Novel crispr dna targeting enzymes and systems

Patent metadata

Field	Value
Publication number	US-2020407715-A1
Application number	US-202017020215-A
Country	US
Kind code	A1
Filing date	Sep 14, 2020
Priority date	Mar 14, 2018
Publication date	Dec 31, 2020
Grant date	—

Abstract

The disclosure describes novel systems, methods, and compositions for the manipulation of nucleic acids in a targeted fashion. The disclosure describes non-naturally occurring, engineered CRISPR systems, components, and methods for targeted modification of nucleic acids such as DNA. Each system includes one or more protein components and one or more nucleic acid components that together target nucleic acids.

First claim

What is claimed is:

1 . An engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)—associated (Cas) system comprising:
(a) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence; and
(b) a CRISPR-Cas effector protein or a nucleic acid encoding the CRISPR-Cas effector protein, wherein the CRISPR-Cas effector protein comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 5,
wherein the CRISPR-Cas effector protein binds to the RNA guide, and wherein the spacer sequence is complementary to at least 15 nucleotides of a target nucleic acid.

2 . The system of claim 1 , wherein the system does not include a tracrRNA.

3 . The system of claim 1 , wherein the CRISPR-Cas effector protein comprises one or more of:
(a) a RuvC domain comprising the amino acid sequence X 1 SHX 4 DX 6 X 7 (SEQ ID NO: 200), wherein X 1 is S or T, X 4 is Q or L, X 6 is P or S, and X 7 is F or L, (b) a RuvC domain comprising the amino acid sequence XIXDXNX 6 X 7 XXXX 11 (SEQ ID NO: 201), wherein X 1 is A, G, or S, X is any amino acid, X 6 is Q or I, X 7 is T, S, or V, and X 10 is T or A; and (c) a RuvC domain comprising the amino acid sequence X 1 X 2 X 3 E (SEQ ID NO: 210), wherein X 1 is C, F, I, L, M, P, V, W, or Y, X 2 is C, F, I, L, M, P, R, V, W, or Y, and X 3 is C, F, G, I, L, M, P, V, W, or Y.

4 . The system of claim 1 , wherein the CRISPR-Cas effector protein comprises the amino acid sequence set forth in SEQ ID NO: 5.

5 . The system of claim 1 , wherein the direct repeat sequence comprises any one of:
(a) 5′-CCGUCNNNNNNUGACGG-3′ (SEQ ID NO: 202) proximal to the 3′ end, wherein N is any nucleobase; (b) 5′-GUGCCNNNNNNUGGCAC-3′ (SEQ ID NO: 203) proximal to the 3′ end, wherein N is any nucleobase; (c) 5′-GUGUCN 5-6 UGACAX 1 -3′ (SEQ ID NO: 204) proximal to the 3′ end, wherein N 5 -6 is a contiguous sequence of any 5 or 6 nucleobases, and X 1 is C or T or U; (d) 5′-UCX 3 UX 5 X 6 X 7 UUGACGG-3′ (SEQ ID NO: 205) proximal to the 3′ end, wherein X 3 is C, T, or U, X 5 is A, T, or U, X 6 is A, C, or G, and X 7 is A or G; (e) 5′-CCX 3 X 4 X 5 CX 7 UUGGCAC-3′ (SEQ ID NO: 206) proximal to the 3′ end, wherein X 3 is C, T, or U, X 4 is A, T, or U, X 5 is C, T, or U, and X 7 is A or G.

6 . The system of claim 1 , wherein the direct repeat sequence comprises a nucleotide sequence with at least 95% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10.

7 . The system of claim 1 , wherein the direct repeat sequence comprises a nucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10.

8 . The system of claim 1 , wherein the direct repeat sequence comprises a stem-loop structure proximal to a 3′ end of the direct repeat sequence, wherein the stem-loop structure comprises:
(a) a first stem nucleotide strand 5 nucleotides in length;
(b) a second stem nucleotide strand 5 nucleotides in length, wherein the first and second stem nucleotide strands bind with each other; and
(c) a loop nucleotide strand arranged between the first and second stem nucleotide strands, wherein the loop nucleotide strand comprises 6, 7, or 8 nucleotides.

9 . The system of claim 1 , wherein the spacer sequence comprises between 15 and 47 nucleotides in length.

10 . The system of claim 9 , wherein the spacer sequence comprises between 24 and 38 nucleotides in length.

11 . The system of claim 9 , wherein the spacer sequence comprises between 20 and 33 nucleotides in length.

12 . The system of claim 9 , wherein the spacer sequence has at least 90% complementarity to the target nucleic acid.

13 . The system of claim 9 , wherein the spacer sequence has at least 95% complementarity to the target nucleic acid.

14 . The system of claim 9 , wherein the spacer sequence has 100% complementarity to the target nucleic acid.

15 . The system of claim 1 , wherein the CRISPR-Cas effector protein recognizes a protospacer adjacent motif (PAM) sequence, wherein the PAM sequence comprises a nucleotide sequence set forth as 5′-TTN-3′, wherein N is any nucleotide.

16 . The system of claim 15 , wherein the PAM sequence comprises a nucleotide sequence set forth as 5′-TTY-3′, wherein Y is C or T, or 5′-TTH-3′, wherein H is A or C or T.

17 . The system of claim 1 , wherein the CRISPR-Cas effector protein further comprises at least one nuclear localization signal (NLS), at least one nuclear export signal (NES), or at least one NLS and at least one NES.

18 . The system of claim 1 , wherein the CRISPR-Cas effector protein further comprises a peptide tag, a fluorescent protein, a base-editing domain, a DNA methylation domain, a histone residue modification domain, a localization factor, a transcription modification factor, a light-gated control factor, a chemically inducible factor, or a chromatin visualization factor.

19 . The system of claim 1 , wherein the nucleic acid encoding the CRISPR-Cas effector protein is codon-optimized for expression in a cell.

20 . The system of claim 1 , wherein the nucleic acid encoding the CRISPR-Cas effector protein is operably linked to a promoter.

21 . The system of claim 1 , wherein the nucleic acid encoding the CRISPR-Cas effector protein is in a vector.

22 . The system of claim 21 , wherein the vector comprises a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

23 . The system of claim 1 , wherein the system is present in a delivery system comprising a nanoparticle, a liposome, an exosome, a microvesicle, or a gene-gun.

24 . A cell comprising the system of claim 1 .

25 . The cell of claim 24 , wherein the cell is a prokaryotic cell or a eukaryotic cell.

26 . The cell of claim 24 , wherein the cell is a mammalian cell or a plant cell.

27 . The cell of claim 26 , wherein the cell is a human cell.

28 . A method of binding the system of claim 1 to the target nucleic acid in a cell comprising:
(a) providing the system; and
(b) delivering the system to the cell,
wherein the cell comprises the target nucleic acid, wherein the CRISPR-Cas effector protein binds to the RNA guide, and wherein the spacer sequence binds to the target nucleic acid.

29 . The method of claim 28 , wherein the target nucleic acid is a single-stranded DNA or a double-stranded DNA.

30 . The method of claim 28 , wherein binding the system to the target nucleic acid results in cleavage of the target nucleic acid.

31 . The method of claim 30 , wherein cleavage of the target nucleic acid results in formation of an insertion or a deletion in the target nucleic acid.

Assignees

• Arbor Biotechnologies Inc

Inventors

• Chong Shaorong
• Yan Winston X
• Scott David A
• Cheng David R
• Hunnewell Pratyusha

Classifications

• C12N2310/20

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

• C12N15/902

  using homologous recombination · CPC title

• C12N15/113

  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)} · CPC title

• C12N9/22 Primary

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

• C12N15/90

  Stable introduction of foreign DNA into chromosome · CPC title