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US 2024/0254464 A1Published application
Cleavage-inactive cas12f1, cleavage-inactive cas12f1-based fusion protein, crispr gene-editing system comprising same, and preparation method and use thereof
AssigneeGenkore Inc.
InventorsYong-Sam KIM, Do Yon KIM
Life of this patent
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Abstract
Disclosed in the present specification are: dead Cas12f1 having the nucleic acid cleavage activity removed; and a fusion protein in which a functional domain is fused to the dead Cas12f1. The dead Cas12f1 and the dCas12f1-based fusion protein may: form a CRISPR gene-editing system together with a guide RNA; and exhibit various functions relating to a target gene such as base editing and expression control.
Claims43
1:
- A wild-type Cas12f1-based dead Cas12f1 protein, represented by the following amino acid sequence:
- (SEQ ID NO:
- 9)
- MAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEA
- CSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEI
- SEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGX1ANASX2VEHYLSDVCY
- TRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLV
- KQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQ
- KSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRG
- SKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIX3VGVKSPLVCAINNAF
- SRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTE
- KSERRFKKLIERWACEIADFFIKNKVGTVQMX4NLESMKRKEDSYFNIR
- LRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNF
- EYX5KKNKFPHFKCEKCNFKENAX6YNAALNISNPKLKSTKEEP,
- wherein X1 is isoleucine or tryptophan, X2 is serine or tyrosine, X3 is aspartic acid, alanine, glutamine, leucine, tryptophan, or valine, X4 is glutamic acid, alanine, glutamine, leucine, tryptophan, or valine, X5 is arginine, alanine, glutamine, leucine, tryptophan, or valine, and X6 is aspartic acid, alanine, glutamine, leucine, tryptophan, or valine, and
- at least one of X3, X4, X5, and X6 is alanine, glutamine, leucine, tryptophan, or valine.
13 more elements
2:
- A dead Cas12f1 protein, comprising:
- a dummy portion comprising 20 to 30 amino acids;
- and
- a wild-type Cas12f1-based dead Cas12f1 protein,
- wherein the dummy portion is represented by an amino acid sequence selected from
- (SEQ ID NO:
- 10)
- MGEKSSRRRRNGKSGAWTAAITSCVGGK,
- (SEQ ID NO:
- 11)
- MAGGPGAGSAAPVSSTSSLPLAALNMRV,
- (SEQ ID NO:
- 12)
- MAGGPGAGSAAPVSSTSSLPLAALNM,
- and
- (SEQ ID NO:
- 13)
- MEKRINKIRKKLSADNATKPVSRSGP;
- the wild-type Cas12f1-based dead Cas12f1 protein is represented by
- (SEQ ID NO:
- 9)
- MAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEA
- CSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEI
- SEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGX1ANASX2VEHYLSDVCY
- TRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLV
- KQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQ
- KSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRG
- SKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIX3VGVKSPLVCAINNAF
- SRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTE
- KSERFRKKLIERWACEIADFFIKNKVGTVQMX4NLESMKRKEDSYFNIR
- LRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNF
- EYX5KKNKFPHFKCEKCNFKENAX6YNAALNISNPKLKSTKEEP,
- wherein X1 is isoleucine or tryptophan, X2 is serine or tyrosine, X3 is aspartic acid, alanine, glutamine, leucine, tryptophan, or valine, X4 is glutamic acid, alanine, glutamine, leucine, tryptophan, or valine, X5 is arginine, alanine, glutamine, leucine, tryptophan, or valine, and X6 is aspartic acid, alanine, glutamine, leucine, tryptophan, or valine,
- at least one of X3, X4, X5, and X6 is alanine, glutamine, leucine, tryptophan, or valine, and
- the dummy portion and the wild-type Cas12f1-based dead Cas12f1 protein are sequentially linked to each other in a direction from the N terminus to the C terminus of the dead Cas12f1 protein.
32 more elements
3:
- The dead Cas12f1 protein of claim 1, wherein the dead Cas12f1 protein is represented by an amino acid sequence selected from SEQ ID NOS:
- 2 to 8.
4:
- The dead Cas12f1 protein of claim 2, wherein the dead Cas12f1 protein is represented by an amino acid sequence selected from SEQ ID NOS:
- 15 to 24, SEQ ID NOS:
- 26 to 29, SEQ ID NOS:
- 31 to 34, and SEQ ID NOS:
- 36 to 39.
2 more elements
5:
- A dCas12f1-base editing fusion protein, comprising:
- the dead Cas12f1 protein of claim 1;
- and
- deaminase,
- wherein the deaminase has an amino acid sequence selected from SEQ ID NO:
- 245, SEQ ID NOS:
- 247 to 249, and SEQ ID NOS:
- 274 to 283.
5 more elements
6:
- The dCas12f1-base editing fusion protein of claim 5, wherein the deaminase has an amino acid sequence selected from SEQ ID NOS:
- 274 to 279.
7:
- The dCas12f1-base editing fusion protein of claim 6, wherein the deaminase is fused to the N terminus of the dead Cas12f1 protein.
8:
- The dCas12f1-base editing fusion protein of claim 6, wherein the deaminase is fused to the C terminus of the dead Cas12f1 protein.
9:
- The dCas12f1-base editing fusion protein of claim 6, wherein the dCas12f1-base editing fusion protein has an amino acid sequence selected from SEQ ID NOS:
- 284 to 324 and SEQ ID NOS:
- 418 to 442.
10:
- The dCas12f1-base editing fusion protein of claim 5, wherein the dCas12f1-base editing fusion protein further comprises at least one uracil glycosylase inhibitor (UGI),
- the deaminase has an amino acid sequence selected from SEQ ID NOS:
- 280 to 283, and
- the at least one UGI is fused to the dead Cas12f1 protein.
1 more element
11:
- The dCas12f1-base editing fusion protein of claim 10, wherein the deaminase is fused to the N terminus of the dead Cas12f1 protein, and
- the at least one UGI is fused to the C terminus of the dead Cas12f1 protein.
12:
- The dCas12f1-base editing fusion protein of claim 10, wherein the deaminase is fused to the C terminus of the dead Cas12f1 protein, and
- the at least one UGI is fused to the N terminus of the dead Cas12f1 protein.
13:
- The dCas12f1-base editing fusion protein of claim 10, which is represented by an amino acid sequence selected from SEQ ID NOS:
- 325 to 328.
14:
- The dCas12f1-base editing fusion protein of claim 5, wherein the dead Cas12f1 protein and the deaminase are linked via a linker, and
- the linker is represented by an amino acid sequence selected from SEQ ID NOS:
- 260 to 273.
15:
- The dCas12f1-base editing fusion protein of claim 5, wherein the dCas12f1-base editing fusion protein further comprises at least one nuclear localization signal (NLS), and
- the NLS is located at the N-terminus, the C-terminus, or both termini.
16:
- A dCas12f1-expression regulating fusion protein, comprising:
- the dead Cas12f1 protein of claim 1;
- and
- at least one expression regulatory domain, each of which is selected from VP64, KRAB, MeCP2, DNMT, HDAC, Tet1, and p300.
1 more element
17:
- The dCas12f1-expression regulating fusion protein of claim 16, wherein the expression regulatory domain is each independently represented by an amino acid sequence selected from SEQ ID NOS:
- 329 to 333.
18:
- The dCas12f1-expression regulating fusion protein of claim 16, wherein the expression regulatory domains are all located at the N terminus of the dead Cas12f1 protein.
19:
- The dCas12f1-expression regulating fusion protein of claim 18, which is represented by an amino acid sequence selected from SEQ ID NOS:
- 511 to 513.
20:
- The dCas12f1-expression regulating fusion protein of claim 16, wherein the expression regulatory domains are all located at the C terminus of the dead Cas12f1 protein.
21:
- The dCas12f1-expression regulating fusion protein of claim 20, which is represented by an amino acid sequence selected from SEQ ID NOS:
- 514 to 518.
22:
- The dCas12f1-expression regulating fusion protein of claim 16, wherein the dCas12f1-expression regulating fusion protein comprises a first expression regulatory domain and a second expression regulatory domain,
- the first expression regulatory domain is located at the N terminus of the dead Cas12f1 protein, and
- the second expression regulatory domain is located at the C terminus of the dead Cas12f1 protein.
23:
- The dCas12f1-expression regulating fusion protein of claim 22, which is represented by an amino acid sequence selected from SEQ ID NOS:
- 519 to 521.
24:
- An engineered CRISPR/Cas12f1 composition, comprising:
- (a) an engineered Cas12f1 protein selected from:
- the dead Cas12f1 protein of claim 1,
- a dCas12f1-base editing fusion protein comprising:
- the dead Cas12f1 protein;
- and
- deaminase,
- wherein the deaminase has an amino acid sequence selected from SEQ ID NO:
- 245, SEQ ID NOS:
- 247 to 249, and SEQ ID NOS:
- 274 to 283,
- a dCas12f1-expression regulating fusion protein 2-3 comprising:
- the dead Cas12f1 protein;
- and
- at least one expression regulatory domain, each of which is selected from VP64, KRAB, MeCP2, DNMT, HDAC, Tet1, and p300,
- or a nucleic acid encoding the engineered Cas12f1 protein;
- and
- (b) at least one guide RNA, or a nucleic acid encoding the guide RNA,
- wherein each of the guide RNAs comprises a scaffold, a spacer, and a U-rich tail,
- the scaffold, the spacer, and the U-rich tail are sequentially linked to each other in a 5′ to 3′ direction,
- the scaffold is represented by a nucleotide sequence selected from SEQ ID NOS:
- 197 to 199,
- the U-rich tail is represented by a nucleotide sequence of (UaN)bUc, where N is each independently selected from A, U, C, and G, a is an integer of between 1 to 5 inclusive, and b is an integer of 0 or more, and
- the spacer comprises nucleosides of between 10 and 50 inclusive and has a nucleotide sequence complementary to a predetermined target sequence.
21 more elements
25:
- The engineered CRISPR/Cas12f1 composition of claim 24, wherein the engineered Cas12f1 protein is a dCas12f1-base editing fusion protein comprising:
- the dead Cas12f1 protein;
- and
- deaminase,
- wherein the deaminase has an amino acid sequence selected from SEQ ID NO:
- 245, SEQ ID NOS:
- 247 to 249, and SEQ ID NOS:
- 274 to 283.
5 more elements
26:
- The engineered CRISPR/Cas12f1 composition of claim 25, wherein the engineered CRISPR/Cas12f1 composition comprises the engineered Cas12f1 protein and the guide RNA in a form of a ribonucleoprotein (RNP).
27:
- The engineered CRISPR/Cas12f1 composition of claim 25, wherein the engineered CRISPR/Cas12f1 composition comprises the nucleic acid encoding the engineered Cas12f1 protein and the nucleic acid encoding the guide RNA in a form of a vector.
28:
- The engineered CRISPR/Cas12f1 composition of claim 27, wherein the vector comprises the nucleic acid encoding the engineered Cas12f1 protein, a nucleic acid encoding a first guide RNA, and a nucleic acid encoding a second guide RNA, and
- a nucleotide sequence of a spacer in the first guide RNA and a nucleotide sequence of a spacer in the second guide RNA are different from each other.
29:
- The engineered CRISPR/Cas12f1 composition of claim 25, wherein the engineered Cas12f1 protein is a dCas12f1-base editing fusion protein comprising:
- the dead Cas12f1 protein;
- and
- at least one expression regulatory domain, each of which is selected from VP64, KRAB, MeCP2, DNMT, HDAC, Tet1, and p300.
1 more element
30:
- The engineered CRISPR/Cas12f1 composition of claim 25, wherein the engineered Cas12f1 protein is a dCas12f1-base editing fusion protein which further comprises at least one uracil glycosylase inhibitor (UGI),
- the deaminase has an amino acid sequence selected from SEQ ID NOS:
- 280 to 283, and
- the at least one UGI is fused to the dead Cas12f1 protein.
1 more element
31:
- The engineered CRISPR/Cas12f1 composition of claim 24, wherein the engineered Cas12f1 protein is the dCas12f1-expression regulating fusion protein of comprising:
- the dead Cas12f1 protein;
- and
- at least one expression regulatory domain, each of which is selected from VP64, KRAB, MeCP2, DNMT, HDAC, Tet1, and p300.
1 more element
32:
- The engineered CRISPR/Cas12f1 composition of claim 31, wherein the dCas12f1-expression regulating fusion protein comprises at least one VP64.
33:
- The engineered CRISPR/Cas12f1 composition of claim 31, wherein the dCas12f1-expression regulating fusion protein comprises at least one expression regulatory domain selected from VP64, KRAB, MeCP2, DNMT, HDAC, Tet1, and p300.
34:
- The engineered CRISPR/Cas12f1 composition of claim 31, wherein the engineered CRISPR/Cas12f1 composition comprises the engineered Cas12f1 protein and the guide RNA in a form of a ribonucleoprotein (RNP).
35:
- The engineered CRISPR/Cas12f1 composition of claim 31, wherein the engineered CRISPR/Cas12f1 composition comprises a vector that comprises the nucleic acid encoding the engineered Cas12f1 protein and the nucleic acid encoding the guide RNA.
36:
- The engineered CRISPR/Cas12f1 composition of claim 35, wherein the vector comprises the nucleic acid encoding the engineered Cas12f1 protein, a nucleic acid encoding a first guide RNA, and a nucleic acid encoding a second guide RNA, and
- a nucleotide sequence of a spacer in the first guide RNA and a nucleotide sequence of a spacer in the second guide RNA are different from each other.
37:
- A method for base editing of a target gene in a cell, comprising:
- introducing the engineered CRISPR/Cas12f1 composition of claim 29 into a living cell,
- wherein the target gene in the cell is double-stranded DNA comprising a target strand and a non-target strand,
- the target strand has a target sequence,
- the non-target strand has a protospacer adjacent motif (PAM) and a protospacer, the protospacer is a nucleotide sequence of 10 to 50 nts which is complementary to the target sequence,
- a spacer in the guide RNA of the engineered CRISPR/Cas12f1 composition is capable of hybridizing with the target sequence in the target strand, and
- the introduction of the engineered CRISPR/Cas12f1 composition into the cell causes a CRISPR-base editing complex to be formed in the cell, and the CRISPR-base editing complex replaces at least one adenine in the protospacer with guanine.
4 more elements
38:
- The method of claim 37, wherein the protospacer in the target gene contains at least one adenine at 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 15th, 16th, 17th, 18th, 19th, and 20th positions from the 5′ end, and performing the method for base editing results in replacement of at least one of the adenines at the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 15th, 16th, 17th, 18th, 19th, and 20th positions with guanine.
39:
- A method for base editing of a target gene in a cell, comprising:
- introducing the engineered CRISPR/Cas12f1 composition of claim 30 into the cell,
- wherein the target gene in the cell is double-stranded DNA comprising a target strand and a non-target strand,
- the target strand has a target sequence,
- the non-target strand has a protospacer adjacent motif (PAM) and a protospacer,
- the protospacer is a nucleotide sequence of 10 to 50 nts which is complementary to the target sequence,
- a spacer in the guide RNA of the engineered CRISPR/Cas12f1 composition is capable of hybridizing with the target sequence in the target strand, and
- the introduction of the engineered CRISPR/Cas12f1 composition into the cell causes a CRISPR-base editing complex to be formed in the cell, and the CRISPR-base editing complex replaces at least one cytosine in the protospacer with thymine.
5 more elements
40:
- The method of claim 39, wherein the protospacer in the target gene contains at least one cytosine at 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, and 9th positions from the 5′ end, and performing the method for base editing results in replacement of at least one of the cytosines at the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, and 9th positions with thymine.
41:
- The method of claim 37, wherein the cell is a eukaryotic cell.
42:
- A method for regulating expression of a target gene in a cell, comprising:
- introducing the engineered CRISPR/Cas12f1 composition of claim 31, into the cell,
- wherein the introduction of the engineered CRISPR/Cas12f1 composition into the cell causes a CRISPR gene regulating complex to be formed, and
- the CRISPR gene regulating complex regulates expression of the target gene.
1 more element
43:
- The method of claim 42, wherein the cell is a eukaryotic cell.