Compositions and methods of nucleic acid-targeting nucleic acids
This disclosure provides for compositions and methods for the use of nucleic acid-targeting nucleic acids and complexes thereof.
1. An engineered CRISPR Cas9 protein comprising:
a modification in a region of a CRISPR Cas9 protein corresponding to a region of amino acid residues 170-312 of a S. pyogenes -Cas9 protein (SEQ ID NO:8), wherein the modification comprises an insertion, deletion, or combination thereof.
2. The engineered CRISPR Cas9 protein of claim 1 , wherein the modification is made in identified regions of sequence similarity between the Cas9 protein and the S. pyogenes -Cas9 protein (SEQ ID NO:8).
3. The engineered CRISPR Cas9 protein of claim 1 , wherein the modification comprises a deletion.
4. The engineered CRISPR Cas9 protein of claim 1 , wherein the modification comprises a deletion of more than 5 amino acid residues.
5. The engineered CRISPR Cas9 protein of claim 1 , wherein the modification comprises a deletion of more than 100 amino acid residues.
6. The engineered CRISPR Cas9 protein of claim 1 , wherein the modification comprises a deletion and an insertion of a non-native amino acid sequence.
7. The engineered CRISPR Cas9 protein of claim 1 , wherein the modification comprises an insertion of a non-native amino acid sequence to form a fusion protein.
8. The engineered CRISPR Cas9 protein of claim 7 , wherein the non-native amino acid sequence comprises a functional domain.
9. The engineered CRISPR Cas9 protein of claim 1 , wherein the engineered CRISPR Cas9 protein is a fusion.
10. The engineered CRISPR Cas9 protein of claim 9 , wherein the engineered CRISPR Cas9 fusion comprises a detectable label.
11. The engineered CRISPR Cas9 protein of claim 9 , wherein the engineered CRISPR Cas9 fusion comprises the engineered CRISPR Cas9 protein and an endonuclease.
12. The engineered CRISPR Cas9 protein of claim 11 , wherein the endonuclease is FokI.
13. The engineered CRISPR Cas9 protein of claim 1 , wherein the engineered CRISPR Cas9 protein is enzymatically inactive.
14. The engineered CRISPR Cas9 protein of claim 1 , wherein the engineered CRISPR Cas9 protein is codon optimized for a host cell of interest.
15. The engineered CRISPR Cas9 protein of claim 14 , wherein the host cell of interest is a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, a fungal cell, an animal cell, a cell from an invertebrate animal, a cell from a vertebrate animal, or a cell from a mammal.
16. The engineered CRISPR Cas9 protein of claim 15 , wherein the host cell is a human cell.
17. A polynucleotide encoding the engineered CRISPR Cas9 protein of claim 1 .
18. The polynucleotide of claim 17 , wherein the modification of the engineered CRISPR Cas9 protein is a deletion of more than 5 amino acid residues.
19. The polynucleotide of claim 17 , wherein the modification of the engineered CRISPR Cas9 protein is a deletion of more than 100 amino acid residues.
20. A kit comprising:
the engineered CRISPR Cas9 protein of claim 1 or a polynucleotide encoding the engineered CRISPR Cas9 protein; and
a reagent.