# US 2023/0054437 A1 · Engineered class 2 type v crispr systems

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- Number: US 2023/0054437 A1
- Document ID: US20230054437A1
- Status: Published application
- Kind: A1
- Application number: 17/572,208
- Filed: 2022-01-10
- Published: 2023-02-23
- Earliest claimed: 2020-12-03
- Assignees: Scribe Therapeutics Inc.
- Applicants: Scribe Therapeutics Inc.
- Inventors: Gayathri VIJAYAKUMAR; Sean HIGGINS; Isabel COLIN; Sarah DENNY; Brett T. STAAHL; Benjamin OAKES; Angus SIDORE; Suraj MAKHIJA
- Web page: https://patentgrep.com/patent/US20230054437A1

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/85: for animal cells
- C12N 15/86: Viral vectors
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/50: Methods for regulating/modulating their activity
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are engineered Class 2, Type V nucleases and guide RNAs useful for the editing of target nucleic acids. Also provided are methods of making and using such variants to modify nucleic acids.

## Claims

### Claim 1

A gene editing pair, comprising a Class 2, Type V CRISPR protein and a guide RNA (gRNA) scaffold wherein:
a. the gRNA scaffold comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 85%, at least about 85%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 2239 and comprises a modification at position C9, U11, U24, A29, U24, U54, G64, A88 or A95 relative to SEQ ID NO: 2239; and
b. a gRNA comprising the gRNA scaffold and the Class 2, Type V CRISPR protein are capable of forming a ribonuclear protein complex (RNP).

### Claim 2 (depends on 1)

The gene editing pair of claim 1, wherein the gRNA comprises one or more modifications at positions relative to the sequence of SEQ ID NO: 2239 selected from the group consisting of C9U, U11C, U24C, A29C, an insertion of G at position 54, an insertion of C at position 64, A88G, and A95G.

### Claim 3 (depends on 2)

The gene editing pair of claim 2, wherein the one or more modifications results in an improved characteristic selected from the group consisting of triplex region stability, scaffold bubble stability, extended stem stability, and binding affinity to a Class 2, Type V CRISPR protein.

### Claim 4 (depends on 2)

The gene editing pair of claim 2, wherein the insertion of C at position 64 and the A88G modification resolves an asymmetrical bulge element of the extended stem, thereby enhancing the stability of the extended stem of the gRNA scaffold.

### Claim 5 (depends on 2)

The gene editing pair of claim 2, wherein the gRNA scaffold comprises a modification of A29C, and wherein the modification results in increased pseudoknot stem stability of a gRNA comprising the gRNA scaffold.

### Claim 6 (depends on 2)

The gene editing pair of claim 2, wherein the modifications of U11C, U24C, and A95G increase the stability of the triplex region of the gRNA scaffold.

### Claim 7 (depends on 2)

The gene editing pair of claim 2, wherein the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 2281, 2282, 2283, 2284, 2285, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2301, 2302, 2320, 2321, 2322, 2323, 2324, 2326, 2328, and 2330, or a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

### Claim 8 (depends on 2)

The gene editing pair of claim 2, wherein the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 2281, 2282, 2283, 2284, 2285, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2301, 2302, 2320, 2321, 2322, 2323, 2324, 2326, 2328, and 2330.

### Claim 9 (depends on 1)

The gene editing pair of claim 1 comprising a targeting sequence at the 3′ end of the gRNA scaffold, wherein the targeting sequence is complementary to a target nucleic acid sequence.

### Claim 10 (depends on 9)

The gRNA of claim 9, wherein the targeting sequence has 18, 19, or 20 nucleotides.

### Claim 11 (depends on 1)

The gene editing pair of claim 1, wherein the Class 2, Type V CRISPR protein comprises
a. a NTSB domain comprising a sequence of QPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFG RCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQ (SEQ ID NO: 2335), or a sequence having at least 80% at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto;
b. a helical I-II domain comprising a sequence of RALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIE HQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWV NLNLWQKLKLSRDDAKPLLRLKGFPSF (SEQ ID NO: 2336), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto;
c. a helical II domain comprising a sequence of PLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEE DRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEE RRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAI EAE (SEQ ID NO: 2351), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto; and
d. a RuvC-I domain comprising a sequence of SSNIKPMNLIGVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQ AKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRG FGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTC (SEQ ID NO: 2352), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto.

### Claim 12 (depends on 11)

The gene editing pair of claim 11, comprising:
a. a NTSB domain comprising a sequence of SEQ ID NO: 2335;
b. a helical I-II domain comprising a sequence of SEQ ID NO: 2336;
c. a helical II domain comprising a sequence of SEQ ID NO: 2351; and
d. a RuvC-I domain comprising a sequence of SEQ ID NO: 2352.

### Claim 13 (depends on 11)

The gene editing pair of claim 11, wherein the CRISPR protein comprises:
a. an OBD-I domain comprising a sequence of QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPE NIPQ (SEQ ID NO: 2342), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto;
b. an OBD-II domain comprising a sequence of NSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYT VINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVI EKTLYNRRTRQDEPALFVALTFERREVLD (SEQ ID NO: 2347), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto;
c. a helical I-I domain comprising a sequence of PISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA (SEQ ID NO: 2343), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto;
d. a TSL domain comprising a sequence of SNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDL SVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETH (SEQ ID NO: 2349), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and
e. a RuvC-II domain comprising a sequence of ADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWK PAV (SEQ ID NO: 2350), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

### Claim 14 (depends on 11)

The gene editing pair of claim 11, comprising:
a. an OBD-I domain comprising a sequence of SEQ ID NO: 2342;
b. an OBD-II domain comprising a sequence of SEQ ID NO: 2347;
c. a helical I-I domain comprising a sequence of SEQ ID NO: 2343;
d. a TSL domain comprising a sequence of SEQ ID NO: 2349; and
e. a RuvC-II domain comprising a sequence of SEQ ID NO: 2350.

### Claim 15 (depends on 13)

The gene editing pair of claim 13, comprising the sequence of SEQ ID NO: 416, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

### Claim 16 (depends on 15)

The gene editing pair of claim 15, wherein the Class 2, Type V CRISPR protein comprises at least one modification in one or more domains selected from the group consisting of an NTSB domain, helical I-II domain, helical II domain, RuvC-I domain, OBD-I domain, and OBD-II domain, wherein the at least one modification comprises:
a. at least one amino acid substitution;
b. at least one amino acid deletion;
c. at least one amino acid insertion; or
d. any combination of (a)-(c);
wherein the Class 2, Type V CRISPR protein exhibits an improved characteristic compared to the sequence of SEQ ID NO: 416.

### Claim 17 (depends on 16)

The gene editing pair of claim 16, wherein
a. the one or more modifications to the NTSB domain are selected from the group consisting of an insertion of G at position 2, an insertion of I at position 4, an insertion of L at position 4, Q9P, E15S, G20D, a deletion of S at position 30, G33T, L41A, Y51T, F55V, L68D, L68E, L68K, A70Y, A70S, E75A, E75D, E75P, K88Q, and G90Q relative to SEQ ID NO: 2335;
b. the one or more modifications to the helical I-II domain are selected from the group consisting of an insertion of T at position 24, an insertion of C at position 25, Y29F,G32Y, G32N, G32H, G32S, G32T, G32A, G32V, a deletion of G at position 32, G32S, G32T, G44L, G44H, S48H, S48T, S51T, Q54H, I56T, V63T, S73H, L74Y, K97G, K97S, K97D, K97E, V100L, M112T, M112W, M112R, M112K, L116K, G137R, G137K, G137N, an insertion of Q at position 138, and S140Q relative to SEQ ID NO: 2336.
c. wherein the one or more modifications to the helical II domain are selected from the group consisting of an insertion of A at position 2, an insertion of H at position 2, a deletion of L at position 2 and a deletion of V at position 3, V3E, V3Q, V3F, a deletion of V at position 3, an insertion of D at position 3, V3P, E4P, a deletion of E at position 4, E4D, E4L, E4R, R5N, Q6V, an insertion of Q at position 6, an insertion of G at position 7, an insertion of H at position 9, an insertion of A at position 9, VD10, an insertion of T1 at position 0, a deletion of V at position 10, an insertion of F at position 10, an insertion of D at position 11, a deletion of D at position 11, D11S, a deletion of W at position 12, W12T, W12H, an insertion of P at position 12, an insertion of Q at position 13, an insertion of G at position 12, an insertion of R at position 13, W13P, W13D, an insertion of D at position 13, W13L, an insertion of P at position 14, an insertion of D at position 14, a deletion of D at position 14 and a deletion of M at position 15, a deletion of M at position 15, an insertion of T at position 16, an insertion of P at position 17, N18I, V19N, V19H, K20D, L22D, I23S, E25C, E25P, an insertion of G at position 25, K26T, K27E, K31L, K31Y, Q35D, Q35P, an insertion of S at position 37, a deletion of L at position 37 and a deletion of A at position 38, K41L, an insertion of R at position 42, a deletion of Q at position 43 and a deletion of E at position 44, L46N, K57Q, Y65T, G68M, L70V, L71C, L72D, L72N, L72W, L72Y, E75F, E75L, E75Y, G79P, an insertion of E at position 79, an insertion of T at position 81, an insertion of R at position 81, an insertion of W at position 81, an insertion of Y at position 81, an insertion of W at position 82, an insertion of Y at position 82, W82G, W82R, K84D, K84H, K84P, K84T, V85L, V85A, an insertion of L at position 85, Y86C, D87G, D87M, D87P, I93C, K95T, K96R, E98G, L100A, K102H, I104T, I104S, I104Q, K105D, an insertion of K at position 109, E109L, R110D, a deletion of R at position 110, D114E, an insertion of D at position 114, K118P, A120R, L121T, W124L, L125C, R126D, A127E, A127L, A129T, A129K, 1133E, an insertion of C at position 133, an insertion of S at position 134, an insertion of G at position 134, an insertion of R at position 135, G135P, L136K, L136D, L136S, L136H, a deletion of E at position 138, D140R, an insertion of D at position 140, an insertion of P at position 141, an insertion of D at position 142, a deletion of E at position 143+a deletion of F at position 144, an insertion of Q at position 143, F144K, a deletion of F at position 144, a deletion of F at position 144 and a deletion of C at position 145, C145R, an insertion of G at position 145, C145K, C147D, an insertion of V at position 148, E148D, an insertion of H at position 149, L149R, K150R, L151H, Q152C, K153P, L158S, E166L, and an insertion of F at position 167 relative to SEQ ID NO: 2351;
d. the one or more modifications to the RuvC-I domain are selected from the group consisting of an insertion of I at position 4, an insertion of S at position 5, an insertion of T at position 6, an insertion of N at position 6, an insertion of R at position 7, an insertion of K at position 7, an insertion of H at position 8, an insertion of S at position 8, V12L, G49W, G49R, S51R, S51K, K62S, K62T, K62E, V65A, K80E, N83G, R90H, R90G, M125S, M125A, L137Y, an insertion of P at position 137, a deletion of L at position 141, L141R, L141D, an insertion of Q at position 142, an insertion of R at position 143, an insertion of N at position 143, E144N, an insertion of P at position 146, L146F, P147A, K149Q, T150V, an insertion of R at position 152, an insertion of H153, T155Q, an insertion of H at position 155, an insertion of R at position 155, an insertion of L at position 156, a deletion of L at position 156, an insertion of W at position 156, an insertion of A at position 157, an insertion of F at position 157, A157S, Q158K, a deletion of Y at position 159, T160Y, T160F, an insertion of I at position 161, S161P, T163P, an insertion of N at position 163, C164K, and C164M relative to SEQ ID NO: 2351;
e. the one or more modifications to the OBD-I domain are selected from the group consisting of an insertion of G at position 3, I3G, I3E, an insertion of G at position 4, K4G, K4P, K4S, K4W, K4W, R5P, an insertion of P at position 5, an insertion of G at position 5, RSS, an insertion of S at position 5, R5A, R5P, R5G, R5L, I6A, I6L, an insertion of G at position 6, N7Q, N7L, N7S, K8G, K15F, D16W, an insertion of F at position 16, an insertion of F18, an insertion of P at position 27, M28P, M28H, V33T, R34P, M36Y, R41P, L47P, an insertion of P at position 48, E52P, an insertion of P at position 55, a deletion of P at position 55 and a deletion of Q at position 56, Q56S, Q56P, an insertion of D at position 56, an insertion of T at position 56, and Q56P relative to SEQ ID NO: 2342;
f. the one or more modifications to the OBD-II domain are selected from the group consisting of a deletion of S at position 2, I3R, I3K, a deletion of I at position 3 and a deletion of L4, a deletion of L at position 4, K11T, an insertion of P at position 24, K37G, R42E, an insertion of S at position 53, an insertion of R at position 58, a deletion of K at position 63, M70T, I82T, Q92I, Q92F, Q92V, Q92A, an insertion of A at position 93, K110Q, R115Q, L121T, an insertion of A at position 124, an insertion of R at position 141, an insertion of D at position 143, an insertion of A at position 143, an insertion of W at position 144, and an insertion of A at position 145 relative to SEQ ID NO: 2342;
g. the one or more modifications to the OBD-II domain are selected from the group consisting of an insertion of M at position 1, a deletion of N at position 2, an insertion of V at position 2, C3S, an insertion of G at position 4, an insertion of W at position 4, F5P, an insertion of W at position 7, K18G, V58D, an insertion of A at position 67, T76E, T76D, T76N, G78D, a deletion of S at position 80, a deletion of G at position 81, an insertion of E at position 82, an insertion of N at position 82, S85I, V96C, V96T, and E98D relative to SEQ ID NO: 2349; or
h. any combination of (a)-(g).

### Claim 18 (depends on 17)

The gene editing pair of claim 17, wherein the improved characteristic comprises increased cleavage activity at a target nucleic sequence comprising an TTC, ATC, GTC, or CTC PAM sequence.

### Claim 19 (depends on 17)

The gene editing pair of claim 17, wherein the improved cleavage activity comprises an enrichment score in log2 of at least about 1.5, at least about 2.0, at least about 2.5, at least about 3, at least about 3.5, at least about 4, at least about 4.5, at least about 5, at least about 6, at least about 7, at least about 8 or more greater compared to score of the sequence of SEQ ID NO: 416 in an in vitro assay.

### Claim 20 (depends on 17)

The gene editing pair of claim 17, wherein the improved characteristic comprises increased specificity, wherein the increased specificity is an enrichment score of at least about 2.0, at least about 2.5, at least about 3, at least about 3.5, at least about 4, at least about 4.5, at least about 5, or at least about 6 log2 or more greater compared to the sequence of SEQ ID NO: 416 in an in vitro assay.

### Claim 21 (depends on 17)

The gene editing pair of claim 17, wherein the improved characteristic comprises decreased off-target cleavage.

### Claim 22 (depends on 17)

The gene editing pair of claim 17, wherein the Class 2, Type V CRISPR protein comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 428, 434-439, 567, 570 and 576, 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.

### Claim 23 (depends on 17)

The gene editing pair of claim 17, comprising one or more nuclear localization signals (NLS), wherein the one or more NLS are positioned at or near at the N-terminus and/or at or near the C-terminus of the protein.

### Claim 24 (depends on 23)

The gene editing pair of claim 23, wherein the one or more NLS are 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: (359), 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), PKKKRKVPPPPKKKRKV (SEQ ID NO: 389), PAKRARRGYKC (SEQ ID NO: 63), KLGPRKATGRW (SEQ ID NO: 64), PRRKREE (SEQ ID NO: 65), PYRGRKE (SEQ ID NO: 66), PLRKRPRR (SEQ ID NO: 67), PLRKRPRRGSPLRKRPRR (SEQ ID NO:68), PAAKRVKLDGGKRTADGSEFESPKKKRKV (SEQ ID NO:69), PAAKRVKLDGGKRTADGSEFESPKKKRKVGIHGVPAA (SEQ ID NO:70), PAAKRVKLDGGKRTADGSEFESPKKKRKVAEAAAKEAAAKEAAAKA (SEQ ID NO:71), PAAKRVKLDGGKRTADGSEFESPKKKRKVPG (SEQ ID NO:72), KRKGSPERGERKRHW (SEQ ID NO:73), KRTADSQHSTPPKTKRKVEFEPKKKRKV (SEQ ID NO:74), and PKKKRKVGGSKRTADSQHSTPPKTKRKVEFEPKKKRKV (SEQ ID NO: 75).

### Claim 25 (depends on 24)

The gene editing pair of claim 24, wherein the one or more NLS are linked to the Class 2, Type V CRISPR protein or to an adjacent NLS with a linker peptide wherein the linker peptide is selected from the group consisting of SR, RS, (G)n (SEQ ID NO: 1023), (GS)n (SEQ ID NO: 1024), (GSGGS)n (SEQ ID NO: 399), (GGSGGS)n (SEQ ID NO: 400), (GGGS)n (SEQ ID NO: 401), GGSG (SEQ ID NO: 402), GGSGG (SEQ ID NO: 403), GSGSG (SEQ ID NO: 404), GSGGG (SEQ ID NO: 405), GGGSG (SEQ ID NO: 406), GSSSG (SEQ ID NO: 407), GPGP (SEQ ID NO: 408), GGP, PPP, PPAPPA (SEQ ID NO: 409), PPPG (SEQ ID NO: 24), PPPGPPP (SEQ ID NO: 410), PPP(GGGS)n (SEQ ID NO: 25), (GGGS)nPPP (SEQ ID NO: 26), AEAAAKEAAAKEAAAKA (SEQ ID NO: 1025), and TPPKTKRKVEFE (SEQ ID NO: 27), wherein n is 1 to 5.

### Claim 26 (depends on 17)

The gene editing pair of claim 17, wherein the RNP comprising the Class 2, Type V CRISPR and the gRNA exhibit a higher percent editing of the target nucleic acid in a timed in vitro assay that is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 100-fold higher compared to an RNP comprising any one of the reference proteins of SEQ ID NOS: 1-3 and the gRNA of SEQ ID NO: 4 or SEQ ID NO:5, when assessed in a comparable in vitro assay system.

### Claim 27

A Class 2, Type V CRISPR protein comprising
a. a NTSB domain comprising a sequence of SEQ ID NO: 2335, or a sequence having at least 80% at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto;
b. a helical I-II domain comprising a sequence of SEQ ID NO: 2336, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto;
c. a helical II domain comprising a sequence of SEQ ID NO: 2351, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto; and
d. a RuvC-I domain comprising a sequence of SEQ ID NO: 2352, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto.

### Claim 28

A gRNA scaffold comprising a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of the sequences selected from the group consisting of SEQ ID NOS: 2292, 2291, 2307, 2281-2290, 2293-2306, 2308-2332 and 2353-2398.

### Claim 29 (depends on 1)

A method of editing or modifying a target nucleic acid in a cell, comprising contacting the target nucleic acid with the gene editing pair of claim 1.