CLASS II, TYPE V CRISPR SYSTEMS
Described herein are methods, compositions, and systems derived from uncultivated microorganisms useful for gene editing.
1 .- 164 . (canceled)
165 . An engineered nuclease system comprising:
(a) an endonuclease having at least 75% sequence identity to SEQ ID NO: 215 or a variant thereof; and
(b) an engineered guide ribonucleic acid (RNA), wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a spacer sequence configured to hybridize to a target nucleic acid sequence.
166 . The engineered nuclease system of claim 165 , wherein said engineered guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3610, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, and 3851-3857.
167 . The engineered nuclease system of claim 165 , wherein said endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence comprising any one of SEQ ID NOs: 3863-3913.
168 . The engineered nuclease system of claim 165 , wherein said engineered guide RNA comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence.
169 . The engineered nuclease system of claim 165 , wherein said engineered guide RNA is 30-250 nucleotides in length.
170 . The engineered nuclease system of claim 165 , wherein said endonuclease comprises at least one of an S168R mutation, an E172R mutation, an N577R mutation, or a Y170R mutation when a sequence of said endonuclease is optimally aligned based on a pairwise alignment to SEQ ID NO: 215.
171 . The engineered nuclease system of claim 170 , wherein said endonuclease comprises said S168R mutation and said E172R mutation when a sequence of said endonuclease is optimally aligned based on a pairwise alignment to SEQ ID NO: 215.
172 . The engineered nuclease system of claim 170 , wherein said endonuclease comprises said N577R mutation or said Y170R mutation when a sequence of said endonuclease is optimally aligned based on a pairwise alignment to SEQ ID NO: 215.
173 . The engineered nuclease system of claim 170 , wherein said endonuclease comprises said S168R mutation when a sequence of said endonuclease is optimally aligned based on a pairwise alignment to SEQ ID NO: 215
174 . The engineered nuclease system of claim 165 , wherein said endonuclease does not comprise a mutation of E172, N577, or Y170.
175 . The engineered nuclease system of claim 165 , further comprising a single- or double-stranded deoxyribonucleic acid (DNA) repair template comprising from 5′ to 3′: a first homology arm comprising a sequence of at least 20 nucleotides 5′ to said target nucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and a second homology arm comprising a sequence of at least 20 nucleotides 3′ to said target sequence.
176 . The engineered nuclease system of claim 175 , wherein said first homology arm or said second homology arm comprises a sequence of at least 40 nucleotides.
177 . The engineered nuclease system of claim 175 , wherein said first homology arm or said second homology arm is homologous to a genomic sequence of a prokaryote, bacteria, fungus, or eukaryote.
178 . The engineered nuclease system of claim 175 , wherein said single- or double-stranded DNA repair template comprises a transgene donor.
179 . The engineered nuclease system of claim 165 , further comprising a deoxyribonucleic acid (DNA) repair template comprising a double-stranded DNA segment further comprising one single-stranded DNA segment or two single-stranded DNA segments flanking said double-stranded DNA segment.
180 . The engineered nuclease system of claim 179 , wherein said one single-stranded DNA segment or said two single-stranded DNA segments have a length from 4 to 10 nucleotide bases.
181 . The engineered nuclease system of claim 179 , wherein said double-stranded DNA segment comprises a barcode, an open reading frame, an enhancer, a promoter, a protein-coding sequence, a miRNA coding sequence, an RNA coding sequence, or a transgene.
182 . The engineered nuclease system of claim 179 , wherein said double-stranded DNA segment is flanked by a nuclease cut site.
183 . The engineered nuclease system of claim 165 , wherein said endonuclease comprises a sequence at least 80% identical to SEQ ID NO: 215 or a variant thereof.
184 . The engineered nuclease system of claim 165 , wherein said endonuclease comprises a sequence at least 90% identical to SEQ ID NO: 215 or a variant thereof.
185 . The engineered nuclease system of claim 165 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of said endonuclease.
186 . The engineered nuclease system of claim 165 , wherein said endonuclease is a class II, type V Cas endonuclease.