Methods and systems for guide RNA design and use
The present disclosure provides a method for designing a set of guide RNAs for hybridizing a genomic region of interest. The present disclosure further provides methods of editing at least one genomic region of interest with at least one set of guide RNAs.
1. A method for editing a gene within a genomic region of interest, the method comprising:
(a) contacting a cell comprising the gene within genomic region of interest with: a set of guide RNAs (gRNAs) comprising
(i) a first gRNA configured to hybridize to a first site of the genomic region of interest and capable of interacting with a nuclease and generating a first double stranded break;
(ii) a second gRNA configured to hybridize to a second site of the genomic region of interest and capable of interacting with the nuclease and generating a second double stranded break, and
(iii) a third gRNA configured to hybridize to a third site of the genomic region of interest and capable of interacting with the nuclease and generating a third double stranded break, wherein the first gRNA, second gRNA, and third gRNA are different and each hybridize to a site that is at least 15 base pairs and at most 2000 base pairs apart from each other; and
(b) introducing the set of gRNA and the nuclease into the cell to generate an edit that modifies the gene; wherein an editing efficiency of the set of gRNA is higher than an individual editing efficiency of each of the gRNAs.
2. The method of claim 1 , wherein the first gRNA, the second gRNA, and the third gRNA are configured to hybridize to the gene.
3. The method of claim 2 , wherein the first gRNA, the second gRNA, and the third gRNA are configured to hybridize an exon of the gene, a regulatory element, a cis-regulatory element or a trans-regulatory element.
4. The method of claim 3 , wherein the regulatory element is a cis-regulatory element and selected from the group consisting of: a promoter, an enhancer, and a silencer.
5. The method of claim 1 , further comprising introducing into the cell a donor polynucleotide.
6. The method of claim 5 wherein the donor polynucleotide comprises a point mutation, allele, tag or exogenous exon relative to a wild-type genotype of the cell.
7. The method of claim 1 , wherein the edit is a knock-out of the gene, an insertion or a deletion relative to wild-type.
8. The method of claim 7 , wherein the edit is an insertion and improves a function of the gene.
9. The method of claim 1 , wherein each of the first, second and third gRNA is hybridizable to a target site that is at least 30 bases apart from the target site of the other gRNA from the set of gRNAs.
10. The method of claim 1 , further comprising introducing a plurality of sets of gRNA targeting a plurality of genomic regions of interest.
11. The method of claim 1 , further comprising screening the population of cells for a phenotype.
12. The method of claim 1 , wherein the on-target efficiency threshold value for each guide RNA of the initial set of gRNAs is an azimuth score greater than 0.4.
13. The method of claim 1 , wherein the contacting comprises transfecting the cell with the set of gRNAs as ribonucleoprotein complexes.
14. The method of claim 1 , wherein the introducing comprises transfecting the cell with the set of gRNAs as ribonucleoprotein complexes.
15. The method of claim 1 , wherein the nuclease is Cas9 nuclease, C2c1 nuclease, C2c3 nuclease, or Cpf1 nuclease.
16. The method of claim 1 , wherein the first gRNA, the second gRNA, and the third guide RNA comprise a 5′ end modification and a 3′ end modification.
17. The method of claim 16 , wherein the 5′ end modification comprises a phosphorothioate internucleotide linkage and a 2′-O-methyl sugar modification and the 3′ end modification comprises a phosphorothioate internucleotide linkage and a 2′-O-methyl sugar modification.
18. The method of claim 17 , wherein the first gRNA, the second gRNA, and the third gRNA are single guide RNAs (sgRNAs), wherein the first binding site and the second binding site are separated by 15-80 base pairs, wherein the second binding site and the third binding site are separated by 15-80 base pairs, wherein the first binding site, the second binding site, and the third binding site are in an exon of a gene, wherein the exon is in a first half of the gene.
19. The method of claim 18 , wherein the first gRNA is configured to hybridize to a binding-site that is 30-80 base pairs apart from a binding site hybridizable to the second gRNA and the second gRNA is configured to hybridize to a binding-site that is 30-80 base pairs apart from a binding site hybridizable to the third gRNA.