Method of target cleaving using CRISPR hybrid DNA/RNA polynucleotides
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 site specific cleaving of a target deoxyribonucleic acid (DNA) molecule in vitro or in an isolated cell, the method comprising:
contacting the target DNA molecule having a target sequence with
a single polynucleotide comprising a targeting region that comprises a mixture of DNA and ribonucleic acid (RNA) and an activating region that comprises a mixture of DNA and RNA, wherein the activating region is adjacent to the targeting region, wherein the targeting region is configured to hybridize with the target sequence, and wherein the activating region comprises a stem loop structure; and
a Cpf1 protein,
wherein the Cpf1 protein binds to the activating region of the single polynucleotide, thereby cleaving the target DNA molecule.
2. The method of claim 1 , wherein the contacting of the target DNA molecule with the single polynucleotide and the Cpf1 protein occurs in an isolated cell.
3. The method of claim 2 , wherein the isolated 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.
4. The method of claim 1 , further comprising providing a donor polynucleotide.
5. The method of claim 1 , wherein the single polynucleotide and the Cpf1 protein form a complex.
6. The method of claim 1 , wherein the Cpf1 protein comprises a nuclear localization signal (NLS).
7. The method of claim 2 , wherein the single polynucleotide and the Cpf1 protein are introduced into the cell by lipofection, electroporation, nucleofection, microinjection, biolistics, liposomes, immunoliposomes, polycation, lipid:nucleic acid conjugates, or combinations thereof.
8. The method of claim 1 , wherein the modifying results in cleavage of the target DNA molecule.
9. The method of claim 1 , wherein the targeting region comprises a compound selected from the group consisting of phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, 5′-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, 3′-amino phosphoramidate, amino alkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates.