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 single polynucleotide comprising:
a targeting region that comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA); and,
an activating region adjacent to said targeting region that comprises DNA; wherein the activating region comprises a stem and a bulge, and wherein said activating region is capable of binding with a Cas9.
2. The polynucleotide of claim 1 , wherein the targeting region comprises a mixture of DNA and RNA.
3. The polynucleotide of claim 1 , wherein the targeting region comprises a mixture of DNA and RNA and the activating 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.
4. The polynucleotide of claim 1 , wherein said activating region comprises a lower stem, a bulge, an upper stem, a nexus, and a hairpin.
5. A Class 2 CRISPR system consisting essentially of:
(i) a single polynucleotide comprising (a) a targeting region that comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA); and (b) an activating region adjacent to said targeting region that comprises DNA; wherein the activating region comprises a stem and a bulge, and wherein said activating region is capable of binding with a Cas9; and
(ii) a Cas9.
6. The Class 2 CRISPR system of claim 5 , wherein said activating region comprises a lower stem, a bulge, an upper stem, a nexus, and a hairpin.
7. The Class 2 CRISPR system of claim 5 , wherein said targeting region comprises a mixture of DNA and RNA.
8. The Class 2 CRISPR system of claim 5 , wherein said activating region comprises a mixture of DNA and RNA.
9. The Class 2 CRISPR system of claim 5 , wherein said targeting region comprises a mixture of DNA and RNA and said activating 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.
10. The Class 2 CRISPR system of claim 5 , further comprising a donor polynucleotide.