MODIFIED NUCLEASES
Provided herein are methods and compositions utilizing modified nucleases and/or other components, such as guide nucleic acids and donor templates, for use in a CRISPR system.
1 .- 122 . (canceled)
123 . A composition comprising a nucleic acid-guided nuclease comprising a Type V CRISPR nuclease polypeptide comprising at least three nuclear localization signals (NLS) at or near the N-terminus or the C-terminus of the polypeptide, wherein at least two of the NLSs are at or near the N-terminus of the polypeptide, or a polynucleotide encoding the nuclease.
124 . The composition of claim 123 wherein the nuclease is a Type Va nuclease.
125 . The composition of claim 123 wherein the Type V CRISPR nuclease polypeptide has at least 80% sequence identity with SEQ ID NO: 1.
126 . The composition of claim 123 wherein the Type V CRISPR nuclease polypeptide comprises four NLSs, each of which is at or near the N-terminus or the C-terminus of the polypeptide.
127 . The composition of claim 123 wherein the Type V CRISPR nuclease polypeptide comprises at least five NLSs, each of which is at or near the N-terminus or the C-terminus of the polypeptide.
128 . The composition of claim 123 wherein at least three of the NLSs are at or near the N-terminus of the polypeptide.
129 . The composition of claim 126 wherein at least four of the NLSs are at or near the N-terminus of the polypeptide.
130 . The composition of claim 127 wherein the 5 NLSs are at or near the N-terminus of the polypeptide.
131 . The composition of claim 130 wherein the polypeptide comprises a sequence at least 80%, identical to any one of SEQ ID NOs: 109-112.
132 . The composition of claim 123 wherein at least two of the NLSs have different nuclear localization mechanisms.
133 . The composition of claim 123 wherein one or more of the NLSs comprises an NLS of the SV40 virus large T-antigen, an NLS from nucleoplasmin, e.g. a nucleoplasmin bipartite NLS, a c-myc NLS; a hRNPA1 M9 NLS; an IBB domain of importin-alpha NLS; a myoma T protein NLS; a sequence from human p53 NLS; a sequence of mouse c-abl IV NLS; a sequence of influenza virus NS1 NLS; a sequence of Hepatitis virus delta antigen NLS; a sequence of mouse Mx1 protein NLS; a sequence of human poly(ADP-ribose) polymerase NLS; a sequence of steroid hormone receptors (human) glucocorticoid NLS; and/or a sequence of EGL-13 NLS.
134 . The composition of claim 123 wherein the Type V CRISPR nuclease polypeptide further comprises a purification tag at or near the N-terminus of the nuclease polypeptide.
135 . The composition claim 134 wherein the Type V CRISPR nuclease polypeptide comprises a cleavage site at or near the N-terminus of the nuclease polypeptide.
136 . The composition of claim 135 comprising 5 NLSs at or near the N-terminus of the polypeptide, a purification tag, and the cleavage site, wherein the cleavage site is after the purification tag.
137 . The composition of claim 136 comprising a sequence at least 80%, identical to SEQ ID NO: 111 or 112.
138 . The composition of claim 123 further comprising a guide nucleic acid (gNA), comprising a spacer sequence that targets a target nucleotide sequence within a polynucleotide, or a polynucleotide coding for the gNA, wherein the gNA, is compatible with the Type V CRISPR nuclease.
139 . The composition of claim 138 wherein the gRNA is a dual gRNA.
140 . A method for modifying a target polynucleotide comprising:
(A) contacting the polynucleotide with a composition comprising
(i) a nucleic acid-guided nuclease comprising a Type V CRISPR nuclease polypeptide comprising at least three nuclear localization signals (NLS) at or near the N-terminus or the C-terminus of the polypeptide, wherein at least two of the NLSs are at or near the N-terminus of the polypeptide, or a polynucleotide encoding the nuclease, and
(ii) a guide RNA compatible with the nuclease and comprising a spacer sequence complementary to the target polynucleotide or a portion thereof; and
(B) allowing the nuclease to modify the target polynucleotide.
141 . The method of claim 140 wherein the target polynucleotide is a genome or a portion of a genome within a cell.
142 . The method of claim 140 wherein the guide RNA is a dual guide RNA.
143 . The method of claim 140 wherein the nuclease is a Type Va nuclease.
144 . The method of claim 140 wherein the Type V CRISPR nuclease polypeptide has at least 80% sequence identity with SEQ ID NO: 1.
145 . The method of claim 140 wherein the Type V CRISPR nuclease polypeptide comprises four NLSs, each of which is at or near the N-terminus or the C-terminus of the polypeptide.
146 . The method of claim 140 wherein the Type V CRISPR nuclease polypeptide comprises at least five NLSs, each of which is at or near the N-terminus or the C-terminus of the polypeptide.
147 . The method of claim 146 wherein at least three of the NLSs are at or near the N-terminus of the polypeptide.
148 . The method of claim 146 wherein at least four of the NLSs are at or near the N-terminus of the polypeptide.
149 . The method of claim 146 wherein the 5 NLSs are at or near the N-terminus of the polypeptide.