Engineered casx systems

Abstract

Provided herein are engineered CasX systems and components thereof, including variant CasX proteins and variant guide nucleic acids (gNAs). The variant CasX proteins and variant gNAs of the disclosure display at least one improved characteristic when compared to a reference CasX protein or reference gNA of the disclosure. In some instances, the variants have one or more improved CasX ribonucleoprotein complex functions. Also provided are methods of making and using said variants.

1 . A variant of a reference CasX protein (CasX variant), wherein:
 a. the CasX variant comprises at least one modification in the reference CasX protein wherein the reference CasX protein comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and   b. the CasX variant exhibits at least one improved characteristic as compared to the reference CasX protein.   
     
     
         2 . The CasX variant of  claim 1 , wherein the improved characteristic of the CasX variant is selected from the group consisting of: improved folding of the CasX variant; improved binding affinity to a guide nucleic acid (gNA); improved binding affinity to a target DNA; improved ability to utilize a greater spectrum of one or more PAM sequences, including ATC, CTC, GTC, or TTC, in the editing of target DNA; improved unwinding of the target DNA; increased editing activity; improved editing efficiency; improved editing specificity; increased nuclease activity; increased target strand loading for double strand cleavage; decreased target strand loading for single strand nicking; decreased off-target cleavage; improved binding of non-target DNA strand; improved protein stability; improved protein solubility; improved protein:gNA complex (RNP) stability; improved protein:gNA complex solubility; improved protein yield; improved protein expression; improved fusion characteristics or a combination thereof. 
     
     
         3 . The Cas X variant of  claim 1 , wherein the at least one modification comprises:
 a. at least one amino acid substitution in a domain of the CasX variant;   b. at least one amino acid deletion in a domain of the CasX variant;   c. at least one amino acid insertion in a domain of the CasX variant;   d. a substitution of all or a portion of a domain from a different CasX;   e. a deletion of all or a portion of a domain of the CasX variant; or f. any combination of (a)-(e).   
     
     
         4 . (canceled) 
     
     
         5 . The CasX variant of  claim 1 , wherein the at least one modification is in a domain selected from:
 a. a non-target strand binding (NTSB) domain;   b. a target strand loading (TSL) domain;   c. a helical I domain;   d. a helical II domain;   e. an oligonucleotide binding domain (OBD); or   f. a RuvC DNA cleavage domain.   
     
     
         6 . The CasX variant of  claim 5  wherein:
 the at least one modification in the TSL domain comprises an amino acid substitution of one or more of amino acids Y857, S890, and S932 of SEQ ID NO: 2; 
 the at least one modification in the helical I domain comprises an amino acid substitution of one or more of amino acids S219, L249, E259, Q252, E292, L307, or D318 of SEQ ID NO: 2; 
 the at least one modification in the helical II domain comprises an amino acid substitution of one or more of amino acids D361, L379, E385, E386, D387, F399, L404, R458, C477, or D489 of SEQ ID NO: 2; 
 the at least one modification in the OBD comprises an amino acid substitution of one or more of amino acids F536, E552, T620, or I658 of SEQ ID NO: 2; and/or 
 the at least one modification in the RuvC DNA cleavage domain comprises an amino acid substitution of one or more of amino acids K682, G695, A708, V711, D732, A739, D733, L742, V747, F755, M771, M779, W782, A788, G791, L792, P793, Y797, M799, Q804, S819, or Y857 or a deletion of amino acid P793 of SEQ ID NO: 2. 
 
     
     
         7 - 16 . (canceled) 
     
     
         17 . The CasX variant of  claim 5 , wherein the modification results in an increased ability to edit the target DNA. 
     
     
         18 . The CasX variant of  claim 1 , wherein the CasX variant is capable of forming a ribonuclear protein complex (RNP) with a guide nucleic acid (gNA). 
     
     
         19 . The CasX variant of  claim 1 , wherein the at least one modification comprises:
 a. a substitution of 1 to 100 consecutive or non-consecutive amino acids in the CasX variant;   b. a deletion of 1 to 100 consecutive or non-consecutive amino acids in the CasX variant;   c. an insertion of 1 to 100 consecutive or non-consecutive amino acids in the CasX; or   d. any combination of (a)-(c).   
     
     
         20 . (canceled) 
     
     
         21 . The CasX variant of  claim 1 , wherein the CasX variant comprises two or more modifications in one domain or wherein the CasX variant comprises modifications in two or more domains. 
     
     
         22 - 32 . (canceled) 
     
     
         33 . The CasX variant of  claim 1 , wherein the at least one modification compared to the reference CasX sequence of SEQ ID NO: 2 is selected from one or more of:
 a. an amino acid substitution of L379R;   b. an amino acid substitution of A708K;   c. an amino acid substitution of T620P;   d. an amino acid substitution of E385P;   e. an amino acid substitution of Y857R;   f. an amino acid substitution of I658V;   g. an amino acid substitution of F399L;   h. an amino acid substitution of Q252K;   i. an amino acid substitution of L404K; and   j. an amino acid deletion of P793.   
     
     
         34 . The CasX variant of  claim 1 , wherein the CasX variant has a sequence selected from the group consisting of the sequences of Tables 3, 8, 9, 10 and 12, a sequence selected from the group consisting of SEQ ID NOS: 258-327, 3508-3520, and 4412-4415, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity thereto. 
     
     
         35 . (canceled) 
     
     
         36 . The CasX variant of  claim 1 , further comprising a substitution of an NTSB and/or a helical lb domain from a different CasX, wherein the substituted NTSB and/or the helical lb domain is from the reference CasX of SEQ ID NO: 1. 
     
     
         37 - 38 . (canceled) 
     
     
         39 . The CasX variant of  claim 1 , further comprising a heterologous protein or domain thereof fused to the CasX, wherein the heterologous protein or domain thereof comprises one or more nuclear localization signals (NLS) selected from the group of sequences consisting of PKKKRKV (SEQ ID NO: 352), KRPAATKKAGQAKKKK (SEQ ID NO: 353), PAAKRVKLD (SEQ ID NO: 354), RQRRNELKRSP (SEQ ID NO: 355), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 356), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 357), VSRKRPRP (SEQ ID NO: 358), PPKKARED (SEQ ID NO: 35), PQPKKKPL (SEQ ID NO: 360), SALIKKKKKMAP (SEQ ID NO: 361), DRLRR (SEQ ID NO: 362), PKQKKRK (SEQ ID NO: 363), RKLKKKIKKL (SEQ ID NO: 364), REKKKFLKRR (SEQ ID NO: 365), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 366), RKCLQAGMNLEARKTKK (SEQ ID NO: 367), PRPRKIPR (SEQ ID NO: 368), PPRKKRTVV (SEQ ID NO: 369), NLSKKKKRKREK (SEQ ID NO: 370), RRPSRPFRKP (SEQ ID NO: 371), KRPRSPSS (SEQ ID NO: 372), KRGINDRNFWRGENERKTR (SEQ ID NO: 373), PRPPKMARYDN (SEQ ID NO: 374), KRSFSKAF (SEQ ID NO: 375), KLKIKRPVK (SEQ ID NO: 376), PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 377), PKTRRRPRRSQRKRPPT (SEQ ID NO: 378), SRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 379), KTRRRPRRSQRKRPPT (SEQ ID NO: 380), RRKKRRPRRKKRR (SEQ ID NO: 381), PKKKSRKPKKKSRK (SEQ ID NO: 382), HKKKHPDASVNFSEFSK (SEQ ID NO: 383), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 384), LSPSLSPLLSPSLSPL (SEQ ID NO: 385), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 386), PKRGRGRPKRGRGR (SEQ ID NO: 387), and PKKKRKVPPPPKKKRKV (SEQ ID NO: 389) or any one of SEQ ID NOS: 3540-3549; wherein the one or more NLS are positioned at or near the C-terminus of the CasX protein, at or near at the N-terminus of the CasX protein, or at or near the N-terminus and at or near the C-terminus of the CasX protein. 
     
     
         40 - 43 . (canceled) 
     
     
         44 . The CasX variant of  claim 2 , wherein one or more of the improved characteristics of the CasX variant is at least about 1.1 to about 100-fold or more improved relative to the reference CasX proteins of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. 
     
     
         45 - 46 . (canceled) 
     
     
         47 . The CasX variant of  claim 1 , wherein a ribonucleoprotein (RNP) comprising the CasX variant exhibits greater editing efficiency and/or binding of a target sequence in the target DNA when any one of the PAM sequences TTC, ATC, GTC, or CTC is located 1 nucleotide 5′ to the non-target strand of the protospacer having identity with the targeting sequence of the gNA in a cellular assay system compared to the editing efficiency and/or binding of an RNP comprising a reference CasX protein in a comparable assay system. 
     
     
         48 - 53 . (canceled) 
     
     
         54 . The CasX variant of  claim 1 , wherein the CasX variant protein comprises a nuclease domain having nickase activity, the CasX variant protein comprises a nuclease domain having double-stranded cleavage activity, or wherein the CasX protein is a catalytically inactive CasX (dCasX) protein, and wherein the dCasX and the gNA retain the ability to bind to the target DNA. 
     
     
         55 - 56 . (canceled) 
     
     
         57 . The CasX variant of  claim 54 , wherein the CasX protein is catalytically inactive (dCasX) and wherein the dCasX comprises a mutation at residues:
 a. D672, and/or E769, and/or D935 corresponding to the CasX protein of SEQ ID NO:1; or   b. D659, and/or E756, and/or D922 corresponding to the CasX protein of SEQ ID NO: 2.   
     
     
         58 . (canceled) 
     
     
         59 . The CasX variant of  claim 1 , wherein the CasX variant comprises a first domain from a first CasX protein and second domain from a second CasX protein different from the first CasX protein. 
     
     
         60 - 62 . (canceled) 
     
     
         63 . The CasX variant of  claim 59 , wherein the first domain comprises a portion of the sequence selected from the group consisting of amino acids 1-56, 57-100, 101-191, 192-332, 333-509, 510-660, 661-824, 825-934, and 935-986 of SEQ ID NO: 1 and the second domain comprises a portion of the sequence selected from the group consisting of amino acids 1-58, 59-102,103-192, 193-333, 334-501, 502-647, 648-812, 813-921, and 922-978 of SEQ ID NO: 2. 
     
     
         64 . (canceled) 
     
     
         65 . The CasX variant of  claim 1 , wherein the CasX variant comprises at least one chimeric domain comprising a first part from a first CasX protein and a second part from a second CasX protein different from the first CasX protein. 
     
     
         66 - 70 . (canceled) 
     
     
         71 . The CasX variant of  claim 1 , comprising a sequence selected from the group consisting of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. 
     
     
         72 - 76 . (canceled) 
     
     
         77 . A variant of a reference guide nucleic acid scaffold (gNA variant) capable of binding a reference CasX protein or a CasX variant, wherein:
 a. the gNA variant comprises at least one modification compared to the reference guide nucleic acid scaffold sequence; and   b. the gNA variant exhibits one or more improved characteristics compared to the reference guide nucleic acid scaffold comprising a sequence selected from the group consisting of SEQ ID NOS: 4-16.   
     
     
         78 . The gNA variant of  claim 77 , wherein the one or more improved characteristics is selected from the group consisting of: improved stability; improved solubility; improved transcription of the gNA; improved resistance to nuclease activity; increased folding rate of the gNA; decreased side product formation during folding; increased productive folding; improved binding affinity to a CasX protein; improved binding affinity to a target DNA when complexed with the CasX protein; improved gene editing when complexed with the CasX protein; improved specificity of editing when complexed with the CasX protein; and improved ability to utilize a greater spectrum of one or more PAM sequences, including ATC, CTC, GTC, or TTC, in the editing of target DNA when complexed with the CasX protein. 
     
     
         79 . (canceled) 
     
     
         80 . The gNA variant of  claim 77 , wherein the at least one modification comprises:
 a. at least one nucleotide substitution in a region of the gNA variant;   b. at least one nucleotide deletion in a region of the gNA variant;   c. at least one nucleotide insertion in a region of the gNA variant;   d. a substitution of all or a portion of a region of the gNA variant;   e. a deletion of all or a portion of a region of the gNA variant; or   f. any combination of (a)-(e); wherein the region of the gNA variant is selected from the group consisting of extended stem loop, scaffold stem loop, triplex, and pseudoknot.   
     
     
         81 . (canceled) 
     
     
         82 . The gNA variant of  claim 80 , wherein the scaffold stem further comprises a bubble, a triplex loop region, or a 5′ unstructured region. 
     
     
         83 - 84 . (canceled) 
     
     
         85 . The gNA variant of  claim 77 , wherein the at least one modification comprises:
 a. a substitution of 1 to 15 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions;   b. a deletion of 1 to 10 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions;   c. an insertion of 1 to 10 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions;   d. a substitution of the scaffold stem loop or the extended stem loop with an RNA stem loop sequence from a heterologous RNA source with proximal 5′ and 3′ ends; or   e. any combination of (a)-(d).   
     
     
         86 . The gNA variant of  claim 77 , comprising an extended stem loop region comprising at least 10, at least 100, at least 500, at least 1000, or at least 10,00010,000 nucleotides. 
     
     
         87 - 88 . (canceled) 
     
     
         89 . The gNA variant of  claim 85 , wherein the extended RNA stem loop comprises a sequence selected from MS2, Qβ, U1 hairpin II, Uvsx, or PP7 stem loops. 
     
     
         90 . The gNA variant of  claim 85 , wherein the at least one modification compared to the reference guide scaffold of SEQ ID NO: 5 is selected from one or more of:
 a. a C18G substitution in the triplex loop;   b. a G55 insertion in the stem bubble;   c. a U1 deletion;   d. a modification of the extended stem loop wherein
 i. a 6 nt loop and 13 loop-proximal base pairs are replaced by a Uvsx hairpin; and 
 ii. a deletion of A99 and a substitution of G64U that results in a loop-distal base that is fully base-paired. 
   
     
     
         91 - 92 . (canceled) 
     
     
         93 . The gNA variant of  claim 77 , wherein the gNA variant further comprises a targeting sequence wherein the targeting sequence is complementary to the target DNA sequence. 
     
     
         94 . The gNA variant of  claim 93 , wherein the targeting sequence has 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides. 
     
     
         95 . (canceled) 
     
     
         96 . The gNA variant of  claim 93 , wherein the gNA is a single-guide gNA comprising the scaffold sequence linked to the targeting sequence. 
     
     
         97 . The gNA variant of  claim 77 , wherein the one or more of the improved characteristics of the CasX variant is at least about 1.1 to about 100-fold or more improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5. 
     
     
         98 - 102 . (canceled) 
     
     
         103 . The gNA variant of  claim 77 , wherein the scaffold of the gNA variant sequence comprises a sequence selected from the group of sequences of SEQ ID NOS: 2101-2280, or having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. 
     
     
         104 . The gNA variant of  claim 103 , wherein the scaffold of the gNA variant sequence consists of a sequence selected from the group of sequences of SEQ ID NOS: 2101-2280. 
     
     
         105 - 107 . (canceled) 
     
     
         108 . The gNA variant of  claim 77 , further comprising a protein binding motif and/or a thermostable stem loop. 
     
     
         109 - 119 . (canceled) 
     
     
         120 . A gene editing pair comprising:
 a variant of a reference CasX protein (CasX variant) wherein:
 a. the CasX variant comprises at least one modification in the reference CasX protein; and 
 b. the CasX variant exhibits at least one improved characteristic as compared to the reference CasX protein; and 
   a guide nucleic acid (gNA variant) comprising a variant of a reference guide nucleic acid scaffold capable of binding a reference CasX protein or the CasX variant, wherein:
 c. the gNA variant comprises at least one modification compared to the reference guide nucleic acid scaffold sequence; and 
 d. the gNA variant exhibits one or more improved characteristics compared to the reference guide nucleic acid scaffold comprising a sequence selected from the group consisting of SEQ ID NOS: 4-16; and 
 e. the gNA variant comprises a targeting sequence, wherein the targeting sequence is complementary to a target DNA sequence. 
   
     
     
         121 - 136 . (canceled) 
     
     
         137 . A method of editing a target DNA, comprising contacting the target DNA with a gene editing pair comprising:
 a variant of a reference CasX protein (CasX variant), wherein:
 a. the CasX variant comprises at least one modification in the reference CasX protein; and 
 b. the CasX variant exhibits at least one improved characteristic as compared to the reference CasX protein; and 
   a guide nucleic acid variant (gNA variant) comprising a variant of a reference guide nucleic acid scaffold (gNA variant) capable of binding a reference CasX protein or the CasX variant, wherein:
 c. the gNA variant comprises at least one modification compared to the reference guide nucleic acid scaffold sequence; and 
 d. the gNA variant exhibits one or more improved characteristics compared to the reference guide nucleic acid scaffold comprising a sequence selected from the group consisting of SEQ ID NOS: 4-16; and 
 e. the gNA variant comprises a targeting sequence, wherein the targeting sequence is complementary to the target DNA sequence; 
   wherein the contacting results in editing or modification of the target DNA.   
     
     
         138 - 159 . (canceled) 
     
     
         160 . The method of  claim 137 , wherein the method comprises contacting the eukaryotic cell with a vector encoding or comprising the CasX protein and the gNA, and optionally further comprising the donor template. 
     
     
         161 . The method of  claim 160 , wherein the vector is an Adeno-Associated Viral (AAV) vector; a lentiviral vector, a non-viral particle or a virus-like particle (VLP). 
     
     
         162 - 177 . (canceled) 
     
     
         178 . A polynucleotide encoding:
 a variant of a reference CasX protein (CasX variant), wherein:
 a. the CasX variant comprises at least one modification in the reference CasX protein, wherein the reference CasX protein; and 
 b. the CasX variant exhibits at least one improved characteristic as compared to the reference CasX protein; and/or 
   a variant of a reference guide nucleic acid scaffold (gNA variant) capable of binding a reference CasX protein or a CasX variant, wherein:
 c. the gNA variant comprises at least one modification compared to the reference guide nucleic acid scaffold sequence; and 
 d. the gNA variant exhibits one or more improved characteristics compared to the reference guide nucleic acid scaffold comprising a sequence selected from the group consisting of SEQ ID NOS: 4-16. 
   
     
     
         179 . (canceled) 
     
     
         180 . A vector comprising the polynucleotide of  claim 178 . 
     
     
         181 - 189 . (canceled) 
     
     
         190 . The gene editing pair of  claim 120 , wherein the CasX protein and the gNA are associated together in a ribonuclear protein complex (RNP). 
     
     
         191 - 200 . (canceled) 
     
     
         201 . A kit, comprising:
 a variant of a reference CasX protein (CasX variant) wherein:
 a. the CasX variant comprises at least one modification in the reference CasX protein; and 
 b. the CasX variant exhibits at least one improved characteristic as compared to the reference CasX protein and/or 
   a variant of a reference guide nucleic acid scaffold (gNA variant) capable of binding a reference CasX protein or a CasX variant, wherein:
 c. the gNA variant comprises at least one modification compared to the reference guide nucleic acid scaffold sequence; and 
 d. the gNA variant exhibits one or more improved characteristics compared to the reference guide nucleic acid scaffold comprising a sequence selected from the group consisting of SEQ ID NOS: 4-16; 
   and a container.   
     
     
         202 - 222 . (canceled)