Class II, type V CRISPR systems
Described herein are methods, compositions, and systems derived from uncultivated microorganisms useful for gene editing.
1. An engineered nuclease system comprising:
a. an endonuclease configured to be selective for a protospacer adjacent motif (PAM) sequence comprising the sequence 5′-YYn-3′ (SEQ ID NO: 3871), wherein said endonuclease is a class 2, type V Cas endonuclease, wherein said endonuclease comprises a WED II domain and a PAM-interacting domain having at least 80% amino acid sequence identity to the WED II domain and the PAM-interacting domain of the amino acid sequence of SEQ ID NO: 215; and
b. an engineered guide 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 nucleotide sequence.
2. The engineered nuclease system of claim 1 , wherein said WED II domain and PAM-interacting domain comprise an amino acid sequence having at least 90% amino acid sequence identity to amino acid residues 575-645 of the amino acid sequence of SEQ ID NO: 215.
3. The engineered nuclease system of claim 1 , wherein said engineered guide RNA comprises a nucleotide sequence with at least 80% nucleotide sequence identity to the non-degenerate nucleotides of the nucleotide sequence of SEQ ID NO: 3609.
4. The engineered nuclease system of claim 1 , wherein said endonuclease comprises a RuvC domain comprising an amino acid sequence having at least 80% amino acid sequence identity to the RuvCI, RuvCII and RuvCIII domains of the amino acid sequence of SEQ ID NO: 215, and wherein said endonuclease comprises the catalytic residues D886, E976, and D1129 of the amino acid sequence of SEQ ID NO: 215.
5. The engineered nuclease system of claim 1 , wherein said endonuclease comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 215, wherein said endonuclease comprises the catalytic residues D886, E976, and D1129 of the amino acid sequence of SEQ ID NO: 215.
6. The engineered nuclease system of claim 1 , wherein said engineered guide RNA comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence.
7. The engineered nuclease system of claim 1 , wherein said engineered guide RNA is 30-250 nucleotides in length.
8. The engineered nuclease system of claim 1 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of said endonuclease.
9. The engineered nuclease system of claim 1 , wherein said endonuclease further comprises at least one of the following mutations: S168R, E172R, N577R, or Y170R when an amino acid sequence of said endonuclease is optimally aligned based on a pairwise alignment to the amino acid sequence of SEQ ID NO: 215.
10. The engineered nuclease system of claim 1 , wherein said endonuclease further comprises the mutations S168R and E172R when an amino acid sequence of said endonuclease is optimally aligned based on a pairwise alignment to the amino acid sequence of SEQ ID NO: 215.
11. The engineered nuclease system of claim 1 , wherein said endonuclease further comprises the mutations N577R or Y170R when an amino acid sequence of said endonuclease is optimally aligned based on a pairwise alignment to the amino acid sequence of SEQ ID NO: 215.
12. The engineered nuclease system of claim 1 , wherein said endonuclease further comprises the mutation S168R when an amino acid sequence of said endonuclease is optimally aligned based on a pairwise alignment to the amino acid sequence of SEQ ID NO: 215.
13. The engineered nuclease system of claim 12 , wherein said endonuclease does not comprise a mutation of E172, N577, or Y170 of the amino acid sequence of SEQ ID NO: 215.
14. The engineered nuclease system of claim 1 , further comprising a single- or double-stranded DNA repair template comprising from 5′ to 3′: a first homology arm comprising a sequence of at least 20 nucleotides 5′ to said target nucleotide 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 nucleotide sequence.
15. The engineered nuclease system of claim 14 , wherein said first or second homology arm comprises a nucleotide sequence of at least 40 nucleotides.
16. The engineered nuclease system of claim 14 , wherein said first and second homology arms are homologous to a genomic sequence of a eukaryote.
17. The engineered nuclease system of claim 14 , wherein said single- or double-stranded DNA repair template comprises a transgene donor.
18. The engineered nuclease system of claim 1 , further comprising a DNA repair template comprising a double-stranded DNA segment flanked by one or two single-stranded DNA segments.
19. The engineered nuclease system of claim 18 , wherein said single-stranded DNA segments are conjugated to the 5′ ends of said double-stranded DNA segment.
20. The engineered nuclease system of claim 18 , wherein said single-stranded DNA segments are conjugated to the 3′ ends of said double-stranded DNA segment.
21. The engineered nuclease system of claim 18 , wherein said single-stranded DNA segments have a nucleotide length from 4 to 10 nucleotide bases.
22. The engineered nuclease system of claim 18 , wherein said single-stranded DNA segments have a nucleotide sequence complementary to a nucleotide sequence within said spacer sequence.
23. The engineered nuclease system of claim 18 , wherein said double-stranded DNA sequence 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.
24. The engineered nuclease system of claim 18 , wherein said double-stranded DNA sequence is flanked by a nuclease cut site.
25. The engineered nuclease system of claim 1 , wherein said amino acid sequence identity is determined by a BLASTP, CLUSTALW, MUSCLE, MAFFT algorithm or a CLUSTALW algorithm with the Smith-Waterman homology search algorithm parameters.
26. The engineered nuclease system of claim 25 , wherein said amino acid sequence identity is determined by said BLASTP homology search algorithm using parameters of a wordlength (W) of 3, a expectation (E) of 10, and a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1 and using a conditional compositional score matrix adjustment.