Improved gene editing systems, guides, and methods

CN123036382APending Publication Date: 2026-11-03RENAGADE THERAPEUTICS MANAGEMENT INC
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Patent Information

Application Number
CN202580020907.0
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Priority Date
2024-10-11
Filing Date
2025-01-16
Publication Date
2026-11-03

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Abstract

The present disclosure provides methods and compositions comprising novel V-type Cas programmable nucleases and lipid nanoparticles capable of delivering the V-type Cas programmable nucleases, and genome editing systems comprising the same. For therapeutic applications as well as plant and industrial biotechnology.
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Claims

1. A type V Cas polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with any of the following amino acid sequences: SEQ ID NO: 334 (ID405), SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415), and SEQ ID NO: 445 (ID419). The polypeptide comprises one or more substitutions selected from the following positions: K292, Q492, T306, L748, A739, F297, N536, D851, N770, D675, K955, S972, N798, K298, H853, E377, V295, K852, F854, N1015, T845, E1019, K745, Q565, K745, I723, K821, K721, A810, K768, S16, I28, V312, V929, L474, H470, E402, K207, V294. R212, F79, Q299, Q465, F537, K1284, V929, D1192, Q1203, T838, N735, I1063, K933, N1249, I97, R212, A1251, N1264, R1138, N588, I584, V453, Q475, N76, R304, I471, E544, V254, D231, H496, V534, D577, I28, A183, D341, K595, V501, S568, H470, V254, D231, and F197, based on SEQ The amino acid sequence provided in ID NO: 334 (ID405), or one or more corresponding substitutions selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415) and SEQ ID NO: 445 (ID419).

2. The V-type Cas polypeptide of claim 1, wherein the V-type Cas polypeptide comprises one or more substitutions selected from the following: Q492X, T306X, L748X, A739X, F297X, N536X, D851X, N770X, D675X, K955X, S972X, N798X, K298X, H853X, E377X, V295X, K852X, F854X, N1015X, T845X, E1019X, K745X, and K292X, based on the amino acid sequence provided in SEQ ID NO: 334 (ID405), or one or more corresponding substitutions selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415) and SEQ ID NO: 445 (ID419), wherein X is arginine, histidine or lysine.

3. The V-type Cas polypeptide as described in claim 1 or 2, wherein, In addition to one or more of the aforementioned substitutions, the V-type Cas polypeptide is at least 95% identical to any of the following: SEQ ID NO: 334 (ID405), SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415), or SEQ ID NO: 445 (ID419).

4. The V-type Cas polypeptide as described in any of the preceding claims, wherein the V-type Cas polypeptide comprises one or more substitutions selected from the group consisting of K955X, F297X, T306X, E1018X, E377X, K852X, K298X, T845X, V295X, N536X, Q492X, N770X, N1015X, and K292X, based on the amino acid sequence provided in SEQ ID NO: 334 (ID405), or one or more corresponding substitutions selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415), and SEQ ID NO: 445 (ID419), wherein X is arginine, histidine, or lysine.

5. The V-type Cas polypeptide as described in any of the preceding claims, wherein the V-type Cas polypeptide comprises one or more substitutions selected from the group consisting of K292X, Q492X, N536X, N770X, and N1015X, based on the amino acid sequence provided in SEQ ID NO: 334 (ID405), or one or more corresponding substitutions selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415), and SEQ ID NO: 445 (ID419), wherein X is arginine, histidine, or lysine.

6. The V-type Cas polypeptide as claimed in any of the preceding claims, said V-type Cas polypeptide comprising substituted K292X and D169X, based on the amino acid sequence provided in SEQ ID NO: 334 (ID405), or one or more corresponding substitutions selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415) and SEQ ID NO: 445 (ID419), wherein X is arginine, histidine or lysine.

7. The V-type Cas polypeptide as described in any of the preceding claims, wherein the V-type Cas polypeptide comprises one or more substitution combinations selected from: N536X and N770X; N536X and N1015X; K292X, N536X and N1015X; K292X and N770X; K292X, N536X and N770X; K292X, N770X and N1015X; K292X and N1015X; K292X and Q492X; K292X, Q492X and N536X; K292X, Q492X and N770X; K292X, Q492X and N1015X; N536X, N770X and N1015X; Q492X, N536X and N770X; Q492X, N536X and N1015X; Q492X and N770X; Q492X, N770X and N1015X; Q492X and N536X; K292X and N536X; N770X and N1015X; Q492X and N1015X; K292X, Q492X, N536X, N770X and N1015X; K292X, N536X, N770X and N1015X; K292X, Q492X, N536X and N770X; K292X, Q492X, N536X and N1015X; K292X, Q492X, N770X and N1015X; and / or Q492X, N536X, N770X and N1015X; Based on the amino acid sequence provided in SEQ ID NO: 334 (ID405), or one or more corresponding substitutions selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415) and SEQ ID NO: 445 (ID419), wherein X is arginine, histidine or lysine.

8. The V-type Cas polypeptide as claimed in any of the preceding claims, said V-type Cas polypeptide comprising a substitution D169X, based on the amino acid sequence provided in SEQ ID NO: 334 (ID405), or a corresponding substitution selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415) and SEQ ID NO: 445 (ID419), wherein X is arginine, histidine or lysine.

9. The V-type Cas polypeptide as described in any of the preceding claims, wherein X is arginine.

10. The V-type Cas polypeptide as claimed in any of the preceding claims, the V-type Cas polypeptide comprising a D169R substitution based on the amino acid sequence provided in SEQ ID NO: 334 (ID405), or a corresponding substitution selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415), and SEQ ID NO: 445 (ID419).

11. The V-type Cas polypeptide as described in any of the preceding claims, wherein the V-type Cas polypeptide comprises one or more substitutions selected from the following positions: Q565, K745, I723, K821, K721, A810, K768, S16, I28, V312, V929, L474, H470, E402, K207, V294, R212, F79, Q299, Q465, F537, K1284, V929, D1192, Q120 3. T838, N735, I1063, K933, N1249, I97, R212, A1251, N1264, R1138, N588, I584, V453, Q475, N76, R304, I471, E544, V254, D231, H496, V534, D577, I28, A183, D341, K595, V501, S568, H470, V254, D231, and F197, based on SEQ The amino acid sequence provided in ID NO: 334 (ID405), or one or more corresponding substitutions selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415) and SEQ ID NO: 445 (ID419).

12. The V-type Cas polypeptide as described in any of the preceding claims, wherein the V-type Cas polypeptide comprises: (a) One or more substitutions selected from the following positions: Q565, K745, I723, K821, K721, A810, K768, S16, I28, V312, V929; and / or (b) One or more substitution combinations at the following locations: L474, H470, and E402; K207, V294, R212, F79, and Q299; Q465 and F537; K1284, V929, D1192 and Q1203; T838 and N735; I1063, K933 and N1249; I97 and R212; A1251, N1264 and R1138; N588, I584, V453 and Q475; N76 and R304; I471 and E544; V254 and D231; H496, V534, and D577; I28, A183, and D341; K595, V501, S568 and H470; V254, D231, and F197; V254 and D341; and / or V254, D341, and F197; Based on the amino acid sequence provided in SEQ ID NO: 334 (ID405), or one or more corresponding substitutions or combinations thereof selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415) and SEQ ID NO: 445 (ID419).

13. The V-type Cas polypeptide as claimed in any of the preceding claims, wherein the V-type Cas polypeptide comprises one or more substitutions selected from the group consisting of: Q565K, K745M, I723V, K821I, K721M, A810V, K768M, S16T, I28F, V312E, V929I, L474Q, H470N, E402V, K207N, V294M, R212Q, F79S, Q299E, Q465R, F537I, K1284R, V929D, D1192E, Q1203H, T838S, N735I, I1063N, K933M, N1249S, I97T, R212L, A1251V, N1264I, R1138S, N588 I, I584S, V453E, Q475H, N76D, R304S, I471T, E544D, V254M, D231V, H496N, V534F, D577G, I28F, A183T, D341V, K595T, V501D, S568I, H470L, V254M, D231V, and F197V, based on SEQ The amino acid sequence provided in ID NO: 334 (ID405), or one or more corresponding substitutions selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415) and SEQ ID NO: 445 (ID419).

14. The V-type Cas polypeptide as described in any of the preceding claims, wherein the V-type Cas polypeptide comprises: (a) One or more substitutions selected from the following: Q565K, K745M, I723V, K821I, K721M, A810V, K768M, S16T, I28F, V312E, V929I; and / or (b) One or more substitution combinations selected from the following: L474Q, H470N and E402V; K207N, V294M, R212Q, F79S and Q299E; Q465R and F537I; K1284R, V929D, D1192E and Q1203H; T838S and N735I; I1063N, K933M and N1249S; I97T and R212L; A1251V, N1264I, and R1138S; N588I, I584S, V453E and Q475H; N76D and R304S; I471T and E544D; V254M and D231V; H496N, V534F, and D577G; I28F, A183T, and D341V; K595T, V501D, S568I and H470L; V254M, D231V, and F197V; V254M and D341V; and / or V254M, D341V, and F197V, Based on the amino acid sequence provided in SEQ ID NO: 334 (ID405), or one or more corresponding substitutions or combinations thereof selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415) and SEQ ID NO: 445 (ID419).

15. The V-type Cas polypeptide as claimed in any of the preceding claims, the V-type Cas polypeptide comprising the following substitutions: K207N, V294M, R212Q, F79S, and Q299F, based on the amino acid sequence provided in SEQ ID NO: 334 (ID405), or one or more corresponding substitutions or combinations thereof selected from any of the following amino acid sequences: SEQ ID NO: 58 (ID414), SEQ ID NO: 564 (ID418), SEQ ID NO: 335 (ID406), SEQ ID NO: 331 (ID411), SEQ ID NO: 20 (ID415), and SEQ ID NO: 445 (ID419).

16. The V-type Cas polypeptide as described in any of the preceding claims, wherein the substitution is based on the amino acid sequence provided in SEQ ID NO:334 (ID405).

17. The V-type Cas polypeptide as described in any of the preceding claims, wherein the V-type Cas polypeptide comprises or is composed of the following: (a) An amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with one or more regions corresponding to the following positions in any of SEQ ID NO: 2306-2351: 18-23; 49-57; 100-105; 128-132; 158-182; 252-311; 359-405; 440-450; 485-499; 528-569; 609-628; 672-677; 734-750; 768-776; 790-812; 840-864; 926-977; and 996, based on the numbering of SEQ ID NO: 334 (ID405); or (b) An amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with any of the following regions corresponding to the following positions in any of SEQ ID NO: 2306-2351: 18-23; 49-57; 100-105; 128-132; 158-182; 252-311; 359-405; 440-450; 485-499; 528-569; 609-628; 672-677; 734-750; 768-776; 790-812; 840-864; 926-977; and 996, based on the numbering of SEQ ID NO: 334 (ID405); or (c) An amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity with any of SEQ ID NO: 2306-2351, 1570-1600, 1632-1670 or 2354-2380.

18. The V-type Cas polypeptide of any of the preceding claims, wherein the V-type Cas polypeptide induces insertion / deletion formation when contacted with a target sequence population and a guide RNA for targeting the V-type Cas polypeptide to the target sequence under conditions suitable for inducing insertion / deletion formation, and wherein when the population is contacted with a reference polypeptide comprising the sequence of SEQ ID NO: 1554 (ID 405-1) and a guide RNA for targeting the reference polypeptide to the target polynucleotide sequence, the contact results in an increase of at least 20%, at least 30%, at least 40%, or at least 50% in the percentage of the target polynucleotide sequence containing the insertion / deletion in the target sequence population compared to the percentage of the target polynucleotide sequence containing the insertion / deletion in the population.

19. A fusion protein comprising the V-type Cas polypeptide of any of the preceding claims fused with a heterologous amino acid sequence.

20. A gene editing system, said gene editing system comprising: (a) One or more V-type Cas polypeptides as described in any of the preceding claims or one or more polynucleotides encoding said V-type Cas polypeptide; and (b) One or more polynucleotide sequences containing a guide RNA (gRNA) or one or more polynucleotides encoding the gRNA, wherein the gRNA contains a sequence complementary to the target polynucleotide sequence.

21. One or more polynucleotides encoding the V-type Cas polypeptide, fusion protein, or gene editing system as described in any of the preceding claims.

22. One or more vectors, said vector comprising one or more polynucleotides as described in claim 21.

23. A cell comprising one or more carriers as described in claim 22 or one or more polynucleotides as described in claim 21.

24. A pharmaceutical composition comprising the V-type Cas polypeptide of any one of claims 1-18, the fusion protein of claim 19, the gene editing system of claim 20, one or more polynucleotides of claim 21, one or more vectors of claim 22, or the cell of claim 23.

25. A method for modifying a targeted polynucleotide sequence, the method comprising introducing the gene editing system of claim 20 into a cell.

Citation Information

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