Targeted trans-splicing using CRISPR/Cas13
This disclosure provides compositions and methods of using these compositions to mediate a targeted trans-splicing event on a pre-mRNA in a cell.
1. A system for targeting a nucleic acid for trans-splicing, the system comprising:
(i) a CRISPR/Cas system comprising a Type VI Cas polypeptide and a nucleic acid guide;
(ii) a specific RNA-binding polypeptide that associates with the CRISPR/Cas system; and
(iii) a trans-splicing template comprising a splice donor and/or acceptor and an RNA sequence that binds to the specific RNA-binding polypeptide, thereby targeting the nucleic acid for trans-splicing.
2. The system of claim 1 , wherein the nucleic acid guide is an RNA guide.
3. The system of claim 1 , wherein the nucleic acid guide is a DNA guide.
4. The system of claim 1 , wherein the CRISPR/Cas system comprises one nucleic acid guide.
5. The system of claim 1 , wherein the CRISPR/Cas system comprises more than one nucleic acid guide.
6. The system of claim 5 , wherein the nucleic acid guides recognize multiple targets.
7. The system of claim 1 , wherein the nucleic acid guide targets a splice acceptor (SA) site.
8. The system of claim 1 , wherein the nucleic acid guide targets a splice donor (SD) site.
9. The system of claim 1 , wherein the nucleic acid guide targets a region near a splice site.
10. The system of claim 9 , wherein the nucleic acid guide targets a region within 200 nucleotides of a splice site.
11. The system of claim 5 , wherein the more than one nucleic acid guides target one nucleic acid of interest.
12. The system of claim 5 , wherein the more than one nucleic acid guides target multiple nucleic acids of interest.
13. The system of claim 1 , wherein the Cas polypeptide is selected from the group consisting of Cas13a, Cas13b, and Cas13d.
14. The system of claim 1 , wherein the specific RNA-binding polypeptide comprises a viral protein.
15. The system of claim 1 , comprising one trans-splicing template.
16. The system of claim 1 , wherein the trans-splicing activity is regulated by a small molecule.
17. The system of claim 1 , wherein the Cas polypeptide is a nuclease-inactive Cas13 polypeptide.
18. The system of claim 1 , comprising one or more polynucleotides encoding the Cas polypeptide, the RNA guide, the specific RNA-binding polypeptide, and/or the trans-splicing template.
19. The system of claim 18 , comprising one or more vectors comprising the one or more polynucleotides.