Compositions and methods for cas9 molecules with improved gene editing properties
AssigneeBoard of Regents, The University of Texas System
InventorsDavid Taylor, Kenneth A. Johnson, Jack P. K. Bravo, Tyler L. Dangerfield
Life of this patent
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Abstract
Disclosed herein are methods and compositions relating to a mutated version of Cas9. This mutation or mutations can be in the Rec3 clamp of Cas9. The mutated Cas9 can have advantages compared Cas9s which don't have mutations in the Rec3 clamp.
Claims79
A method of increasing fidelity, specificity, and/or speed of processivity in a functional Cas9 molecule, the method comprising mutating one or more amino acid residues within a clamp region of Cas9 Recognition Lobe (REC3 clamp), wherein the REC3 clamp is 80% or more identical to SEQ ID NO:
- 2, and further wherein the one or more mutations increase fidelity, specificity, and/or speed of processivity of the Cas9 molecule.
An isolated functional Cas9 variant comprising a REC3 clamp, wherein the REC3 clamp is 80% or more identical to SEQ ID NO:
- 2, and further wherein the REC3 clamp comprises one or more amino acid mutations which increase fidelity, specificity, and/or speed of processivity compared to a REC3 clamp without said one or more mutations.
A method of performing gene editing, the method comprising contacting a target site with a functional Cas9 molecule, wherein the Cas9 molecule comprises a REC3 clamp, wherein the REC3 clamp is 80% or more identical to SEQ ID NO:
- 2, and further wherein the REC3 clamp comprises one or more amino acid mutations which increase fidelity, specificity, and/or speed of processivity compared to a REC3 clamp without said one or more mutations.
A method of treating a subject with a disease or disorder which is treatable with gene editing, the method comprising contacting a target site of one or more genes in need of editing within the genome of the subject with a functional Cas9 molecule comprising a REC3 clamp, wherein the REC3 clamp is 80% or more identical to SEQ ID NO:
- 2, and further wherein the REC3 clamp comprises one or more amino acid mutations which increase fidelity, specificity, and/or speed of processivity compared to a REC3 clamp without said one or more mutations;
- wherein said Cas9 molecule edits one or more genes in a manner which effectively treats said disease or disorder.
The method of 54 or 55, wherein said contacting occurs via transfection by a vector encoding said Cas9 molecule.
A method of modifying an organism to produce a non-naturally occurring product, or a naturally occurring product in a non-naturally occurring amount, the method comprising contacting a target site of one or more genes within the genome of the organism with a functional Cas9 molecule comprising a REC3 clamp, wherein the REC3 clamp is 80% or more identical to SEQ ID NO:
- 2, and further wherein the REC3 clamp comprises one or more amino acid mutations which increase fidelity, specificity, and/or speed of processivity compared to a REC3 clamp without said one or more mutations;
- wherein said Cas9 molecule edits one or more genes of interest so that the organism produces a non-naturally occurring product, or a naturally occurring product in a non-naturally occurring amount.
A kit comprising an isolated functional Cas9 molecule comprising a REC3 clamp, wherein the REC3 clamp is 80% or more identical to SEQ ID NO:
- 2, and further wherein the REC3 clamp comprises one or more amino acid mutations which increase fidelity, specificity, and/or speed of processivity compared to a REC3 clamp without said one or more mutations.
Dependencies are inferred from the claim text.