CRISPR hybrid DNA/RNA polynucleotides and methods of use

A Class 2 CRISPR system consisting essentially of:

• (i) a set of two Class 2 CRISPR polynucleotides comprising (a) a first Class 2 CRISPR polynucleotide wherein the first Class 2 CRISPR polynucleotide comprises a targeting region comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and an activating region adjacent to said targeting region;
• and (b) a second Class 2 CRISPR polynucleotide wherein the second Class 2 CRISPR polynucleotide comprises an activating region wherein the activating region comprises DNA and a sequence that is complementary to a sequence in said activating region of the first Class 2 CRISPR polynucleotide, wherein said activating region of the first Class 2 CRISPR polynucleotide and said activating region of the second Class 2 CRISPR polynucleotide are capable of hybridizing to each other to form an activating duplex region, wherein said activating duplex region comprises a stem and a bulge, and wherein said activating duplex region is capable of binding with a Cas9;
• and
• (ii) a Cas9.

A method of modifying a target nucleic acid molecule in a non-human organism, an isolated cell, or in vitro, wherein said method comprises:

• introducing into a cell
• (i) a set of two Class 2 CRISPR polynucleotides comprising (a) a first Class 2 CRISPR polynucleotide wherein the first Class 2 CRISPR polynucleotide comprises a targeting region configured to hybridize to a target sequence within the target nucleic acid and an activating region adjacent to said targeting region;
• and (b) a second Class 2 CRISPR polynucleotide wherein the second Class 2 CRISPR polynucleotide comprises an activating region wherein the activating region comprises DNA and a sequence that is complementary to a sequence in said activating region of the first Class 2 CRISPR polynucleotide, wherein said activating region of the first Class 2 CRISPR polynucleotide and said activating region of the second Class 2 CRISPR polynucleotide are capable of hybridizing to each other to form an activating duplex region, and wherein said activating duplex region comprises a stem and a bulge;
• and
• (ii) a Cas9, wherein the Cas9 binds with said activating duplex region of the two Class 2 CRISPR polynucleotides, wherein said targeting region of the first Class 2 CRISPR polynucleotide hybridizes to said target sequence, and wherein said target nucleic acid is cleaved.

A method of modulating transcription of at least one gene within a target nucleic acid molecule in a non-human organism, an isolated cell, or in vitro, wherein said method comprises:

• introducing into a cell
• (i) a set of two Class 2 CRISPR polynucleotides comprising (a) a first Class 2 CRISPR polynucleotide wherein the first Class 2 CRISPR polynucleotide comprises a targeting region configured to hybridize to a target sequence within the open reading frame of the at least one gene or a target sequence within the promoter sequence of the at least one gene and an activating region adjacent to said targeting region;
• and (b) a second Class 2 CRISPR polynucleotide wherein the second Class 2 CRISPR polynucleotide comprises an activating region wherein the activating region comprises DNA and a sequence that is complementary to a sequence in said activating region of the first Class 2 CRISPR polynucleotide, wherein said activating region of the first Class 2 CRISPR polynucleotide and said activating region of the second Class 2 CRISPR polynucleotide are capable of hybridizing to each other to form an activating duplex region, and wherein said activating duplex region comprises a stem and a bulge;
• and
• (ii) a Cas9, wherein the Cas9 has no nuclease activity, wherein the Cas9 binds with said activating duplex region of the two Class 2 CRISPR polynucleotides, wherein said targeting region of the first Class 2 CRISPR polynucleotide hybridizes to said target sequence, and wherein the transcription of at least one gene within the target nucleic acid molecule is modulated.