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US 2026/0218149 A1Published application
Class ii, type v crispr systems
AssigneeMetagenomi Therapeutics, Inc.
InventorsBrian C. Thomas, Lisa Alexander, Alan Brooks, Christopher Brown, Cristina Noel Butterfield, Cindy Castelle, Audra Devoto, Daniela S.A. Goltsman, Paula B. Matheus Carnevali, Isabel Nocedal, Morayma Temoche-Diaz
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Abstract
Described herein are methods, compositions, and systems derived from uncultivated microorganisms useful for gene editing.
Claims171
A method of disrupting a CD38 locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said CD38 locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 4466-4503 and 5686;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS:
- 4428-4465 and 5685.
5 more elements
A method of disrupting a TIGIT locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said TIGIT locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 4521-4537;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 4504-4520.
5 more elements
A method of disrupting an AAVS1 locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said AAVS1 locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 4569-4599;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 4538-4568.
5 more elements
A method of disrupting a B2M locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said B2M locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 4676-4751;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 4600-4675.
5 more elements
A method of disrupting a CD2 locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said CD2 locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 4837-4921;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 4752-4836.
5 more elements
A method of disrupting a CD5 locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said CD5 locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 4946-4969;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS:
- 4922-4945.
5 more elements
A method of disrupting a mouse TRAC locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said mouse TRAC locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5126-5195, 5682, or 5684;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5056-5125, 5681, or 5683.
5 more elements
A method of disrupting a mouse TRBC1 or TRBC2 locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said mouse TRBC1 or TRBC2 locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5211-5225 or 5247-5267;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5196-5210 or 5226-5246.
5 more elements
A method of disrupting a human TRBC1 or TRBC2 locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said human TRBC1 or TRBC2 locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5661-5679;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5642-5660.
5 more elements
A method of disrupting an HPRT locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said HPRT locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5562-5641;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS:
- 5482-5561.
5 more elements
The method of any one of claims 73-80, wherein said cell is a eukaryotic cell, hepatocyte, T-cell, hematopoietic stem cell, or precursor thereof.
A method of disrupting an APO-A1 locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said APO-A1 locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5861-5874;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5847-5860.
5 more elements
A method of disrupting an ANGPTL3 locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said ANGPTL3 locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5953-6030;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5875-5952.
5 more elements
A method of disrupting a human Rosa26 locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said human Rosa26 locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5013-5055;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 4970-5012.
5 more elements
A method of disrupting a FAS locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said FAS locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5367-5465;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5268-5366.
5 more elements
A method of disrupting a PD-1 locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said PD-1 locus,
- wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5474-5481;
- or
- wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 5466-5473.
5 more elements
An engineered nuclease system comprising:
- (a) an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs:
- 215 or a variant thereof;
- 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 nucleic acid sequence,
- wherein said system has reduced immunogenicity when administered to a human subject compared to an equivalent system comprising a Cas9 enzyme.
2 more elements
A method of disrupting a mouse HAO-1 locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said mouse HAO-1 locus,
- wherein said engineered guide RNA comprises the nucleotides of guide RNAs mH29-1_37, mH29-15_37, mH29-29_37 (SEQ ID NOs:
- 5779-5781) comprising the nucleotide modifications described in SEQ ID NOs:
- 5779-5781;
- or wherein said engineered guide RNA comprises any one of SEQ ID NOs:
- 4184-4225.
5 more elements
A method of disrupting a human TRAC locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said human TRAC locus,
- wherein said engineered guide RNA comprises the nucleotides of MG29-1-TRAC-sgRNA-35 (SEQ ID NOs:
- 5681 or 5683) comprising the nucleotide modifications described in SEQ ID NOs:
- 5681 or 5683.
3 more elements
A method of disrupting an albumin locus in a cell, comprising introducing to said cell:
- (a) a class 2, type V Cas endonuclease;
- 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 region of said albumin locus,
- Wherein said engineered guide RNA comprises the nucleotides of mAlb298-37, mAlb2912-37, mAlb2918-37, or mAlb298-34 (SEQ ID NOs:
- 5756-5759) comprising the nucleotide modifications described in SEQ ID NOs:
- 5756-5759;
- or
- wherein said engineered guide RNA comprises the nucleotides of mAlb29-8-44, mAlb29-8-50, mAlb29-8-50b, mAlb29-8-51b, mAlb29-8-52b, mAlb29-8-53b, or mAlb29-8-54b comprising the nucleotide modifications described in Table 5.
5 more elements
An engineered guide RNA comprising:
- a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a target sequence in a target DNA molecule;
- and
- b) a protein-binding segment configured to bind to a class 2, type V Cas endonuclease, and
- wherein said guide RNA comprises a nucleotide modification pattern depicted in any one of SEQ ID NOs:
- 5695-5701.
2 more elements
An engineered nuclease system comprising:
- (a) an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs:
- 1-3470 or a variant thereof, or a nucleotide sequence encoding said endonuclease;
- and
- (b) a polynucleotide sequence encoding a CRISPR array, wherein said CRISPR array is configured to be processed by said endonuclease to 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 nucleic acid sequence,
- wherein said spacer sequence is configured to hybridize to an albumin gene.
2 more elements
An engineered nuclease system comprising:
- (a) an endonuclease having at least 75% sequence identity to SEQ ID NOs:
- 470 or a variant thereof;
- 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 nucleic acid sequence.
1 more element
The engineered nuclease system of claim 151 or 152, wherein said endonuclease is configured to be selective for a 5′ PAM sequence comprising SEQ ID NO:
- 6032.
An engineered nuclease system comprising:
- (a) an endonuclease having at least at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 2824, 2841, or 2896, or a variant thereof;
- 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 nucleic acid sequence,
- wherein said engineered guide RNA comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs:
- 6033, 6034, or 6035.
3 more elements
A lipid nanoparticle comprising:
- (a) any of the endonucleases described herein;
- (b) any of the engineered guide RNAs described herein:
- (c) a cationic lipid;
- (d) a sterol;
- (e) a neutral lipid;
- and
- (f) a PEG-modified lipid.
4 more elements
An engineered nuclease system comprising:
- (a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs:
- 6274-6281 or 6340-6551, or a variant thereof, wherein said endonuclease is a class 2, type V endonuclease, or a nucleotide sequence encoding said endonuclease;
- 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 nucleic acid sequence.
1 more element
An engineered nuclease system comprising:
- (a) an endonuclease comprising a PI (PAM interacting) domain having at least 80% sequence identity to a PI domain of any one of SEQ ID NOs:
- 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, or 6281, or a variant thereof, or a nucleotide sequence encoding said endonuclease, wherein said endonuclease is a class 2, type V endonuclease and said endonuclease is configured to be selective for a 5′ PAM of any one of SEQ ID NOs:
- 6326-6339;
- 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 nucleic acid sequence.
2 more elements
Dependencies are inferred from the claim text.