Mutant Cas9 proteins
Methods of making mutant Cas9 proteins are described.
1. A method of making a mutant Cas9 protein having DNA binding activity comprising
aligning full length amino acid sequences of a family of Cas9 proteins,
identifying one or more diverging amino acid sequence portions among the full length amino acid sequences of the family of Cas9 proteins,
identifying a nucleic acid sequence for the one or more diverging amino acid sequences portions,
generating a nucleic acid sequence for a target Cas9 protein which lacks the nucleic acid sequence for the one or more diverging amino acid sequences portions, and
generating a mutant Cas9 protein from the generated nucleic acid sequence.
2. The method of claim 1 wherein the target Cas9 protein has nuclease activity.
3. The method of claim 1 wherein the target Cas9 protein is a nickase.
4. The method of claim 1 wherein the target Cas9 protein is nuclease null.
5. The method of claim 1 wherein the nucleic acid sequence encoding the one or more diverging amino acid sequences portions is deleted from a nucleic acid sequence encoding a full length Cas9 protein.
6. The method of claim 1 wherein the nucleic acid sequence encoding the one or more diverging amino acid sequences portions is deleted from a nucleic acid sequence encoding a full length Cas9 protein and replaced with a linker.
7. The method of claim 1 wherein the nucleic acid sequence encoding the one or more diverging amino acid sequences portions is deleted from a nucleic acid sequence encoding the target Cas9 protein and replaced with a SGGGS linker.
8. The method of claim 1 wherein the mutant Cas9 protein is a mutant Cas9 protein encoded by the nucleic acid sequence of SEQ ID NO:1.
9. The method of claim 1 wherein the mutant Cas9 protein is a mutant Cas9 protein encoded by the nucleic acid sequence of SEQ ID NO:2.