# US 12,760,331 B2 · CAS12 proteins and uses thereof

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- Number: US 12,760,331 B2
- Document ID: US12760331B2
- Status: Granted
- Kind: B2
- Application number: 19/303,600
- Filed: 2025-08-19
- Published: 2026-01-08
- Granted: 2026-10-13
- Earliest claimed: 2023-08-19
- Assignees: Reforgene Medicine; Zhejiang Synsorbio Technology Co., Ltd.
- Applicants: Reforgene Medicine; Zhejiang Synsorbio Technology Co., Ltd.
- Inventors: Junbin Liang; Liancheng Huang; Chongjian Chen; Yang Sun; Weiye Pan; Hui Xu; Kaiwei Si; Jinxiu Cai; Qing Liao; Desheng Huangfu
- Web page: https://patentgrep.com/patent/US12760331B2

## CPC

- C12N 15/907: {in mammalian cells}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/1082: {Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors}
- 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 15/90: Stable introduction of foreign DNA into chromosome
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Cas12 protein, guide polynucleotide, inactivated Cas12 mutant, fusion protein or conjugate including the Cas12 protein, isolated nucleic acid, CRISPR-Cas12 system, vector system, delivery system, cell, pharmaceutical composition, and kit, and the use thereof are provided.

## Claims

### Claim 1

A non-naturally occurring Cas12 protein, wherein the Cas12 protein comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence shown in SEQ ID NO: 696, wherein the Cas12 protein has one or more mutations at amino acid residues with corresponding positions in the sequence shown in SEQ ID NO: 696;
the Cas12 protein forms a complex with a guide polynucleotide, the guide polynucleotide comprises a guide sequence that is reverse complementary to a target nucleic acid, and
the guide polynucleotide comprises a scaffold sequence that interacts with the Cas12 protein.

### Claim 2 (depends on 1)

The Cas12 protein of claim 1, wherein the Cas12 protein has at least one mutation in at least one of the amino acid residues corresponding to positions 1, 2, 3, 4, 5, 7, 10, 24, 30, 48, 51, 55, 58, 59, 66, 108, 118, 138, 141, 175, 178, 185, 186, 257, 333, 352, 356, 375, 376, 378, 379, 383, 397, 400, 416, 426, 443, 449, 456, 459, 462, 469, 484, 485, 509, 561, 597 607, 609, 623, 638, 639, 640, 697, 722, 731, 733, 755, 758, 771, 773, 779, 781, 784, 785, 786, 789, 792, 794, 798, 822, 823, 825, 826, 829, 830, 833, 834, 836, 842, 845, 846, 847, 850, 851, 853, 855, 856, 858, 859, 860, 866, 884, 892, 893, 900, 904, 926, 956, 985, 988, 989, 992, 993, 996, 1016, 1033, 1045, 1050, 1073, 1074, 1095, 1100, 1124, 1129, or 1132 of the amino acid sequence shown in SEQ ID NO: 696.

### Claim 3 (depends on 1)

The Cas12 protein of claim 1, wherein the Cas12 protein has a mutation at an amino acid residue corresponding to any position selected from I33, G184, S185, Q186, G194, N195, G196, G197, N245, G256, L260, Y278, S285, Y316, H350, D352, A355, A356, C385, P386, H387, G390, K391, N392, D429, Q461, Q462, Q469, E485, S491, K521, P525, L611, K629, K631, N633, D841, N898, K987, A988, G989, Q990, T991, D1010, E1013, A1136, K1138, or T1139, in the amino acid sequence shown in SEQ ID NO: 696.

### Claim 4 (depends on 1)

The Cas12 protein of claim 1, wherein the Cas12 protein has any mutation combination of the amino acid sequence shown in SEQ ID NO: 696 at positions corresponding to the amino acid sequence shown in SEQ ID NO: 696, and the mutation combination is selected from 186+352+1+426+846+858+860, 186+352+1+426+846+860, 186+352+1+5+426+858+860, 186+352+1+3+426+858+860, 186+352+3+426+860, 186+352+1+333+426+858+860, 186+352+1+426+485+858+860, 186+352+1+5+426+860, 186+352+3+426+858+860, 186+352+5+426+858+860, 186+352+333+426+858+860, 186+352+333+426+860, 186+352+426+846+860, 186+352+5+426+860, 186+352+1+3+426+860, 5+426+860, 186+352+426+846+85+860, 186+352+1+426+485+860, 426+858+860, 7+426+858, 186+352+426+860, 186+352+426+485+858+860, 186+352+426+485+860, 426+846+858+860, 7+426+846, 186+352+860, 184+186+352+376+1132, 5+333+426, 5+426+858, 186+352+1+333+426+860, 184+186+352+3+107+426, 186+352+5+426, 333+376+426, 186+352+5, 2+5+846+858, 186+352+3+426, 5+846+858, 186+352+426+846, 186+352+7, 186+352+3+376+426, 186+352+376+426+860+865, 186+352+376+426, 3+846+860, 846+858+988, 3+426+858, 3+846+858+860, 3+858+860, 186+352+858, 184+186+352+860, 333+426, 184+186+352+3+376, 3+426+860, 846+860+988, 3+860, 184+186+352+3+639, 186+352+333+426, 846+585+860, 186+352+426, 333+426+846, 333+426+485, 5+846+860, 3+846+988, 184+186+352+376, 3+333+426, 186+352+426+485, 333+426+858, 333+426+1132, 428+485, 186+352+333+352+376+426, 858+860, 186+352+376+426+485+860, 184+186+352+426, 186+352+639, 5+858+988, 3+858+988, 5+858, 3+858, 184+186+352+846, 184+186+352+639, 858+988, 184+186+352+426+1132, 186+352+1132, 184+186+352+5, 184+186+352+858, 858+860+1132, 3+5, 426+649, 186+352+426+485+860, 186+352+333, 184+186+352, 186+376, 846+860, 858+988+1132, 846+858, 333+376, 376+426, 184+186+352+3, 3+846+1132, 5+846+1132, 186+352+426+1132, 376+426+485+660, 426, 5+846, 846+860+1132, 333+376+485, 184+186+352+639+1132, 352+426, 333+485, 184+186+352+333, 846+858+1132, 333+426+860, 186+352+988, 5+860, 846+988, 186+352, 3+846, 846+1132, 184+186+352+1132, 186+485, 988+1132, 184+186+352+485, 376+485, 5+1132, 3+7, 186+352+485, 184+186+352+7, 184+186+352+333+336, 3+1132, 426+858+988, 186+352+376, 186+352+3, and 186+352+333+336+352+376+426.

### Claim 5 (depends on 1)

A nuclease-inactivated mutant of the Cas12 protein of claim 1, wherein an inactivating mutation of the nuclease-inactivated mutant is selected from one or more of D651A, E891A, and D1082A corresponding to the amino acid sequence shown in SEQ ID NO: 696.

### Claim 6

A fusion protein or conjugate, comprising:
(1) a Cas12 protein, wherein the Cas12 protein comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence shown in SEQ ID NO: 696; and
(2) a homologous or heterologous functional domain.

### Claim 7 (depends on 6)

The fusion protein or conjugate of claim 6, wherein the homologous or heterologous functional domain is selected from any one, two, three, four, or more of the following: a subcellular positioning signal, a DNA binding domain, a protease domain, a transcriptional activation domain, a transcriptional repression domain, a nuclease domain, a deaminase domain, a uracil DNA glycosylase domain (UDG), a uracil DNA glycosylase inhibitory domain (UGI), a DNA methyltransferase, a DNA demethylase, a histone methyltransferase, a histone demethylase, a transcription release factor, a histone acetylase domain, a histone deacetylase domain, a DNA ligase, an affinity tag, a reporter tag, an affinity domain, and a reporter domain.

### Claim 8 (depends on 7)

The fusion protein or conjugate of claim 7, wherein the subcellular positioning signal is selected from a nuclear localization signal, a nuclear export signal, a mitochondrial localization signal, and a chloroplast localization signal.

### Claim 9 (depends on 1)

An isolated nucleic acid, wherein the isolated nucleic acid encodes the Cas12 protein of claim 1.

### Claim 10 (depends on 1)

A kit, comprising the Cas12 protein of claim 1.