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 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 targeter 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,
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 wherein the nuclease complex retains activity compared to a nuclease complex with the same gNA without the modifications,
and the Type V-A nuclease.
2. The composition of claim 1 wherein the modulator nucleic acid comprises one to ten phosphorothioate modifications to internucleotide linkages.
3. 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.
4. The composition of claim 3 wherein the modulator nucleic acid comprises one to five phosphorothioate modifications to internucleotide linkages between one or more of the first five nucleotides from the 5′ end.
5. The composition of claim 1 , wherein the Type VA nucleic acid-guided nuclease comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of an ABW, ART, or MAD nuclease.
6. The composition of claim 5 wherein the Type VA nucleic acid nuclease comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of MAD1, MAD2, MAD3, MAD4, MAD5, MAD6, MAD7, MAD8, MAD9, MAD10, MAD11, MAD12, MAD13, MAD14, MAD15, MAD16, MAD17, MAD18, MAD19, or MAD20.
7. The composition of claim 6 wherein the Type VA nucleic acid nuclease comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1.
8. The composition of claim 5 wherein the Type VA nucleic acid nuclease comprises an amino acid sequence at least 80% identical to an amino acid sequence of ART1, ART2, ART3, ART4, ART5, ART6, ART7, ART8, ART9, ART10, ART11, ART12, ART13, ART14, ART15, ART16, ART17, ART18, ART19, ART20, ART21, ART22, ART23, ART24, ART25, ART26, ART27, ART28, ART28, ART30, ART31, ART32, ART33, ART34, ART35, or ART11*.
9. 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) according to claim 1 , 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.
10. The method of claim 9 wherein the modulator nucleic acid comprises one to ten phosphorothioate modifications to internucleotide linkages.
11. The method of claim 9 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.
12. The method of claim 11 wherein the modulator nucleic acid comprises one to five phosphorothioate modifications to internucleotide linkages between one or more of the first five nucleotides from the 5′ end.
13. The method of claim 11 wherein the modulator nucleic acid comprises one to five 2′-O-methoxy modifications to nucleotides within the first five nucleotides from the 5′ end.
14. The method of claim 11 wherein the modulator nucleic acid comprises one to five fluoro modifications to nucleotides within the first five nucleotides from the 5′ end.
15. The method of claim 9 , wherein the Type VA nucleic acid-guided nuclease comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of an ABW, ART, or MAD nuclease.
16. The method of claim 15 wherein the Type VA nucleic acid nuclease comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of MAD1, MAD2, MAD3, MAD4, MAD5, MAD6, MAD7, MAD8, MAD9, MAD10, MAD11, MAD12, MAD13, MAD14, MAD15, MAD16, MAD17, MAD18, MAD19, or MAD20.
17. The method of claim 16 wherein the Type VA nucleic acid nuclease comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1.
18. The method of claim 15 wherein the Type VA nucleic acid nuclease comprises an amino acid sequence at least 80% identical to an amino acid sequence of ART1, ART2, ART3, ART4, ART5, ART6, ART7, ART8, ART9, ART10, ART11, ART12, ART13, ART14, ART15, ART16, ART17, ART18, ART19, ART20, ART21, ART22, ART23, ART24, ART25, ART26, ART27, ART28, ART28, ART30, ART31, ART32, ART33, ART34, ART35, or ART11*.
19. 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) according to claim 1 , 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) 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 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.