RNA-guided nucleases and active fragments and variants thereof and methods of use
Compositions and methods for binding to a target sequence of interest are provided. The compositions find use in cleaving or modifying a target sequence of interest, visualization of a target sequence of interest, and modifying the expression of a sequence of interest. Compositions comprise RNA-guided nuclease polypeptides, CRISPR RNAs, trans-activating CRISPR RNAs, guide RNAs, and nucleic acid molecules encoding the same. Vectors and host cells comprising the nucleic acid molecules are also provided. Further provided are CRISPR systems for binding a target sequence of interest, wherein the CRISPR system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs. Methods and kits for detecting a target DNA sequence are also provided.
1 . A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein said RGN polypeptide is selected from the group consisting of:
a) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117, 30, 75, 1, 9, 16, 23, 38, 46, 61, 69, 82, 89, 95, 103, or 110; and
b) an RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 54;
wherein said polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to said polynucleotide.
2 . The nucleic acid molecule of claim 1 , wherein said RGN polypeptide of a) is nuclease inactive or is capable of functioning as a nickase.
3 . The nucleic acid molecule of claim 1 , wherein the RGN polypeptide is operably fused to a base-editing polypeptide.
4 . A vector comprising the nucleic acid molecule of claim 1 .
5 . The vector of claim 4 , further comprising at least one nucleotide sequence encoding a guide RNA (gRNA) capable of hybridizing to a target DNA sequence, and wherein the RNA comprises a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 118, 31, 76, 2, 10, 17, 24, 39, 47, 55, 62, 70, 83, 90, 96, 104, or 111.
6 . The vector of claim 5 , wherein said gRNA comprises a tracrRNA having at least 95% sequence identity to SEQ ID NO: 119, 32, 77, 3, 11, 18, 25, 40, 48, 56, 63, 71, 84, 91, 97, 105, or 112.
7 . A cell comprising the nucleic acid molecule of claim 1 .
8 . A nucleic acid molecule comprising
(A) a polynucleotide encoding a CRISPR RNA (crRNA), wherein said crRNA comprises a spacer sequence and a CRISPR repeat sequence, wherein said CRISPR repeat sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 118, 31, 76, 2, 10, 17, 24, 39, 47, 55, 62, 70, 83, 90, 96, 104, or 111, and wherein said CRISPR repeat sequence is capable of hybridizing to a trans-activating CRISPR RNA (tracrRNA);
or
(B) a polynucleotide encoding a tracrRNA comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 119, 32, 77, 3, 11, 18, 25, 40, 48, 56, 63, 71, 84, 91, 97, 105, or 112, wherein said tracrRNA of (B) is capable of hybridizing to a CRISPR repeat sequence of a crRNA;
wherein a guide RNA comprising:
i) said crRNA of (A);
or
ii) said tracrRNA of (B) and a crRNA comprising a spacer sequence and a CRISPR repeat sequence that is capable of hybridizing to the tracrRNA of (B)
is capable of hybridizing to a target DNA sequence of a DNA molecule in a sequence specific manner through the spacer sequence of said crRNA of (A) or (B) when said guide RNA is bound to an RNA-guided nuclease (RGN) polypeptide, and
wherein said polynucleotide encoding said crRNA of (A) or said tracrRNA of (B) is operably linked to a promoter heterologous to said polynucleotide.
9 . A vector comprising the nucleic acid molecule of claim 8 .
10 . A system for binding a target DNA sequence of a DNA molecule, said system comprising:
a) a guide RNA (gRNA), or a polynucleotide comprising a nucleotide sequence encoding the gRNA, wherein the gRNA is capable of hybridizing to said target DNA sequence; and
b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence selected from the group consisting of:
i) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117, 30, 75, 1, 9, 16, 23, 38, 46, 61, 69, 82, 89, 95, 103, or 110; and
ii) the amino acid sequence set forth as SEQ ID NO: 54;
or a polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide;
wherein at least one of said nucleotide sequence encoding the gRNA and encoding the RGN polypeptide is operably linked to a promoter heterologous to said nucleotide sequence.
11 . The system of claim 10 , wherein the target DNA sequence is within a eukaryotic cell.
12 . The system of claim 10 , wherein said RGN polypeptide of b) i) is nuclease inactive or is capable of functioning as a nickase.
13 . The system of claim 10 , wherein said system further comprises one or more donor polynucleotides or one or more nucleotide sequences encoding the one or more donor polynucleotides.
14 . A method for binding a target DNA sequence of a DNA molecule comprising delivering a system according to claim 10 , to said target DNA sequence or a cell comprising the target DNA sequence.
15 . A method for cleaving or modifying a target DNA sequence of a DNA molecule comprising delivering a system according to claim 10 , to said target DNA sequence or a cell comprising the DNA molecule and cleavage or modification of said target DNA sequence occurs.
16 . A method for cleaving and/or modifying a target DNA sequence of a DNA molecule, comprising contacting the DNA molecule with:
a) an RNA-guided nuclease (RGN) polypeptide, wherein said RGN comprises an amino acid sequence selected from the group consisting of:
i) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117, 30, 75, 1, 9, 16, 23, 38, 46, 61, 69, 82, 89, 95, 103, or 110; and
ii) the amino acid sequence set forth as SEQ ID NO: 54;
and
b) one or more guide RNAs capable of targeting the RGN of (a) to the target DNA sequence;
wherein the one or more guide RNAs hybridize to the target DNA sequence, thereby directing said RGN polypeptide to bind to said target DNA sequence and cleavage and/or modification of said target DNA sequence occurs.
17 . The method of claim 16 , wherein said RGN polypeptide is nuclease inactive or functions as a nickase.
18 . The method of claim 16 , wherein the target DNA sequence is within a eukaryotic cell.
19 . The method of claim 16 , further comprising culturing a cell comprising said target DNA sequence under conditions in which the RGN polypeptide is expressed and cleaves the target DNA sequence to produce a DNA molecule comprising a modified DNA sequence; and selecting a cell comprising said modified target DNA sequence.
20 . The nucleic acid molecule of claim 1 , wherein the RGN polypeptide comprises the amino acid sequence of SEQ ID NO: 117, 30, 75, 1, 9, 16, 23, 38, 46, 61, 69, 82, 89, 95, 103, or 110.
21 . The nucleic acid molecule of claim 3 , wherein the base-editing polypeptide is a deaminase.
22 . The nucleic acid molecule of claim 1 , wherein the RGN polypeptide is operably fused to an effector domain.
23 . The nucleic acid molecule of claim 22 , wherein the effector domain is a cleavage domain, a deaminase domain, or an expression modulator domain.
24 . The nucleic acid molecule of claim 23 , wherein the expression modulator domain is an epigenetic modification domain, a transcriptional repressor domain, or a transcriptional activation domain.
25 . The nucleic acid molecule of claim 22 , wherein the effector domain is operably fused at the N-terminus or at the C-terminus of the RGN polypeptide.
26 . The nucleic acid molecule of claim 22 , wherein the effector domain is operably fused at an internal location of the RGN polypeptide.
27 . The system of claim 10 , wherein the RGN polypeptide comprises the amino acid sequence of SEQ ID NO: 117, 30, 75, 1, 9, 16, 23, 38, 46, 61, 69, 82, 89, 95, 103, or 110.
28 . The system of claim 10 , wherein the RGN polypeptide is operably linked to a base-editing polypeptide.
29 . The system of claim 28 , wherein the base-editing polypeptide is a deaminase.
30 . The system of claim 10 , wherein the RGN polypeptide is operably fused to an effector domain.
31 . The system of claim 30 , wherein the effector domain is a cleavage domain, a deaminase domain, or an expression modulator domain.
32 . The system of claim 31 , wherein the expression modulator domain is an epigenetic modification domain, a transcriptional repressor domain, or a transcriptional activation domain.
33 . The system of claim 30 , wherein the effector domain is operably fused at the N-terminus or at the C-terminus of the RGN polypeptide.
34 . The system of claim 30 , wherein the effector domain is operably fused at an internal location of the RGN polypeptide.
35 . The method of claim 14 , wherein the target DNA sequence is cleaved and/or modified.
36 . The method of claim 14 , wherein the target DNA sequence is not cleaved and/or modified.
37 . The method of claim 16 , wherein the RGN polypeptide comprises the amino acid sequence of SEQ ID NO: 117, 30, 75, 1, 9, 16, 23, 38, 46, 61, 69, 82, 89, 95, 103, or 110.
38 . The method of claim 16 , wherein the RGN polypeptide is operably linked to a base-editing polypeptide.