspect, the present disclosure provides a Cas12i polypeptide, the Cas12i polypeptide comprising: (1) As shown in the amino acid sequence of the xCas12i-N243R+E336R+D892R mutant, (2) xCas12i-N243R + E336R + D892R mutant amino acid sequence, or (3) An amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to the amino acid sequence of the xCas12i-N243R+E336R+D892R mutant.

[0045] According to one aspect, the present disclosure provides a Cas12i polypeptide, the Cas12i polypeptide comprising: (1) As shown in the amino acid sequence of the xCas12i-N243R+E336R+G883R mutant, (2) comprising the amino acid sequence of the xCas12i-N243R + E336R + G883R mutant, or (3) An amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to the amino acid sequence of the xCas12i-N243R+E336R+G883R mutant.

[0046] In some embodiments, the Cas12i polypeptide can recognize a target adjacent motif (TAM) adjacent to the protospacer sequence 5' on the non-target strand of the target dsDNA, and wherein the TAM is 5'-NTTN-3', where N is A, T, G, or C.

[0047] In some embodiments, the Cas12i polypeptide further comprises a functional domain associated with the Cas12i polypeptide.

[0048] In some embodiments, the functional domain has transposase activity, methylase activity, demethylase activity, translation activating activity, translation inhibiting activity, transcription activating activity, transcription inhibiting activity, transcription release factor activity, chromatin modifying or remodeling activity, histone modifying activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, nucleic acid binding activity, detectable activity, or any combination thereof.

[0049] According to one aspect, the present disclosure provides a fusion protein, the fusion protein comprising a Cas12i polypeptide of the present disclosure and a functional domain.

[0050] In some embodiments, the functional domain is fused N-terminally, C-terminally, or internally to the Cas12i polypeptide.

[0051] In some embodiments, the functional domain is fused to the Cas12i polypeptide via a linker, such as an XTEN linker (SEQ ID NO:442), a GS linker comprising multiple glycine and serine residues, a GS linker comprising multiple glycine and serine residues and an XTEN linker (SEQ ID NO:442), a GS linker comprising multiple glycine and serine residues and a BP NLS (SEQ ID NO:443).

[0052] In some embodiments, the functional domain is a nuclear localization signal (NLS), a nuclear export signal (NES), a deaminase or a catalytic domain thereof, a uracil glycosidase inhibitor (UGI), a uracil glycosidase (UNG), a methylpurine glycosidase (MPG), a methylase or a catalytic domain thereof, a demethylase or a catalytic domain thereof, a transcription activation domain (e.g., VP64 or VPR), a transcription inhibition domain (e.g., a KRAB portion or a SID portion), a reverse transcriptase or a catalytic domain thereof, an exonuclease or a catalytic domain thereof, a histone residue modifying domain, a nuclease catalytic domain (e.g., FokI), a transcriptional modifier, a light gating factor ... factors), chemical inducers, chromatin visualization factors, targeting polypeptides to provide binding to cell surface moieties on a target cell or target cell type, reporter (e.g., fluorescent) polypeptides or detectable markers (e.g., GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP), localization signals, polypeptide targeting moieties, DNA binding domains (e.g., MBP, Lex A DBD, Gal4The target DNA modifying activity is selected from the group consisting of a functional domain exhibiting an activity to modify a target DNA, a catalytic domain thereof, a functional fragment thereof, and any combination thereof, and the activity to modify the target DNA is selected from the group consisting of a methyltransferase activity, a DNA repair activity, a DNA damage activity, a dismutase activity, an alkylation activity, a dealkylation activity, a depurination activity, an oxidation activity, a deoxidation ... The enzyme activity is selected from the group consisting of ubiquitin ligase activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosidase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity (e.g., from O-GlcNAc transferase), and deglycosylation activity.

[0053] In some embodiments, the NLS comprises or is SV40 NLS (SEQ ID NO:444), bpSV40 NLS (BP NLS, bpNLS, SEQ ID NO:443) or NP NLS (Xenopus laevis nucleoplasmic protein NLS, nucleoplasmic protein NLS, SEQ ID NO:445).

[0054] In some embodiments, the functional domain comprises a deaminase or a catalytic domain thereof.

[0055] In some embodiments, the deaminase or catalytic domain thereof is an adenine deaminase (e.g., TadA, e.g., TadA8e, TadA8.17, TadA8.20, TadA9) or catalytic domain thereof.

[0056] In some embodiments, the deaminase, or catalytic domain thereof, is a cytidine deaminase (e.g., an APOBEC, e.g., an APOBEC3, e.g., APOBEC3A, APOBEC3B, APOBEC3C, DddA) or catalytic domain thereof.

[0057] In some embodiments, the functional domain comprises a uracil glycosidase inhibitor (UGI).

[0058] In some embodiments, the functional domain comprises uracil glycosidase (UNG).

[0059] In some embodiments, the functional domain comprises methylpurine glycosidase (MPG).

[0060] In some embodiments, the adenine deaminase domain is wild-type TadA or a mutant thereof, which is (1) As shown in SEQ ID NO: 439, (2) comprising the amino acid sequence of SEQ ID NO: 439, or (3) An amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:439.

[0061] In some embodiments, the adenine deaminase domain is TadA8e-V106W of SEQ ID NO:439 or TadA8e.

[0062] In some embodiments, the UGI domain comprises: (1) As shown in SEQ ID NO: 441, (2) comprising the amino acid sequence of SEQ ID NO: 441, or (3) An amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:441.

[0063] In some embodiments, the cytidine deaminase domain is APOBEC3 or a variant thereof, which is (1) As shown in SEQ ID NO: 440, (2) comprising the amino acid sequence of SEQ ID NO: 440, or (3) An amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:440.

[0064] In some embodiments, the cytidine deaminase domain is human APOBEC3-W104A of SEQ ID NO:440.

[0065] In some embodiments, the functional domain comprises a reverse transcriptase or a catalytic domain thereof.

[0066] In some embodiments, the functional domain comprises a methylase or a catalytic domain thereof.

[0067] In some embodiments, the functional domain comprises a transcriptional activation domain.

[0068] In some embodiments, the functional domain comprises an exonuclease or a catalytic domain thereof, such as T5 exonuclease (T5E) (SEQ ID NO:449).

[0069] In some embodiments, the exonuclease is fused to the Cas12i polypeptide at the N-terminus or C-terminus.

[0070] In some embodiments, the exonuclease is fused to the Cas12i polypeptide at the C-terminus.

[0071] In some embodiments, the T5 exonuclease is (1) As shown in SEQ ID NO: 449, (2) comprising the amino acid sequence of SEQ ID NO: 449, or (3) An amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:449.

[0072] According to one aspect, the present disclosure provides a fusion protein, the fusion protein comprising: (1) a Cas12i polypeptide, and (2) Contains an adenine deaminase domain.

[0073] In some embodiments, the adenine deaminase domain is an adenine deaminase (e.g., TadA, e.g., TadA8e, TadA8.17, TadA8.20, TadA9) or a catalytic domain thereof.

[0074] In some embodiments, the adenine deaminase domain is wild-type TadA or a mutant thereof, which is (1) As shown in SEQ ID NO: 439, (2) comprising the amino acid sequence of SEQ ID NO: 439, or (3) An amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:439.

[0075] In some embodiments, the adenine deaminase domain is TadA8e-V106W of SEQ ID NO:439 or TadA8e.

[0076] According to one aspect, the present disclosure provides a fusion protein, the fusion protein comprising: (1) a Cas12i polypeptide, and (2) Contains a cytidine deaminase domain.

[0077] In some embodiments, the fusion protein further comprises a uracil glycosidase inhibitor (UGI) domain.

[0078] In some embodiments, the UGI domain comprises: (1) As shown in SEQ ID NO: 441, (2) comprising the amino acid sequence of SEQ ID NO: 441, or (3) An amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:441.

[0079] In some embodiments, the cytidine deaminase domain is a cytidine deaminase (e.g., APOBEC (Apolipoprotein B mRNA Editing Catalytic Polypeptide-like), such as APOBEC3, such as APOBEC3A, APOBEC3B, APOBEC3C, DddA) or a catalytic domain thereof.

[0080] In some embodiments, the cytidine deaminase domain is APOBEC3 or a variant thereof, which is (1) As shown in SEQ ID NO: 440, (2) comprising the amino acid sequence of SEQ ID NO: 440, or (3) An amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:440.

[0081] In some embodiments, the cytidine deaminase domain is human APOBEC3-W104A of SEQ ID NO:440.

[0082] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:85 or 184.

[0083] According to one aspect, the present disclosure provides a fusion protein, the fusion protein comprising: (1) a Cas12i polypeptide, and (2) Contains a non-LTR reverse transcription transposon domain.

[0084] According to one aspect, the present disclosure provides a fusion protein, the fusion protein comprising: (1) a Cas12i polypeptide, and (2) Contains a transcription activation domain.

[0085] In some embodiments, the Cas12i polypeptide is a Cas12i polypeptide of the present disclosure.

[0086] In some embodiments, the adenine deaminase domain is fused to the Cas12i polypeptide at the N-terminus or C-terminus.

[0087] In some embodiments, the cytidine deaminase domain is fused to the Cas12i polypeptide at the N-terminus or C-terminus.

[0088] In some embodiments, the uracil glycosidase inhibitor domain is fused to the Cas12i polypeptide at the N-terminus or C-terminus.

[0089] In some embodiments, the uracil glycosidase inhibitor domain is fused to the cytidine deaminase domain at the N-terminus or C-terminus.

[0090] In some embodiments, the non-LTR reverse transcription transposon domain is fused to the Cas12i polypeptide at the N-terminus or C-terminus.

[0091] In some embodiments, the fusion protein comprises one, two, three or more UGI domains.

[0092] In some embodiments, the fusion protein comprises one, two, three or more UGI domains linked in tandem with or without a linker.

[0093] In some embodiments, the fusion protein comprises one, two, three, four or more NLSs and / or NESs.

[0094] In some embodiments, the fusion protein comprises an NLS or NES at the N-terminus and / or C-terminus of the Cas12i polypeptide.

[0095] In some embodiments, the fusion protein comprises an NLS or NES at the N-terminus and / or C-terminus of the adenine deaminase domain.

[0096] In some embodiments, the fusion protein comprises an NLS or NES at the N-terminus and / or C-terminus of the cytidine deaminase domain.

[0097] In some embodiments, the fusion protein comprises an NLS or NES at the N-terminus and / or C-terminus of the UGI domain.

[0098] In some embodiments, the fusion protein comprises an NLS or NES at the N-terminus and / or C-terminus of the reverse transcriptase domain.

[0099] In some embodiments, the fusion protein comprises an NLS or NES at the N-terminus and / or C-terminus of the non-LTR reverse transcription transposon domain.

[0100] In some embodiments, the fusion is via a linker.

[0101] In some embodiments, the linker is a GS linker, an XTEN linker (SEQ ID NO:442), a linker comprising an XTEN, a linker comprising an NLS or an NES, a GS linker comprising an XTEN, or a GS linker comprising an NLS or an NES.

[0102] In some embodiments, the fusion protein includes an inducible element, such as an inducible polypeptide.

[0103] In some embodiments, the NLS comprises or is SV40 NLS (SEQ ID NO:444), bpSV40 NLS (BP NLS, bpNLS, SEQ ID NO:443) or NP NLS (Xenopus nucleoplasmic protein NLS, nucleoplasmic protein NLS, SEQ ID NO:445).

[0104] According to one aspect, the present disclosure provides a vector, wherein the vector is an AAV vector genome, the AAV vector genome comprising: (1) a polynucleotide encoding and comprising a fusion protein of the present disclosure operably linked to a promoter; (2) a polynucleotide of a guide RNA operably linked to a promoter, the guide RNA comprising: (i) a direct repeat sequence capable of forming a complex with the Cas12i polypeptide or the fusion protein; (ii) a spacer sequence capable of directing the complex to the target dsDNA by hybridizing to a target sequence on a target strand of the target dsDNA.

[0105] In some embodiments, the fusion protein has an increased efficiency (e.g., base editing efficiency, methylation efficiency, transcription activation efficiency) relative to an otherwise identical control fusion protein or control complex or control fusion protein comprising a reference polypeptide of any one of SEQ ID NOs: 1-3, 6, and 10, e.g., an efficiency of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or more increase.

[0106] According to one aspect, the present disclosure provides a guide RNA, the guide RNA comprising: (1) a direct repeat sequence capable of forming a complex with a Cas12i polypeptide or a fusion protein comprising the Cas12i polypeptide and a functional domain; (2) a spacer sequence capable of directing the complex to the target dsDNA by hybridizing to a target sequence on a target strand of the target dsDNA.

[0107] In some embodiments, the direct repeat sequence is at the 5' end of the spacer sequence.

[0108] In some embodiments, the guide RNA further comprises an aptamer.

[0109] In some embodiments, the guide RNA further comprises an extension to add an RNA template.

[0110] In some embodiments, the guide RNA further comprises a donor sequence for insertion into a target dsDNA.

[0111] In some embodiments, the direct repeat sequence comprises: (1) As shown in any one of SEQ ID NOs: 11-13, 16, 20, and 501-507; (2) SEQ ID NO: 11-13, 16, 20, and 501-507, or (3) A polynucleotide sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to any one of the polynucleotide sequences of SEQ ID NOs: 11-13, 16, 20 and 501-507.

[0112] In some embodiments, the direct repeat sequence is a direct repeat sequence comprising a nucleotide sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%) but less than 100% sequence identity to a polynucleotide sequence of any one of SEQ ID NOs: 11-13, 16, 20 and 501-507.

[0113] In some embodiments, the direct repeat sequence has a secondary structure substantially the same as the secondary structure of any one of SEQ ID NOs: 11-13, 16, 20, and 501-507.

[0114] In some embodiments, the direct repeat sequence is not any of SEQ ID NOs: 11-13, 16 and 20.

[0115] In some embodiments, when a guide RNA is used in combination with a Cas12i polypeptide (e.g., a Cas12i polypeptide of the present disclosure), the guide RNA may be selected from the group consisting of SEQ ID NOs. Compared to a control guide RNA that is otherwise identical, including any one of NOs:11-13, 16, 20, and 501-507, the guide RNA exhibits increased spacer sequence-specific dsDNA and / or ssDNA cleavage activity, for example, an increase of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or more.

[0116] In some embodiments, the guide RNA, when used in combination with a Cas12i polypeptide (e.g., a Cas12i polypeptide of the present disclosure), exhibits reduced spacer sequence-specific dsDNA and / or ssDNA cleavage activity, e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% reduction, compared to an otherwise identical control guide RNA, including any one of SEQ ID NOs: 11-13, 16, 20, and 501-507, used in combination with a Cas12i polypeptide.

[0117] In some embodiments, when the guide RNA is used in combination with a fusion protein (e.g., a fusion protein of the disclosure) comprising a Cas12i polypeptide (e.g., a Cas12i polypeptide of the disclosure) and a functional domain (e.g., a functional domain of the disclosure), the SEQ ID NO: Compared to an otherwise identical control guide RNA, including any one of NOs:11-13, 16, 20 and 501-507, the efficiency (e.g., base editing efficiency, methylation efficiency, transcription activation efficiency) is increased, for example, by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or more.

[0118] In some embodiments, the direct repeat sequence comprises one or more mutations (e.g., insertions, deletions, or substitutions) in one or more nucleotides, which correspond to one or more nucleotides in any one of the polynucleotide sequences of SEQ ID NOs:11-13, 16, 20, and 501-507.

[0119] In some embodiments, the one or more mutations are in a stem-loop region, wherein the stem-loop region corresponds to a stem-loop region (e.g., the R1 region, the R2 region, the R3 region, the R4 region) of any one of the polynucleotide sequences of SEQ ID NOs:11-13, 16, 20, and 501-507.

[0120] In some embodiments, the direct repeat sequence comprises one or more mutations (e.g., insertions, deletions, or substitutions) in one or more nucleotides, the one or more nucleotides being selected from the group consisting of any one of the polynucleotide sequences of SEQ ID NOs: 11-13, 16, 20, and 501-507. Corresponding to one or more nucleotides at any one of positions from position 1 to the end, e.g., 36, of any one of SEQ ID NOs:11-13, 16, 20 and 501-507, for example, one or more nucleotides at one or more of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36.

[0121] In some embodiments, the direct repeat sequence comprises one or more mutations (e.g., insertions, deletions, or substitutions) in one or more nucleotides of the polynucleotide sequence of SEQ ID NO:11. This corresponds to one or more nucleotides at any one of positions 1 to 36, for example, one or more of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36.

[0122] In some embodiments, the mutation is a deletion.

[0123] In some embodiments, the mutation is a substitution.

[0124] In some embodiments, the mutation is a substitution with A, U, G, or C.

[0125] In some embodiments, the direct repeat sequence comprises a deletion.

[0126] In some embodiments, the deletion is within a stem loop region of the direct repeat sequence (eg, the R1 region, the R2 region, the R3 region, the R4 region, the R5 region).

[0127] In some embodiments, the deletion is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides.

[0128] In some embodiments, the stem loop region comprising the deletion retains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs.

[0129] In some embodiments, the stem loop region containing the deletion retains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs.

[0130] In some embodiments, the stem loop region comprising the deletion contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 non-AU or non-GC mismatches.

[0131] In some embodiments, the direct repeat sequence comprises a substitution of one or more thermodynamically unstable base pairs with one or more GC or CG base pairs.

[0132] In some embodiments, the thermodynamically unstable base pair is an AU or UA base pair, an AG or GA base pair, or an UG or GU base pair.

[0133] In some embodiments, the thermodynamically unstable base pair is within the stem of the stem-loop region of the direct repeat sequence.

[0134] In some embodiments, the thermodynamically unstable base pairs begin with a base pair common to both the stem and the loop of the stem-loop region and include the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, or 30th base pair of said base pairs.

[0135] In some embodiments, the direct repeat sequence comprises: (1) As shown in any one of SEQ ID NOs:501 to 507; (2) comprising any one of the polynucleotide sequences of SEQ ID NO:501 to 507; or (3) A polynucleotide sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to any one of the polynucleotide sequences of SEQ ID NOs:501 to 507.

[0136] In some embodiments, the target sequence comprises at least about 16 contiguous nucleotides of the target gene, e.g., about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 contiguous nucleotides of the target gene, or a range of values ​​between any two of the foregoing values, e.g., from about 16 to about 50 contiguous nucleotides of the target gene, consisting essentially of, or consisting of.

[0137] In some embodiments, the length of the target sequence is at least about 16 nucleotides, e.g., the length is about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 nucleotides, or the length is a range of values ​​between any two of the above values, e.g., the length is from about 16 to about 50 nucleotides.

[0138] In some embodiments, the protospacer sequence comprises at least about 16 contiguous nucleotides of the target gene, e.g., about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 contiguous nucleotides of the target gene, or a range of values ​​between any two of the foregoing values, e.g., from about 16 to about 50 contiguous nucleotides of the target gene, consisting essentially of, or consisting of.

[0139] In some embodiments, the length of the protospacer sequence is at least about 16 nucleotides, e.g., the length is about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 nucleotides, or the length is a numerical range between any two of the above values, e.g., the length is from about 16 to about 50 nucleotides.

[0140] In some embodiments, the target sequence comprises a protospacer adjacent motif (PAM) sequence that is 5' to the target sequence.

[0141] In some embodiments, the target sequence comprises a protospacer adjacent motif (PAM) sequence that is reverse complementary to the protospacer sequence 5' to the target sequence.

[0142] In some embodiments, the length of the spacer sequence is at least about 16 nucleotides, e.g., the length is about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 nucleotides, or the length is a numerical range between any two of the above values, e.g., the length is from about 16 to about 50 nucleotides.

[0143] In some embodiments, the spacer sequence is about 90%-100% complementary to the target sequence and / or contains no more than 1, 2, 3, 4, or 5 mismatches to the target sequence.

[0144] In some embodiments, the guide RNA comprises multiple (e.g., 2, 3, 4, 5 or more) spacer sequences capable of hybridizing to multiple target sequences, respectively.

[0145] In some embodiments, the multiple target sequences are on the same polynucleotide or on a single polynucleotide.

[0146] In some embodiments, the spacer sequence comprises at least 16 consecutive nucleotides of any one of SEQ ID NOs: 82-125, 130, 131-381, 382, ​​391, 398-438.

[0147] In some embodiments, the dsDNA is intracellular.

[0148] According to one aspect, the disclosure provides a polynucleotide, the polynucleotide encoding a Cas12i polypeptide or fusion protein of the disclosure.

[0149] According to one aspect, the present disclosure provides a polynucleotide, wherein the polynucleotide encodes a guide RNA of the present disclosure.

[0150] In some embodiments, the polynucleotide is codon-optimized for expression in eukaryotic (eg, mammalian, eg, human) cells.

[0151] In some embodiments, the polynucleotide is a polydeoxyribonucleotide or a polyribonucleotide.

[0152] In some embodiments, one or more nucleotides of the polynucleotide are modified.

[0153] According to an aspect, the present disclosure provides a system or composition, the system or composition comprising: (1) A Cas12i polypeptide, or a fusion protein comprising the Cas12i polypeptide and a functional domain, or a polynucleotide encoding the Cas12i polypeptide or the fusion protein; and (2) A guide RNA (also called "CRISPR RNA" or "crRNA"), or a polynucleotide encoding the guide RNA, wherein the guide RNA comprises: (i) a direct repeat sequence capable of forming a complex with the Cas12i polypeptide or the fusion protein; (ii) a spacer sequence capable of directing the complex to the target dsDNA by hybridizing to a target sequence on a target strand of the target dsDNA.

[0154] In some embodiments, the system or composition is a non-naturally occurring, engineered system or composition.

[0155] In some embodiments, the Cas12i polypeptide or the fusion protein is a Cas12i polypeptide or fusion protein of the present disclosure.

[0156] In some embodiments, the guide RNA is a guide RNA of the present disclosure.

[0157] In some embodiments, the direct repeat sequence is a direct repeat sequence of the present disclosure.

[0158] In some embodiments, the spacer sequence is a spacer sequence of the present disclosure.

[0159] In some embodiments, the system or composition further comprises an inducible system, such as TMP, DOX, Degron.

[0160] In some embodiments, the inducible system comprises an inducing agent capable of activating a fusion protein that comprises an inducible element.

[0161] In some embodiments, the inducible system comprises an inducer capable of activating expression of a Cas12i polypeptide or a fusion protein comprising an inducible element.

[0162] In some embodiments, the system or composition comprises an activator capable of activating a fusion protein that comprises a transcription activation domain.

[0163] In some embodiments, the coding sequence is a DNA coding sequence or an RNA coding sequence.

[0164] In some embodiments, the system or composition further comprises a serine or tyrosine recombinase.

[0165] In some embodiments, the system or composition further comprises a donor construct, the donor construct comprising a donor polynucleotide for insertion into a target dsDNA and positioned between two binding elements capable of forming a complex with a non-LTR reverse transcription transposon protein.

[0166] In some embodiments, the Cas12i polypeptide is fused to the N-terminus of a non-LTR reverse transposon protein.

[0167] In some embodiments, the Cas12i polypeptide is a nickase.

[0168] In some embodiments, the guide RNA guides the fusion protein to a target sequence 5' to the targeted insertion site, and where the Cas12i polypeptide generates a double-stranded break at the targeted insertion site.

[0169] In some embodiments, the guide RNA guides the fusion protein to a target sequence 5' or 3' to the targeted insertion site, and wherein the Cas12i polypeptide generates a double-stranded break at the targeted insertion site.

[0170] In some embodiments, the donor polynucleotide further comprises a polymerase processing element to facilitate processing of the 5' or 3' end of the donor polynucleotide sequence.

[0171] In some embodiments, the donor polynucleotide further comprises a region of homology to the target sequence on the 5' end of the donor construct, the 3' end of the donor construct, or both ends.

[0172] In some embodiments, the region of homology is between 8 and 25 base pairs.

[0173] According to one aspect, the present disclosure provides a vector, the vector comprising a polynucleotide encoding a Cas12i polypeptide or fusion protein of the present disclosure.

[0174] In some embodiments, the polynucleotide is operably linked to a promoter.

[0175] According to one aspect, the present disclosure provides a vector, the vector comprising a polynucleotide encoding a guide RNA of the present disclosure.

[0176] In some embodiments, the polynucleotide is operably linked to a promoter.

[0177] In one aspect, the present disclosure provides a vector, the vector comprising a polynucleotide encoding a Cas12i polypeptide or fusion protein of the present disclosure and a polynucleotide encoding a guide RNA of the present disclosure.

[0178] In some embodiments, the polynucleotide encoding the Cas12i polypeptide or fusion protein of the disclosure and the polynucleotide encoding the guide RNA of the disclosure are operably linked to the same promoter.

[0179] In some embodiments, the polynucleotide encoding the Cas12i polypeptide or fusion protein of the present disclosure and the polynucleotide encoding the guide RNA of the present disclosure are each operably linked to a promoter.

[0180] In some embodiments, the promoter is selected from a broad spectrum promoter, a tissue specific promoter, a cell type specific promoter, a constitutive promoter and an inducible promoter.

[0181] In some embodiments, the promoter is a (human) U6 promoter (e.g., SEQ ID NO:446), elongation factor 1 alpha short (EFS) promoter, (human) Cbh promoter, MHCK7 promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, H1 promoter, retroviral Rous sarcoma virus LTR promoter, (human) cytomegalovirus (CMV) promoter (e.g., SEQ ID NO:447), SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (Ie) enhancer and / or promoter, chicken β-actin (CBA) promoter or derivatives thereof, such as the CAG promoter (e.g., SEQ ID NO:448), NO:500), CB promoter, (human) elongation factor 1 α-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B chain (PDGF-β) promoter, synapsin (Syn) promoter, synapsin 1 (Syn1) promoter, methyl-CpG-binding polypeptide 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent polypeptide kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, β-globin The promoter may include or be selected from the group consisting of minigene nβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic polypeptide (GFAP) promoter and myelin basic polypeptide (MBP) promoter.

[0182] In some embodiments, a polynucleotide encoding a Cas12i polypeptide or fusion protein of the disclosure is 5' or 3' to a polynucleotide encoding a guide RNA of the disclosure.

[0183] In some embodiments, the vector is a plasmid.

[0184] In some embodiments, the vector is a viral vector.

[0185] In some embodiments, the vector is a retroviral vector, a phage vector, an adenoviral vector, a herpes simplex virus (HSV) vector, an AAV vector, or a lentiviral vector.

[0186] In some embodiments, the AAV vector is a DNA-encapsidated AAV vector or an RNA-encapsidated AAV vector.

[0187] In some embodiments, the AAV vector comprises a capsid, and the capsid is of a serotype AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV.PHP.eB (a member of the clade to which any one of AAV1-AAV13 belongs), or a functional truncation variant or functional mutant thereof.

[0188] According to one aspect, the present disclosure provides a recombinant AAV (rAAV) particle, the rAAV particle comprising a vector of the present disclosure.

[0189] In some embodiments, the rAAV particle comprises a capsid, the capsid being of a serotype AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV.PHP.eB (a member of the clade to which any one of AAV1-AAV13 belongs), a functional truncation variant thereof, or a functional mutant thereof, thereby encapsidating the vector.

[0190] According to one aspect, the present disclosure provides a lipid nanoparticle (LNP), the lipid nanoparticle (LNP) comprising a polynucleotide encoding a Cas12i polypeptide or fusion protein of the present disclosure and a guide RNA of the present disclosure.

[0191] In some embodiments, the polynucleotide encoding the Cas12i polypeptide or fusion protein of the present disclosure is in the form of mRNA.

[0192] In some embodiments, the polynucleotide encoding the Cas12i polypeptide or fusion protein comprises a 5'UTR.

[0193] In some embodiments, the polynucleotide encoding the Cas12i polypeptide or fusion protein comprises a 3' polyA tail.

[0194] According to one aspect, the present disclosure provides a method for modifying a target dsDNA, the method comprising contacting a target dsDNA with a system, vector, rAAV particle, or LNP of the present disclosure, wherein the spacer sequence is capable of hybridizing to a target sequence of a target strand of the target dsDNA, and wherein the target sequence is modified by the complex.

[0195] According to one aspect, the present disclosure provides the use of a system, vector, rAAV particle or LNP of the present disclosure in the manufacture of a medicament for modifying a target dsDNA, wherein the spacer sequence is capable of hybridizing to a target sequence of a target strand of the target dsDNA, and wherein the target sequence is modified by the complex.

[0196] According to one aspect, the present disclosure provides a system, vector, rAAV particle, or LNP of the present disclosure for use in modifying a target dsDNA, wherein the spacer sequence is capable of hybridizing to a target sequence of a target strand of the target dsDNA, and wherein the target sequence is modified by the complex.

[0197] In some embodiments, the target dsDNA is the human TRAC gene.

[0198] In some embodiments, the spacer sequence comprises at least consecutive nucleotides of any one of SEQ ID NOs:123-125.

[0199] According to one aspect, the present disclosure provides a cell or its progeny, wherein the cell or its progeny comprises a Cas12i polypeptide, fusion protein, guide RNA, system, polynucleotide, vector, rAAV particle and / or LNP of the present disclosure.

[0200] In one aspect, the present disclosure provides a modified cell or its progeny, wherein said modified cell is modified by a method of the present disclosure.

[0201] In some embodiments, the cell is in vivo, ex vivo, or in vitro.

[0202] In some embodiments, the cell is a eukaryotic cell (e.g., an animal cell, a vertebrate cell, a mammalian cell, a non-human mammalian cell, a non-human primate cell, a rodent (e.g., a mouse or rat) cell, a human cell, a plant cell, or a yeast cell) or a prokaryotic cell (e.g., a bacterial cell).

[0203] In some embodiments, the cells are cultured cells, isolated primary cells, or in vivo cells.

[0204] In some embodiments, the cell is a T cell (e.g., a CAR-T cell), a B cell, a NK cell (e.g., a CAR-NK cell), or a stem cell (e.g., an iPS cell, an HSC cell).

[0205] In some embodiments, the cells are derived from or xenogeneic to the subject.

[0206] In one aspect, the present disclosure provides a host, said host comprising a cell of the present disclosure or a progeny thereof.

[0207] In some embodiments, the host is a non-human animal or a plant.

[0208] In some embodiments, the non-human animal is an animal (eg, a rodent or non-human primate) model for a human genetic disorder.

[0209] According to some aspects, the present disclosure provides (e.g., pharmaceutical) compositions comprising a Cas12i polypeptide, fusion protein, guide RNA, polynucleotide, system, vector, rAAV particle, LNP, and / or cell of the present disclosure or its progeny.

[0210] In some embodiments, the composition comprises a pharma- ceutically acceptable excipient.

[0211] In some embodiments, the compositions are formulated for delivery via nanoparticles, e.g., lipid nanoparticles, liposomes, exosomes, microvesicles, nucleic acid (e.g., DNA) nanoassemblies, gene guns, or implantable devices.

[0212] According to one aspect, the present disclosure provides a delivery system, the delivery system comprising: (1) a delivery vehicle; (2) A Cas12i polypeptide, fusion protein, guide RNA, polynucleotide, system, vector, rAAV particle, LNP, cell or progeny thereof, and / or composition of the present disclosure.

[0213] In some embodiments, the delivery vehicle is a nanoparticle, such as a lipid nanoparticle, a liposome, an exosome, a microvesicle, a nucleic acid (eg, DNA) nanoassembly, a gene gun, or an implantable device.

[0214] According to one aspect, the present disclosure provides a kit, the kit comprising a Cas12i polypeptide, a fusion protein, a guide RNA, a polynucleotide, a system, a vector, a rAAV particle, a LNP, a cell or its progeny, a composition and / or a delivery system of the present disclosure.

[0215] In some embodiments, the kit further comprises instructions for modifying the target dsDNA.

[0216] According to certain aspects, the disclosure provides a method for diagnosing, preventing, or treating a disease or disorder in a subject, the method comprising administering to the subject (e.g., an effective amount of) a system, vector, rAAV particle, LNP, cell or progeny, composition, delivery system, and / or kit of the disclosure.

[0217] According to certain aspects, the disclosure provides the use of (e.g., an effective amount of) a system, vector, rAAV particle, LNP, cell or progeny, composition, delivery system, and / or kit of the disclosure in the manufacture of a medicament or kit for diagnosing, preventing or treating a disease or disorder in a subject.

[0218] According to certain aspects, the disclosure provides for the use of (e.g., an effective amount of) a system, vector, rAAV particle, LNP, cell or progeny, composition, delivery system, and / or kit of the disclosure in the diagnosis, prevention, or treatment of a disease or disorder in a subject.

[0219] In some embodiments, the disease or disorder is associated with distortion of a target dsDNA in a subject.

[0220] In some embodiments, the spacer sequence is capable of hybridizing to a target sequence of a target strand of a target dsDNA, wherein distortion of the target dsDNA is modified by the complex.

[0221] In some embodiments, the method or use further comprises administering to the subject an effective amount of the same recombinant donor template, wherein the same recombinant donor template comprises a donor sequence for insertion into the target dsDNA, wherein insertion of the donor sequence corrects the distortion in the target dsDNA.

[0222] In some embodiments, the modified cells or their progeny prevent or treat a disease or disorder.

[0223] In some embodiments, the disease or disorder is a TTR-related disease or disorder, such as ATTR.

[0224] In some embodiments, the spacer sequence comprises at least 16 contiguous nucleotides of SEQ ID NO:107.

[0225] In some embodiments, the disease or disorder is a PCSK9-associated disease or disorder.

[0226] In some embodiments, the spacer sequence comprises at least 16 contiguous nucleotides of SEQ ID NO:122.

[0227] In some embodiments, the system further comprises a same recombinant donor template, the same recombinant donor template comprising a donor sequence for insertion into a target dsDNA.

[0228] In some embodiments, directing the complex to the target dsDNA causes binding of the complex to the target dsDNA.

[0229] In some embodiments, said directing the complex to the target dsDNA causes modification of the target dsDNA.

[0230] In some embodiments, the modification of the target dsDNA comprises a double strand break (DSB) in the target dsDNA.

[0231] In some embodiments, the DSB results in a deletion and / or an insertion mutation (an indel mutation).

[0232] In some embodiments, the indel mutation modifies the transcription and / or expression of the target dsDNA.

[0233] In some embodiments, the donor DNA template is inserted at the site of the DSB.

[0234] In some embodiments, the modification of the target dsDNA comprises a single strand break (SSB) at a target sequence on the target strand of the target dsDNA.

[0235] In some embodiments, the modification of the dsDNA comprises the substitution of one or more nucleotides of the protospacer sequence that is reverse complementary to the target sequence.

[0236] In some embodiments, the substitution is A to T, A to G, A to C, C to A, C to T, C to G, T to A, T to G, T to C, G to A, G to T, and / or G to C.

[0237] In some embodiments, the modification of the dsDNA comprises a single-stranded break (SSB) in the non-target strand of the target dsDNA.

[0238] In some embodiments, modifications of the dsDNA include the insertion, deletion and / or substitution of one or more nucleotides in the non-target strand.

[0239] In some embodiments, the modification is: a. introducing one or more base edits, b. correcting or introducing a premature stop codon, c. disrupting a splice site, d. inserting or restoring a splice site, e. inserting a gene or gene fragment into one or both alleles of the target polynucleotide, or f. a combination thereof.

[0240] In some embodiments, the complex targets a reverse transcriptase domain to the target sequence, and the reverse transcriptase promotes insertion of the donor sequence from the guide RNA into the target dsDNA.

[0241] In some embodiments, the insertion of the donor sequence a. introduces one or more base edits, b. corrects or introduces a premature stop codon, c. disrupts a splice site, d. inserts or restores a splice site, e. inserts a gene or gene fragment into one or two alleles of the target polynucleotide, or f. a combination thereof.

[0242] In some embodiments, the complex guides a non-LTR reverse transposon protein to a target sequence, and the non-LTR reverse transposon protein promotes insertion of a donor polynucleotide sequence from a donor construct into a target dsDNA.

[0243] In some embodiments, the insertion of the donor sequence a. introduces one or more base edits, b. corrects or introduces a premature stop codon, c. disrupts a splice site, d. inserts or restores a splice site, e. inserts a gene or gene fragment into one or two alleles of the target polynucleotide, or f. a combination thereof.

[0244] In some embodiments, said targeting of the complex to the target dsDNA results in transcriptional modification of the target dsDNA.

[0245] In some embodiments, the transcribed modification is upregulated transcription, downregulated transcription, activated transcription, or inhibited transcription.

[0246] In some embodiments, modification of the target dsDNA comprises methylation or demethylation of one or more nucleotides of the target dsDNA.

[0247] These and other aspects, objects, features and advantages of the exemplary embodiments will become apparent to those of ordinary skill in the art upon consideration of the following detailed description of the exemplary embodiments presented.

[0248] It should be understood that any one embodiment of the present disclosure described herein, including those embodiments described only in the examples or claims, or only in one aspect / portion below, may be combined with any other one or more embodiments of the present disclosure, unless expressly denied or deemed unjustified. An understanding of certain features and advantages of the present disclosure will be obtained by reference to the following detailed description which sets forth illustrative embodiments in which the principles of the present disclosure may be utilized and in which: [Brief description of the drawings]

[0249]

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[0250] The drawings herein are for illustrative purposes only and are not necessarily drawn to scale. overview In this study, applicants demonstrated that the type VI Cas12i system can achieve versatile and efficient genome editing in mammalian cells. Applicants discovered a Cas12i, xCas12i (also referred to herein as "SiCas12i"), that exhibits high editing efficiency at TTN-PAM sites. Through semi-rational design and protein engineering of its PI, REC, and RuvC domains, applicants obtained a highly efficient, high-fidelity mutant hfCas12Max that contains N243R, E336R, and D892R substitutions. Consistent with the hypothesis that the introduction of arginine at critical sites can reinforce the binding between Cas and DNA, the introduction of N243R in the PI domain and E336R in the REC domain significantly increased the editing activity and expanded the PAM recognition. Interestingly, D892R or G883R substitutions in the RuvC domain reduced off-target and retained on-target cleavage activity, whereas alanine substitutions to reduce off-target activity. 28,29 However, this was not the case (Figure S6C). D892R-substituted hfCas12Max was obviously more sensitive to mismatches, indicating that D892R or G883R improved sgRNA binding specificity. According to the sequence comparison and predicted structure of xCas12i and Cas12i2, asparagine 892 is located on the NUC domain, which forms a cleft together with the RuvC domain, and the crRNA:DNA heteroduplex is located in the cleft. The mutant with D892R did not modify the on-target activity, but eliminated the off-target activity, possibly because the arginine substitution of asparagine affected the binding of non-target crRNA. Our data indicate that the semi-rational engineering strategy of arginine substitution based on the EGFP-activated reporter system can be used as a versatile method to improve the activity of CRISPR editing tools.

[0251] Through engineering, the disclosed Cas12i system achieves high editing activity, high specificity and broad PAM range, comparable to SpCas9 and superior to other Cas12 systems due to its relatively small size, relatively short crRNA guide and self-processing characteristics. 4,8,10Considering these, the VI-Cas12i system is 30 or LNP 12,13 Indeed, the data disclosed herein indicate that the type VI Cas12i system mediates robust ex vivo or in vivo genome editing efficiency via ribonucleoprotein (RNP) delivery and lipid nanoliposome (LNP) delivery, respectively, thereby demonstrating its enormous potential for therapeutic genome editing applications.

[0252] In addition, the applicant has already confirmed that the type VI Cas12i system can be used for base editing applications. As for the base editor, the dCas12i system shows high editing from A to G at the A9-A11 site and even the A19 site of the KLF locus, and high editing from C to T at the A7-A10 site, which is similar to the dCas12a system but different from the dCas9 / nCas9 system. Comparable to dCas12a, dCas12i-BE shows higher base editing activity at the KLF4, PCSK9 and DYRK1A loci (Figure 1K, Figure S13A, Figure S15A), thereby indicating that it may have greater potential as a base editor. This indicates that the dCas12i system may be used for a wide range of genome engineering applications, including epigenome editing, genome activation and chromatin imaging. 1,31-34 .

[0253] In summary, the Cas12i system described here has robust editing activity and high specificity, making it a versatile platform for genome or base editing in mammalian cells and potentially useful for in vivo or ex vivo therapeutic applications.

[0254] Cas12i is a programmable RNA-guided dsDNA endonuclease, which can generate double-strand breaks (DSBs) on target dsDNA as guided by programmable RNA, called guide RNA (gRNA), which includes a spacer sequence and a direct repeat sequence. Without wishing to be bound by theory, it is believed that the direct repeat sequence is responsible for forming a complex with Cas12i, and the spacer sequence is responsible for guiding the complex of gRNA and Cas12i to the target dsDNA by hybridizing to the target sequence of the target dsDNA.

[0255] Referring to FIG. 20, the target dsDNA is depicted as including a 5' to 3' upstrand and a 3' to 5' downstrand. The guide RNA is depicted as including a green spacer sequence and an orange direct repeat sequence. The spacer sequence is designed to hybridize to a portion of the downstrand, so that the spacer sequence "targets" a portion of the downstrand. Thus, the downstrand is referred to as the "target DNA strand" or "target strand (TS)" of the target dsDNA, while the upstrand is referred to as the "non-target DNA strand" or "non-target strand (NTS)" of the target dsDNA. The portion of the target strand on which the spacer sequence is designed and to which the spacer sequence can hybridize is referred to as the "target sequence," while the corresponding portion of the target strand on the non-target strand is referred to as the "reverse complement of the target sequence" or "reverse complement" or "protospacer sequence." In case of conflict with other parts of this disclosure, the definitions in this paragraph shall prevail.

[0256] Unless otherwise indicated, the present invention will be practiced using conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, cell biology, stem cell technology, cell culture and transgenic biology within the skill of the art, many of which are described below for illustrative purposes, and such techniques are fully described in the literature.

[0257] All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

[0258] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. For purposes of the present invention, the following terms are defined as consistent with their commonly understood meaning in the art.

[0259] The articles "a / an" and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" refers to one element or to more than one element.

[0260] The use of the alternative (eg, "or") should be understood to mean either one of, both, or any combination thereof.

[0261] The term "and / or" should be understood to mean either one or both of these alternatives.

[0262] As used herein, the term "about" or "approximately" refers to an amount, level, value, quantity, frequency, percentage, dimension, size, mass, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference amount, level, value, quantity, frequency, percentage, dimension, size, mass, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of an amount, level, value, quantity, frequency, percentage, dimension, size, mass, weight, or length that is about ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference amount, level, value, frequency, percentage, scale, size, mass, quantity, weight, or length.

[0263] As used herein, the term "substantially / essentially" refers to about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more of the degree, amount, level, value, quantity, frequency, percentage, dimension, size, mass, weight or length of a reference.

[0264] Numeric ranges include the end values ​​of the range and each specific value within the range; for example, "16 to 100 nucleotides" includes 16 and 100 and each specific value between 16 and 100.

[0265] Throughout this description, unless the context requires otherwise, the terms "comprise", "include", "contain" and "have" are understood to imply the inclusion of a described step or element or set of steps or elements, but not the exclusion of any other steps or elements or other sets of steps or elements. In some embodiments, the terms "comprise", "include", "contain" and "have" are used interchangeably.

[0266] "Consisting of" means the inclusion, but not limitation, of any of the elements of the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or essential and that other elements may not be present.

[0267] "Consisting essentially of" is intended to include any elements recited in the phrase "consisting essentially of," and is limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure of the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are essential or essential, but that other elements are not optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0268] Throughout the specification, references to "one embodiment," "an embodiment," "one particular embodiment," "one related embodiment," "one embodiment," "another embodiment," or "a further embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in different places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0269] "Sequence identity" between two polypeptide or nucleic acid sequences refers to the percentage of the number of the same residues between the sequences relative to the total number of residues, and the calculation of the total number of residues is determined based on the type of mutation. The type of mutation includes insertion (extension) at either or both ends of the sequence, deletion (truncation) at either or both ends of the sequence, substitution / replacement of one or more amino acids / nucleotides, insertion within the sequence, and deletion within the sequence. For example, for polypeptides (similarly for nucleotides), if the type of mutation is one or more of substitution / replacement of one or more amino acids / nucleotides, insertion within the sequence, and deletion within the sequence, the number of residues in the larger molecule of the compared molecules is considered to be the total number of residues. If the type of mutation further includes insertion (extension) at either or both ends of the sequence, or deletion (truncation) at either or both ends of the sequence, the number of amino acids inserted or deleted at either or both ends (e.g., insertion or deletion of less than 20 at either end) is not counted in the total number of residues. When calculating the percentage of identity, the sequences being compared are compared in a way that gives the largest match between the sequences and gaps in the comparison (if any) are analyzed using a specific algorithm.

[0270] Conservative substitutions of non-essential amino acids can be made without affecting the normal function of the protein. Conservative substitution refers to the replacement of an amino acid with a chemically or functionally similar amino acid. It is well known in the art to provide conservative substitution tables of similar amino acids. For example, in some embodiments, the amino acid groups provided below are considered conservative substitutions for each other.

[0271] In some embodiments, selected amino acids that are considered to be conservatively substituted for one another are as follows:

[0272] [Table 1]

[0273] Other selected amino acids that, in some embodiments, are considered to be conservative substitutions for one another are:

[0274] [Table 2]

[0275] Other selected amino acids that, in some embodiments, are considered to be conservative substitutions for one another are:

[0276] [Table 3]

[0277] The term "amino acid" refers to the 20 common, naturally occurring amino acids, including alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine, M, phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0278] As used herein, the term "Cas12i protein" is used in its broadest sense and includes a parent or reference Cas12i protein (e.g., a Cas12i protein comprising any one of SEQ ID NOs:1-10), derivatives or variants thereof, and functional fragments thereof, such as oligonucleotide-binding fragments thereof.

[0279] As used herein, the term "crRNA" may be used interchangeably with guide molecule, gRNA, and guide RNA, and refers to a nucleic acid-based molecule, including but not limited to, an RNA-based molecule that can form a complex (e.g., via a direct repeat sequence, DR) with a CRISPR-Cas protein (e.g., any Cas12i protein described herein) and that is sufficiently complementary to a target nucleic acid sequence to hybridize to the target nucleic acid sequence and includes a sequence (e.g., a spacer region) that guides sequence-specific binding of the complex to the target nucleic acid sequence.

[0280] As used herein, the term "CRISPR array" includes the nucleic acid (e.g., DNA) fragment of CRISPR repeat sequence and spacer region, which starts from the first nucleotide of the first CRISPR repeat sequence and ends with the last nucleotide of the last (terminal) CRISPR repeat sequence. Generally, each spacer region in a CRISPR array is located between two repeat sequences. As used herein, the term "CRISPR repeat sequence" or "CRISPR direct repeat sequence" or "direct repeat sequence" refers to multiple short direct repeat sequences that show little or no sequence variation in a CRISPR array. Suitably, the VI direct repeat sequence can form a stem-loop structure.

[0281] A "stem-loop structure" is a nucleic acid having a secondary structure, which includes a region of nucleotides known or predicted to form a double strand (a stem) connected at one end through a region (a loop) that is primarily single-stranded nucleotides. The terms "hairpin" and "turnback" structures are also used herein to refer to stem-loop structures. Such structures are well known in the art, and these terms are used according to their well-known meanings in the art. As is known in the art, stem-loop structures do not require exact base pairing. Thus, the stem may contain one or more base mismatches. Alternatively, the base pairing may be exact, i.e., without mismatches.

[0282] As used herein, a target nucleic acid can be used interchangeably with a target sequence or a target nucleic acid sequence to refer to a particular nucleic acid that comprises a nucleic acid sequence complementary to all or a portion of a spacer region in a crRNA. In some examples, a target nucleic acid comprises a gene or a sequence within a gene. In some examples, a target nucleic acid comprises a non-coding region (e.g., a promoter). In some examples, a target nucleic acid is single-stranded. In some examples, a target nucleic acid is double-stranded.

[0283] As used herein, "donor template nucleic acid" or "donor template" can be used interchangeably to refer to a nucleic acid molecule that is used to modify the structure of a target nucleic acid by one or more cellular proteins after a CRISPR enzyme described herein has modified the target nucleic acid. In some examples, the donor template nucleic acid is a double-stranded nucleic acid. In some examples, the donor template nucleic acid is a single-stranded nucleic acid. In some examples, the donor template nucleic acid is linear. In some examples, the donor template nucleic acid is circular (e.g., a plasmid). In some examples, the donor template nucleic acid is an exogenous nucleic acid molecule. In some examples, the donor template nucleic acid is an endogenous nucleic acid molecule (e.g., a chromosome).

[0284] The target nucleic acid should be associated with a PAM (protospacer adjacent motif), a short sequence recognized by the CRISPR complex. Depending on the nature of the CRISPR-Cas protein, the target sequence should be selected so that its complementary sequence in the DNA duplex (the complementary sequence of the target sequence) is upstream or downstream of the PAM. In one embodiment of the invention, the complementary sequence of the target sequence is downstream or 3' of the PAM. The exact sequence and length requirements of the PAM vary depending on the Cas12i protein used.

[0285] As one of skill in the art would understand, rather than uracil being represented by "u" and thymine being represented by "t", both uracil and thymine may be represented by "t", and it should be understood that in the context of ribonucleic acid, unless otherwise indicated, "t" is used to represent uracil.

[0286] As used herein, the term "cleavage" refers to the DNA cleavage in the target nucleic acid produced by the nuclease of the CRISPR system described herein.In some instances, the cleavage is double-stranded DNA cleavage.In some instances, the cleavage is single-stranded DNA cleavage.

[0287] As used herein, the meanings of "cleaving a target nucleic acid" or "modifying a target nucleic acid" may overlap. Modifying a target nucleic acid not only includes the modification of a single nucleotide, but also includes the insertion or deletion of a nucleic acid fragment.

[0288] The present application provides Cas12i proteins having single-stranded or double-stranded DNA cleavage activity, such as SEQ ID NOs:1-10. The Cas12i proteins described herein have less than about 50% sequence identity with other known Cas12is, and are smaller and have better delivery efficiency than other Cass, such as Cas9 or Cas12. In some embodiments, the Cas12i protein comprises any one of SEQ ID NOs:1-10, such as any one of SEQ ID NOs:1-3, 6 and 10, or SEQ ID NO:1. In some embodiments, the Cas12i protein is isolated. In some embodiments, the Cas12i protein is engineered. In some embodiments, the Cas12i protein is artificial.

[0289] The Cas12i proteins described herein, such as SiCas12i, Si2Cas12i, WiCas12i, and SaCas12i, have excellent cleavage activity against exogenous or endogenous genes in vitro or at the cellular level, which is equal to or greater than the cleavage activity of SpCas9, LbCas12a, and Cas12i.3. The Cas12i proteins described herein, such as SiCas12i, Si2Cas12i, WiCas12i, and SaCas12i, may have cleavage activity against a specific target sequence of an exogenous or endogenous gene at the cellular level that is greater than any one of about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even greater than 99%. In general, the Cas12i proteins described herein have superior cleavage activity against specific target sequences of exogenous or endogenous genes at the cellular level compared to Cas12i.3.

[0290] SiCas12i has a cleavage activity against an exogenous or endogenous gene at an in vitro or cellular level that is equal to or even greater than SpCas9 or LbCas12a, and is significantly superior to Cas12i.3. That is, the cleavage activity against a specific target sequence of an exogenous or endogenous gene at a cellular level may be greater than any one of about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even greater than 99%. In general, SiCas12i has a cleavage activity against a specific target sequence of an exogenous or endogenous gene at a cellular level that is significantly superior to Cas12i.3.

[0291] The Cas12i protein may further comprise amino acid mutations that do not essentially affect the catalytic activity (endonuclease cleavage activity) or nucleic acid binding function of Cas12i (e.g., an effect of no more than about any one of 5%, 4%, 3%, 2%, 1% or less).

[0292] In some embodiments, the Cas12i protein (including mutants, dCas, nickases, etc.) of the invention, e.g., SiCas12i, comprises one or more nuclear localization sequences (NLSs) at its N-terminus and / or C-terminus, preferably comprises one NLS at its N-terminus and one NLS at its C-terminus. In some embodiments, the NLS is an SV40 NLS (e.g., as shown in SEQ ID NO:444), preferably when the Cas12i protein is used for cleavage. In some embodiments, the NLS is a BP NLS, e.g., as shown in SEQ ID NO:443, preferably when the Cas12i protein is used for base editing, more preferably when the Cas12i protein is fused to the BP NLS of SEQ ID NO:443 at its N-terminus and to the BP NLS of SEQ ID NO:443 at its C-terminus.

[0293] The present invention further provides variants of any of the Cas12i proteins described herein, such as Cas12i variants having a sequence that is at least about 80% (e.g., at least about any one of 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) but less than 100% identical to any one of SEQ ID NOs:1-10 (preferably, SEQ ID NOs:1-3, 6 and 10, more preferably, SEQ ID NO:1). In some embodiments, the Cas12i variants include one or more substitutions, insertions, deletions or truncations relative to the amino acid sequence of a reference Cas12i protein (e.g., a Cas12i protein comprising the amino acid sequence of any one of SEQ ID NOs:1-10).

[0294] As used herein, a "variant" is a polynucleotide or polypeptide that differs from a reference (e.g., parent) polynucleotide or polypeptide, respectively, but retains required properties. A typical variant of a polynucleotide differs in nucleic acid sequence from a reference polynucleotide. Nucleotide changes may or may not alter the amino acid sequence of a polypeptide encoded by a reference polynucleotide. Nucleotide changes can result in substitution, addition, deletion, or truncation of amino acids in a polypeptide encoded by a reference polynucleotide. A typical variant of a polypeptide differs in amino acid sequence from a reference polypeptide. Generally, such differences are limited such that the sequences of the reference and variant polypeptides are generally very similar and identical in many regions. The amino acid sequences of variant and reference polypeptides may differ by any combination of one or more of substitutions, additions, deletions, or truncations. The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. A variant of a polynucleotide or polypeptide may be naturally occurring (e.g., allelic variant) or non-naturally occurring. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, direct synthesis, or other recombinant methods known to those skilled in the art.

[0295] As used herein, the term "wild-type" has the meaning commonly understood by those of skill in the art and refers to the typical form of an organism, strain, gene or trait that can be isolated from a natural source and that has not been intentionally modified.

[0296] As used herein, the terms "non-naturally occurring" and "engineered" can be used interchangeably and refer to human involvement. When these terms are used to describe a nucleic acid molecule or polypeptide, it means that the nucleic acid molecule or polypeptide is at least essentially free from at least one other component with which it is naturally associated or occurs in nature.

[0297] In some embodiments, the Cas12i variant is isolated. In some embodiments, the Cas12i variant is engineered or does not occur in nature. In some embodiments, the Cas12i variant is artificially synthesized. In some embodiments, the Cas12i variant has one or more amino acid mutations (e.g., insertions, deletions, or substitutions) in one or more domains, such as the PI domain, the helical domain, the RuvC domain, the WED domain, the Nuc domain, etc., relative to a reference Cas12i protein (e.g., a parent Cas12i protein).

[0298] In some embodiments, the Cas12i mutant is a mutant of SiCas12i (SEQ ID NO:1). This means that a Cas12i mutant (e.g., a mutant of Si2Cas12i) can be compared to the original SiCas12i (SEQ ID NO:1) in its original sequence (e.g., Si2Cas12i, SEQ ID NO:2), and one or more positions with amino acid mutations (e.g., insertions, deletions, or substitutions) can be identified. In some embodiments, the Cas12i mutant is an engineered SiCas12i.

[0299] In some embodiments, compared to a corresponding reference Cas12i protein (e.g., a Cas12i protein comprising any one of SEQ ID NOs: 1-10), the Cas12i variant (e.g., a SiCas12i variant) has a higher spacer region-specific endonuclease cleavage activity against a target sequence of a target DNA complementary to a guide sequence, e.g., at least about 1.2-fold (e.g., at least about any one of 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 5, 10, 20, 50-fold or more) of the corresponding reference Cas12i protein.

[0300] In some embodiments, compared to a corresponding Cas12i mutant (e.g., a SiCas12i mutant), the original reference Cas12i protein (e.g., a Cas12i protein comprising any one of SEQ ID NOs: 1-10) has a higher spacer region-specific endonuclease cleavage activity against a target sequence of a target DNA complementary to a guide sequence, for example at least about 1.2-fold (e.g., at least about any one of 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 5, 10, 20, 50-fold or more) of the Cas12i mutant.

[0301] In some embodiments, the spacer region-specific endonuclease cleavage activity of the Cas12i variant (e.g., a SiCas12i variant) against the target sequence of the target DNA complementary to the guide sequence is the same as or not significantly different (e.g., within about 1.2-fold) from the corresponding original Cas12i protein (e.g., a Cas12i protein comprising any one of SEQ ID NOs: 1-10). For example, in some embodiments, the spacer region-specific endonuclease cleavage activity of the Cas12i variant against the target sequence of the target DNA complementary to the guide sequence is the same as the corresponding original Cas12i protein. In some embodiments, the spacer region-specific endonuclease cleavage activity of the Cas12i variant against the target sequence of the target DNA complementary to the guide sequence is about 1.2-fold or less (e.g., about any one of about 1.2, 1.19, 1.15, 1.1, 1.01, 1.001-fold or less, etc.) than the corresponding original Cas12i protein. In some embodiments, the spacer region-specific endonuclease cleavage activity of the original Cas12i protein against a target sequence of a target DNA complementary to the guide sequence is about 1.2-fold or less (e.g., about 1.2, 1.19, 1.15, 1.1, 1.01, 1.001-fold or less, etc.) than that of the corresponding Cas12i mutant.

[0302] The present invention further provides dead Cas12i (dCas12i) proteins that have no or essentially no catalytic activity. For example, in some embodiments, the dCas12i protein retains less than about 50% (e.g., less than about any one of 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2.5%, 2%, 1%, or less) of the spacer region-specific endonuclease cleavage activity of a corresponding parent Cas12i protein (e.g., a Cas12i protein comprising any one of SEQ ID NOs: 1-10) against a target sequence of a target DNA complementary to a guide sequence. In some embodiments, the dCas12i protein has essentially no catalytic activity by containing one or more amino acid substitutions in the RuvC domain (e.g., the RuvC domain of a Cas12i protein comprising any one of SEQ ID NOs: 1-10). In some embodiments, the DNA cleavage activity of dCas12i is zero or negligible compared to non-mutated Cas12i forms. In some embodiments, the dCas12i is a catalytically inactive Cas12i protein that contains a mutation in the RuvC domain that allows for successful CRISPR complex formation and binding to a target nucleic acid, but does not allow for successful nuclease activity (catalysis / cleavage activity).

[0303] In some embodiments, dCas12i is a dSiCas12i that has essentially no catalytic activity. In some embodiments, dSiCas12i comprises one or more substitutions at amino acid residues 650, 700, 875, and / or 1049 relative to SEQ ID NO:1. In some embodiments, dSiCas12i comprises one or more substitutions selected from D700A, D700V, D650A, D650V, E875A, E875V, D1049A, and D1049V relative to SEQ ID NO:1. In one embodiment, dSiCas12i comprises any one of the amino acid sequences of dSiCas12i-D700A, dSiCas12i-D650A, dSiCas12i-E857A, and dSiCas12i-D1049A, respectively. In some embodiments, the dSiCas12i comprises one or more substitutions selected from D650A, D700A, E875A, D1049A, D650A+D700A, D700A+E875A, D700A+D1049A, D650A+E875A, D650A+D1049A, E875A+D1049A, D650A+D700A+E875A, D650A+D700A+D1049A, D650A+E875A+D1049A, D700A+E875A+D1049A and D650A+D700A+E875A+D1049A with respect to SEQ ID NO:1.

[0304] dCas12i may also contain mutations other than those previously described, which do not essentially affect the catalytic activity or nucleic acid binding function of the dCas12i protein (e.g., an effect of about 5%, 4%, 3%, 2%, 1% or less). dCas12i proteins that do not essentially have catalytic activity may be used as DNA binding proteins.

[0305] In some embodiments, the dCas12i described herein can be fused to adenosine deaminase (ADA) or cytidine deaminase (CDA) or catalytic domains thereof to achieve single base editing. In some embodiments, the single base editing efficiency of a fusion protein comprising any of the dCas12i proteins described herein and ADA or CDA (or catalytic domains thereof) is at least about 10% (e.g., at least about any one of 20%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 100%, 120%, 150%, 200%, 500%, 1000% or more) higher than that of a fusion protein comprising a dCas12i not derived from the present invention and a healthy ADA or CDA (or catalytic domains thereof).

[0306] The number of amino acids in the full-length sequence of any of the above Cas12i or dCas12i proteins is significantly fewer than that of other types of Cas12 proteins, and their small molecular size facilitates subsequent assembly and delivery of the Cas system in vivo.

[0307] In some embodiments, the adenosine deaminase is TadA8e, for example, a TadA8e comprising the sequence of SEQ ID NO:439.

[0308] In some embodiments, the C' terminus of the deaminase (e.g., adenosine deaminase) is fused to the N' terminus of dCas12i via an optional peptide linker (e.g., a peptide linker comprising SEQ ID NO:442). In some embodiments, the N' terminus of the deaminase (e.g., adenosine deaminase) is fused to the C' terminus of dCas12i via an optional peptide linker (e.g., a peptide linker comprising SEQ ID NO:442). In some embodiments, a fusion protein is provided that includes dSiCas12i and an adenosine deaminase (e.g., TadA8e), such as the fusion protein TadA8e-dSiCas12i-D1049A, or the fusion protein TadA8e-dSiCas12i-E875A.

[0309] Unless otherwise specified, "Cas12i" or "Cas12i protein" as used herein includes any Cas12i protein described herein and variants (e.g., mutants), derivatives (e.g., Cas12i fusion proteins), and dCas12i proteins and derivatives (e.g., dCas12i fusion proteins, e.g., dCas12i-TadA) that have essentially no catalytic activity. The present invention further provides a nucleotide sequence encoding any Cas12i protein and variants and derivatives thereof, e.g., any one of the polynucleotide sequences of SEQ ID NOs:21-40.

[0310] Generally, the crRNA (interchangeable with guideRNA / gRNA) described herein comprises a direct repeat sequence (DR) and a spacer region, consists essentially of a direct repeat sequence (DR) and a spacer region, or consists of a direct repeat sequence (DR) and a spacer region. In some embodiments, the crRNA comprises, consists essentially of, or consists of a DR linked to a spacer region. In some embodiments, the crRNA comprises a DR, a spacer region and a DR (DR-spacer region-DR). This is a typical arrangement for pre-crRNA. In some embodiments, the crRNA comprises a DR, a spacer region, a DR and a spacer region (DR-spacer region-DR-spacer region). In some embodiments, the crRNA comprises two or more DRs and two or more spacer regions. In some embodiments, the crRNA comprises a truncated DR and a spacer region. This is typical for processed or mature crRNA. In some embodiments, the CRISPR-Cas12i effector protein forms a complex with the crRNA, and the spacer region guides the complex to a target nucleic acid complementary to the spacer region for sequence-specific binding.

[0311] In some embodiments, the CRISPR-Cas12i system described herein comprises one or more crRNAs (e.g., 1, 2, 3, 4, 5, 10, 15 or more) or their encoding nucleic acids. In some embodiments, the two or more crRNAs target different target sites, e.g., two target sites in the same target DNA or gene, or two target sites in two different target DNAs or genes.

[0312] The sequence and length of the crRNA described herein can be optimized. In some embodiments, the optimal length of the crRNA may be determined by identifying the processed form of the crRNA or by empirical length studies of the crRNA. In some embodiments, the crRNA comprises base modifications. Direct repeat (DR)

[0313] Table A provides examples of DR sequences for the relevant Cas12i proteins of the present invention. For example, the DR sequence corresponding to SiCas12i (or a variant or derivative thereof, or dSiCas12i or a fusion protein thereof) may comprise the nucleotide sequence set forth in SEQ ID NO:11 or a functional variant thereof. Any DR sequence capable of mediating binding of the Cas12i protein to the relevant crRNA described herein may be used in the present invention. In some embodiments, the DR comprises an RNA sequence of any one of SEQ ID NOs:11-20 and 501-507. In some embodiments, the DR is a "functional variant" of any RNA sequence of SEQ ID NOs:11-20, e.g., a "functional truncated version", a "functional extended version", or a "functional replacement version". For example, the DR sequence of SEQ ID NO:501 or 502 is a part of SEQ ID NO:11 (truncated form), which is a functional variant or a functionally truncated DR variant since it still has DR function as shown in the examples. A "functional variant" of a DR is a variant of a reference DR (e.g., parent DR) that is 5' and / or 3' extended (functional extended version) or truncated (functional truncated version), or that contains one or more insertions, deletions and / or substitutions (functional replacement version) of one or more nucleotides relative to the reference DR (e.g., parent DR), while still retaining at least about 20% (e.g., at least about any one of 30%, 40%, 50%, 60%, 60%, 70%, 80%, 90%, 95% or more) functionality of the reference DR, i.e., the ability to mediate binding of the Cas12i protein to the corresponding crRNA. A DR functional variant typically retains a stem-loop-like secondary structure or a portion thereof that can be used for binding of the Cas12i protein. As shown in FIG. 21, DR-T2 (SEQ ID NO:502) is one of the functional truncation versions of the DR shown in SEQ ID NO:11. In some embodiments, the DR or a functional variant thereof comprises a stem-loop-like secondary structure or a portion thereof that can be used for binding of a Cas12i protein.In some embodiments, the DR or functional variant thereof comprises at least two (e.g., 2, 3, 4, 5 or more) stem-loop-like secondary structures or portions thereof that can be used for binding of a Cas12i protein.

[0314] In some embodiments, the DR or functional variant thereof comprises at least about 16 nucleotides (nt), e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides. In some embodiments, the DR comprises about 20 nt to about 40 nt, e.g., about 20 nt to about 30 nt, about 22 nt to about 40 nt, about 23 nt to about 38 nt, about 23 nt to about 36 nt, or about 30 nt to about 40 nt. In some embodiments, the DR comprises 22 nt, 23 nt, or 24 nt. In some embodiments, the DR comprises 35 nt, 36 nt, or 37 nt.

[0315] In some embodiments, the DR sequence comprises a stem-loop structure near the 3' end (immediately adjacent to the spacer sequence). A "stem-loop structure" is a nucleic acid having a secondary structure, which comprises a region of nucleotides connected at one end via a linking region (loop) of essentially single-stranded nucleotides that is known or predicted to form a double-stranded (stem) portion. The term "hairpin" structure is also used herein to refer to a stem-loop structure. Such structures are well known in the art, and these terms are used according to their generally known meaning in the art. A stem-loop structure does not require exact base pairing. Thus, the stem may contain one or more base mismatches. Alternatively, the base pairing may be exact, i.e., without any mismatches.

[0316] The crRNA of the present invention comprises a DR comprising a stem-loop structure near the 3' end of the DR sequence. The DR stem-loop structure of SiCas12i is shown in FIG. 11. In some embodiments, the stem in the DR comprises 5 pairs of complementary bases that hybridize with each other, and the loop length is 6, 7, 8, or 9 nucleotides. In some embodiments, the loop length is 7 nucleotides. In some embodiments, the stem may comprise at least 2, at least 3, at least 4, or at least 5 base pairs. In some embodiments, the DR comprises two complementary nucleotide segments that are about 5 nucleotides in length and spaced apart by about 7 nucleotides. In some embodiments, the stem-loop structure comprises a first stem nucleotide strand having a length of 5 nucleotides, a second stem nucleotide strand having a length of 5 nucleotides, where the first stem nucleotide strand and the second stem nucleotide strand are capable of hybridizing to each other, and a cyclic nucleotide strand comprising 6, 7 or 8 nucleotides arranged between the first stem nucleotide strand and the second stem nucleotide strand.

[0317] As used herein, the secondary structures of two or more crRNAs being essentially the same or without substantial differences means that the crRNAs contain stems and / or loops that differ in length by no more than 1, 2 or 3 nucleotides, and the nucleotide sequences of the crRNAs differ by no more than 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides in terms of nucleotide type (A, U, G or C) when compared by sequence comparison. In some embodiments, the secondary structures of two or more crRNAs being essentially the same or without substantial differences means that the crRNAs contain stems that differ in length by at most one pair of complementary bases, and / or loops that differ in length by at most one nucleotide, and / or contain stems that have the same length but with mismatched bases. In some embodiments, the stem-loop structure is 5'-X 1 X 2 X3 X 4 X 5 NNNnNNNX 6 X 7 X 8 X 9 X 10 -3', where X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 and X 10 may be any base, n may be any base or a deletion, and N may be any base, where X 1 X 2 X 3 X 4 X 5 and X 6 X 7 X 8 X 9 X 10 can hybridize to each other to form a stem and NNNnNNN to form a loop. In some embodiments, the stem-loop structure comprises any one of SEQ ID NOs:503-507.

[0318] In some embodiments, a DR sequence capable of directing any Cas12i of the present invention to a target site comprises one or more nucleotide changes selected from nucleotide additions, insertions, deletions and substitutions, which nucleotide changes do not result in a substantial difference in secondary structure compared to the DR sequence set forth in any one of SEQ ID NOs: 11-20 and 501-507 or a functionally truncated version thereof. Spacer Region

[0319] In some embodiments, the length of the spacer sequence is at least about 16 nucleotides, preferably about 16 to about 100 nucleotides, more preferably about 16 to about 50 nucleotides (e.g., any one of about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides). In some embodiments, the spacer region is about 16 to about 27 nucleotides, e.g., any one of about 17 to about 24 nucleotides, about 18 to about 24 nucleotides, or about 18 to about 22 nucleotides.

[0320] In some embodiments, the spacer region is at least about 70% (e.g., at least about any one of 75%, 80%, 85%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) complementary to the target sequence. In some embodiments, there are at least about 15 (e.g., at least about any one of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more) nucleotides between the spacer sequence and the target sequence of the target nucleic acid (e.g., DNA).

[0321] Perfect complementarity is not required for the spacer region, provided that the crRNA has sufficient complementarity to function (i.e., guide the Cas12i protein to the target site). The cleavage efficiency of Cas12i mediated by the crRNA can be adjusted by introducing one or more mismatches (e.g., one or two mismatches between the spacer sequence and the target sequence, including the position of the mismatch along the spacer region / target sequence). If the mismatch (e.g., double mismatch) is located more centrally in the spacer region (i.e., not at the 3' or 5' end of the spacer region), it will have a greater impact on the cleavage efficiency. Therefore, the cleavage efficiency of Cas12i can be adjusted by selecting the position of the mismatch along the spacer sequence. For example, if less than 100% cleavage of the target sequence is desired (e.g., in a cell population), one or two mismatches between the spacer sequence and the target sequence can be introduced into the spacer sequence.

[0322] In some embodiments, the Cas12i protein of the present invention can recognize a PAM (protospacer adjacent motif) and act on a target sequence. In some embodiments, the PAM comprises or consists of 5'-NTTN-3' (wherein N is A, T, G, or C). In some embodiments, the PAM comprises or consists of 5'-TTC-3', 5'-TTA-3', 5'-TTT-3', 5'-TTG-3', 5'-ATA-3', or 5'-ATG-3'. In some embodiments, the PAM comprises or consists of 5'-TTC-3'.

[0323] 1. A Cas12i protein comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10 (preferably SEQ ID NOs:1-3, 6 and 10, and more preferably SEQ ID NO:1). The Cas12i protein may further contain amino acid mutations that do not essentially affect the catalytic activity (endonuclease cleavage activity) or nucleic acid binding function of Cas12i.

[0324] 2. The Cas12i protein according to any one of the preceding embodiments, wherein the Cas12i protein essentially does not have the spacer region-specific endonuclease cleavage activity (e.g., less than 50%, 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2.5%, 2%, 1% or less) of the corresponding parent Cas12i protein (e.g., a Cas12i protein comprising any one of SEQ ID NOs: 1 to 10) against a target sequence of a target DNA complementary to a guide sequence. In one embodiment, the Cas12i essentially does not have the spacer region-specific endonuclease cleavage activity or spacer region-nonspecific bypass activity (e.g., retains less than or equal to 50%, 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2.5%, 2%, 1%) of the corresponding parent Cas12i protein (e.g., a Cas12i protein comprising any one of SEQ ID NOs: 1-10).

[0325] 3. The Cas12i protein according to any one of the preceding embodiments, wherein the Cas12i protein comprises one or more amino acid mutations in its RuvC domain such that the Cas12i protein essentially does not have the spacer region-specific endonuclease cleavage activity (e.g., retains less than or equal to 50%, 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2.5%, 2%, 1%) of a corresponding parent Cas12i protein (e.g., a Cas12i protein comprising any one of SEQ ID NOs: 1-10) against a target sequence of a target DNA complementary to a guide sequence.

[0326] 4. The Cas12i protein of any one of the preceding embodiments, wherein the amino acid mutation is selected from an amino acid addition, insertion, deletion and substitution.

[0327] 5. The Cas12i protein of any one of the preceding embodiments, wherein the Cas12i protein comprises an amino acid substitution at one or more positions corresponding to positions 700 (D700), 650 (D650), 875 (E875) or 1049 (D1049) of the sequence shown in SEQ ID NO:1. The amino acids at the above amino acid positions (D700, D650, E875 or D1049) may be mutated to another amino acid different from the corresponding amino acid in the parent sequence (e.g., a parent Cas12i protein comprising any one of SEQ ID NOs:1-10) so as to essentially lose endonuclease cleavage activity. The Cas12i protein may further contain other mutations that do not substantially affect the catalytic activity or nucleic acid binding function of Cas12i.

[0328] 6. The Cas12i protein of any one of the preceding embodiments, wherein the amino acid substitutions are selected from D700A / V, D650A / V, E875A / V and D1049A / V.

[0329] 7. The Cas12i protein of any one of the preceding embodiments, wherein the amino acid substitutions are selected from D700A, D650A, E875A and D1049A.

[0330] 8. The Cas12i protein of any one of the preceding embodiments, wherein the amino acid substitutions are selected from D700A, D650A, E875A, D1049A, D700A+D650A, D700A+E875A, D700A+D1049A, D650A+E875A, D650A+D1049A, E875A+D1049A, D700A+D650A+E875A, D700A+D650A+D1049A, D650A+E875A+D1049A and D700A+D650A+E875A+D1049A.

[0331] 10. A Cas12i protein described in any one of the preceding embodiments, wherein the Cas12i protein is linked to one or more functional domains.

[0332] 11. The Cas12i protein of any one of the preceding embodiments, wherein the functional domain is linked to the N-terminus and / or C-terminus of the Cas12i protein. The linkage may be a direct linkage or an indirect linkage via a linker.

[0333] 12. The Cas12i protein according to any one of the preceding embodiments, wherein the functional domain is selected from a nuclear localization signal (NLS), a nuclear export signal (NES), a deaminase (e.g., adenosine deaminase or cytidine deaminase) catalytic domain, a DNA methylation catalytic domain, a DNA demethylation catalytic domain, a histone residue modifying domain, a nuclease catalytic domain, a fluorescent protein, a transcriptional modifier (e.g., a transcriptional activation catalytic domain, a transcriptional inhibition catalytic domain), an optical gating factor, a chemical inducer, a chromatin visualization factor, a targeting polypeptide for providing binding to a cell surface moiety on a target cell or a target cell type.

[0334] 13. The functional domain exhibits an activity of modifying a target DNA, and the activity is selected from the group consisting of nuclease activity, methylation activity, demethylation activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosidase activity, acetyltransferase activity, deacetylase activity, and acetyltransferase activity. the Cas12i protein according to any one of the preceding embodiments, wherein the Cas12i protein has an activity selected from a ubiquitin ligase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitination activity, an adenylation activity, a deadenylation activity, a sumoylation activity, a desumoylation activity, a ribosylation activity, a deribosylation activity, a myristoylation activity, a demyristoylation activity, a glycosylation activity (e.g., from an O-GlcNAc transferase), a deglycosylation activity, a transcriptional inhibition activity, and a transcriptional activation activity.

[0335] 14. The Cas12i protein of any one of the preceding embodiments, wherein the functional domain is selected from an adenosine deaminase catalytic domain or a cytidine deaminase catalytic domain.

[0336] 15. The Cas12i protein of any one of the preceding embodiments, wherein the functional domain is a full-length or functional fragment of TadA8e.

[0337] 17. A Cas12i protein described in any one of the preceding embodiments, wherein the Cas12i protein is modified to reduce or eliminate spacer region non-specific endonuclease bypass activity.

[0338] 18. A polynucleotide encoding the Cas12i protein of any one of the preceding embodiments.

[0339] 19. The polynucleotide of any one of the preceding embodiments, wherein the polynucleotide is codon-optimized for expression in a eukaryotic cell.

[0340] 20. The polynucleotide according to any one of the preceding embodiments, comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to a nucleotide sequence set forth in any one of SEQ ID NOs:21 to 40.

[0341] 21. A vector comprising a polynucleotide according to any one of the preceding embodiments.

[0342] 22. A vector according to any one of the preceding embodiments, wherein the polynucleotide is operably linked to a promoter.

[0343] 23. The vector of any one of the preceding embodiments, wherein the promoter is a constitutive promoter, an inducible promoter, a broad-spectrum promoter, a cell type-specific promoter, or a tissue-specific promoter.

[0344] 24. A vector according to any one of the preceding embodiments, wherein the vector is a plasmid.

[0345] 25. The vector of any one of the preceding embodiments, wherein the vector is a retroviral vector, a phage vector, an adenoviral vector, a herpes simplex virus (HSV) vector, an adeno-associated virus (AAV) vector, or a lentiviral vector.

[0346] 26. The vector of any one of the preceding embodiments, wherein the AAV vector is selected from recombinant AAV vectors of serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV13.

[0347] 27. A delivery system comprising: (1) a delivery vehicle; and (2) a Cas12i protein, polynucleotide or vector described in any one of the preceding embodiments.

[0348] 28. A delivery system according to any one of the preceding embodiments, wherein the delivery vehicle is a nanoparticle, a liposome, an exosome, a microvesicle, or a gene gun.

[0349] 29. An engineered, non-naturally occurring CRISPR-Cas system, comprising: A Cas12i protein according to any one of the above embodiments, or a polynucleotide encoding the Cas12i protein; and a CRISPR RNA (crRNA), or a polynucleotide encoding the crRNA, wherein the crRNA comprises: a spacer region capable of hybridizing to a target sequence of a target DNA; a direct repeat (DR) linked to the spacer region and capable of guiding binding of the Cas12i protein and the crRNA to form a CRISPR-Cas complex that targets the target sequence. The Cas12i protein can bind to the crRNA and target the target sequence, where the target sequence is single-stranded or double-stranded DNA or RNA.

[0350] 30. A CRISPR-Cas system comprising one or more vectors, the one or more vectors comprising: A first regulatory element operably linked to a nucleotide sequence encoding a Cas12i protein according to any one of the preceding embodiments; and a second regulatory element operably linked to a polynucleotide encoding a CRISPR RNA (crRNA), said crRNA comprising: a spacer region capable of hybridizing to a target sequence of a target DNA; A direct repeat (DR) linked to the spacer region that can guide the binding of the Cas12i protein and the crRNA to form a CRISPR-Cas complex that targets the target sequence; wherein the first regulatory element and the second regulatory element are located on the same or different vectors of the CRISPR-Cas vector system.

[0351] 31. An engineered, non-naturally occurring CRISPR-Cas complex, comprising: A Cas12i protein according to any one of the above embodiments, and CRISPR RNA (crRNA), wherein the crRNA comprises a spacer region capable of hybridizing to a target sequence of a target DNA; and a direct repeat (DR) linked to the spacer region, the DR guiding the binding of the Cas12i protein to the crRNA.

[0352] 32. The CRISPR-Cas system or complex according to any one of the preceding embodiments, wherein the length of the spacer region is greater than 16 nucleotides, preferably between 16 and 100 nucleotides, more preferably between 16 and 50 nucleotides (e.g. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides), more preferably between 16 and 27 nucleotides, more preferably between 17 and 24 nucleotides, more preferably between 18 and 24 nucleotides, and most preferably between 18 and 22 nucleotides.

[0353] 33. A CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the DR has substantially the same secondary structure as the secondary structure of a DR shown in any one of SEQ ID NOs:11 to 20.

[0354] 34. The CRISPR-Cas system or complex of any one of the preceding embodiments, wherein the DR has a nucleotide addition, insertion, deletion or substitution that does not result in a substantial difference in secondary structure compared to the DR shown in any one of SEQ ID NOs:11-20.

[0355] 35. The DR comprises a stem-loop structure near the 3' end of the DR; wherein the stem-loop structure is 5'-X 1 X 2 X 3 X 4 X 5 NNNnNNNX 6 X 7 X 8 X 9 X 10 -3'(X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 and X 10 is any base, n is any nucleobase or a deletion, and N is any nucleobase, 1 X 2 X 3 X 4 X 5 and X 6 X 7 X 8 X 9 X 10 are capable of hybridizing to each other.

[0356] 36. The DR comprises a stem-loop structure selected from any of the following: near the 3' end of the DR, 5'-CUCCCNNNNNNUGGGAG-3' (SEQ ID NO:), where N is any nucleobase; near the 3' end of the DR, 5'-CUCCUNNNNNNUGGGAG-3' (SEQ ID NO:), where N is any nucleobase; near the 3' end of the DR is 5'-GUCCCNNNNNNUGGGAC-3' (SEQ ID NO:), where N is any nucleobase; near the 3' end of the DR, 5'-GUGUCNNNNNNUGACAC-3' (SEQ ID NO:), where N is any nucleobase; near the 3' end of the DR, 5'-GUGCCNNNNNNUGGCAC-3' (SEQ ID NO:), where N is any nucleobase; near the 3' end of the DR is 5'-UGUGUNNNNNNUCACAC-3' (SEQ ID NO:), where N is any nucleobase; near the 3' end of the DR, 5'-CCGUCNNNNNNUGACGG-3' (SEQ ID NO:), where N is any nucleobase; near the 3' end of the DR, 5'-GUUUCNNNNNNUGAAAC-3' (SEQ ID NO:), where N is any nucleobase; 5'-GUGUUNNNNNNUAACAC-3' (SEQ ID NO:), where N is any nucleobase, near the 3' end of the DR; and 5'-UUGUCNNNNNNUGACAA-3' (SEQ ID NO:), where N is any nucleobase, near the 3' end of the DR.

[0357] 37. A CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the CRISPR-Cas system or complex further comprises a target DNA capable of hybridizing to the spacer region.

[0358] 38. A CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the target DNA is eukaryotic DNA.

[0359] 39. The CRISPR-Cas system or complex of any one of the preceding embodiments, wherein the target DNA is in a cell, preferably the cell is selected from a prokaryotic cell, a eukaryotic cell, an animal cell, a plant cell, a fungal cell, a vertebrate cell, an invertebrate cell, a rodent cell, a mammalian cell, a primate cell, a non-human primate cell and a human cell.

[0360] 40. A CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the crRNA hybridizes to a target sequence of the target DNA to form a complex and causes cleavage of the target sequence by the Cas12i protein.

[0361] 41. The CRISPR-Cas system or complex of any one of the preceding embodiments, wherein the target sequence is at the 3' end of a protospacer adjacent motif (PAM).

[0362] 42. A CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the PAM comprises a 5'-T-rich motif.

[0363] 43. The CRISPR-Cas system or complex of any one of the preceding embodiments, wherein the PAM is 5'-TTA, 5'-TTT, 5'-TTG, 5'-TTC, 5'-ATA or 5'-ATG.

[0364] 44. The CRISPR-Cas system or complex of any one of the preceding embodiments, wherein the one or more vectors comprise one or more of a retroviral vector, a phage vector, an adenoviral vector, a herpes simplex virus (HSV) vector, an adeno-associated virus (AAV) vector, or a lentiviral vector.

[0365] 45. The CRISPR-Cas system or complex of any one of the preceding embodiments, wherein the AAV vector is selected from recombinant AAV vectors of serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV13.

[0366] 46. ​​The CRISPR-Cas system or complex of any one of the preceding embodiments, wherein the regulatory element comprises a promoter.

[0367] 47. The CRISPR-Cas system or complex of any one of the preceding embodiments, wherein the promoter is selected from a constitutive promoter, an inducible promoter, a broad-spectrum promoter, a cell type-specific promoter or a tissue-specific promoter.

[0368] 48. A CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the promoter is functional in eukaryotic cells.

[0369] 49. The CRISPR-Cas system or complex of any one of the preceding embodiments, wherein the eukaryotic cell includes an animal cell, a plant cell, a fungal cell, a vertebrate cell, an invertebrate cell, a rodent cell, a mammalian cell, a primate cell, a non-human primate cell and a human cell.

[0370] 50. The CRISPR-Cas system or complex of any one of the preceding embodiments, optionally further comprising a DNA donor template that is inserted into the locus of interest by homology-directed repair (HDR).

[0371] 51. A cell or a progeny thereof comprising the Cas12i protein, polynucleotide, vector, delivery system, CRISPR-Cas system or complex according to any one of the preceding embodiments, wherein preferably the cell is selected from a prokaryotic cell, a eukaryotic cell, an animal cell, a plant cell, a fungal cell, a vertebrate cell, an invertebrate cell, a rodent cell, a mammalian cell, a primate cell, a non-human primate cell and a human cell, or a progeny thereof.

[0372] 52. A non-human multicellular organism, comprising a cell or a progeny thereof according to any one of the preceding embodiments, wherein preferably said non-human multicellular organism is an animal (e.g., a rodent or non-human primate) model of a human gene-associated disease.

[0373] 53. A method for modifying a target DNA, comprising contacting the target DNA with a CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the contacting causes modification of the target DNA by a Cas12i protein.

[0374] 54. The method of any one of the preceding embodiments, wherein the modification occurs outside a cell in vitro.

[0375] 55. The method of any one of the preceding embodiments, wherein the modification occurs inside a cell in vitro.

[0376] 56. A method according to any one of the preceding embodiments, wherein the modification occurs inside a cell in vivo.

[0377] 57. The method of any one of the preceding embodiments, wherein the cell is a eukaryotic cell.

[0378] 58. The method of any one of the preceding embodiments, wherein the eukaryotic cell is selected from an animal cell, a plant cell, a fungal cell, a vertebrate cell, an invertebrate cell, a rodent cell, a mammalian cell, a primate cell, a non-human primate cell and a human cell.

[0379] 59. The modification is to cleave the target DNA; Optionally, the cleavage is performed in a manner that cleaves single-stranded DNA, or, optionally, in a manner that sequentially cleaves the same or different sites in double-stranded DNA.

[0380] 60. A method according to any one of the preceding embodiments, wherein the cleavage causes a deletion of a nucleotide sequence and / or an insertion of a nucleotide sequence.

[0381] 61. A method according to any one of the preceding embodiments, wherein the cleavage comprises cleaving the target nucleic acid at two sites, causing a deletion or inversion of the sequence between the two sites.

[0382] 62. A method according to any one of the preceding embodiments, wherein the modification is a base mutation, preferably an A→G or C→T base mutation.

[0383] 63. A cell or progeny thereof from a method according to any one of the preceding embodiments, which comprises a modification not present in a cell that has not been subjected to said method.

[0384] 64. The cell or progeny thereof of any one of the preceding embodiments, wherein the cell not subjected to the method contains an abnormality and the abnormality in the cell by the method has already been resolved or corrected.

[0385] 65. A cellular product from a cell according to any one of the preceding embodiments or a progeny thereof, which is modified relative to the nature or amount of the cellular product from a cell not subjected to the method.

[0386] 66. A cell product described in any one of the preceding embodiments, wherein a cell that has not been subjected to the method contains an abnormality and the cell product reflects that the abnormality has already been resolved or corrected by the method.

[0387] 67. A method for non-specifically cleaving non-target DNA, comprising contacting the target DNA with a CRISPR-Cas system or complex described in any one of the preceding embodiments, thereby hybridizing the spacer region with a target sequence of the target DNA and cleaving the target sequence by the Cas12i protein, whereby the Cas12i protein cleaves the non-target DNA by spacer region non-specific endonuclease bypass activity.

[0388] 68. A method for detecting target DNA in a sample, comprising: The sample is contacted with the CRISPR-Cas system or complex according to any one of the above-mentioned embodiments and a reporter nucleic acid capable of emitting a detectable signal after cleavage, whereby the spacer region is hybridized with a target sequence of the target DNA and the target sequence is cleaved by the Cas12i protein, whereby the Cas12i protein cleaves the reporter nucleic acid by spacer region non-specific endonuclease bypass activity; detecting the presence of said target DNA in said sample by measuring a detectable signal produced by cleavage of said reporter nucleic acid.

[0389] 69. A method according to any one of the preceding embodiments, wherein the method further comprises comparing the level of the detectable signal with a level of a reference signal, and determining the level of the target DNA in the sample based on the level of the detectable signal.

[0390] 70. The method of any one of the preceding embodiments, wherein the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloidal phase transition / dispersion, electrochemical detection or semiconductor-based detection.

[0391] 71. The method of any one of the preceding embodiments, wherein the reporter nucleic acid comprises a fluorescent dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher / fluor pair, and wherein cleavage of the reporter nucleic acid by the Cas12i protein increases or decreases the level of the detectable signal produced by cleavage of the reporter nucleic acid.

[0392] 72. A method for treating a condition or disease in a subject in need thereof, comprising administering to the subject a CRISPR-Cas system described in any one of the preceding embodiments.

[0393] 73. The disease or disorder is cancer, an infectious disease, or a neurological disease; Optionally, the cancer is: Selected from Wilms' tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, uterine cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myelocele, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma and bladder cancer, Optionally, the infectious disease is: It is caused by human immunodeficiency virus (HIV), herpes simplex virus-1 (HSV1) and herpes simplex virus-2 (HSV2), Optionally, the neuropathy is: 4. The method of any one of the preceding embodiments, wherein the condition is selected from glaucoma, age-related loss of RGCs, optic nerve damage, retinal ischemia, Leber's hereditary optic neuropathy, a neurological disease associated with degeneration of RGC neurons, a neurological disease associated with degeneration of functional neurons of the striatum in a subject in need thereof, Parkinson's disease, Alzheimer's disease, Huntington's disease, schizophrenia, depression, drug addiction, movement disorders (e.g. chorea, choreoid symptoms and dyskinesia), bipolar disorder, autism spectrum disorder (ASD) or dysfunction.

[0394] 74. The method of any one of the preceding embodiments, wherein the disease or disorder is selected from cystic fibrosis, progressive pseudohypertrophic muscular dystrophy, Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, fragile X syndrome, Friedreich ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, hypercholesterolemia, Leber congenital amaurosis, sickle cell disease, and beta thalassemia.

[0395] 75. A method according to any one of the preceding embodiments, wherein the disease or condition is caused by the presence of a pathogenic point mutation.

[0396] 76. A kit comprising the CRISPR-Cas system of any one of the preceding embodiments, preferably with the components of the system in the same container or in separate containers.

[0397] 77. A sterile container comprising the CRISPR-Cas system of any one of the preceding embodiments, preferably wherein the sterile container is a syringe.

[0398] 78. An implantable device comprising the CRISPR-Cas system of any one of the preceding embodiments, preferably wherein the CRISPR-Cas system is stored in a reservoir. Bypass activity

[0399] The Cas12i protein may have bypass activity, i.e., under some conditions, the activated Cas12i protein remains active after binding to the target sequence and continues to non-specifically cleave non-target oligonucleotides. Such bypass activity allows the Cas12i system to be used to detect the presence of a specific target oligonucleotide. In one embodiment, the Cas12i system is engineered to non-specifically cleave ssDNA or transcripts. In some embodiments, Cas12i is provided or expressed transiently or stably in an in vitro system or cell and targeted or induced to non-specifically cleave cellular nucleic acid, e.g., ssDNA, e.g., viral ssDNA. In some embodiments, the Cas12i protein described herein is modified to reduce (e.g., reduce by at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or eliminate the spacer region non-specific endonuclease cleavage activity. In some embodiments, the Cas12i proteins described herein essentially lack (e.g., at least about any one of 50%, 60%, 70%, 80%, 90%, 95% or 100%) the spacer region non-specific endonuclease bypass activity of a parent / reference Cas12i protein (e.g., a Cas12i protein of any one of SEQ ID NOs: 1-10) against non-target DNA.

[0400] Bypass activity has recently been exploited in a highly sensitive and specific nucleic acid detection platform called SHERLOCK, which may be used in many clinical diagnostics (Gootenberg, JS et al., Nucleic acid detection with CRISPR-Cas13a / C2c2. Science 356, 438-442 (2017)). Reporter Nucleic Acid

[0401] A "reporter nucleic acid" is a molecule that can be cleaved or inactivated by an activated CRISPR system protein as described herein. A reporter nucleic acid comprises a nucleic acid element that can be cleaved by a CRISPR protein. Cleavage of the nucleic acid element releases an agent or causes a conformational change, thereby producing a detectable signal. The reporter nucleic acid prevents the production or detection of a positive detectable signal before cleavage or when the reporter nucleic acid is in an "active" state. It should be understood that in some exemplary embodiments, minimal background signal can be produced in the presence of an active reporter nucleic acid. A positive detectable signal can be any signal that is detectable using optical, fluorescent, chemiluminescent, electrochemical methods, or other detection methods known in the art. For example, in some embodiments, a first signal (i.e., a negative detectable signal) can be detected when the reporter nucleic acid is present, and a target molecule is detected and the reporter nucleic acid is cleaved or inactivated by an activated CRISPR protein, and the first signal is converted to a second signal (e.g., a positive detectable signal). Functional domains

[0402] A functional domain is used in its broadest sense and includes a protein, such as an enzyme or factor itself or a specific functional fragment (domain) thereof.

[0403] The Cas12i protein (e.g., dCas12i) is associated with one or more functional domains selected from a deaminase (e.g., adenosine deaminase or cytidine deaminase) catalytic domain, a DNA methylation catalytic domain, a DNA demethylation catalytic domain, a histone residue modification domain, a nuclease catalytic domain, a fluorescent protein, a transcriptional modifier (e.g., a transcriptional activation catalytic domain, a transcriptional inhibition catalytic domain), a nuclear localization signal (NLS), a nuclear export signal (NES), an optical gating factor, a chemical inducer, or a chromatin visualization factor, preferably the functional domain is selected from an adenosine deaminase catalytic domain or a cytidine deaminase catalytic domain.

[0404] In some embodiments, the functional domain may be a transcription activation domain. In some embodiments, the functional domain is a transcription inhibition domain. In some embodiments, the functional domain is an epigenetic modification domain, thereby providing an epigenetic modification enzyme. In some embodiments, the functional domain is an activation domain. In some embodiments, the Cas12i protein is associated with one or more functional domains, and the Cas12i protein contains one or more mutations in the RuvC domain, and the resulting CRISPR complex can deliver epigenetic modifications or transcribe or translate activation or inhibition signals.

[0405] In some embodiments, the functional domain exhibits an activity that modifies a target DNA or a protein associated with a target DNA, wherein the activity is a nuclease activity (e.g., HNH nuclease, RuvC nuclease, Trex1 nuclease, Trex2 nuclease), a methylation activity, a demethylation activity, a DNA repair activity, a DNA damage activity, a deamination activity, a dismutase activity, an alkylation activity, a depurination activity, an oxidation activity, a pyrimidine dimer formation activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a lig ... The target DNA-associated protein is one or more selected from the group consisting of adenase activity, helicase activity, photolyase activity, glycosidase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity (e.g., from O-GlcNAc transferase), deglycosylation activity, transcription inhibitory activity, and transcription activating activity. Target DNA-associated proteins include, but are not limited to, proteins capable of binding to target DNA or proteins capable of binding to proteins bound to target DNA, such as histones, transcription factors, mediators, and the like.

[0406] The functional domain may be, for example, a domain having one or more selected from methylase activity, demethylase activity, transcription activation activity, transcription inhibition activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switch (e.g., light induction). When one or more functional domains are included, these functional domains may be the same or different. BasesEdit

[0407] In some exemplary embodiments, Cas12i (e.g., dCas12i) may be fused to adenosine deaminase or cytidine deaminase for base editing purposes. Adenosine deaminase

[0408] As used herein, the term "adenosine deaminase" or "adenosine deaminase protein" refers to a protein, polypeptide, or one or more functional domains of a protein or polypeptide that can catalyze a hydrolytic deamination reaction to convert adenine (or the adenine portion of a molecule) to hypoxanthine (or the hypoxanthine portion of a molecule), as shown below. In some embodiments, the adenine-containing molecule is adenosine (A) and the hypoxanthine-containing molecule is inosine (I). The adenine-containing molecule may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0409] According to the present disclosure, adenosine deaminases that may be used in combination with the present disclosure include, but are not limited to, members of the enzyme family called adenosine deaminases acting on RNA (ADAR), adenosine deaminases acting on tRNA (ADAT), and other family members that contain an adenosine deaminase domain (ADAD). According to the present disclosure, adenosine deaminases can target adenines in RNA / DNA and RNA duplexes. Indeed, Zheng et al. (Nucleic Acids Res. 2017, 45(6):3369-3377) have demonstrated that ADARs can edit adenosines to inosines in RNA / DNA and RNA / RNA duplexes. In certain embodiments, as described in detail below, adenosine deaminases are modified to increase their ability to edit DNA in RNA / DNA heteroduplexes of RNA duplexes.

[0410] In some embodiments, the adenosine deaminase is from one or more metazoan species, including but not limited to mammals, birds, frogs, squid, fish, flies and worms, hi some embodiments, the adenosine deaminase is human, squid or Drosophila adenosine deaminase.

[0411] In some embodiments, the adenosine deaminase is a human ADAR, including hADAR1, hADAR2, and hADAR3. In some embodiments, the adenosine deaminase is a Caenorhabditis elegans ADAR protein, including ADR-1 and ADR-2. In some embodiments, the adenosine deaminase is a Drosophila ADAR protein, including dAdar. In some embodiments, the adenosine deaminase is a squid (Loligo pealeii) ADAR protein, including sqADAR2a and sqADAR2b. In some embodiments, the adenosine deaminase is a human ADAT protein. In some embodiments, the adenosine deaminase is a Drosophila ADAT protein. In some embodiments, the adenosine deaminase is a human ADAD protein, including TENR (hADAD1) and TENRL (hADAD2).

[0412] In some embodiments, the adenosine deaminase is a TadA protein, e.g., E. coli TadA. See Kim et al., Biochemistry 45:6407-6416 (2006); Wolf et al., EMBO J. 21:3841-3851 (2002). In some embodiments, the adenosine deaminase is mouse ADA. See Grunebaum et al., Curr. Opin. Allergy Clin. Immunol. 13:630-638 (2013). In some embodiments, the adenosine deaminase is human ADAT2. See Fukui et al., J. Nucleic Acids 2010:260512 (2010). In some embodiments, the deaminase (e.g., an adenosine deaminase or a cytidine deaminase) is one or more of those deaminases described in the following references: Cox et al., Science. 2017 Nov. 24; 358(6366):1019-1027; Komore et al., Nature. 2016 May 19; 533(7603):420-4; and Gaudelli et al., Nature. 2017 Nov. 23; 551(7681):464-471.

[0413] In some embodiments, the adenosine deaminase protein recognizes one or more target adenosine residues in a double-stranded nucleic acid substrate and converts them to inosine residues. In some embodiments, the double-stranded nucleic acid substrate is an RNA-DNA heteroduplex. In some embodiments, the adenosine deaminase protein recognizes a binding window on the double-stranded substrate. In some embodiments, the binding window comprises at least one target adenosine residue. In some embodiments, the binding window is in the range of about 3 bp to about 100 bp. In some embodiments, the binding window is in the range of about 5 bp to about 50 bp. In some embodiments, the binding window is in the range of about 10 bp to about 30 bp. In some embodiments, the binding window is about 1 bp, 2 bp, 3 bp, 5 bp, 7 bp, 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, or 100 bp.

[0414] In some embodiments, the adenosine deaminase protein comprises one or more deaminase domains. Without wishing to be bound by any particular theory, it is contemplated that the deaminase domain is used to recognize one or more target adenosine (A) residues contained in a double-stranded nucleic acid substrate and convert them to inosine (I) residues. In some embodiments, the deaminase domain comprises an active center. In some embodiments, the active center comprises a zinc ion. In some embodiments, during AI editing, base pairs at the target adenosine residue are disrupted and the target adenosine residue is "flipped" out of the double helix to become accessible by adenosine deaminase. In some embodiments, amino acid residues at or near the active center interact with one or more nucleotides 5' of the target adenosine residue. In some embodiments, amino acid residues at or near the active center interact with one or more nucleotides 3' of the target adenosine residue. In some embodiments, an amino acid residue at or near the active center also interacts with a nucleotide complementary to the target adenosine residue on the opposite strand, hi some embodiments, the amino acid residue forms a hydrogen bond with the 2' hydroxyl group of the nucleotide.

[0415] In some embodiments, the adenosine deaminase comprises the whole human ADAR2 protein (hADAR2) or its deaminase domain (hADAR2-D). In some embodiments, the adenosine deaminase is a member of the ADAR family that is homologous to hADAR2 or hADAR2-D.

[0416] Specifically, in some embodiments, the homologous ADAR protein is human ADAR1 (hADAR1) or its deaminase domain (hADAR1-D). In some embodiments, glycine 1007 of hADAR1-D corresponds to glycine 487 of hADAR2-D, and glutamic acid 1008 of hADAR1-D corresponds to glutamic acid 488 of hADAR2-D.

[0417] In some embodiments, the adenosine deaminase comprises a wild-type amino acid sequence of hADAR2-D, hi some embodiments, the adenosine deaminase comprises one or more mutations in the hADAR2-D sequence such that the editing efficiency and / or substrate editing preference of hADAR2-D is altered as desired.

[0418] In some embodiments, the adenosine deaminase is TadA8e, e.g., TadA8e comprising the sequence of SEQ ID NO: 182. In some embodiments, a Cas12i protein described herein (e.g., dCas12i) is fused to TadA8e or a functional fragment thereof (i.e., capable of single base editing from A to I). Cytidine deaminase

[0419] In some embodiments, the deaminase is a cytidine deaminase. As used herein, the term "cytidine deaminase" or "cytidine deaminase protein" refers to a protein, polypeptide, or one or more functional domains of a protein or polypeptide that can catalyze a hydrolytic deamination reaction to convert cytosine (or a cytosine portion of a molecule) to uracil (or a uracil portion of a molecule), as shown below. In some embodiments, the cytosine-containing molecule is cytidine (C) and the uracil-containing molecule is uridine (U). The cytosine-containing molecule may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0420] According to the present disclosure, cytidine deaminases that may be used in combination with the present disclosure include, but are not limited to, members of the enzyme family called apolipoprotein B mRNA editing complex (APOBEC) family deaminases, activation-induced deaminases (AIDs) or cytidine deaminase 1 (CDA1), and in the embodiments for carrying out the invention, are deaminases selected from APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, and APOBEC3D deaminase, APOBEC3E deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, or APOBEC4 deaminase.

[0421] In the methods and systems of the invention, the cytidine deaminase can target cytosines in a single strand of DNA. In some exemplary embodiments, the cytidine deaminase can be edited on a single strand that is external to a binding moiety, e.g., bound to Cas13. In other exemplary embodiments, the cytidine deaminase can be edited in a local bubble, e.g., edited in a bubble that is formed at the target editing site but has a guide sequence mismatch. In some exemplary embodiments, the cytidine deaminase can include a mutation that contributes to focusing activity, such as those described in Kim et al., Nature Biotechnology (2017) 35(4):371-377 (doi:10.1038 / nbt.3803).

[0422] In some embodiments, the cytidine deaminase is from one or more metazoan species, including but not limited to mammals, birds, frogs, squid, fish, flies and worms, hi some embodiments, the cytidine deaminase is a human, primate, bovine, canine, rat or mouse cytidine deaminase.

[0423] In some embodiments, the cytidine deaminase is a human APOBEC, including hAPOBEC1 or hAPOBEC3, hi some embodiments, the cytidine deaminase is human AID.

[0424] In some embodiments, the cytidine deaminase protein recognizes one or more target cytosine residues in a single-stranded bubble of an RNA duplex and converts them to uracil residues. In some embodiments, the cytidine deaminase protein recognizes a binding window on the single-stranded bubble of an RNA duplex. In some embodiments, the binding window comprises at least one target cytosine residue. In some embodiments, the binding window is in the range of about 3 bp to about 100 bp. In some embodiments, the binding window is in the range of about 5 bp to about 50 bp. In some embodiments, the binding window is in the range of about 10 bp to about 30 bp. In some embodiments, the binding window is about 1 bp, 2 bp, 3 bp, 5 bp, 7 bp, 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, or 100 bp.

[0425] In some embodiments, the cytidine deaminase protein comprises one or more deaminase domains. Without wishing to be bound by theory, it is contemplated that the deaminase domain is used to recognize one or more target cytosine (C) residues contained in a single-stranded bubble of an RNA duplex and convert them to uracil (U) residues. In some embodiments, the deaminase domain comprises an active center. In some embodiments, the active center comprises a zinc ion. In some embodiments, amino acid residues in or near the active center interact with one or more nucleotides 5' to the target cytosine residue. In some embodiments, amino acid residues in or near the active center interact with one or more nucleotides 3' to the target cytosine residue.

[0426] In some embodiments, the cytidine deaminase comprises the human APOBEC1 whole protein (hAPOBEC1) or its deaminase domain (hAPOBEC1-D) or its C-terminally truncated form (hAPOBEC-T). In some embodiments, the cytidine deaminase is a member of the APOBEC family homologous to hAPOBEC1, hAPOBEC-D, or hAPOBEC-T. In some embodiments, the cytidine deaminase comprises the human AID1 whole protein (hAID) or its deaminase domain (hAID-D) or its C-terminally truncated form (hAID-T). In some embodiments, the cytidine deaminase is a member of the AID family homologous to hAID, hAID-D, or hAID-T. In some embodiments, hAID-T is hAID truncated by about 20 amino acids at the C-terminus.

[0427] In some embodiments, the cytidine deaminase comprises a wild-type amino acid sequence of a cytosine deaminase, hi some embodiments, the cytidine deaminase comprises one or more mutations in the cytosine deaminase sequence such that the editing efficiency and / or substrate editing preference of the cytosine deaminase is altered as desired.

[0428] As used herein, "associated" is used in its broadest sense and covers the situation where two of the functional modules directly or indirectly (through a linker) form a fusion protein, and where two of the functional modules are independently linked by covalent (e.g., disulfide) or non-covalent bonds.

[0429] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted to effect replication of the inserted segment. Generally, when combined with the proper control elements, a vector is capable of replication.

[0430] In some cases, the vector system comprises a single vector. Alternatively, the vector system comprises multiple vectors. The vector may be a viral vector.

[0431] Vectors include, but are not limited to, single-stranded, double-stranded or partially double-stranded nucleic acid molecules, nucleic acid molecules with one or more free ends or no free ends (e.g., circular), nucleic acid molecules containing DNA, RNA or both, and other polynucleotide variants known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA circle into which other DNA segments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which there are DNA or RNA sequences from a virus for packaging into a virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses and adeno-associated viruses). Viral vectors further include polynucleotides carried by the virus for transfection into a host cell. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and replicated along with the host genome after introduction of these vectors into a host cell. Some vectors can also direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." Vectors that are expressed in eukaryotic cells and vectors that cause expression in eukaryotic cells may be referred to herein as "eukaryotic expression vectors." Common expression vectors that can be used in recombinant DNA techniques are usually in the form of plasmids.

[0432] A recombinant expression vector may comprise the nucleic acid of the present invention in a form suitable for expression in a host cell, which means that the recombinant expression vector comprises one or more regulatory elements, which regulatory elements can be selected according to the host cell for expression, and the nucleic acid is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory element in a manner that allows expression of the nucleotide sequence (e.g., expressed in an in vitro transcription / translation system or expressed in a host cell when the vector is introduced into the host cell). Advantageous vectors include lentiviruses and adeno-associated viruses, and these types of vectors can also be selected to target specific types of cells.

[0433] The term "regulatory elements" is intended to include promoters, enhancers, internal ribosome entry sites (IRES) and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those elements that direct constitutive expression of a nucleotide sequence in many types of host cells and those elements that direct expression of a nucleotide sequence only in some host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired target tissue, such as muscle, neurons, bone, skin, blood, a specific organ (e.g., liver, pancreatic gland) or a specific cell type (e.g., lymphocytes). Regulatory elements may also direct expression in a time-dependent manner, such as a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type specific.

[0434] In some embodiments, the vector encodes a Cas12i protein that includes one or more nuclear localization sequences (NLSs), such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. More particularly, the vector includes one or more NLSs that are not naturally present in the Cas12i protein. Most particularly, the NLSs are present 5' and / or 3' of the vector to the Cas12i protein sequence. In some embodiments, the protein of the targeting RNA includes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the amino terminus and about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the carboxyl terminus, or a combination thereof (e.g., zero or at least one or more NLSs at the amino terminus and zero or one or more NLSs at the carboxyl terminus). When more than one NLS is present, each of them may be selected independently of the others, such that a single NLS can be present in one or more copies and / or in one or more copies in combination with one or more other NLSs. In some embodiments, an NLS is considered to be attached to the N-terminus or C-terminus if the nearest amino acid to the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more amino acids along the polypeptide chain from the N-terminus or C-terminus.

[0435] "Codon optimization" refers to a method of modifying a nucleic acid sequence in a target host cell to enhance expression while maintaining the native amino acid sequence by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the native sequence with a codon that is more frequently or most frequently used in the host cell's genes. Several species show a specific bias for some codons of specific amino acids. Codon bias (differences in codon usage in organisms) is usually related to the translation efficiency of messenger RNA (mRNA), which is believed to depend on the characteristics of the translated codon and the availability of specific transfer RNA (tRNA) molecules. The dominance of the tRNA selected in a cell usually reflects the codons most commonly used in peptide synthesis. Therefore, genes can be customized to optimize gene expression in a given organism based on codon optimization. Codon usage tables are easy to obtain, for example from the "Codon Usage Database" at www.kazusa.orjp / codon / , and can be modified in various ways. See Nakamura, Y. et al., "Codon usage tabulated from the international DNA Sequence databases: status for the year 2000," Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimization of a particular sequence to be expressed in a particular host cell are also available, e.g., Gene Forge (Aptagen, Jacobus, PA). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more or all codons) in the sequence encoding the DNA / RNA targeting the Cas protein correspond to the most commonly used codon for a particular amino acid.For codon usage in yeast, see the online Yeast Genome Database available at www.yeastgenome.org / community / codon_usage.shtml, or Codon selection in yeast, Bennetzen and Hall, J Biol Chem. 1982 Mar. 25;257(6):3026-31. For codon usage in plants (including algae), see Codon usage in higher plants, green algae, and cyanobacteria, Campbell and Gowri, Plant Physiol. 1990 Jan;92(1):1-11., and Codon usage in plant genes, Murray et al., Nucleic Acids Res. 1989 Jan 25;17(2):477-98, or Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages, Morton BR, J Mol Evol. 1998 Apr;46(4):449-59. Delivery System

[0436] In some embodiments, the components of the CRISPR-Cas system may be delivered in various formats, such as DNA / RNA or RNA / RNA or protein-RNA combinations. For example, the Cas12i protein may be delivered as a DNA-encoding polynucleotide or an RNA-encoding polynucleotide, or as a protein. The guide may be delivered as a DNA- or RNA-encoding polynucleotide. All possible combinations are contemplated, including mixed delivery formats.

[0437] According to some aspects, the present invention provides methods for delivering one or more polynucleotides, such as one or more vectors described herein, one or more transcripts thereof, and / or one or more proteins transcribed therefrom, to a host cell.

[0438] In some embodiments, expression of the elements of the nucleic acid targeting system guides the formation of a nucleic acid targeting complex at one or more target sites by introducing one or more vectors driving the expression of one or more elements of the nucleic acid targeting system into a host cell. For example, the nucleic acid encoding the effector enzyme and the nucleic acid encoding the guide RNA can each be operably linked to a single regulatory element on a single vector. The RNA of the nucleic acid targeting system can be delivered to a transgenic nucleic acid targeting effector protein animal or mammal, such as an animal or mammal that constitutively, inducibly or conditionally expresses the nucleic acid targeting effector protein, or an animal or mammal that otherwise expresses the nucleic acid targeting effector protein or has cells that contain the nucleic acid targeting effector protein, for example by prior administration thereto of one or more vectors that encode and express the in vivo nucleic acid targeting effector protein. Alternatively, two or more elements regulated by the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the nucleic acid targeting system not included in the first vector. The elements of the nucleic acid targeting system combined in a single vector may be arranged in any suitable orientation, for example, one element may be located 5' ("upstream") to the second element or 3' ("downstream") to the second element. The coding sequence of one element may be on the same or opposite strand to the coding sequence of the second element and oriented in the same or opposite orientation. In some embodiments, a single promoter drives expression of transcripts encoding the nucleic acid targeting effector protein and the nucleic acid targeting guide RNA, and the transcripts are embedded in one or more intronic sequences (e.g., each in a single intron, two or more in at least one intron, or all in a single intron).In some embodiments, the nucleic acid targeting effector protein and the nucleic acid targeting guide RNA may be operably linked to and expressed by the same promoter. Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expressing one or more elements of the nucleic acid targeting system are as described in previous documents, e.g., WO2014 / 093622 (PCT / US2013 / 074667, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the vector comprises one or more insertion sites, e.g., restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct may be used to target nucleic acids to a variety of relevant target sequences in active target cells. For example, a single vector may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more guide sequences. In some embodiments, a vector containing about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more such guide sequences may be provided and, optionally, delivered to a cell. In some embodiments, the vector comprises a regulatory element operably linked to an enzyme coding sequence encoding a nucleic acid targeting effector protein. The nucleic acid targeting effector protein or one or more nucleic acid targeting guide RNAs may be delivered alone, and advantageously, at least one of them is delivered via a particle complex. The nucleic acid targeting effector protein mRNA may be delivered before the nucleic acid targeting guide RNA to allow time for expression of the nucleic acid targeting effector protein. The nucleic acid targeting effector protein mRNA may be administered 1-12 hours (preferably about 2-6 hours) before administration of the nucleic acid targeting guide RNA.Alternatively, the nucleic acid targeting effector protein mRNA and the nucleic acid targeting guide RNA may be administered together. Advantageously, a second booster dose of guide RNA may be administered 1-12 hours (preferably about 2-6 hours) after the first administration of the nucleic acid targeting effector protein mRNA + guide RNA. Additional doses of nucleic acid targeting effector protein mRNA and / or guide RNA may be used to achieve the most effective level of genome modification.

[0439] Common viral or non-viral based gene transfer methods may be used to introduce nucleic acids into mammalian cells or target tissues. Such methods may be used to administer nucleic acids encoding components of a nucleic acid targeting system to cells in culture or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have episomal or integrated genomes after delivery to cells. For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel and Felgner, TIBTECH 11:211-217 (1993); Mitani and Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); and Kremer and Perricaudet, British Medical Journal. Bulletin 51(1):31-44 (1995), Haddada et al., Current Topics in Microbiology and Immunology, Doerfler and Bohm (eds.) (1995), and Yu et al., Gene Therapy 1:13-26 (1994).

[0440] Non-viral methods of delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virions, liposomes, immunoliposomes, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, and reagent-assisted DNA uptake. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for effective receptor-recognition lipofection of polynucleotides include those of Felgner, WO91 / 17424, WO91 / 16024, and it is contemplated that these lipids may be delivered to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

[0441] Plasmid delivery involves cloning guide RNA into a plasmid expressing CRISPR-Cas proteins and transfecting the DNA in cell culture. Plasmid backbones are commercially available and do not require specific equipment. Advantageously, they are modular and can carry different sizes of CRISPR-Cas coding sequences (including sequences of proteins coding for larger sizes), and selection markers. Plasmids are also advantageous because they ensure transient but continuous expression. However, plasmid delivery is not direct and usually causes low in vivo efficiency. Continuous expression can also be a disadvantage, since it can increase off-target editing. Excessive accumulation of CRISPR-Cas proteins can also be toxic to cells. Finally, plasmids always carry the risk of randomly integrating dsDNA into the host genome, and more specifically, consider the risk of double-strand breaks (on-target and off-target).

[0442] The preparation of lipid:nucleic acid complexes (including targeted liposomes, e.g., immunolipid complexes) is well known to those of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. [Cancer Research] 52:4817-4820 (1992); see U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028 and 4,946,787, as discussed in more detail below.

[0443] Delivering nucleic acids using RNA or DNA virus-based systems takes advantage of the highly evolved process of targeting viruses to specific cells in vivo and transporting the viral payload to the cell nucleus. Viral vectors may be administered directly to patients (in vivo) or they may be used to treat cells in vitro and, optionally, modified cells (ex vivo) may be administered to patients. Common virus-based systems may include retroviral, lentiviral, adenoviral, adeno-associated viral and herpes simplex viral vectors for gene transfer. Integration into the host genome by retroviral, lentiviral and adeno-associated viral gene transfer methods always results in long-term expression of the inserted transgenic. Also, high transduction efficiency is observed in many different cell types and target tissues.

[0444] The tropism of retroviruses may be modified by the incorporation of exogenous envelope proteins to expand the potential target population of target cells. Lentiviral vectors are retroviral vectors that transduce or infect non-dividing cells and usually produce high viral titers. Therefore, the choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats of foreign sequences with a packaging capacity of up to 6-10 kb. The minimal cis-acting LTRs are sufficient for vector replication and packaging, and the vector is used to integrate therapeutic genes into target cells to provide permanent transgenic expression. Widely used retroviral vectors include vectors based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. [Journal of Virology] 66:2731-2739 (1992); Johann et al., J. Virol. [Journal of Virology] 66:1635-1640 (1992); Sommnerfelt et al., Virol. [Journal of Virology] 176:58-59 (1990); Wilson et al., J. Virol. [Journal of Virology] 63:2374-2378 (1989); Miller et al., J. Virol. [Journal of Virology] 65:2220-2224 (1991); PCT / US94 / 05700).

[0445] Preferably, for transient expression applications, adenovirus-based systems can be used. Adenovirus-based vectors provide high transduction efficiency in many cell types and do not require cell division. High titers and high expression levels have already been achieved using such vectors. The vectors can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors may be used, for example, in the in vitro production of nucleic acids and peptides, and to transduce target nucleic acids into cells in in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994)). The construction of recombinant AAV vectors has been described in many publications, including U.S. Pat. No. 5,173,414, Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985), Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984), Hermonat and Muzyczka, PNAS 81:6466-6470 (1984), and Samulski et al., J. Virol. 63:03822-3828 (1989).

[0446] The present invention provides an AAV, the AAV comprising or consisting essentially of an exogenous nucleic acid molecule encoding a CRISPR system, the exogenous nucleic acid molecule being, for example, a plurality of cassettes comprising or consisting of the following cassettes: a first cassette comprising or consisting of a promoter, a nucleic acid molecule encoding a CRISPR-associated (Cas) protein (putative nuclease or helicase protein) (e.g., Cas12i) and a terminator, and one or more cassettes, advantageously up to the packaging size limit of the vector, for example five cassettes in total (including the first cassette), the cassettes comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a guide RNA (gRNA) and a terminator (for example, each cassette may be arranged as promoter-gRNA1-terminator, promoter-gRNA2-terminator... promoter-gRNA(N)-terminator). where N is the upper limit of the packaging size limit of the insertable vector), or the invention provides two or more single rAAVs, each of which contains one or more cassettes of a CRISPR system, e.g., a first rAAV contains a first cassette, the first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a Cas, e.g., Cas (Cas12i), and a terminator, and a second rAAV contains one or more cassettes, each of which comprises or consists essentially of a promoter, a nucleic acid molecule encoding a guide RNA (gRNA), and a terminator (e.g., each cassette is represented diagrammatically as promoter-gRNA1-terminator, promoter-gRNA2-terminator... promoter-gRNA(N)-terminator, where N is the upper limit of the packaging size limit of the insertable vector). Alternatively, since Cas12i can process its own crRNA / gRNAs, a single crRNA / gRNA array may be used for multiplex gene editing.Thus, rather than comprising multiple cassettes for delivering gRNAs, rAAV may contain a single cassette that comprises or is composed of a promoter, multiple crRNAs / gRNAs, and a terminator (e.g., represented diagrammatically as promoter-gRNA1-gRNA2...gRNA(N)-terminator, where N is the upper packaging size limit of the insertable vector). See Zetsche et al., Nature Biotechnology 35, 31-34 (2017), which is incorporated herein by reference in its entirety. Since rAAVs are DNA viruses, the nucleic acid molecules in the AAV or rAAV discussion herein are advantageously DNA. In some embodiments, the promoter is advantageously the human synaptophysin I promoter (hSyn). Other methods of delivering nucleic acids to cells are known to those of skill in the art. See, for example, US20030087817, which is incorporated herein by reference.

[0447] In another embodiment, cocal vesiculovirus enveloped pseudoretroviral vector particles are contemplated (see, e.g., U.S. Patent Disclosure No. 20120164118, assigned to Fred Hutchinson Cancer Research Center). Cocalvirus belongs to the genus Cocal vesiculovirus and is the causative agent of vesicular stomatitis in mammals. Cocalvirus was originally isolated from ticks in Trinidad (Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964)), and cocalvirus infections have been identified in insects, cattle and horses in Trinidad, Brazil and Argentina. Many cocal vesiculoviruses that infect mammals have already been isolated from naturally infected arthropods, indicating that they are vector-transmitted. Antibodies to varicella virus are widespread in rural areas and laboratories that have acquired the virus locally, and their infection in humans usually results in influenza-like symptoms. The envelope glycoprotein of Cocalvirus has 71.5% identity at the amino acid level with VSV-G Indiana strain, and phylogenetic comparison of the varicella virus envelope gene indicates that Cocalvirus is serologically distinct from, but most closely related to, VSV-G Indiana strain of Varicella virus. Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964) and Travassos da Rosa et al., Am. J. Tropical Med. & Hygiene 33:999-1006 (1984). Cocal vesicle virus envelope pseudo-retroviral vector particles may include, for example, lentivirus, alpha retrovirus, beta retrovirus, gamma retrovirus, delta retrovirus and epsilon retrovirus vector particles, which may comprise retroviral Gag, Pol and / or one or more accessory proteins and Cocal vesicle virus envelope protein.In some aspects of these embodiments, the Gag, Pol and accessory proteins are lentivirus and / or gamma retrovirus.

[0448] In some embodiments, host cells are transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, cells are transfected and optionally reintroduced into a subject if the cells are naturally present in the subject. In some embodiments, the transfected cells are taken from the subject. In some embodiments, the cells are derived from cells, e.g., cell lines, from the subject. Various cell lines used for tissue culture are known in the art. Examples of cell lines are C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375 , ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB 55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial cells, BALB / 3T3 mouse fetal fibroblasts, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts, 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B 16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR293, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR / 0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR and transgenic varieties thereof. Cell lines are available from a variety of sources known to those of skill in the art (see, e.g., American Type Culture Collection (ATCC), Manassus, Va.).

[0449] In certain embodiments, the transient expression and / or presence of one or more components of the AD-functionalized CRISPR system may be of interest, for example, to reduce off-target effects. In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines that contain sequences derived from one or more vectors. In some embodiments, cells that have been transiently transfected with components of the AD-functionalized CRISPR system described herein (e.g., transiently transfected with one or more vectors or transfected with RNA) and modified by the activity of the CRISPR complex are used to establish new cell lines that include cells that contain these modifications but without any other exogenous sequences. In some embodiments, cells that are transiently or non-transiently transfected with one or more vectors described herein or cell lines derived from such cells are used to evaluate one or more test compounds.

[0450] In some embodiments, it is contemplated that RNA and / or proteins may be directly introduced into the host cell. For example, CRISPR-Cas proteins may be delivered as encoded mRNA with guide RNA from in vitro transcription. Such methods can reduce and ensure the action time of CRISPR-Cas proteins and further prevent long-term expression of components of the CRISPR system.

[0451] In some embodiments, the RNA molecules of the invention can be delivered in liposomal or lipofectin formulations, and can be prepared by methods well known to those of skill in the art, such as those described in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are incorporated herein by reference in their entireties. Specifically designed delivery systems have been developed to enhance and improve siRNA delivery into mammalian cells (see, e.g., Shen et al., FEBS Let. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010; Reich et al., Mol. Vision. 2003, 9:210-216; Sorensen et al., J. Mol. Biol. 2003, 327:761-766; Lewis et al., Nat. Gen. 2002, 32:107-108; and Simeoni et al., NAR [Nucleic Acids Research] 2003, 31, 11:2717-2724) and are applicable to the present invention. siRNA has recently been used successfully to inhibit gene expression in primates (see, for example, Tolentino et al., Retina 24(4):660), and is also applicable to the present invention.

[0452] Indeed, RNA delivery is a useful in vivo delivery method. Liposomes or particles can be used to deliver Cas12i, adenosine deaminase and guide RNA to cells. Therefore, the delivery of the CRISPR-Cas protein (e.g., Cas12i), adenosine deaminase (which can be fused to the CRISPR-Cas protein or adapter protein) and / or RNA of the present invention can be in the form of RNA and can be carried out via microvesicles, liposomes or particles or nanoparticles. For example, for in vivo delivery, Cas12i mRNA, adenosine deaminase mRNA and guide RNA are packaged into liposomal particles. Liposomal transfection reagents, such as lipofectamine from Life Technologies and other reagents on the market, can efficiently deliver RNA molecules to the liver. In some embodiments, the lipid nanoparticles (LNPs) comprise ALC-0315:cholesterol:PEG-DMG:DOPE in a molar ratio of 50 mM:50 mM:10 mM:20 mM. In some embodiments, the LNPs encapsulate Cas12i and its corresponding crRNA (e.g., SiCas12i:crRNA, 1:1 weight ratio) or its encoding nucleic acid. In some embodiments, the LNPs comprising Cas12i and / or crRNA (or its encoding nucleic acid) are administered to an individual (e.g., a human) by intravenous infusion.

[0453] Delivery of RNA is also preferably via particles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticles for small interfering RNA delivery to endothelial cells, Advanced Functional Materials, 19:3112-3118, 2010) or exosomes (Schroeder, A., Levins, C., Cortez, C., Langer, R. and Anderson, D., Lipid-based nanotherapeutics for siRNA delivery, Journal of Internal Medicine, 19:3112-3118, 2010). Medicine, 267:9-21, 2010, PMID:20059641). In fact, it has been shown that exosomes are particularly useful for siRNA delivery, and that the system has some similarities to the CRISPR system. For example, El-Andaloussi S et al. ("Exosome-mediated delivery of siRNA in vitro and in vivo." Nat Protoc. 2012 December;7(12):2112-26. doi:10.1038 / nprot.2012.131. Electronically available on November 15, 2012) described how exosomes are a promising tool for drug delivery to cross different biological barriers and for siRNA delivery in vitro and in vivo. Their method involves producing targeted exosomes by transfecting an expression vector containing an exosomal protein fused to a peptide conformation, purifying and characterizing exosomes from transfected cell supernatants, and loading RNA into the exosomes.Delivery or administration according to the present invention can be performed using exosomes, particularly (but not limited to) to the brain. Vitamin E (α-tocopherol) can be combined with CRISPR Cas and delivered to the brain with high density lipoprotein (HDL), similar to how Uno et al. (HUMAN GENE THERAPY 22:711-719 (June 2011)) delivers short interfering RNA (siRNA) to the brain. Mice are injected via osmotic minipumps (model 1007D, Alzet, Cupertino, CA) loaded with phosphate buffered saline (PBS) or free TocsiBACE or Toc-siBACE / HDL and connected to a Brain Infusion Kit 3 (Alzet). A brain infusion cannula is placed about 0.5 mm posterior to the bregma at the midline to inject into the dorsal third ventricle. Uno et al. found that Toc-siRNA containing as low as 3 nmol of HDL can induce large target reduction by the same ICV injection method. In the present invention, for humans, a similar dose of CRISPR Cas can be considered, for example, about 3 nmol to about 3 μMol of CRISPR Cas, which is combined with α-tocopherol and administered with HDL to target the brain. Zou et al. (HUMAN GENE THERAPY 22:465-475 (April 2011)) described a lentivirus-mediated delivery method of short hairpin RNA targeting PKCγ for in vivo gene silencing in rat spinal cord. Zou et al. administered about 10 μl of recombinant lentivirus via an intrathecal catheter, with a titer of 1×10. 9 In the present invention, a similar dose of CRISPR Cas expressed in a lentiviral vector targeted to the brain can be considered for humans, for example, about 10-50 ml of lCRISPR Cas targeted to the brain in a lentivirus can be considered, with a titer of 1×10 9 Transducing units (TU) / ml.

[0454] Other suitable modifications and variations of the methods of the invention described herein will be apparent to those skilled in the art and can be made, using appropriate equivalents, without departing from the scope of the invention or the embodiments disclosed herein. Illustrative Embodiments

[0455] Embodiment 1. A Cas12i protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10 (preferably SEQ ID NOs:1-3, 6 and 10, and more preferably SEQ ID NO:1).

[0456] Embodiment 2. The Cas12i protein described in any one of the preceding embodiments, wherein the Cas12i protein essentially does not have the spacer region-specific endonuclease cleavage activity (e.g., less than 50%, 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2.5%, 2%, 1% or less) of the corresponding parent Cas12i protein (e.g., a Cas12i protein having any one of SEQ ID NOs: 1 to 10) against a target sequence of a target DNA complementary to a guide sequence.

[0457] Embodiment 3. The Cas12i protein according to any one of the preceding embodiments, wherein the Cas12i protein comprises one or more amino acid mutations in its RuvC domain such that the Cas12i protein essentially does not have the spacer region-specific endonuclease cleavage activity (e.g., retains less than or equal to 50%, 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2.5%, 2%, 1%) of a corresponding parent Cas12i protein (e.g., a Cas12i protein comprising any one of SEQ ID NOs: 1-10) against a target sequence of a target DNA complementary to a guide sequence.

[0458] Embodiment 4. The Cas12i protein of any one of the preceding embodiments, wherein the amino acid mutation is selected from addition, insertion, deletion and substitution of amino acids.

[0459] Embodiment 5. The Cas12i protein of any one of the preceding embodiments, wherein the Cas12i protein comprises an amino acid substitution at one or more positions corresponding to positions 700 (D700), 650 (D650), 875 (E875) or 1049 (D1049) of the sequence shown in SEQ ID NO:1.

[0460] Embodiment 6. The Cas12i protein of any one of the preceding embodiments, wherein the amino acid substitutions are selected from D700A / V, D650A / V, E875A / V and D1049A / V.

[0461] Embodiment 7. The Cas12i protein of any one of the preceding embodiments, wherein the amino acid substitutions are selected from D700A, D650A, E875A and D1049A.

[0462] Embodiment 8. The Cas12i protein of any one of the preceding embodiments, wherein the amino acid substitutions are selected from D700A, D650A, E875A, D1049A, D700A+D650A, D700A+E875A, D700A+D1049A, D650A+E875A, D650A+D1049A, E875A+D1049A, D700A+D650A+E875A, D700A+D650A+D1049A, D650A+E875A+D1049A and D700A+D650A+E875A+D1049A.

[0463] Embodiment 10. A Cas12i protein according to any one of the preceding embodiments, wherein the Cas12i protein is linked to one or more functional domains.

[0464] Embodiment 11. A Cas12i protein according to any one of the preceding embodiments, wherein the functional domain is linked to the N-terminus and / or C-terminus of the Cas12i protein.

[0465] Embodiment 12. The Cas12i protein according to any one of the preceding embodiments, wherein the functional domain is selected from a nuclear localization signal (NLS), a nuclear export signal (NES), a deaminase (e.g., adenosine deaminase or cytidine deaminase) catalytic domain, a DNA methylation catalytic domain, a histone residue modifying domain, a nuclease catalytic domain, a fluorescent protein, a transcriptional modifier, an optical gating factor, a chemical inducer, a chromatin visualization factor, a targeting polypeptide for providing binding to a cell surface moiety on a target cell or target cell type.

[0466] Embodiment 13 The functional domain exhibits an activity of modifying a target DNA, and the activity is a nuclease activity, a methylation activity, a demethylation activity, a DNA repair activity, a DNA damage activity, a deamination activity, a dismutase activity, an alkylation activity, a depurination activity, an oxidation activity, a pyrimidine dimer formation activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, a photolyase activity, a glycosidase activity, or an acetyltransferase activity. , deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, meat myristoylation activity, demyristoylation activity, glycosylation activity (e.g. from O-GlcNAc transferase), deglycosylation activity, transcription inhibition activity, and transcription activation activity.

[0467] Embodiment 14. The Cas12i protein according to any one of the preceding embodiments, wherein the functional domain is selected from an adenosine deaminase catalytic domain or a cytidine deaminase catalytic domain.

[0468] Embodiment 15. The Cas12i protein of any one of the preceding embodiments, wherein the functional domain is a full-length or functional fragment of TadA8e.

[0469] Embodiment 17. A Cas12i protein according to any one of the preceding embodiments, wherein the Cas12i protein is modified to reduce or eliminate spacer region non-specific endonuclease bypass activity.

[0470] Embodiment 18. A polynucleotide encoding the Cas12i protein according to any one of the preceding embodiments.

[0471] Embodiment 19. The polynucleotide of any one of the preceding embodiments, wherein the polynucleotide is codon-optimized for expression in a eukaryotic cell.

[0472] Embodiment 20. The polynucleotide according to any one of the preceding embodiments, wherein the polynucleotide comprises a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 99.5% or 100% identity to any one of the nucleotide sequences set forth in SEQ ID NOs:21 to 40.

[0473] Embodiment 21. A vector comprising a polynucleotide according to any one of the preceding embodiments.

[0474] Embodiment 22. A vector according to any one of the preceding embodiments, wherein the polynucleotide is operably linked to a promoter.

[0475] Embodiment 23. A vector described in any one of the preceding embodiments, wherein the promoter is a constitutive promoter, an inducible promoter, a broad spectrum promoter, a cell type specific promoter or a tissue specific promoter.

[0476] Embodiment 24. A vector according to any one of the preceding embodiments, wherein the vector is a plasmid.

[0477] Embodiment 25. A vector described in any one of the preceding embodiments, wherein the vector is a retroviral vector, a phage vector, an adenoviral vector, a herpes simplex virus (HSV) vector, an adeno-associated virus (AAV) vector or a lentiviral vector.

[0478] Embodiment 26. The vector described in any one of the preceding embodiments, wherein the AAV vector is selected from recombinant AAV vectors of serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV13.

[0479] Embodiment 27. A delivery system comprising: (1) a delivery vehicle; and (2) a Cas12i protein, polynucleotide or vector described in any one of the preceding embodiments.

[0480] Embodiment 28. A delivery system described in any one of the preceding embodiments, wherein the delivery vehicle is a nanoparticle, liposome, exosome, microvesicle or gene gun.

[0481] Embodiment 29. An engineered, non-naturally occurring CRISPR-Cas system, comprising: A Cas12i protein according to any one of the above embodiments, or a polynucleotide encoding the Cas12i protein; and a CRISPR RNA (crRNA), or a polynucleotide encoding the crRNA, wherein the crRNA comprises: a spacer region capable of hybridizing to a target sequence of a target DNA; a direct repeat (DR) linked to the spacer region and capable of guiding binding of the Cas12i protein and the crRNA to form a CRISPR-Cas complex that targets the target sequence.

[0482] Embodiment 30. A CRISPR-Cas system comprising one or more vectors, the one or more vectors comprising: A first regulatory element operably linked to a nucleotide sequence encoding a Cas12i protein according to any one of the preceding embodiments; and a second regulatory element operably linked to a polynucleotide encoding a CRISPR RNA (crRNA), said crRNA comprising: a spacer region capable of hybridizing to a target sequence of a target DNA; A direct repeat (DR) linked to the spacer region and capable of guiding the binding of the Cas12i protein and the crRNA to form a CRISPR-Cas complex that targets the target sequence; wherein the first regulatory element and the second regulatory element are located on the same or different vectors of the CRISPR-Cas vector system.

[0483] Embodiment 31. An engineered, non-naturally occurring CRISPR-Cas complex, comprising: A Cas12i protein according to any one of the preceding embodiments, and CRISPR RNA (crRNA), wherein the crRNA comprises a spacer region capable of hybridizing to a target sequence of a target DNA; and a direct repeat (DR) linked to the spacer region, the DR guiding the binding of the Cas12i protein to the crRNA.

[0484] Embodiment 32. The CRISPR-Cas system or complex according to any one of the preceding embodiments, wherein the length of the spacer region is greater than 16 nucleotides, preferably between 16 and 100 nucleotides, more preferably between 16 and 50 nucleotides, more preferably between 16 and 27 nucleotides, more preferably between 17 and 24 nucleotides, more preferably between 18 and 24 nucleotides, and most preferably between 18 and 22 nucleotides.

[0485] Embodiment 33. A CRISPR-Cas system or complex according to any one of the preceding embodiments, wherein the DR has substantially the same secondary structure as the secondary structure of a DR shown in any one of SEQ ID NOs: 11 to 20.

[0486] Embodiment 34. A CRISPR-Cas system or complex according to any one of the preceding embodiments, wherein the DR has a nucleotide addition, insertion, deletion or substitution that does not result in a substantial difference in secondary structure compared to the DR shown in any one of SEQ ID NOs: 11 to 20.

[0487] Embodiment 35. The DR comprises a stem-loop structure near the 3' end of the DR, wherein the stem-loop structure is 5'-X 1 X 2 X 3 X 4 X 5 NNNnNNNX 6 X 7 X 8 X 9 X 10 -3'(X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 and X 10 is any base, n is any nucleobase or a deletion, and N is any nucleobase, 1 X 2 X 3 X 4 X 5 and X 6 X 7 X 8 X 9 X 10 are capable of hybridizing to each other.

[0488] The DR comprises a stem-loop structure selected from any one of the following: near the 3' end of the DR is 5' CUCCCNNNNNNUGGGAG 3', where N is any nucleobase; near the 3' end of the DR is 5' CUCCUNNNNNNUGGGAG 3', where N is any nucleobase; near the 3' end of the DR is 5' GUCCCNNNNNNUGGGAC 3', where N is any nucleobase; near the 3' end of the DR is 5' GUGUCNNNNNNUGACAC 3', where N is any nucleobase; near the 3' end of the DR is 5' GUGCCNNNNNNUGGCAC 3', where N is any nucleobase; proximal to the 3' end of the DR is 5' UGUGUNNNNNNUCACAC 3', where N is any nucleobase, and near the 3' end of the DR is 5' CCGUCNNNNNNUGACGG 3', where N is any nucleobase; near the 3' end of the DR is 5' GTTTCNNNNNNUGAAAC 3', where N is any nucleobase; near the 3' end of the DR is 5' GTGTTNNNNNNUAACAC 3', where N is any nucleobase; 3. The CRISPR-Cas system or complex of any one of the preceding embodiments, wherein the DR has a 5' TTGTCNNNNNNUGACAA 3' sequence adjacent the 3' end, where N is any nucleobase.

[0489] Embodiment 37. A CRISPR-Cas system or complex according to any one of the preceding embodiments, further comprising a target DNA capable of hybridizing to the spacer region.

[0490] Embodiment 38. A CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the target DNA is eukaryotic DNA.

[0491] Embodiment 39. A CRISPR-Cas system or complex according to any one of the preceding embodiments, wherein the target DNA is in a cell, preferably the cell is selected from a prokaryotic cell, a eukaryotic cell, an animal cell, a plant cell, a fungal cell, a vertebrate cell, an invertebrate cell, a rodent cell, a mammalian cell, a primate cell, a non-human primate cell and a human cell.

[0492] Embodiment 40. A CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the crRNA hybridizes to a target sequence of the target DNA to form a complex and causes cleavage of the target sequence by the Cas12i protein.

[0493] Embodiment 41. A CRISPR-Cas system or complex according to any one of the preceding embodiments, wherein the target sequence is at the 3' end of a protospacer adjacent motif (PAM).

[0494] Embodiment 42. A CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the PAM comprises a 5'-T rich motif.

[0495] Embodiment 43. A CRISPR-Cas system or complex according to any one of the preceding embodiments, wherein the PAM is 5'-TTA, 5'-TTT, 5'-TTG, 5'-TTC, 5'-ATA or 5'-ATG.

[0496] Embodiment 44. A CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the one or more vectors include one or more retroviral vectors, phage vectors, adenoviral vectors, herpes simplex virus (HSV) vectors, adeno-associated virus (AAV) vectors, or lentiviral vectors.

[0497] Embodiment 45. The CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the AAV vector is selected from recombinant AAV vectors of serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV13.

[0498] Embodiment 46. A CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the regulatory element comprises a promoter.

[0499] Embodiment 47. A CRISPR-Cas system or complex according to any one of the preceding embodiments, wherein the promoter is selected from a constitutive promoter, an inducible promoter, a broad-spectrum promoter, a cell type-specific promoter or a tissue-specific promoter.

[0500] Embodiment 48. A CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the promoter is functional in eukaryotic cells.

[0501] Embodiment 49. The CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein the eukaryotic cell includes an animal cell, a plant cell, a fungal cell, a vertebrate cell, an invertebrate cell, a rodent cell, a mammalian cell, a primate cell, a non-human primate cell and a human cell.

[0502] Embodiment 50. The CRISPR-Cas system or complex of any one of the preceding embodiments, optionally further comprising a DNA donor template that is inserted into the locus of interest by homology directed repair (HDR).

[0503] Embodiment 51. A cell or a progeny thereof comprising the Cas12i protein, polynucleotide, vector, delivery system, CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein preferably the cell is selected from a prokaryotic cell, a eukaryotic cell, an animal cell, a plant cell, a fungal cell, a vertebrate cell, an invertebrate cell, a rodent cell, a mammalian cell, a primate cell, a non-human primate cell and a human cell, or a progeny thereof.

[0504] Embodiment 52. A non-human multicellular organism comprising a cell or a progeny thereof according to any one of the preceding embodiments, preferably said non-human multicellular organism being an animal (e.g., a rodent or non-human primate) model of a human gene-associated disease.

[0505] Embodiment 53. A method for modifying a target DNA, comprising contacting the target DNA with a CRISPR-Cas system or complex described in any one of the preceding embodiments, wherein said contacting causes modification of the target DNA by a Cas12i protein.

[0506] Embodiment 54. The method of any one of the preceding embodiments, wherein the modification occurs outside a cell in vitro.

[0507] Embodiment 55. The method of any one of the preceding embodiments, wherein the modification occurs inside a cell in vitro.

[0508] Embodiment 56 The method of any one of the preceding embodiments, wherein the modification occurs inside a cell in vivo.

[0509] Embodiment 57. The method of any one of the preceding embodiments, wherein the cell is a eukaryotic cell.

[0510] Embodiment 58. A method according to any one of the preceding embodiments, wherein the eukaryotic cell is selected from an animal cell, a plant cell, a fungal cell, a vertebrate cell, an invertebrate cell, a rodent cell, a mammalian cell, a primate cell, a non-human primate cell and a human cell.

[0511] Embodiment 59. The method of any one of the preceding embodiments, wherein the modification cleaves the target DNA.

[0512] Embodiment 60. A method according to any one of the preceding embodiments, wherein the truncation causes a deletion of a nucleotide sequence and / or an insertion of a nucleotide sequence.

[0513] Embodiment 61. A method according to any one of the preceding embodiments, wherein the cleavage comprises cleaving the target nucleic acid at two sites, causing a deletion or inversion of the sequence between the two sites.

[0514] Embodiment 62. The method of any one of the preceding embodiments, wherein the modification is a base mutation, preferably an A→G or C→T base mutation.

[0515] Embodiment 63. A cell or a progeny thereof from a method according to any one of the preceding embodiments, comprising a modification not present in a cell that has not been subjected to said method.

[0516] Embodiment 64. A cell or a progeny thereof described in any one of the preceding embodiments, wherein the cell not subjected to the method contains an abnormality and the abnormality in the cell by the method has already been resolved or corrected.

[0517] Embodiment 65. A cellular product from a cell or a progeny thereof according to any one of the preceding embodiments, which is modified relative to the nature or amount of a cellular product from a cell not subjected to the method.

[0518] Embodiment 66. A cell product described in any one of the preceding embodiments, wherein a cell that has not been subjected to the method contains an abnormality and the cell product reflects that the abnormality has already been resolved or corrected by the method.

[0519] Embodiment 67. A method for non-specifically cleaving non-target DNA, comprising contacting the target DNA with a CRISPR-Cas system or complex described in any one of the preceding embodiments, thereby hybridizing the spacer region with a target sequence of the target DNA and cleaving the target sequence by the Cas12i protein, whereby the Cas12i protein cleaves the non-target DNA by spacer region non-specific endonuclease bypass activity.

[0520] Embodiment 68. A method for detecting target DNA in a sample, comprising: (1) contacting the sample with the CRISPR-Cas system or complex according to any one of the above-mentioned embodiments and a reporter nucleic acid capable of emitting a detectable signal after cleavage, the spacer region hybridizes with a target sequence of the target DNA and the target sequence is cleaved by the Cas12i protein, whereby the Cas12i protein cleaves the reporter nucleic acid by spacer region non-specific endonuclease bypass activity; (2) detecting the presence of the target DNA in the sample by measuring a detectable signal produced by cleavage of the reporter nucleic acid.

[0521] Embodiment 69. A method according to any one of the preceding embodiments, wherein the method further comprises comparing the level of the detectable signal with the level of a reference signal, and determining the content of the target DNA in the sample based on the level of the detectable signal.

[0522] Embodiment 70. A method according to any one of the preceding embodiments, wherein the measurement is performed using gold nanoparticle detection, fluorescence polarization, colloidal phase transition / dispersion, electrochemical detection or semiconductor-based detection.

[0523] Embodiment 71. The method of any one of the preceding embodiments, wherein the reporter nucleic acid comprises a fluorescent dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher / fluor pair, and wherein cleavage of the reporter nucleic acid by the Cas12i protein increases or decreases the level of the detectable signal produced by cleavage of the reporter nucleic acid.

[0524] Embodiment 72. A method for treating a condition or disease in a subject in need thereof, comprising administering to the subject a CRISPR-Cas system described in any one of the preceding embodiments.

[0525] The disease or disorder is cancer, an infectious disease, or a neurological disease; Optionally, the cancer is: Selected from Wilms' tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, uterine cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myelocele, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma and bladder cancer, Optionally, the infectious disease is: It is caused by human immunodeficiency virus (HIV), herpes simplex virus-1 (HSV1) and herpes simplex virus-2 (HSV2), Optionally, the neurological disorder is: The method according to any one of the preceding embodiments, wherein the condition is selected from glaucoma, age-related loss of RGCs, optic nerve damage, retinal ischemia, Leber's hereditary optic neuropathy, a neurological disease associated with degeneration of RGC neurons, a neurological disease associated with degeneration of functional neurons of the striatum in a subject in need thereof, Parkinson's disease, Alzheimer's disease, Huntington's disease, schizophrenia, depression, drug addiction, movement disorders (e.g. chorea, choreoid symptoms and dyskinesia), bipolar disorder, autism spectrum disorder (ASD) or dysfunction.

[0526] Embodiment 74. The method of any one of the preceding embodiments, wherein the disease or disorder is selected from cystic fibrosis, progressive pseudohypertrophic muscular dystrophy, Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, fragile X syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, hypercholesterolemia, Leber's congenital amaurosis, sickle cell disease and beta thalassemia.

[0527] Embodiment 75. The method of any one of the preceding embodiments, wherein the disease or condition is caused by the presence of a pathogenic point mutation.

[0528] Embodiment 76. A kit comprising a CRISPR-Cas system according to any one of the preceding embodiments, preferably wherein the components of the system are in the same container or in separate containers.

[0529] Embodiment 77. A sterile container comprising a CRISPR-Cas system according to any one of the preceding embodiments, preferably wherein the sterile container is a syringe.

[0530] Embodiment 78. An implantable device comprising a CRISPR-Cas system described in any one of the preceding embodiments, preferably wherein the CRISPR-Cas system is stored in a reservoir.

[0531] The present disclosure further provides the following embodiments.

[0532] Item 1. An engineered, non-naturally occurring CRISPR-Cas system, (1) A Cas12i protein having an amino acid sequence having at least about 90% identity to any one of SEQ ID NOs: 1 to 3 and 6, or a polynucleotide encoding the Cas12i protein; (2) CRISPR RNA (crRNA) or a polynucleotide encoding the crRNA, wherein the crRNA (i) a spacer region capable of hybridizing to a target sequence of a target DNA; (ii) a direct repeat (DR) linked to the spacer region and capable of guiding binding of the Cas12i protein and the crRNA to form a CRISPR-Cas complex that targets the target sequence.

[0533] Item 2. The engineered, non-naturally occurring CRISPR-Cas system of item 1, wherein the Cas12i protein essentially does not have the spacer region-specific endonuclease cleavage activity of a parent Cas12i protein corresponding to any one of SEQ ID NOs: 1-3 and 6 against the target sequence of the target DNA.

[0534] Item 3. The engineered, non-naturally occurring CRISPR-Cas system of item 2, wherein the Cas12i protein comprises an amino acid substitution at a position selected from one or more of D700, D650, E875, and D1049 of the parent Cas12i protein sequence of SEQ ID NO:1.

[0535] Item 4. The engineered, non-naturally occurring CRISPR-Cas system of item 3, wherein the amino acid substitutions are selected from D700A, D700V, D650A, D650V, E875A, E875V, D1049A, D1049V, D700A+D650A, D700A+E875A, D700A+D1049A, D650A+E875A, D650A+D1049A, E875A+D1049A, D700A+D650A+E875A, D700A+D650A+D1049A, D650A+E875A+D1049A and D700A+D650A+E875A+D1049A.

[0536] Item 5. The engineered, non-naturally occurring CRISPR-Cas system of item 3, wherein the Cas12i protein comprises any one of the amino acid sequences of SEQ ID NOs:79-82.

[0537] Item 6. The engineered, non-naturally occurring CRISPR-Cas system of item 2, wherein the Cas12i protein is fused to one or more functional domains to form a fusion protein.

[0538] Item 7. The engineered, non-naturally occurring CRISPR-Cas system of item 6, wherein the functional domain is selected from an adenosine deaminase catalytic domain, a cytidine deaminase catalytic domain, a DNA methylation catalytic domain, a DNA demethylation catalytic domain, a transcription activation catalytic domain, a transcription inhibition catalytic domain, a nuclear export signal, and a nuclear localization signal.

[0539] Item 8. The engineered, non-naturally occurring CRISPR-Cas system of item 7, wherein the Cas12i protein is fused to TadA8e or a functional fragment thereof to form the fusion protein.

[0540] Item 9. The engineered, non-naturally occurring CRISPR-Cas system of item 8, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 85 or 184.

[0541] Item 10. The engineered, non-naturally occurring CRISPR-Cas system of item 1, wherein the Cas12i protein essentially does not have the spacer region non-specific endonuclease bypass activity against non-target DNA of the parent Cas12i protein of any one of SEQ ID NOs: 1-3 and 6.

[0542] Item 11. The engineered, non-naturally occurring CRISPR-Cas system of item 1, wherein the DR has substantially the same secondary structure as the secondary structure of any one of the DRs of SEQ ID NOs: 21-23, 26, and 101-106.

[0543] Item 12. The engineered, non-naturally occurring CRISPR-Cas system of item 11, wherein the DR comprises a stem-loop structure near the 3' end of the DR selected from any one of SEQ ID NOs: 114-123, where N is any nucleobase.

[0544] Item 13. The engineered, non-naturally occurring CRISPR-Cas system of item 1, wherein the target sequence is at the 3' end of a protospacer adjacent motif (PAM).

[0545] Item 14. The engineered, non-naturally occurring CRISPR-Cas system of item 13, wherein the PAM is selected from 5'-TTA, 5'-TTT, 5'-TTG, 5'-TTC, 5'-ATA and 5'-ATG.

[0546] Item 15. The engineered, non-naturally occurring CRISPR-Cas system of item 1, wherein the engineered, non-naturally occurring CRISPR-Cas system comprises a polynucleotide encoding the Cas12i protein and a polynucleotide encoding the crRNA, the polynucleotide being located on the same or different vectors.

[0547] Item 16. The engineered, non-naturally occurring CRISPR-Cas system of item 15, wherein the polynucleotide encoding the Cas12i protein and the polynucleotide encoding the crRNA, located on the same vector, are each operably linked to a regulatory element.

[0548] Item 17. The engineered, non-naturally occurring CRISPR-Cas system of item 1, wherein the length of the spacer region is at least about 16 nucleotides.

[0549] Item 18. A method for modifying a target DNA, comprising contacting the target DNA with the engineered, non-naturally occurring CRISPR-Cas system described in item 1, wherein the crRNA hybridizes to a target sequence of the target DNA via a spacer region of the crRNA, and wherein the Cas12i protein binds to the crRNA to form a CRISPR-Cas complex and modify the target sequence of the target DNA.

[0550] Item 19. The method according to Item 18, wherein the modification comprises one or more of cleavage, single base editing, and repair of the target DNA.

[0551] Item 20. The method according to Item 19, wherein the modification comprises repair of the target DNA, and wherein the method further comprises introducing a repair template DNA.

[0552] Item 21. The method according to item 18, wherein the modification occurs in vitro, ex vivo or in vivo.

[0553] Item 22. A cell obtained by the method according to item 18 or a progeny thereof.

[0554] Item 23. A non-human multicellular organism comprising the cell according to item 22 or a descendant thereof.

[0555] Item 24. A method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the engineered, non-naturally occurring CRISPR-Cas system of item 1, wherein the disease or disorder is associated with a mutation in a target DNA, wherein the crRNA hybridizes to a target sequence of the target DNA comprising the mutation via a spacer region of the crRNA, wherein the Cas12i protein binds to the crRNA to form a CRISPR-Cas complex and modify the target sequence of the target DNA, and wherein modifying the mutation in the target DNA treats the disease or disorder.

[0556] Item 25. The method according to Item 24, wherein the disease or disorder is selected from transthyretin amyloidosis (ATTR), cystic fibrosis, hereditary angioedema, diabetes mellitus, progressive pseudohypertrophic muscular dystrophy, Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, fragile X syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, hypercholesterolemia, Leber's congenital amaurosis, sickle cell disease and beta thalassemia.

[0557] Item 26. The method of item 25, wherein the disease or disorder is ATTR.

[0558] Item 27. The method of item 24, wherein the engineered, non-naturally occurring CRISPR-Cas system is administered in a lipid nanoparticle.

[0559] Further embodiments are described in the following examples, which are for illustrative purposes only and are not intended to limit the scope of the disclosure. EXAMPLES

[0560] Working Example Materials and Methods Unless otherwise specified, the experimental methods used in the examples are conventional.

[0561] Unless otherwise specified, materials, reagents and the like used in the examples are commercially available.

[0562] Unless otherwise specified, the following materials and experimental methods are used in the examples.

[0563] Plasmid vector construction. Human codon-optimized Cas12i, TadA8e and human APOBEC3A genes were synthesized by GenScript Co., Ltd. and cloned by Gibson assembly to generate pCAG_NLS-Cas12i-NLS_pA_pU6_BpiI_pCMV_mCherry_pA. crRNA oligomer was synthesized, annealed and engineered into the BpiI site by HuaGene Co., Ltd. to generate pCAG_NLS-Cas12i-NLS_pA_pU6_crRNA_pCMV_mCherry_pA.

[0564] Cell culture, transfection and flow cytometric analysis. The mammalian cell lines used in this study were HEK293T and N2A. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, penicillin / streptomycin, and GlutMAX. Transfection was performed using polyetherimide (PEI). For mutant screening, HEK293T cells were cultured in 24-well plates, and after 12 hours, these cells were transfected with 2 μg of plasmids (1 μg of expression plasmid and 1 μg of reporter plasmid) in 4 μL PEI. 48 hours after transfection, BFP, mCherry, and EGFP fluorescence were analyzed using a Beckman CytoFlex flow cytometer. For assays of mutations at the target site of endogenous genes, 1 μg of expression plasmid was transfected into HEK293T or N2A cells, and they were sorted 48 hours after transfection using a BD FACS Aria III or BD LSRFortessa X-20 flow cytometer.

[0565] Detecting gene editing frequency. 6000 sorted cells were cleaved in 20 μl of cleavage buffer (Vazyme). Targeting sequence primers were synthesized by Phanta Max ultra-high fidelity DNA polymerase (Vazyme) and used for nested PCR amplification. Targeting deep sequence analysis was used to determine indel frequency. A to G or C to T editing frequency was calculated by targeted deep sequence analysis or Sanger sequencing and EditR. A to G editing purity was calculated as A to G editing efficiency / (A to T editing efficiency + A to C editing efficiency + A to G editing efficiency). C to T editing purity was calculated as C to T editing efficiency / (C to A editing efficiency + C to G editing efficiency + C to T editing efficiency).

[0566] PEM-seq. PEM-seq was performed in HEK293 cells as previously described. 23Briefly, LbCas12a, Ultra-AsCas12a, hfCas12Max, ABR001 or Cas12i2HiFi and the integrated plasmid containing the crRNA targeting TTR.2 were transfected into HEK293 cells via PEI, respectively, and the positive cells were harvested after 48 hours for DNA extraction. 20 μg of genomic DNA was fragmented by Covaris sonication, with peak lengths of 300-700 bp. DNA fragments were labeled with biotin at the 5' end by a single loop of biotinylated primer extension, and the primers were removed by AMPure XP beads and purified by streptavidin beads. The single-stranded DNA on the streptavidin beads was ligated to a bridge adapter containing a 14-bp RMB, and the PCR products were subjected to nested PCR to enrich DNA fragments containing the bait DSB and labeled with an illumine adapter sequence. The produced sequencing libraries were sequenced at 2x150bp on a Hi-seq 2500.

[0567] RNP delivery and ex vivo editing. RNPs were complexed by mixing purified hfCas12Max protein with chemically synthesized RNA oligonucleotides (Jinsrui) at a molar ratio of 1:2 in 1X PBS. RNPs were incubated at room temperature for >15 min and electroporated in a Lonza® 4D-Nucleofector™. 0.2×10 6 The cells were resuspended in 20 μL of Lonza buffer and mixed with 5 μL of RNPs of different concentrations electroporated according to the Lonza standard. 72 hours after electroporation, HEK293 or CD3+ T cells were harvested and subjected to targeting deep sequence analysis.

[0568] LNP delivery and in vivo editing. LNPs were prepared with ALC0315, cholesterol, DMG-PEG2k, and DSPC in 100% ethanol, carrying in vitro transcribed (IVT) mRNA and chemically synthesized RNA oligonucleotides (Jinsrui Co., Ltd.), with a weight ratio of 1:1. According to the manufacturer's protocol, lipids and RNA solution microfluidics were mixed to form LNPs using Precision Nano Systems NanoAssemblr benchtop instrument. 0.1, 0.3, 0.5, and 1 μg RNA from LNPs diluted with PBS were transfected into N2a cells or delivered to C57 mice at different doses by tail vein injection. 48 hours after transfection, cells were harvested for cleavage and targeting deep sequence analysis. For in vivo editing, liver tissue was collected from the left lobe or median lobe of each mouse 7 days after injection and used for DNA extraction and targeting deep sequence analysis.

[0569] Fertilized egg injection and embryo culture. Superovulated female C57 mice (7-8 weeks old) were mated with B6D2F1 males, and fertilized embryos were collected from the oviducts 20 hours after hCG injection, after injection with 5IU pregnant mare serum gonadotropin (PMSG). For fertilized egg injection, hfCas12Max mRNA (100ng / μL) and sgRNA (100ng / μL) were mixed and injected into the cytoplasm of fertilized eggs in a HEPES-CZB medium droplet containing 5mg / ml cytochalasin B (CB) using a FemtoJet microsyringe (Eppendorf) with a constant flow setting. The injected fertilized eggs were incubated at 37°C in KSOM medium with amino acids, with 5% CO. 2 The embryos were cultured under air until blastocyst stage and harvested for targeted deep sequence analysis.

[0570] Example 1 Identification of Cas12i protein and evaluation of dsDNA cleavage activity of CRISPR-Cas12i system containing Cas12i protein To identify more Cas12i, the applicant developed and employed a bioinformatics pipeline to annotate Cas12i proteins, CRISPR arrays and predicted PAM preferences, and identified 10 CRISPR-Cas12i systems in Table 1 below.

[0571] [Table 4]

[0572] To evaluate the activity of these Cas12i in mammalian cells, Applicants designed a dual-plasmid fluorescent reporter system that detects the increase in enhanced green fluorescent protein (EGFP) signal intensity activated by Cas-mediated dsDNA cleavage or double-strand breaks (Figure 3A). This system is based on co-transfection of an expression plasmid encoding mCherry, a nuclear localization signal (NLS)-tagged Cas protein and its guide RNA (gRNA) or crRNA, and a reporter plasmid encoding a BFP and an activatable EGxxFP cassette (i.e., EGxx-target site-xxFP). 11 EGFP activation was dependent on Cas-mediated DSB and single-strand annealing (SSA)-mediated repair.

[0573] In particular, referring to FIG. 3A, the reporter plasmid comprises a polynucleotide encoding 5' to 3' BFP-P2A-activatable EGxxxxFP (SEQ ID NO:41) (EGxx-insertion sequence (SEQ ID NO:42) (5' to 3' TTC-containing protospacer adjacent motif (PAM) of Cas12i, protospacer sequence (SEQ ID NO:43) (which is the reverse complement of the target sequence (SEQ ID NO:44)) and GGG protospacer adjacent motif (PAM) of Cas9)-xxFP), followed by a bGH polyA (SEQ ID NO:448) coding sequence, which was operably linked to a human CMV promoter (SEQ ID NO:447). The protospacer sequence (SEQ ID NO:43) contained a premature stop codon TAG that prevented EGFP expression and thus green fluorescent signal emission. The BFP coding sequence expressed BFP to indicate successful transfection of the reporter plasmid into host cells by blue fluorescence.

[0574] While most known Cas12i proteins recognize 5'-T-rich PAMs in dsDNA, Cas9 recognizes 3'-G-rich PAMs in dsDNA. The coexistence of the TTC 5' PAM of Cas12i and the GGG 3' PAM of Cas9 flanked by protospacer sequences (SEQ ID NO:43) allowed for a simultaneous comparison of the dsDNA cleavage activities of Cas12i and Cas9.

[0575] Activatable EGxxxxFP coding sequence, SEQ ID NO:41 CCATTACAGTAGGAGCATAC GGGAGACAAGCTTTGgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa Inserted sequence, SEQ ID NO:42 GGATCCGTGTCTTTC CCATTACAGTAGGAGCATAC GGGAGACAAGCTTTG Protospacer sequence (reverse complementary sequence of the target sequence), 20bp, SEQ ID NO:43 CCATTACAGTAGGAGCATAC Target sequence of Cas12i, 20nt, SEQ ID NO:44 GTATGCTCCTACTGTAATGG EGxxxxFP targeting spacer sequence, 20nt, SEQ ID NO:45 CCATTACAGTAGGAGCATAC Non-targeting ("NT") spacer sequence, 20 nt, SEQ ID NO:46 GGTCTTCGATAAGAAGACCT Cas12i PAM TTC Cas9 PAM GGG

[0576] With further reference to FIG. 3A , the expression plasmid comprises, from 5′ to 3′, i) a Cas12i coding sequence (SEQ ID NO:31-40) that is codon-optimized for expression in mammalian cells, which is flanked at the 5′ end by an SV40 NLS (SEQ ID NO:444) coding sequence and at the 3′ end by a NP NLS (SEQ ID NO:445) coding sequence, which is then operably linked to a CAG promoter (SEQ ID NO:500); ii) a sequence encoding a guide RNA (gRNA) configured in a 5′-DR sequence-spacer sequence-3′ format and operably linked to a human U6 promoter (SEQ ID NO:446); and iii) a coding sequence for mCherry, which is then operably linked to a human CMV promoter (SEQ ID NO:447). and a coding sequence for mCherry (ID NO:448), which expressed mCherry to indicate successful transfection of the plasmid into a host cell by red fluorescence.

[0577] When the target sequence on the target strand and the protospacer sequence on the non-target strand of the targeted dsDNA were both successfully cleaved by the gRNA-guided Cas12i polypeptide to generate double-stranded breaks (DSBs), subsequent DNA repair (e.g., repair mediated by DSB-induced single-strand annealing (SSA)) restored the EGFP coding sequence to express EGFP, and green fluorescence emission indicated the dsDNA cleavage activity.

[0578] For the test group, the spacer sequence for each test Cas12i polypeptide (SEQ ID NO:1-10) contained in the gRNA (SEQ ID NO:51-60) was an EGxxxxFP targeting spacer sequence (SEQ ID NO:45), which was designed to target and hybridize to the target sequence (SEQ ID NO:44), and the DR sequence in the gRNA (SEQ ID NO:51-60) was the DR sequence (SEQ ID NO:11-20) corresponding to each test Cas12i polypeptide (SEQ ID NO:1-10), as shown in Table 2.

[0579] [Table 5]

[0580] For negative controls ("NT") for each tested CRISPR-Cas system (Cas12i, SpCas9, LbCas12a), the EGxxxxFP targeting spacer sequence (SEQ ID NO:45) was replaced with a non-targeting spacer sequence ("NT", SEQ ID NO:46) that cannot hybridize to the target sequence (SEQ ID NO:44), while retaining the other elements of each CRISPR system.

[0581] For positive controls, the CRISPR-Cas12i system tested was substituted for the CRISPR-SpCas9 and CRISPR-LbCas12a systems shown in Table 3 below, where the same EGxxxxFP targeting spacer sequence (SEQ ID NO: 45) and the corresponding crRNA (SEQ ID NO: 48 or 50) were used. Also, for the CRISPR-SpCas9 system, the gRNA presented the following configuration: 5'-spacer sequence-scaffold sequence-3'.

[0582] [Table 6]

[0583] HEK293T cells were cultured in 24-well tissue culture plates for 12 hours according to standard methods, and the reporter and expression plasmids were transfected into the cells using standard polyethyleneimine (PEI) transfection. The transfected cells were then incubated at 37°C in 5% CO 2 The cells were then cultured under 5% CO2 for 48 h. The BFP, EGFP and mCherry fluorescence signals of the cultured cells were analyzed by flow cytometry. A "blank" control group was also set up, in which only the reporter plasmid was transfected, and no expression plasmid was introduced.

[0584] The dsDNA cleavage activity of the Cas protein was evaluated by the percentage of EGFP-positive cells in BFP and mCherry double positive cells ("EGFP"). + " directs dsDNA cleavage at designated target sites on a reporter plasmid, and "mCherry + BFP + ", which indicated successful co-transfection and co-expression of the expression plasmid and the reporter plasmid. + / mCherry + BFP + The higher the concentration, the higher the dsDNA cleavage activity.

[0585] Using this dual-plasmid fluorescent reporter system, five Cas12i (Cas12i3, Cas12i7, Cas12i10, Cas12i11, and Cas12i12) were observed to exhibit significant activation of EGFP expression upon targeting gRNA induction, thereby directing significant dsDNA cleavage (Figure 1A, Figure 3B), where Cas12i12 (also referred to herein as SiCas12i or xCas12i) showed higher dsDNA cleavage than LbCas12a or SpCas9 as determined by fluorescence-activated cell sorting (FACS) analysis (Figure 1A). Compared to SpCas9 and LbCas12a, the dimensions of xCas12i were smaller (Figure 4A).

[0586] Example 2 Evaluation of the effective spacer sequence length of xCas12i To test the effective spacer sequence length of xCas12i using the dual plasmid fluorescent reporter system in Example 1, spacer sequences of different lengths (SEQ ID NO: 45 and 61-81, shown in Table 4 below) with a range of 10-50 nt were designed to target and hybridize with the reverse complement of the corresponding protospacer sequence (also SEQ ID NO: 45 and SEQ ID NO: 61-81) of the insert sequence (SEQ ID NO: 42) of the GFxxxxFP reporter plasmid in Example 1, and the 20 nt spacer sequence is exactly the EGxxxxFP targeting spacer sequence (SEQ ID NO: 45) in Example 1. To evaluate the length of the different spacer region, the EGxxxxFP targeting spacer sequence (SEQ ID NO: 45) in the reporter plasmid was replaced with the corresponding length of the spacer sequence (SEQ ID NO: 61-81) while retaining other elements of the dual plasmid fluorescent reporter system.

[0587] [Table 7]

[0588] Using the experimental procedures in Example 1, it was observed that a spacer sequence length range of at least 16 nucleotides was effective for xCas12i activity, and within this range, 17-22 nt was optimal (Figure 4B).

[0589] Example 3 Evaluation of PAM recognition of xCas12i Given the 5'-TTN PAM preference of Cas12i, Applicants performed a NTTN PAM identification assay using the dual plasmid fluorescent reporter system in Example 1, in which various 5' PAMs were substituted for the original 5' PAM of TTC while retaining other elements of the dual plasmid fluorescent reporter system.

[0590] Using the experimental procedures in Example 1, it was observed that xCas12i consistently showed high EGFP activation frequency at target sites with a 5'-NTTN PAM sequence, where N was A, T, C, or G, and LbCas12a had comparable activity at a 5'-TTTN PAM, respectively (Figure 4C).

[0591] Example 4 Effect of DR sequence on dsDNA cleavage activity of xCas12i To test whether the original DR sequence of xCas12i (SEQ ID NO: 11) can tolerate mutation, the applicant truncated the original DR sequence without destroying the secondary structure of the original DR sequence to generate two functional fragments, DR-T1 and DR-T2, of SEQ ID NOs: 501 and 502, respectively, and then designed five DR mutants of DR-T2 to generate DR-A, DR-B, DR-C, DR-D and DR-E sequences, of SEQ ID NOs: 503-507, respectively, each of which contained 5%-30% mutations in the stem-loop region without destroying the secondary structure of the original DR sequence (i.e., the secondary structure of the DR mutants is basically the same as that of the original DR sequence).

[0592] SEQ ID NO:501 DR-T1, 30nt ATGACTCAGAAATGTGTCCCCAGTTGACAC SEQ ID NO:502 DR-T2 sequence, 23 nt AGAAATGTGTCCCCAGTTGACAC SEQ ID NO:503 DR-A sequence, 23 nt AGAAATCCGTCCTTAGTTGACGG SEQ ID NO:504 DR-B sequence, 22nt AGACATGTGTCCCCAGTGACAC SEQ ID NO:505 DR-C sequence, 23 nt AGAAATGTTTCCCCAGTTGAAAC SEQ ID NO:506 DR-D sequence, 23 nt AGAAATGTGTTCCCAGTTAACAC SEQ ID NO:507 DR-E sequence, 23 nt AGAAATTTGTCCCCAGTTGACAA

[0593] By using the dual plasmid fluorescent reporter system of xCas12i in Example 1, the original DR sequence was replaced with various DR mutants (DR-T1, DR-T2, DR-A, DR-B, DR-C, DR-D and DR-E) while retaining other elements of the reporter system (Figure 21), which showed that xCas12i still exhibited high dsDNA cleavage activity mediated by gRNAs with various DR sequence mutants. As can be seen from the above, under the condition that the secondary structure of the DR sequence is maintained (i.e., the secondary structure of the DR mutant is basically the same as that of the original DR sequence), the CRISPR-SiCas12i system had no loss of dsDNA cleavage activity, could tolerate mismatches or deletions on the DR sequence, and had broad adaptability to DR sequence changes. These data further demonstrated that two functional truncated versions of the original xCas12i DR sequence of SEQ ID NO:11 (36 nt), namely DR-T1 (SEQ ID NO:501, 30 nt) and DR-T2 (SEQ ID NO:502, 23 nt), still had high dsDNA cleavage activity that could mediate xCas12i.

[0594] Example 5 Evaluation of dsDNA cleavage activity of xCas12i in endogenous genes To further verify the dsDNA cleavage activity (genomic cleavage) of xCas12i in endogenous genes in mammalian cells, Applicant conducted a genomic cleavage assay using human TTR in HEK293T (human embryonic kidney 293 cells). 12 Gene and human PCSK9 13The expression plasmid encoding the NLS-tagged xCas12i in Example 1 was transfected with a 37-site gRNA targeting the mouse Ttr gene in genes or N2a cells (Neuro2a cells, a fast-growing mouse neuroblastoma cell line) (Figure 3A, Figure 4D). The EGxxxxFP targeting spacer sequence (SEQ ID NO: 45) in Example 1 was replaced with the corresponding gene targeting spacer sequence (SEQ ID NO: 82-126 in Table 5), and the DR-T1 sequence (SEQ ID NO: 501) replaced the original DR sequence (SEQ ID NO: 11) (and the same in the following examples unless otherwise specified), while retaining other elements of the CRISPR-xCas12i system in Example 1. 48 hours after transfection, the dsDNA cleavage activity, i.e., the formation of indels (insertions and / or deletions), at these loci was measured by FACS and targeted deep sequencing.

[0595] We observed that xCas12i mediated high frequency (up to 90%) indel formation at most sites derived from Ttr, TTR and PCSK9, with the average indel formation rate exceeding 50% (Figure 4E-F). These data indicated that xCas12i exhibited robust genome editing efficiency in mammalian cells, thereby indicating that it has excellent potential in therapeutic genome editing applications.

[0596] [Table 8-1] [Table 8-2]

[0597] Example 6 Development of xCas12i mutants and evaluation of their dsDNA cleavage activity To modify the activity of xCas12i and expand its PAM site recognition range, Applicants engineered the xCas12i protein by mutagenesis and screened for mutants with more efficient and broader PAMs using a dual plasmid fluorescent reporter system similar to that in Example 1, except that the EGxxxxFP targeting guide RNA (SEQ ID NO:51) coding sequence is located on a reporter plasmid together with the BFP-P2A-EGxxxxFP coding sequence (SEQ ID NO:41) rather than on an expression plasmid together with the xCas12i coding sequence (SEQ ID NO:31) (see "On-target reporter gene" in Figure 1B). Combined with the predictive structural analysis of xCas12i, the applicant generated a library of more than 500 types of xCas12i mutants by performing arginine (R) scanning mutagenesis in the PI domain (amino acid residue positions 173-291), REC-I domain (amino acid residue positions 427-473), and RuvC-II domain (amino acid residue positions 800-1082) of xCas12i, where a single non-R amino acid substitution was made with R. The xCas12i (SEQ ID NO:1) coding sequence on the expression plasmid was replaced with a sequence encoding each type of xCas12i mutant, and the DR-T1 sequence (SEQ ID NO:501) was substituted for the original DR sequence (SEQ ID NO:11), while retaining other elements of the reporter system. The applicant then transfected the expression plasmid and the reporter plasmid alone into HEK293T cells and analyzed them by FACS (Figure 1B).

[0598] For the negative control ("NT"), a non-targeting spacer sequence ("NT", SEQ ID NO:46) that cannot hybridize to the target sequence (SEQ ID NO:44) was substituted for the EGxxxxFP targeting spacer sequence (SEQ ID NO:45) and used in combination with xCas12i (SEQ ID NO:1), while retaining the other elements of the reporter system.

[0599] For the positive control ("WT"), the original xCas12i (SEQ ID NO:1) was used.

[0600] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0601] Based on the fluorescence intensity with EGFP-activated cells, it was observed that 192 xCas12i mutants showed increased dsDNA cleavage activity (Figure 5A, Table 6) relative to wild-type (WT) xCas12i (SEQ ID NO:1), and one of them, mutant xCas12i-N243R (referred to as Cas12Max), showed about 3.6-fold improvement (Figure 5A). In addition, 51 xCas12i mutants had 5% or less dsDNA cleavage activity relative to WT xCas12i (SEQ ID NO:1).

[0602] Applicants then performed saturation mutagenesis on N243 and observed that mutation to R indeed exhibited the highest dsDNA cleavage activity (FIG. 6A).

[0603] Applicants then targeted the DMD or Ttr sites using a fluorescent reporter system (replacing the insert sequence (SEQ ID NO:42) with an insert sequence containing a DMD or Ttr protospacer and the corresponding 5' PAM listed in Table 5) and observed that they displayed a clearly increased EGFP activation frequency relative to WT xCas12i, Cas12Max (Figure 1C, Figure 6B-C).

[0604] To further test the efficacy of Cas12Max in targeting loci, Applicant designed a total of eight gRNAs to target TTR and PCSK9 sites in HEK293T cells, and another three gRNAs to target Ttr (Table 5) in N2a cells, and used DR-T2 (SEQ ID NO:502). Consistent with previous results, compared to WT xCas12i, Cas12Max showed a significantly increased insertion and deletion frequency (Figure 1D).

[0605] Example 7 Further development of Cas12Max-based mutants and evaluation of their off-target dsDNA cleavage activity To test the specificity of Cas12Max, Applicant 12 A construct designed to express Cas12Max was transfected with gRNA targeting the TTR (TTR targeting (on-target) spacer sequence having SEQ ID NO: 130) and Cas-OFFinder 17 We performed indel frequency analysis of the on-target and off-target (OT) sites predicted by the gene expression vector.

[0606] [Table 10]

[0607] A dual plasmid fluorescent reporter system for evaluating off-target dsDNA cleavage activity (off-target reporter system, see "off-target reporter gene" in FIG. 1B) was established, which is similar to the dual plasmid fluorescent reporter system for evaluating dsDNA cleavage activity in Example 5, with the difference that the insert sequence of EGxxxxFP coding sequence contains TTR off-target protospacer sequence (SEQ ID NO:127-129), which contains one or more mismatches (bold, underlined) with TTR targeting spacer sequence (SEQ ID NO:130) instead of containing TTR protospacer sequence (also SEQ ID NO:130), and uses DR-T1 sequence (SEQ ID NO:501).

[0608] Using an off-target reporter system (Figure 7A) or targeting deep sequence analysis of endogenous genes (Figure 7B), Applicants observed that Cas12Max effectively edited the target site ("ON.1") and simultaneously directed off-target dsDNA cleavage activity by causing indel formation at two ("OT.1" and "OT.2") of three predicted off-target sites ("OT.1", "OT.2", and "OT.3").

[0609] To eliminate the off-target activity of Cas12Max, Applicant performed single mutations in the REC and RuvC domains in Example 5. 18 We selected those mutants with TTR OT1 and OT2, respectively, that had undiminished on-target cleavage activity (corresponding to WT), and tested their off-target dsDNA cleavage activity using the above two off-target reporter systems with TTR OT1 and OT2 (Figure 1B).

[0610] It was observed that the four xCas12i mutants (xCas12i-V880R (v4.1), xCas12i-M923R (v4.2), xCas12i-D892R (v4.3) and xCas12i-G883R (v4.4)) maintained high levels of on-target dsDNA cleavage activity and displayed essentially no off-target dsDNA cleavage activity at the two TTR OT1 and OT2 sites (Figure 8A).

[0611] Applicants further combined one or more of these four amino acid substitutions with N243R or N243R+E336R (Figure 8B) and observed that mutant v6.3 (N243R+E336R+D892R) showed the lowest off-target EGFP activation at OT.1 and OT.2 sites, but high on-target activation at ON.1 site (Figure 8B-C). Targeting deep sequencing analysis of endogenous TTR.2 sites and their off-target sites in HEK293T showed that v6.3 (N243R+E336R+D892R) significantly reduced off-target indel frequency at six OT sites and maintained on-target activation at ON sites compared to Cas12Max (Figure 1E). In addition, Cas12Max (v1.1) and v6.3 (N243R+E336R+D892R) retained equivalent or even higher on-target activity at the DMD.1, DMD.2, and DMD.3 sites (Figure 8D). Therefore, applicants have named v6.3 high-fidelity Cas12Max (hfCas12Max).

[0612] [Table 11]

[0613] To study the PAM preference of hfCas12Max, Applicants also performed a 5'-NNN PAM recognition assay by designing a reporter plasmid with the same target sequence but different PAMs, similar to Example 3. In addition to showing identical or even better cleavage activity at sites with 5'-TTN PAM, hfCas12Max also showed similar cleavage activity to the commonly used Cas12 7,19 (LbCas12a, Ultra-AsCas12a) and the recently reported improved Cas12i2 20,21 (ABR001, Cas12i2 HiFi ), hfCas12Max and Cas12Max showed similar high cleavage activity against targets with TNN, ATN, GTN, and CTN PAM sites (Figure 1F). Collectively, these results demonstrated that hfCas12Max exhibited efficient editing activity with highly flexible 5'-TN or 5'-TNN PAM recognition.

[0614] Example 8: Verification and comparison of on-target and off-target dsDNA cleavage activities of hfCas12Max in the TTR gene To fully evaluate the performance of hfCas12Max in human cells, Applicants designed multiple target sites in the exons of TTR for various Cas nucleases, and DR-T2 (SEQ ID NO:502) was used in this and subsequent examples unless otherwise specified.

[0615] 43 sites in hfCas12Max with TTN PAM, 43 sites in ABR001 (derived from Professor ZHANG Feng's engineered Cas12i2) with TTN PAM, Cas12i2 with TTN PAM HiFiIn total, we monitored the editing activity at 43 sites of hfCas12Max (Prof. LI Wei), 45 sites of SpCas9 with NGG PAM, 12 sites of LbCas12a with TTTN PAM, 12 sites of Ultra AsCas12a with TTTN PAM, and 20 sites of KKH-saCas9 with NNNRRT PAM (Table 9). Insertion and deletion analysis showed that hfCas12Max exhibited an average on-target dsDNA cleavage activity approximately 70% higher than other Cas nucleases and Cas12Max (Figure 1G, Figure 9).

[0616] [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7]

[0617] To further evaluate the specificity of hfCas12Max for endogenous genes in human cells, Applicants will investigate the insertion and deletion frequencies of the P2RX5 and NLRC4 on-target sites and their corresponding computationally predicted off-target sites. 22Targeting deep sequence analysis showed that hfCas12Max had higher on-target editing efficiency and similarly little indel activity at potential off-target sites compared to Ultra AsCas12a and LbCas12a (Figures 10A-B, protospacer sequences / spacer sequences of SEQ ID NO: 382-390 from top to bottom in Figure 10A, protospacer sequences / spacer sequences of SEQ ID NO: 391-397 from top to bottom in Figure 10B).

[0618] To fully detect off-targets of hfCas12Max and compare it to other Cas proteins, Applicants used PEM-seq. 23 We quantified indels and translocation events, germline events (uncut or complete recombination), and editing events in the TTR.2 library. Collectively, these results demonstrate that hfCas12Max has high efficiency and specificity, and is superior to SpCas9 and other Cas12 nucleases.

[0619] Example 9. Development and evaluation of a base editor based on dead xCas12i Applicants further explored base editing with xCas12i by generating xCas12i with inactivated nuclease (dead xCas12i, dxCas12i). 8 and Cas12i2 10 This was accomplished by introducing single mutations (D650A, D700A, E875A, or D1049A) into the conserved active site of xCas12i based on a comparison of the two (Figures 12A-B).

[0620] dxCas12i-D1049A is linked to TadA8e at the C-terminus via a GS linker containing an XTEN linker (SEQ ID NO: 442) or a GS linker containing a BP NLS (SEQ ID NO: 443). V106W(SEQ ID NO: 439, TadA8e.1) to form the adenine base editor TadA8e.1-dxCas12i, and dxCas12i-D1049A is fused to a GS linker containing an XTEN linker (SEQ ID NO: 442) or a GS linker containing a BP NLS (SEQ ID NO: 443) and one UGI (SEQ ID NO: 441) at the C-terminus of human APOBEC3A. W104A (SEQ ID NO: 440, hA3A.1) and the cytidine base editor hA3A.1-dxCas12i 24-26 (FIG. 1H and FIG. 1J). For the adenine base editor, it contained an N-terminal SV40 NLS (SEQ ID NO:444) and a C-terminal BP NLS (SEQ ID NO:443). For the cytidine base editor, it contained an N-terminal BP NLS (SEQ ID NO:443) and a C-terminal BP NLS (SEQ ID NO:443).

[0621] TadA8e V106W , SEQ ID NO:439 SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN hAPOBEC3 W104A , SEQ ID NO:440 MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISYSPCFSAGCAGEVRAFLQENTHVRLRIFAARIFDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN UGI, SEQ ID NO:441 TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML XTEN linker, SEQ ID NO:442 SGSETPGTSESATPES bpNLS (also called BP NLS or bpSV40 NLS), (doi:10.1038 / nature20565.), SEQ ID NO:443 KRTADGSEFESPKKKRKV SV40 NLS, from Betapolyomavirus macacae, SEQ ID NO:444 PKKKRKV NP NLS (also called Xenopus nucleoplasmic protein NLS or nucleoplasmic protein NLS), (doi:10.1126 / science.abj6856.), also a two-component NLS, SEQ ID NO:445 KRPAATKKAGQAKKKK Human U6 promoter, 241bp, SEQ ID NO:446 gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaagatattagtacaaaatacgtgacgtaga aagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggac Human CMV promoter, 204bp, SEQ ID NO:447 gtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttgg caccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagct bGH polyA signal, 208bp, SEQ ID NO:448 ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaa attgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctgggga T5 EXO, SEQ ID NO:449 MSKSWGKFIEEEEAEMASRRNLMIVDGTNLGFRFKHNNSKKPFASSYVSTIQSLAKSYSARTTIVLGDKGKSVFRLEHLPEYKGNRDEKYAQRTEEEKALDEQFFEYLKDAFELCKTTFPTFTIRGVEADDMAAYIVKLIGHLYD HVWLISTDGDWDTLLTDKVSRFSFTTRREYHLRDMYEHHNVDDVEQFISLKAIMGDLGDNIRGVEGIGAKRGYNIIREFGNVLDIIDQLPLPGKQKYIQNLNASEELLFRNLILVDLPTYCVDAIAAVGQDVLDKFTKDILEIAEQ CAG promoter (human CMV enhancer + chicken β-actin promoter) (containing hybrid intron), SEQ ID NO:500 cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattGtgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcag

[0622] The initial versions of TadA8e.1-dxCas12i and hA3A.1-dxCas12i showed low base editing activity, with frequencies of about 8% A to G and about 2% C to T, respectively (Figure 1l, Figure 1K). To solve this problem, applicants introduced single and combined mutations for high cleavage activity into the PI and Rec domains of dxCas12i, which significantly increased the editing activity from A to G (Figure 13A). In the improved mutant, TadA8e.1-dxCas12i-v2.2 (N243R + E336R) achieved 50% activity at the A9 and A11 sites of the KLF4 locus, which was obviously higher than the 30% activity of TadA8e.1-dLbCas12a (Figure 1l, Figure 13B-C). At target sites within PCSK9 and TTR, TadA8e.1-dxCas12i-v2.2 showed a similar increase in efficiency in mediating A to G conversions and was higher than TadA8e.1-dLbCas12a at the PCSK9 site (Figure 15). To test whether the orientation of the deaminase fusion affects base editing efficiency, Applicants constructed dxCas12i-ABE by fusing TadA8e.1 to the N-terminus or C-terminus of dxCas12i and found that TadA8e.1 at the C-terminus of dxCas12i showed slightly higher activity than the N-terminus (Figure 14). Applicants then further engineered the NLS, linker and TadA8e.1 protein (restored to TadA8e) (Figure 13A) to produce v3.1-v3.8 and v4.1-v4.4, in which TadA8e-dxCas12i-v4.3 showed close to 80% A-to-G editing efficiency and >95% editing purity, while the editing activity of the other dxCas12i-ABE versions was unchanged (Figures 1H-I, Figure 13D-E). Applicants named TadA8e-dxCas12i-v4.3 dCas12Max-ABE.

[0623] To further characterize the base editing activity of dCas12Max-ABE, Applicants performed experiments on 21 sites with TTN PAM, 13 sites with ATN PAM, and 13 sites with CTN PAM (Table 10). It was observed that dCas12Max-ABE showed significant activity from A to G at sites with TTN PAM (Figure 16).

[0624] Moreover, hA3A.1-dxCas12i-v1.2(N243R), hA3A.1-dxCas12i-v2.2(N243R+E336R) and hA3A.1-dxCas12i-v4.3(N243R+E336R-bpNLS) showed persistently elevated C to T editing efficiency and editing purity of >95% at the C7 and C10 sites of RUNX1, DYRK1A and site 4 loci, which were therefore higher than hA3A.1-dLbCas12a at RUNX1 and DYRK1A (Figure 1J-K).

[0625] These results collectively demonstrated that the engineered editor based on dxCas12i exhibited high base editing activity in mammalian cells.

[0626] [Table 13-1] [Table 13-2]

[0627] Example 10 Evaluation of RNP delivery of hfCas12Max in T cells To explore the therapeutic potential of hfCas12Max, Applicant is developing an hfCas12Max RNP that targets TRAC in CD3+ T cells. 19(Figure 2A). Applicants previously tested hfCas12Max RNPs targeting TTR and TRAC in HEK293 cells and found that after increasing the RNP dosage, gene editing efficiency also increased, and cell vitality and proliferation were unaffected (Figure 18A-C). Applicants achieved approximately 90% dsDNA cleavage activity and >95% vitality for TRAC at a dose of 3.2 μM in HEK293 cells (Figure 18A-C). Three guide sequences were designed to target TRAC (Table 5), and both TRAC sg.2 and sg.3 produced ~90% editing and ~80% vitality at doses of 1.6 μM and 3.2 μM in CD3+ T cells (Figure 2B). Flow cytometry analysis showed that 5 days after electroporation treatment with RNPs targeting sg.2 or sg.3, TRAC expression in CD3+ T cells was detected to be reduced to a level of 2% to 3%, compared to 96.6% in untreated cells (Figure 2C). The guide RNA used in this example exhibited the following configuration: 5' DR-T1-spacer sequence-DR-T2-spacer sequence-3'.

[0628] Example 11 Evaluation of LNP delivery of hfCas12Max in vivo To evaluate the feasibility of hfCas12Max or its base editor for in vivo gene editing, Applicant delivered guide RNA and coding hfCas12Max mRNA via tail vein injection into the liver of C57 mice by LNP packaging. 27(Figure 2D). Applicant targeted exon 3 in the mouse transthyretin (Ttr) gene (Ttr_sg12 in Table 5) by gene editing (dsDNA cleavage) and base editing (Figure 2E). In N2a cells, robust editing efficiency was detected under four different concentrations, with an editing efficiency approaching 100% at a dose of 1 μg (Figure 2F). Similarly, targeting deep sequence analysis indicated that the editing efficiency in mouse liver was about 70% at doses equivalent to saturation of 0.3 and 0.5 mg / kg (mpk) (Figure 2G). Furthermore, delivered by LNP packaging, TadA8e-dxCas12i-v4.3 (dCas12Max-ABE) achieved an A to G efficiency of about 25% of A13 at the Ttr locus in mouse liver at a dose of 3 mpk (Figure 2H). The guide RNA used in this example had the following configuration: 5' DR-T1-spacer sequence-DR-T2-spacer sequence-3'.

[0629] Applicant also injected hfCas12Max mRNA and two gRNAs targeting the Ttr gene (Ttr_sg3 and 12 in Table 5) into mouse fertilized eggs, which were cultured to the blastocyst stage and genotyped (Figure 19A). Targeting deep sequence analysis showed that most fertilized eggs were edited, and some reached 100% (Figure 19B). These results indicated that hfCas12Max exhibited great potential for disease modeling and therapy by mediating robust ex vivo and in vivo gene editing.

[0630] Misfolding and aggregation of transthyretin (TTR) has been associated with amyloid diseases, including transthyretin-associated wild-type amyloidosis (ATTRwt), transthyretin-associated hereditary amyloidosis (ATTRm), familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC). Silencing the TTR gene to reduce the production of TTR protein may have therapeutic effects on amyloid diseases associated with TTR. In this example, the highly efficient cleavage of the TTR target site in mice demonstrated that the SiCas12i-crRNA system of the present invention has a very promising prospect in the treatment of TTR-associated amyloid diseases, such as ATTR (e.g., ATTRwt or ATTRm).

[0631] Example 12: Screening for xCas12i mutants with nickase activity To screen for xCas12i mutants with nickase activity (i.e., having ssDNA cleavage activity and essentially lacking dsDNA cleavage activity), the reporter system with dsDNA cleavage activity in Example 1 and the reporter system with nickase activity established based on the reporter system with dsDNA cleavage activity in Example 1 were used to design xCas12i mutants in Tables 11-14 and test their nickase activity and dsDNA cleavage activity, in which the insertion sequence was replaced with an insertion sequence of a reverse complement sequence containing 5' PAM, protospacer sequence (SEQ ID NO:43), linker, target sequence (SEQ ID NO:44), and 5' PAM from 5' to 3'.

[0632] When the xCas12i mutant only had nickase activity, it could only produce green fluorescence in the reporter system with dsDNA cleavage activity, but could produce green fluorescence in the reporter system with nickase activity. When the xCas12i mutant had dsDNA cleavage activity, it could produce green fluorescence in both reporter systems with nickase activity and dsDNA cleavage activity. Therefore, the reporter system with nickase activity indicated the sum of dsDNA cleavage activity and nickase activity. The nickase activity was calculated as the green fluorescence from the reporter system with nickase activity minus the green fluorescence from the reporter system with dsDNA cleavage activity. The nickase percentage was calculated as nickase activity / dsDNA cleavage activity.

[0633] It was observed that xCas12i-W896R, xCas12i-S924R and xCas12i-S925R exhibited significant nickase activity relative to WT xCas12i.

[0634] [Table 14]

[0635] Further induction was performed at W896, S924, or S925 of xCas12i to generate the mutants in Tables 12-14. It was observed that eight xCas12i mutants W896R, W896P, W896K, S924F, S924D, S924E, S924H, and S925T achieved significantly better nickase preference (nickase activity / dsDNA cleavage activity >1.0) and higher nickase activity (>20%).

[0636] [Table 15]

[0637] [Table 16]

[0638] [Table 17]

[0639] Various modifications and variations of the products, methods and applications of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. Although the present disclosure has been described in connection with specific embodiments, it should be understood that further modifications are possible and the present disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications to carry out the methods described in the present disclosure that are obvious to those skilled in the art are intended to be within the scope of the present disclosure. This application is generally intended to cover any changes, uses or adaptations of the present disclosure that follow the principles of the present disclosure, and includes those that deviate from the content of the present disclosure that are within the known and conventional operations of the art to which the present disclosure pertains and are applicable to the basic features previously set forth.

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Exemplary sequences SEQ ID NO:1 >SiCas12i protein SEQ ID NO:2>Si2Cas12i protein SEQ ID NO:3 >WiCas12i protein SEQ ID NO:4 >Wi2Cas12i protein SEQ ID NO:5 >Wi3Cas12i protein SEQ ID NO:6 > SaCas12i protein SEQ ID NO:7>Sa2Cas12i protein SEQ ID NO:8 >Sa3Cas12i protein SEQ ID NO:9 > WaCas12i protein SEQ ID NO:10 >Wa2Cas12i protein SEQ ID NO:11 >SiCas12i direct repeat sequence CTAGCAATGACTCAGAAATGTGTCCCCAGTTGACAC SEQ ID NO:12 >Si2Cas12i direct repeat sequence ATCGCAACATCTTAGAAATCCGTCCTTAGTTGACGG S EQ ID NO:13 >WiCas12i direct repeat sequence TCTCAACGATAGTCAGACATGTGTCCCCAGTGACAC SEQ ID NO:14 >Wi2Cas12i direct repeat sequence CTCAAAGTGTCAAAAGAATGTCCCTGCTAATGGGAC SEQ ID NO:15 >Wi3Cas12i direct repeat sequence TCCCAAAGTGGCAAAAGAATCTCCCTGTTAATGGGAG SEQ ID NO:16 >SaCas12i direct repeat sequence GTCTAACTGCCATAGAATCGTGCCTGCAATTGGCAC SEQ ID NO:17 >Sa2Cas12i direct repeat sequence TCGGGGCACCAAAATAATCTCCTTGGTAATGGGAG SEQ ID NO:18 >Sa3Cas12i direct repeat sequence CCACAACAACCAAAAGAATGTCCCTGAAAGTGGGAC SEQ ID NO:19 >WaCas12i direct repeat sequence GTAACAGTGGCTAAGTAATGTGTCTTCCAATGACAC SEQ ID NO:20 >Wa2Cas12i direct repeat sequence GAGAGAATGTGTGCAAAGTCACAC SEQ ID NO:21 >SiCas12i coding sequence SEQ ID NO:22 >Si2Cas12i coding sequence SEQ ID NO:23 >WiCas12i coding sequence SEQ ID NO:24 >Wi2Cas12i coding sequence SEQ ID NO:25 >Wi3Cas12i coding sequence SEQ ID NO:26 >SaCas12i coding sequence SEQ ID NO:27 >Sa2Cas12i coding sequence SEQ ID NO:28 >Sa3Cas12i coding sequence SEQ ID NO:29 >WaCas12i coding sequence SEQ ID NO:30 >Wa2Cas12i coding sequence SEQ ID NO:31 >SiCas12i codon-optimized coding sequence SEQ ID NO:32 >Si2Cas12i codon-optimized coding sequence SEQ ID NO:33 >WiCas12i codon-optimized coding sequence SEQ ID NO:34 >Wi2Cas12i codon-optimized coding sequence SEQ ID NO:35 >Wi3Cas12i codon-optimized coding sequence SEQ ID NO:36 > SaCas12i codon-optimized coding sequence SEQ ID NO:37 > Sa2Cas12i codon-optimized coding sequence SEQ ID NO:38 >Sa3Cas12i codon-optimized coding sequence SEQ ID NO:39 >WaCas12i codon-optimized coding sequence SEQ ID NO:40 >Wa2Cas12i codon-optimized coding sequence SpCas9 protein, SEQ ID NO:47 SpCas9 scaffold sequence, SEQ ID NO:48 CCATTACAGTAGGAGCATAC GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC LbCas12a protein, SEQ ID NO:49 LbCas12a DR sequence、SEQ ID NO:50 TAATTTCTACTAAGTTAGAT CCATTACAGTAGGAGCATAC SEQ ID NO:51 >SiCas12i-crRNA CTAGCAATGACTCAGAAATGTGTCCCCAGTTGACAC CCATTACAGTAGGAGCATAC SEQ ID NO:52 >Si2Cas12i-crRNA ATCGCAACATCTTAGAAATCCGTCCTAGTTGACGG CCATTACAGTAGGAGCATAC SEQ ID NO:53 >WiCas12i-crRNA TCTCAACGATAGTCAGACATGTGTCCCCAGTGACAC CCATTACAGTAGGAGCATAC SEQ ID NO:54 >Wi2Cas12i-crRNA CTCAAAGTGTCAAAAAGAATGTCCCTGCTAATGGGAC CCATTACAGTAGGAGCATAC SEQ ID NO:55 >Wi3Cas12i-crRNA TCCCAAAGTGGCAAAAGAATCTCCCTGTTAATGGGAG CCATTACAGTAGGAGCATAC SEQ ID NO:56 >SaCas12i-crRNA GTCTAACTGCCATAGAATCGTGCCTGCAATTGGCAC CCATTACAGTAGGAGCATAC SEQ ID NO:57 >Sa2Cas12i-crRNA TCGGGGCACAAATAATCTCCTTGGTAATGGGAG CCATTACAGTAGGAGCATAC SEQ ID NO:58 >Sa3Cas12i-crRNA CCACAACAACCAAAAGAATGTCCCTGAAAGTGGGAC CCATTACAGTAGGAGCATAC SEQ ID NO:59 >WaCas12i-crRNA GTAACAGTGGCTAAGTAATGTGTCTTCCAATGACAC CCATTACAGTAGGAGCATAC SEQ ID NO:60 >Wa2Cas12i-crRNA GAGAGAATGTGTGCAAAGTCACAC CCATTACAGTAGGAGCATAC

Claims

1. Cas12i polypeptide, (1) SEQ ID NO: As shown in one of 1-3, 6 and 10, (2) SEQ ID NO: Contains any one amino acid sequence from 1 to 3, 6 and 10, or (3) Cas12i polypeptide comprising an amino acid sequence having at least about 60% (for example, at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity with any one amino acid sequence among SEQ ID NO: 1-3, 6, and 10.

2. The Cas12i polypeptide according to claim 1, wherein the Cas12i polypeptide includes substitutions selected from D650A, D700A, E875A and D1049A or a combination thereof, of SEQ ID NO:

1.

3. The Cas12i polypeptide comprises one or more substitutions of one or more amino acids, wherein the one or more amino acids are the amino acid sequence of the reference Cas12i polypeptide of SEQ ID NO:

1. S118, D119, F121, W123, Q136, E138, E143, V146, S155, V158, E161, S162, T163, A165, N166, G178, D180, T185, K189, A193 , D196, N199, N200, E202, L203, S221, V233, E235, N236, S241, N243, S245, K251, D255, L257, N273, D287, S295, V302, S332 , E336, S338, V339, E362, D375, A377, N378, D381, T382, E385, D387, N390, E395, E396, Q398, N399, V400, D403, E406, Q407, V 409, D411, C412, N416, N418, L440, L448, V451, Q455, E464, S806, S817, V818, S819, S832, M833, F835, T836, F837, C839, A84 0, E842, E843, K844, T846, N847, K848, N854, A856, S858, Q862, K863, Y865, L866, G868, K870, M871, D876, D877, V880, G883, K884, G886, K887, A888, A891, D892, M894, A900, K903, K904, N906, V910, M912, S913, C915, Y916, A918, M923, S925, H926, Q9 27, V931, M933, Q934, D935, K936, K937, T938, S939, V940, F945, M946, V948, N949, K950, D951, S952, D955, Y956, A959, G960 , N966, S967, K968, S969, D970, A971, G972, S974, V975, Y976, Q979, A980, L982, H983, C985, E986, A987, G989, V990, S991, P 992, E993, L994, V995, K996, N997, K998, K999, T1000, H1001, A1002, A1003, E1004, G1006, G1010, A1012, M1013, L1014, W1017, V1022, K1028, D1032, K1034, K1037, C1039, G1040, Q1045, H1047, corresponding to one or more amino acids at one or more positions among C1063 and G1069, The Cas12i polypeptide according to claim 1.

4. The Cas12i polypeptide comprises one or more substitutions of one or more amino acids, wherein the one or more amino acids are the amino acid sequence of the reference Cas12i polypeptide of SEQ ID NO:

1. Corresponding to one or more amino acids at one or more positions among N243, E336, V880, G883, D892 and M923, The Cas12i polypeptide according to claim 1.

5. The Cas12i polypeptide according to claim 3, wherein the substitution is carried out with arginine (Arg / R).

6. The Cas12i polypeptide includes (1) substitution of N243R + E336R + D892R, (2) substitution of N243R + E336R + G883R, (3) substitution of N243R + E336R, or (4) substitution of N243R. Furthermore, the position of the amino acid here corresponds to SEQ ID NO:

1. The Cas12i polypeptide according to claim 1.

7. The Cas12i polypeptide according to claim 1, wherein the Cas12i polypeptide is an xCas12i-N243R+E336R+D892R mutant or an xCas12i-N243R+E336R+G883R mutant.

8. A fusion protein comprising a Cas12i polypeptide and a functional domain according to any one of claims 1 to 7.

9. The functional domains include nuclear localization signals (NLS), nuclear export signals (NES), deaminase or its catalytic domain, uracil glycosidase inhibitors (UGI), uracil glycosidase (UNG), methylpurine glycosidase (MPG), methylase or its catalytic domain, demethylase or its catalytic domain, transcriptional activation domains (e.g., VP64 or VPR), transcriptional inhibition domains (e.g., KRAB moiety or SID moiety), reverse transcriptase or its catalytic domain, exonuclease or its catalytic domain, and Stone residue modification domains, nuclease catalytic domains (e.g., FokI), transcription modifiers, photogating factors, chemoinducible factors, chromatin visualization factors, targeting polypeptides for providing binding to cell surface regions on target cells or target cell types, reporter (e.g., fluorescent) polypeptides or detection markers (e.g., GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP), localization signals, polypeptide targeting moieties, DNA binding domains (e.g., MBP, Lex A DBD, Gal4A functional domain exhibiting activity to modify target DNA, its catalytic domain, its functional fragment, and any combination thereof are selected from DBD, epitope tags (e.g., His, myc, V5, FLAG, HA, VSV-G, Trx, etc.), transcription release factors, HDAC, a moiety having ssRNA cleavage activity, a moiety having dsRNA cleavage activity, a moiety having ssDNA cleavage activity, a moiety having dsDNA cleavage activity, a DNA or RNA ligase, a functional domain exhibiting activity to modify target DNA, its catalytic domain, its functional fragment, and any combination thereof, wherein the activity to modify target DNA is selected from methyltransferase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, dealkylation activity, depurination activity, oxidation activity, deacidification activity. Selected from: polymer activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosidase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMO activity, deSUMO activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity (e.g., derived from O-GlcNActransferase), and deglycosylation activity. The fusion protein according to claim 8.

10. A polynucleotide comprising a Cas12i polypeptide according to any one of claims 1 to 7, or a sequence encoding a fusion protein comprising a Cas12i polypeptide according to any one of claims 1 to 7 and a functional domain.

11. A vector comprising the polypeptide of claim 10.

12. A system, (1) A Cas12i polypeptide according to any one of claims 1 to 7, or a fusion protein comprising a Cas12i polypeptide according to any one of claims 1 to 7 and a functional domain, or a polynucleotide encoding the Cas12i polypeptide or the fusion protein, (2) A guide RNA, or a polynucleotide encoding the guide RNA, wherein the guide RNA is (i) A direct repeat sequence that can form a complex with the Cas12i polypeptide or the fusion protein, (ii) A spacer sequence that can lead the complex to the target dsDNA by hybridizing to a target sequence on the target strand of the target dsDNA, system.

13. A method for modifying a target dsDNA, comprising contacting the target dsDNA with the system described in claim 12, wherein the spacer sequence is hybridizable to a target sequence on the target strand of the target dsDNA, and the target sequence is modified by the complex.

14. A cell or its progeny comprising a Cas12i polypeptide according to any one of claims 1 to 7, or a fusion protein comprising a Cas12i polypeptide according to any one of claims 1 to 7 and a functional domain.

15. A cell or its progeny comprising the polynucleotide described in Claim 10.

16. A cell or its progeny comprising the vector described in Claim 11.

17. A cell or its progeny comprising the system described in Claim 12.

18. A composition for use in a method for diagnosing, preventing or treating a disease or disorder, comprising the system described in claim 12.

19. A composition for use in a method for diagnosing, preventing or treating a disease or disorder, comprising the cells or their progeny described in Claim 14.

20. A composition for use in a method for diagnosing, preventing or treating a disease or disorder, comprising the cells or their progeny described in Claim 15.

21. A composition for use in a method for diagnosing, preventing or treating a disease or disorder, comprising the cells or their progeny described in claim 16.

22. A composition for use in a method for diagnosing, preventing or treating a disease or disorder, comprising the cells or their progeny described in Claim 17.

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