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 (RGN) 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 RGN systems for binding a target sequence of interest, wherein the RGN system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs.
1 . A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 117, and wherein the RGN polypeptide is operably fused to a base-editing polypeptide.
2 . The nucleic acid molecule of claim 1 , wherein the RGN polypeptide is operably fused to one or more nuclear localization signals.
3 . The nucleic acid molecule of claim 1 , wherein the RGN polypeptide is codon optimized for expression in a eukaryotic cell.
4 . A cell comprising the nucleic acid molecule of claim 1 .
5 . The nucleic acid molecule of claim 1 , wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117.
6 . The nucleic acid molecule of claim 1 , wherein the RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 117.
7 . The nucleic acid molecule of claim 1 , wherein said RGN polypeptide is nuclease inactive or is a nickase.
8 . The nucleic acid molecule of claim 1 , wherein said polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to said polynucleotide.
9 . A vector comprising a nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 117, wherein said polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to said polynucleotide,
wherein the vector further comprises at least one nucleotide sequence encoding a guide RNA capable of hybridizing to a target DNA sequence, wherein the guide RNA comprises:
i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 118; and
ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 119.
10 . The vector of claim 9 , wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117.
11 . The vector of claim 9 , wherein the RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 117.
12 . The vector of claim 9 , wherein the CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 118.
13 . The vector of claim 9 , wherein the CRISPR repeat sequence is set forth as SEQ ID NO: 118.
14 . The vector of claim 9 , wherein the tracrRNA has at least 95% sequence identity to SEQ ID NO: 119.
15 . The vector of claim 9 , wherein the tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 119.
16 . A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 117, wherein the RGN polypeptide is operably fused to a deaminase, and wherein the RGN polypeptide is capable of recognizing a protospacer adjacent motif (PAM) comprising NGG (SEQ ID NO: 116).
17 . The nucleic acid molecule of claim 16 , wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117.
18 . The nucleic acid molecule of claim 16 , wherein the RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 117.
19 . The nucleic acid molecule of claim 16 , wherein said RGN polypeptide is nuclease inactive or is a nickase.
20 . The nucleic acid molecule of claim 16 , wherein said polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to said polynucleotide.
21 . A cell comprising the nucleic acid molecule of claim 16 .