Compositions and methods for targeting, editing, or modifying genes
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.
1. A nuclease complex composition comprising
a synthetic split guide nucleic acid (gNA) comprising:
(i) a targeter nucleic acid comprising:
(a) a spacer sequence capable of hybridizing 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 comprise four or five nucleotides that base pair with each other, and wherein the modulator and targeter nucleic acids are separate nucleic acids, and
the split gNA is capable of binding to and forming a nucleic acid-guided nuclease complex, 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 the Type V-A nuclease,
wherein the targeter nucleic acid or the modulator nucleic acid, or both, comprise one or more modified nucleotides and/or one or more modified internucleotide linkages at or near its 3′ end, at or near its 5′ end, or both, wherein the modulator nucleic acid comprises a 2′-O-methoxy modification to the 5′ nucleotide, and wherein the nuclease complex retains activity compared to a nuclease complex with the same split gNA without the modifications.
2. The composition of claim 1 , wherein the modulator nucleic acid comprises at least one modified nucleotide and at least two modified internucleotide linkages within the first five nucleotides from the 5′ end.
3. The composition of claim 1 wherein the modulator nucleic acid comprises one to ten phosphorothioate modifications to internucleotide linkages.
4. The composition of claim 1 further comprising a 2′-O-methoxy modification at a nucleotide 3′ to the 5′ nucleotide of the modulator nucleic acid.
5. The composition of claim 1 wherein the targeter nucleic acid comprises one to five fluoro modifications to nucleotides within five nucleotides from the 3′ end of the targeter nucleic acid.
6. The composition of claim 1 wherein the targeter nucleic acid comprises a 2′-O-methoxy modification to the 3′ nucleotide of the targeter nucleic acid and/or the nucleotide adjacent to the 3′nucleotide.
7. The composition of claim 1 wherein the targeter nucleic acid comprises a phosphorothiote internucleotide linkage modification to one to five internucleotide linkages at the 3′end of the targeter nucleic acid.
8. A method of editing a genome of a eukaryotic cell comprising
(I) delivering to the eukaryotic cell
(A) one or more synthetic split guide nucleic acids (gNA), or polynucleotides encoding the one or more split gNAs, comprising
(i) a targeter nucleic acid comprising:
(a) a spacer sequence capable of hybridizing 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 comprise four or five nucleotides that base pair with each other, and wherein the modulator and targeter nucleic acids are separate nucleic acids,
wherein the targeter nucleic acid or the modulator nucleic acid, or both, comprise one or more modified nucleotides and/or one or more modified internucleotide linkages at or near its 3′ end, at or near its 5′ end, or both, wherein the modulator nucleic acid comprises a 2′-O-methoxy modification to the 5′ nucleotide, and wherein the nuclease complex retains activity compared to a nuclease complex with the same split gNA without the modifications, and
the split gNA is capable of binding to and forming a nucleic acid-guided nuclease complex;
(B) one or more Type V-A nucleic acid-guided nucleases, or polynucleotides encoding the one or more nucleases; and, optionally,
(C) one or more donor templates,
wherein the gNA and the Type V-A nucleic acid-guided nuclease form a nucleic acid-guided nuclease complex, 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; and
(II) contacting the genome with the nucleic acid-guided nuclease complex to form one or more strand breaks in the genome, whereby at least a portion of the donor template is inserted into the genome at or near the one or more strand breaks.
9. The method of claim 8 , further comprising treating the eukaryotic cell with a homology-directed repair (HDR) enhancer.
10. The method of claim 9 , wherein the HDR enhancer comprises a DNA-PK antagonist.
11. The method of claim 8 , wherein the method comprises delivering at least two split gNAs, or polynucleotides encoding the split gNAs, wherein each split gNA comprises a different spacer sequence such that when complexed with a nucleic acid-guided nuclease, the nucleic acid-guided nuclease complexes form strand breaks in the genome at or near each of the target nucleotide sequences.
12. A method of editing a genome of a eukaryotic cell comprising
(I) delivering to the eukaryotic cell
(A) one or more synthetic split guide nucleic acids (gNA), or polynucleotides encoding the one or more split gNAs, comprising
(i) a targeter nucleic acid comprising:
(a) a spacer sequence capable of hybridizing 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) a predicted minimum free energy of the targeter stem sequence and the modulator stem sequence as determined by RNAcofold WebServer is between −10 and −4 kcal/mol, and
(3) the gNA is capable of binding to and forming a nucleic acid-guided nuclease complex,
wherein the targeter stem sequence and the modulator stem sequence comprise four or five nucleotides that base pair with each other, and
wherein the targeter nucleic acid or the modulator nucleic acid, or both, comprise one or more modified nucleotides and/or one or more modified internucleotide linkages at or near its 3′ end, at or near its 5′ end, or both, wherein the modulator nucleic acid comprises a 2′-O-methoxy modification to the 5′ nucleotide, and wherein the nuclease complex retains activity compared to a nuclease complex with the same split gNA without the modifications;
(B) one or more Type V-A nucleic acid-guided nucleases, or polynucleotides encoding the one or more nucleases; and, optionally,
(C) one or more donor templates,
wherein the gNA and the Type V-A nucleic acid-guided nuclease form a nucleic acid-guided nuclease complex, 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; and
(II) contacting the genome with the nucleic acid-guided nuclease complex to form one or more strand breaks in the genome, whereby at least a portion of the donor template is inserted into the genome at or near the one or more strand breaks.