CRISPR hybrid DNA/RNA polynucleotides and methods of use
The present disclosure provides DNA-guided CRISPR systems; polynucleotides comprising DNA, RNA and mixtures thereof for use with CRISPR systems; and methods of use involving such polynucleotides and DNA-guided CRISPR systems.
1. A method of modifying a target nucleic acid molecule comprising: contacting the target nucleic acid molecule having a target sequence with (i) a single polynucleotide comprising (a) a targeting region that comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and configured to hybridize to the target sequence; and (b) an activating region adjacent to the targeting region that comprises DNA and wherein the activating region comprises a stem loop structure; and (ii) a Cpf1, wherein the Cpf1 binds with the activating region of the single polynucleotide, wherein the contacting of the target nucleic acid molecule with the single polynucleotide and the Cpf1 occurs (A) in vitro, (B) in a non-human cell, or (C) in an isolated cell and wherein the target nucleic acid molecule is cleaved.
2. The method of claim 1 , wherein the target nucleic acid molecule comprises DNA.
3. The method of claim 1 , wherein the target nucleic acid molecule comprises RNA.
4. The method of claim 1 , wherein the target nucleic acid molecule comprises a mixture of DNA and RNA.
5. The method of claim 1 , wherein the targeting region comprises a mixture of DNA and RNA.
6. The method of claim 1 , wherein the activating region comprises a mixture of DNA and RNA.
7. The method of claim 1 , wherein the targeting region comprises a mixture of DNA and RNA and the activating region comprises a mixture of DNA and RNA.
8. The method of claim 1 , wherein the contacting of the target nucleic acid molecule with the single polynucleotide and the Cpf1 occurs in vitro.
9. The method of claim 1 , wherein the contacting of the target nucleic acid molecule with the single polynucleotide and the Cpf1 occurs in a non-human cell.
10. The method of claim 1 , wherein the contacting of the target nucleic acid molecule with the single polynucleotide and the Cpf1 occurs in an isolated cell.
11. The method of claim 10 , wherein the cell is selected from the group consisting of a bacterial cell, an archaeal cell, a plant cell, an algal cell, a fungal cell, an invertebrate cell, a vertebrate cell, a mammalian cell and a human cell.
12. The method of claim 1 , further comprising providing a donor polynucleotide.
13. A method of modulating transcription of at least one gene within a target nucleic acid molecule comprising: contacting the target nucleic acid molecule having a target sequence with (i) a single polynucleotide comprising (a) a targeting region that comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and configured to hybridize to the target sequence, wherein the target sequence is within the open reading frame sequence of the at least one gene or is within a promoter sequence of the at least one gene; and (b) an activating region adjacent to the targeting region that comprises DNA and wherein the activating region comprises a stem loop structure; and (ii) a Cpf1, wherein the Cpf1 has no nuclease activity, wherein the Cpf1 binds with the activating region of the single polynucleotide, wherein the targeting region of the single polynucleotide hybridizes to the target sequence, wherein the contacting of the target nucleic acid molecule with the single polynucleotide and the Cpf1 occurs (A) in vitro, (B) in a non-human cell, or (C) in an isolated cell and wherein the transcription of at least one gene within the target nucleic acid molecule is modulated.
14. The method of claim 13 , wherein the targeting region comprises a mixture of DNA and RNA.
15. The method of claim 13 , wherein the activating region comprises a mixture of DNA and RNA.
16. The method of claim 13 , wherein the targeting region comprises a mixture of DNA and RNA and the activating region comprises a mixture of DNA and RNA.
17. The method of claim 13 , wherein the contacting of the target nucleic acid molecule with the single polynucleotide and the Cpf1 occurs in vitro.
18. The method of claim 13 , wherein the contacting of the target nucleic acid molecule with the single polynucleotide and the Cpf1 occurs in a non-human cell.
19. The method of claim 13 , wherein the contacting of the target nucleic acid molecule with the single polynucleotide and the Cpf1 occurs in an isolated cell.
20. The method of claim 19 , wherein the cell is selected from the group consisting of a bacterial cell, an archaeal cell, a plant cell, an algal cell, a fungal cell, an invertebrate cell, a vertebrate cell, a mammalian cell and a human cell.