Programmable nucleases and methods of use
AssigneeMammoth Biosciences, Inc.
InventorsLucas Benjamin HARRINGTON, Janice Sha CHEN, Isaac Paterson WITTE
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Abstract
Provided herein are programmable nucleases and methods of genome editing and detection of nucleic acids with said programmable nuclease. In some embodiments, the programmable nuclease is a programmable nickase.
Claims13
1-50. (canceled)
A composition comprising an RNA guide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary to a target sequence within a gene associated with facioscapulohumeral muscular dystrophy and (ii) a repeat sequence;
- wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-TTTN-3′, wherein T is thymine and N is any nucleotide.
A method for modifying a gene associated with facioscapulohumeral muscular dystrophy or the expression thereof, the method comprising introducing one or more CRISPR associated (Cas) proteins to the gene or a regulatory element thereof that results in cleaving the target nucleic acid, deleting one or more nucleotides of the target nucleic acid, inserting one or more nucleotides of the target nucleic acid, mutating one or more nucleotides of the target nucleic acid, or modifying the target nucleic acid, thereby reducing or eliminating the expression or function of an aberrant gene product and/or restoring a wild-type protein activity.
A method of modifying a gene associated with facioscapulohumeral muscular dystrophy comprising a target nucleic acid, the method comprising introducing the break by contacting the target nucleic acid with:
- (a) a first guide nucleic acid comprising a first region that binds to a first Cas protein having a length of no more than 900 amino acids;
- and
- (b) a second guide nucleic acid comprising a first region that binds to a second Cas protein having a length of no more than 900 amino acids,
- wherein the first guide nucleic acid comprises a second region that binds to the target nucleic acid and wherein the second guide nucleic acid comprises a second region that binds to the target nucleic acid and wherein the second region of the first guide nucleic acid and the second region of the second guide nucleic acid bind opposing strands of the target nucleic acid.
A system for regulating aberrant expression of a gene associated with facioscapulohumeral muscular dystrophy, comprising:
- a Cas protein, wherein the Cas protein has a length that is less than or equal to about 900 amino acids;
- and a guide nucleic acid molecule configured to form a complex with the Cas protein, wherein the guide nucleic acid molecule exhibits specific binding to a target polynucleotide sequence, wherein, upon formation of the complex, the complex is capable of binding the target polynucleotide sequence, to effect modification of an expression level and/or a methylation level of the gene.
Dependencies are inferred from the claim text.