Crispr systems with engineered dual guide nucleic acids
AssigneesArtisan Development Labs, Inc., Danmarks Tekniske Universitet
InventorsRyan T. GILL, Tanya WARNECKE, Andrea BARGHETTI, Line Dahl POULSEN
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Abstract
The present invention relates to an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system comprising engineered dual guide nucleic acids (e.g., RNAs) capable of activating a CRISPR-Associated (Cas) nuclease, such as a type V-A Cas nuclease. Also provided are methods of targeting, editing, and/or modifying a nucleic acid using the engineered CRISPR system, and compositions and cells comprising the engineered CRISPR system.
Claims31
1-45. (canceled)
An engineered, non-naturally occurring system comprising:
- (a) a targeter nucleic acid comprising:
- (i) a spacer sequence designed to hybridize with a target nucleotide sequence;
- and
- (ii) a targeter stem sequence;
- and
- (b) a modulator nucleic acid comprising a modulator stem sequence complementary to the targeter stem sequence,
- wherein the targeter nucleic acid and the modulator nucleic acid are separate nucleic acids, and wherein a complex comprising the targeter nucleic acid and the modulator nucleic acid is capable of activating a CRISPR Associated (Cas) nuclease that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA, wherein the Cas nuclease comprises a Type V-A Cas nuclease;
- and
- (c) the Type V-A Cas nuclease.
A cell comprising
- (a) a targeter nucleic acid, or a polynucleotide encoding thereof, comprising
- (i) a spacer sequence designed to hybridize with a target nucleotide sequence and
- (ii) a targeter stem sequence;
- (b) a modulator nucleic acid, or a polynucleotide encoding thereof, comprising a modulator stem sequence complementary to the targeter stem sequence,
- wherein the targeter nucleic acid and the modulator nucleic acid are expressed as separate nucleic acids, and wherein a complex comprising the targeter nucleic acid and the modulator nucleic acid is capable of activating a Cas nuclease that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA, wherein the Cas nuclease comprises a Type V-A nuclease;
- and
- (c) a Type V-A Cas nuclease, or a polynucleotide encoding thereof.
A method of cleaving a target DNA having a target nucleotide sequence, the method comprising contacting the target DNA with an engineered, non-naturally occurring system comprising
- (a) a targeter nucleic acid comprising:
- (i) a spacer sequence designed to hybridize with the target nucleotide sequence;
- and
- (ii) a targeter stem sequence;
- and
- (b) a modulator nucleic acid comprising a modulator stem sequence complementary to the targeter stem sequence,
- wherein the targeter nucleic acid and the modulator nucleic acid are separate nucleic acids, and wherein a complex comprising the targeter nucleic acid and the modulator nucleic acid is capable of activating a CRISPR Associated (Cas) nuclease that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA, wherein the Cas nuclease comprises a type V-A Cas nuclease, and
- (c) the Cas nuclease;
- thereby resulting in cleavage of the target DNA.
A method of editing a genome of a eukaryotic cell, the method comprising delivering an engineered, non-naturally occurring system comprising
- (a) a targeter nucleic acid comprising:
- (i) a spacer sequence designed to hybridize with a target nucleotide sequence within the genome;
- and
- (ii) a targeter stem sequence;
- and
- (b) a modulator nucleic acid comprising a modulator stem sequence complementary to the targeter stem sequence,
- wherein the targeter nucleic acid and the modulator nucleic acid are separate nucleic acids, and wherein a complex comprising the targeter nucleic acid and the modulator nucleic acid is capable of activating a CRISPR Associated (Cas) nuclease that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA, wherein the Cas nuclease comprises a type V-A Cas nuclease, and
- (c) the Cas nuclease;
- and/or a polynucleotide or polynucleotides coding for one or more of (a), (b), and/or (c),
- into the eukaryotic cell, thereby resulting in editing of the genome of the eukaryotic cell.
Dependencies are inferred from the claim text.