Compositions, systems, and methods for base diversification
Described herein are methods of modifying or editing a target nucleic acid such as methods that edit adenine to cytosine, thymine, or guanine. Compositions and systems for modifying or editing a target nucleic acid are also described. Methods, compositions and systems described herein may be used for generating allelic diversity.
1. A method of modifying a target nucleic acid, the method comprising:
contacting the target nucleic acid with:
a CRISPR-Cas effector protein,
an adenine deaminase,
an inosine glycosylase, wherein the inosine glycosylase is active, and
a guide nucleic acid comprising a sequence that is complementary to the target nucleic acid,
thereby modifying the target nucleic acid,
wherein modifying the target nucleic acid comprises modifying an adenine (A) of the target nucleic acid to a cytosine (C), a thymine (T), or a guanine (G), and
wherein the contacting comprises:
contacting the target nucleic acid with a first fusion protein comprising the CRISPR-Cas effector protein, the adenine deaminase, and the inosine glycosylase, wherein the inosine glycosylase is fused to the C-terminus of the CRISPR-Cas effector protein, or
contacting the target nucleic acid with a second fusion protein comprising the CRISPR-Cas effector protein and the adenine deaminase, wherein the inosine glycosylase is provided in trans, or
recruiting the adenine deaminase to the CRISPR-Cas effector protein, wherein the inosine glycosylase is provided in trans.
2. The method of claim 1 , wherein the modifying comprises modifying a first adenine (A) of the target nucleic acid to a cytosine (C) and a second adenine (A) of the target nucleic acid to a thymine (T).
3. The method of claim 1 , wherein the modifying comprises modifying a first adenine (A) of the target nucleic acid to a cytosine (C) and a second adenine (A) of the target nucleic acid to a guanine (G).
4. The method of claim 1 , wherein the modifying comprises modifying a first adenine (A) of the target nucleic acid to a thymine (T) and a second adenine (A) of the target nucleic acid to a guanine (G).
5. The method of claim 1 , wherein the modifying comprises modifying a first adenine (A) of the target nucleic acid to a cytosine (C), a second adenine (A) of the target nucleic acid to a thymine (T), and a third adenine (A) of the target nucleic acid to a guanine (G).
6. The method of claim 1 , wherein the contacting comprises contacting the target nucleic acid with the first fusion protein and wherein the adenine deaminase is linked to the inosine glycosylase via a peptide linker.
7. The method of claim 1 , wherein the contacting comprises recruiting the CRISPR-Cas effector protein to the adenine deaminase, and wherein the inosine glycosylase is overexpressed in a cell in which it is present.
8. The method of claim 1 , wherein the inosine glycosylase comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to any one of SEQ ID NOs:79-91 or 146-276.
9. The method of claim 1 , wherein the contacting comprises recruiting the CRISPR-Cas effector protein to the adenine deaminase, and wherein the inosine glycosylase is recruited to the target nucleic acid via the CRISPR-Cas effector protein and/or via the adenine deaminase.
10. The method of claim 1 , wherein the inosine glycosylase comprises a peptide tag.
11. The method of claim 1 , wherein the CRISPR-Cas effector protein, the adenine deaminase, and the guide nucleic acid form a complex or are comprised in a complex.
12. The method of claim 1 , wherein the guide nucleic acid comprises a RNA recruiting motif.
13. The method of claim 1 , wherein the inosine glycosylase and/or adenine deaminase comprise a MS2 capping protein (MCP) or a portion thereof.
14. The method of claim 1 , wherein the contacting comprises recruiting the CRISPR-Cas effector protein to the adenine deaminase, and wherein the CRISPR-Cas effector protein and/or the adenine deaminase comprises a peptide tag;
wherein the inosine glycosylase comprises an affinity polypeptide capable of binding the peptide tag;
wherein the guide nucleic acid comprises a RNA recruiting motif; and
wherein the peptide tag is recruited to the RNA recruiting motif and the inosine glycosylase is recruited to the peptide tag using the affinity polypeptide.
15. The method of claim 1 , wherein the method is devoid of cleavage of the target nucleic acid and/or is devoid of nucleic acid cleavage.
16. The method of claim 1 , wherein the adenine deaminase comprises all or a portion of an amino acid sequence of any one of SEQ ID NOs:50-60.
17. The method of claim 1 , wherein the contacting comprises contacting the target nucleic acid with the first fusion protein and wherein the first fusion protein comprises from the N-terminus to the C-terminus, the adenine deaminase, the CRISPR-Cas effector protein, and the inosine glycosylase.
18. The method of claim 1 , wherein the contacting comprises contacting the target nucleic acid with the second fusion protein and the inosine glycosylase that is provided in trans.