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US 2023/0265461 A1Published application
Compositions and methods for targeting, editing, or modifying genes
AssigneeArtisan Development Labs, Inc.
InventorsAndrea BARGHETTI, Roland BAUMGARTNER, Tanya WARNECKE, Kurt MARSHALL, John SCHIEL, Alyssa Carlson
Life of this patent
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Abstract
Provided herein are nucleic acids useful as guide nucleic acids (gNAs), e.g., guide ribonucleic acids (gRNAs), in a CRISPR system wherein the guide nucleic acids contain one or more modifications to one or more nucleotides, use of such guide nucleic acids in modifying cells, and other uses wherein CRISPR Cas proteins are utilized.
Claims30
A composition comprising a synthetic guide nucleic acid (gNA) comprising:
- (i) a targeter nucleic acid comprising:
- (a) a spacer sequence configured to hybridize with a target nucleotide sequence, and
- (b) a targeter stem sequence;
- and
- (ii) a modulator nucleic acid comprising:
- (a) a modulator stem sequence complementary to the target stem sequence, and
- (b) a 5′ sequence;
- wherein the targeter stem sequence and the modulator stem sequence each comprise 4-10 nucleotides that base pair with each other, and
- the gNA is capable of binding to a CRISPR Type VA nucleic acid-guided nuclease and forming a nucleic acid-guided nuclease complex,
- wherein the targeter and modulator nucleic acids are separate polynucleotides, and
- wherein the targeter nucleic acid or the modulator nucleic acid, or both, comprise one or more modified nucleotides or one or more modified internucleotide linkages at or near its 3′ end, at or near its 5′ end, or both, and wherein the modulator nucleic acid comprises at least one modified nucleotide and at least two modified internucleotide linkages within the first 10 nucleotides from the 5′ end.
8 more elements
A method of editing a genome of a eukaryotic cell comprising
- (I) delivering to the eukaryotic cell
- (A) one or more synthetic guide nucleic acids (gNA), or polynucleotides encoding the one or more gNAs, comprising
- (i) a targeter nucleic acid comprising:
- (a) a spacer sequence configured to hybridize with a target nucleotide sequence, and
- (b) a targeter stem sequence;
- and
- (ii) a modulator nucleic acid comprising:
- (a) a modulator stem sequence complementary to the target stem sequence, and
- (b) a 5′ sequence;
- wherein the targeter stem sequence and the modulator stem sequence each comprise 4-10 nucleotides that base pair with each other, and
- the gNA is capable of binding to a CRISPR Type VA nucleic acid-guided nuclease and forming a nucleic acid-guided nuclease complex,
- wherein the targeter and modulator nucleic acids are separate polynucleotides, and
- wherein the targeter nucleic acid or the modulator nucleic acid, or both, comprise one or more modified nucleotides or one or more modified internucleotide linkages at or near its 3′ end, at or near its 5′ end, or both, and wherein the modulator nucleic acid comprises at least one modified nucleotide and at least two modified internucleotide linkages within the first 10 nucleotides from the 5′ end;
- (B) the Type VA nucleic acid-guided nuclease, or polynucleotides encoding the nuclease;
- wherein the gNA and the Type V nucleic acid-guided nuclease form a nucleic acid-guided nuclease complex;
- and
- (II) contacting the genome with the nucleic acid-guided nuclease complex to form one or more strand breaks in the genome.
14 more elements
A composition comprising a synthetic guide nucleic acid (gNA) comprising
- (i) a targeter nucleic acid comprising:
- (a) a spacer sequence configured to hybridize with a target nucleotide sequence, and
- (b) a targeter stem sequence;
- and
- (ii) a modulator nucleic acid comprising:
- (a) a modulator stem sequence complementary to the target stem sequence, and
- (b) a 5′ sequence;
- wherein
- (1) the targeter nucleic acid and modulator nucleic acids are separate polynucleotides,
- (2) the predicted minimum free energy of hybridization of the targeter stem sequence and the modulator stem sequence, as determined by the RNAcofold Web Server, is between −10 and −4 kcal/mol, and
- (3) the gNA is capable of binding to a CRISPR Type V nucleic acid-guided nuclease and forming a nucleic acid-guided nuclease complex.
8 more elements
A method of editing a genome of a eukaryotic cell comprising
- (I) delivering to the eukaryotic cell
- (A) one or more synthetic guide nucleic acids (gNA), or polynucleotides encoding the one or more gNAs, comprising
- (i) a targeter nucleic acid comprising:
- (a) a spacer sequence configured to hybridize with a target nucleotide sequence, and
- (b) a targeter stem sequence;
- and
- (ii) a modulator nucleic acid comprising:
- (a) a modulator stem sequence complementary to the target stem sequence, and
- (b) a 5′ sequence;
- wherein
- (1) the targeter nucleic acid and modulator nucleic acids are separate polynucleotides,
- (2) the predicted minimum free energy of the targeter stem sequence and the modulator stem sequence as determined by the RNAcofold Web Server is between −10 and −4 kcal/mol, and
- (3) the gNA is capable of binding to a CRISPR Type VA nucleic acid-guided nuclease and forming a nucleic acid-guided nuclease complex;
- (B) one or more Type VA nucleic acid-guided nucleases, or polynucleotides encoding the one or more nucleases;
- wherein the gNA and the Type V nucleic acid-guided nuclease form a nucleic acid-guided nuclease complex;
- and
- (II) contacting the genome with the nucleic acid-guided nuclease complex to form one or more strand breaks in the genome.
14 more elements
Dependencies are inferred from the claim text.