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 set of two Class 2 Type II CRISPR polynucleotides forming a dual guide Class 2 Type II polynucleotide, comprising:
(i) a first Class 2 Type II CRISPR polynucleotide comprising a targeting region comprising ribonucleic acid (RNA) and an activating region adjacent to said targeting region; and,
(ii) a second Class 2 Type II CRISPR polynucleotide comprising an activating region comprising a sequence that is complementary to a sequence in the activating region of the first Class 2 Type II CRISPR polynucleotide,
wherein the activating region of the first Class 2 Type II CRISPR polynucleotide and the activating region of the second Class 2 Type II CRISPR polynucleotide form a dual guide activating region comprising a mixture of RNA and deoxyribonucleic acid (DNA), wherein the two activating regions are hybridized to each other to form an activating duplex region,
wherein the activating duplex region comprises a lower duplex region adjacent 3′ end of the targeting region and an upper duplex region, wherein a bulge is located between the lower and upper duplex regions, and the second Class 2 Type II CRISPR polynucleotide comprises a nexus 3′ to the lower duplex region, and at least one hairpin 3′ to the nexus, and
wherein the activating duplex region is capable of binding with a Cas9 protein.
2 . The dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , wherein the targeting region comprises a mixture of RNA and DNA.
3 . The dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , wherein the upper duplex region and the at least one hairpin comprise a mixture of DNA and RNA.
4 . The dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , wherein the second Class 2 Type II CRISPR polynucleotide comprises two hairpins 3′ to the nexus, wherein at least one of said hairpins comprises a mixture of RNA and DNA.
5 . The dual guide Class 2 Type II CRISPR polynucleotide of claim 4 , wherein the targeting region, the upper duplex region, and both sides of the hairpin closest to the 3′ end of the second Class 2 Type II CRISPR polynucleotide comprise a mixture of RNA and DNA.
6 . The dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , wherein the targeting region polynucleotide comprises a compound selected from the group consisting of phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphorotriesters, aminoalkylphosphosphorotriesters, alkylphosphonates, 5′-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, 3′-amino phosphoramidate, amino alkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates.
7 . A Class 2 Type II CRISPR complex comprising the dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , and a Cas9 protein.
8 . The Class 2 Type II CRISPR complex of claim 7 , wherein the Cas9 protein comprises a nuclear localization signal (NLS).
9 . A cell comprising the Class 2 Type II CRISPR complex of claim 7 .
10 . The cell of claim 9 , further comprising a donor polynucleotide.
11 . An isolated cell comprising the Class 2 Type II CRISPR complex of claim 7 .
12 . The isolated cell of claim 11 , further comprising a donor polynucleotide.
13 . A Class 2 Type II CRISPR system comprising the dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , and a donor polynucleotide.
14 . A method of modifying a target nucleic acid molecule comprising:
contacting the target nucleic acid molecule having a target sequence with:
(i) a dual guide Class 2 Type II CRISPR polynucleotide comprising:
(a) a first Class 2 Type II CRISPR polynucleotide comprising a targeting region comprising ribonucleic acid (RNA) and an activating region adjacent to said targeting region; and,
(b) a second Class 2 Type II CRISPR polynucleotide comprising an activating region comprising a sequence that is complementary to a sequence in the activating region of the first Class 2 Type II CRISPR polynucleotide,
wherein the activating region of the first Class 2 Type II CRISPR polynucleotide and the activating region of the second Class 2 Type II CRISPR polynucleotide form a dual guide activating region comprising a mixture of RNA and deoxyribonucleic acid (DNA), wherein the two activating regions are hybridized to each other to form an activating duplex region,
wherein the activating duplex region comprises a lower duplex region adjacent 3′ end of the targeting region and an upper duplex region, wherein a bulge is located between the lower and upper duplex regions, and the second Class 2 Type II CRISPR polynucleotide comprises a nexus 3′ to the lower duplex region, and at least one hairpin 3′ to the nexus; and
(ii) a Cas9 protein;
wherein:
the Cas9 protein binds with the activating duplex region of the polynucleotide,
the contacting of the target nucleic acid molecule with the Class 2 Type II CRISPR polynucleotide and the Cas9 occurs in vitro, in a cell, or in an isolated cell, and
the target nucleic acid molecule is cleaved.
15 . The method of claim 14 , wherein the target nucleic acid molecule comprises DNA.
16 . The method of claim 14 , wherein the target nucleic acid molecule comprises RNA.
17 . The method of claim 14 , wherein the targeting region comprises a mixture of DNA and RNA.
18 . The method of claim 14 , wherein the upper duplex region and the at least one hairpin comprise a mixture of DNA and RNA.
19 . The method of claim 14 , wherein the second Class 2 Type II CRISPR polynucleotide comprises two hairpins 3′ to the nexus, wherein at least one of said hairpins comprises a mixture of RNA and DNA.
20 . The method of claim 19 , wherein the targeting region, the upper duplex region, and both sides of the hairpin closest to the 3′ end of the second Class 2 Type II CRISPR polynucleotide comprise a mixture of RNA and DNA.