Three-component CRISPR/Cas complex system and uses thereof
The invention described herein provides compositions and reagents for assembling a tripartite complex at a specific location of a target DNA. The invention also provides methods for using the complex to, for example, label a specific genomic locus, to regulate the expression of a target gene, or to create a gene regulatory network.
1. An RNA polynucleotide comprising:
(1) a DNA-targeting sequence that is complementary to a target polynucleotide sequence;
(2) a Cas9-binding sequence; and,
(3) at least 5 tandem copies of a PUF domain-Binding Sequence (PBS).
2. The polynucleotide of claim 1 , wherein the DNA-targeting sequence is complementary to the target polynucleotide sequence over about 12-22 nucleotides.
3. The polynucleotide of claim 1 , wherein the DNA-targeting sequence has a 5′ end nucleotide, and the 5′ end nucleotide is G.
4. The polynucleotide of claim 1 , further comprising a linker sequence linking the DNA-targeting sequence to the Cas9-binding sequence.
5. The polynucleotide of claim 1 , wherein the Cas9-binding sequence
(1) forms a hairpin structure, or
(2) is about 37-47 nt or about 42 nt.
6. The polynucleotide of claim 1 , wherein each of the multiple at least 5 tandem copies of the PBS has about 8 nucleotides.
7. The polynucleotide of claim 1 , comprising
a PBS of the sequence 5′-UGUAUGUA-3′ that can be bound by the PUF domain PUF(3-2), or
a PBS of the sequence 5′-UUGAUAUA-3′ that can be bound by the PUF domain PUF(6-2/7-2).
8. The polynucleotide of claim 1 , comprising 5-35 tandem copies of identical PUF domain-Binding Sequences.
9. The polynucleotide of claim 1 , comprising 5-35 tandem copies of different PUF domain-Binding Sequences.
10. The polynucleotide of claim 1 , comprising 5-10 tandem copies of identical PUF domain-Binding Sequences.
11. The polynucleotide of claim 1 , comprising 5-10 tandem copies of different PUF domain-Binding Sequences.
12. The polynucleotide of claim 1 , wherein the tandem copies of the PBS are separated from each other by a spacer sequence.
13. A vector comprising the polynucleotide of claim 1 .
14. A plurality of vectors of claim 13 , wherein the polynucleotides of the plurality of vectors have a different DNA-targeting sequence, Cas9-binding sequence, and/or PBS copy number or identity, relative to each other.
15. A complex comprising the polynucleotide of claim 1 and a Cas9 protein.
16. The complex of claim 15 , further comprising a PUF domain bound to a PBS of the RNA polynucleotide.
17. The complex of claim 15 , wherein the Cas9 protein is a nuclease-deficient dCas9 protein.
18. A host cell comprising the vector of claim 13 .