Crispr/cas12f enzyme and system

Patent metadata

Field	Value
Publication number	US-2021395784-A1
Application number	US-201917289204-A
Country	US
Kind code	A1
Filing date	Oct 29, 2019
Priority date	Oct 29, 2018
Publication date	Dec 23, 2021
Grant date	—

Abstract

The application belongs to the field of nucleic acid editing, in particular to the field of clustered regularly interspaced short palindromic repeats (CRISPR) technology. In particular, the application provides a Cas effector protein, a fusion protein with the Cas effector protein, and a nucleic acid molecule encoding the same. Also provided are a compound and a composition for nucleic acid editing (e.g., gene or genome editing) with the protein or the nucleic acid molecule, and a method for nucleic acid editing (e.g., gene or genome editing) using the protein.

First claim

1 .- 50 . (canceled)

51 . A protein comprising an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3 with one or more amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions compared to SEQ ID NOs: 1, 2, or 3) and of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID NOs: 1, 2, and 3;
for example, the protein is an effector protein in the CRISPR/Cas system.

52 . A conjugate or a fusion protein comprising the protein of claim 51 and a modified portion, an additional protein, or an additional polypeptide,
wherein the modified portion, the additional protein, or the additional polypeptide is selected from a protein, a polypeptide, a detectable label, an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting moiety, a transcription activation domain (for example, VP64), a transcription repression domain (for example, KRAB domain or SID domain), a nuclease domain (for example, Fok1), a domain having an activity selected from: nucleotide deaminase, methylase activity, demethylase, transcription activation activity, transcription inhibition activity, transcription release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, and nucleic acid binding activity, and any combinations thereof.

53 . The conjugate or the fusion protein of claim 52 , wherein the modified portion, the additional protein, or the additional polypeptide is connected to the N-terminus or C-terminus of the protein through a linker.

54 . The conjugate or the fusion protein claim 52 , wherein the conjugate or the fusion protein comprises an NLS sequence and
wherein the NLS sequence is shown in SEQ ID NO: 19 and/or the NLS sequence is located at, near, or close to the end of the protein (e.g., N-terminal or C-terminal) or wherein the fusion protein has an amino acid sequence as shown in SEQ ID NO:20

55 . An isolated nucleic acid molecule comprising a sequence selected from the following or consisting of a sequence selected from the following:
(i) a sequence of SEQ ID NO: 7 or 13 with one or more base substitutions, deletions or additions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base substitutions, deletions or additions compared to SEQ ID NO: 7 or 13) and of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% sequence identity with the sequence as shown in SEQ ID NO:7 or 13; (ii) a sequence that hybridizes to the sequence as described in (i) under stringent conditions; or (iii) a complementary sequence of the sequence as described in (i); in addition, the sequence as described in any one of (i)-(iii) substantially retains the biological function of the sequence from which it is derived; for example, the isolated nucleic acid molecule is RNA; for example, the isolated nucleic acid molecule is a direct repeat sequence in the CRISPR/Cas system.

56 . The isolated nucleic acid molecule of claim 55 , wherein the nucleic acid molecule comprises one or more stem loops or optimized secondary structures;
for example, the sequence as described in any one of (i) to (iii) retains the secondary structure of the sequence from which it is derived.

57 . A complex comprising:
(i) a protein component having an amino acid sequence of SEQ ID NOs: 1, 2, or 3 with one or more amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions compared to SEQ ID NOs: 1, 2, or 3) and of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID NOs: 1, 2, and 3; and (ii) a nucleic acid component, which comprises the isolated nucleic acid molecule of claim 55 and a targeting sequence capable of hybridizing to the target sequence from 5′ to 3′ direction, wherein the protein component and the nucleic acid component combine with each other to form a complex; for example, the nucleic acid component is a guide RNA in the CRISPR/Cas system; for example, the nucleic acid molecule is RNA; for example, the complex does not contain trans-activating crRNA (tracrRNA).

58 . The complex of claim 57 , wherein the targeting sequence is attached to the 3′ end of the nucleic acid molecule or wherein the targeting sequence comprises a complementary sequence of the target sequence.

59 . An isolated nucleic acid molecule comprising:
(i) a nucleotide sequence encoding the protein component of claim 57 ; (ii) a nucleotide sequence encoding the nucleic acid component of claim 57 ; and/or (iii) a nucleotide sequence containing (i) and (ii); for example, the nucleotide sequence described in any one of (i) to (iii) is codon-optimized for expression in a prokaryotic cell or an eukaryotic cell.

60 . A vector comprising the isolated nucleic acid molecule of claim 59 .

61 . A host cell comprising the vector of claim 60 .

62 . A composition comprising:
(i) a first component, which is selected from: a protein having an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3 with one or more amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions compared to SEQ ID NOs: 1, 2, or 3) and of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID NOs: 1, 2, and 3, a nucleotide sequence encoding the protein, and any combinations thereof; and (ii) a second component, which is a nucleotide sequence containing a guide RNA, or a nucleotide sequence encoding the nucleotide sequence containing a guide RNA; wherein the guide RNA includes a direct repeat sequence and a targeting sequence from the 5′ to 3′, and the targeting sequence can hybridize with the target sequence; the targeting RNA can form a complex with the protein, conjugate or fusion protein as described in (i); the direct repeat sequence is an isolated nucleic acid molecule as defined claim 55 ; for example, the composition does not contain a trans-activating crRNA (tracrRNA).

63 . A composition comprising one or more vectors comprising:
(i) a first nucleic acid, which is a nucleotide sequence encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 3, with one or more amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions compared to SEQ ID NOs: 1, 2, or 3) and of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at

Assignees

• Univ China Agricultural

Inventors

• Lai Jinsheng
• Zhou Yingsi
• Zhu Jinjie
• Yi Fei
• Zhang Xiangbo
• Zhao Haiming
• Song Weibin

Classifications

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/902 Primary

  using homologous recombination · CPC title