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BR-112017027212-B1 - Manipulated Streptococcus Pyogenes CAS9 Protein, Composition and CRISPR-Cas System

BR112017027212B1BR 112017027212 B1BR112017027212 B1BR 112017027212B1BR-112017027212-B1

Abstract

CRISPR ENZYME MUTATIONS THAT REDUCE OFF-TARGET EFFECTS. Mutation(s) or modification(s) of the CRISPR enzyme are disclosed and claimed, for example, a Cas enzyme, such as a Cas9, that achieves an improvement, for example a reduction, in the off-target effects of a CRISPR-Cas or CRISPR-enzyme or CRISPR-Cas9 system or complex containing or including a mutated or modified Cas or CRISPR or Cas9 enzyme. Methods of manufacturing and use, and uses of such mutated or modified Cas or CRISPR or Cas9 enzymes and systems or complexes containing the same and products of such methods and uses are also disclosed and claimed.

Inventors

  • Feng Zhang
  • Linyi Gao
  • Bernd Zetsche
  • Ian Slaymaker

Assignees

  • MASSACHUSETTS INSTITUTE OF TECHNOLOGY
  • THE BROAD INSTITUTE INC

Dates

Publication Date
20260317
Application Date
20160617
Priority Date
20150618

Claims (12)

  1. 1. Manipulated Streptococcus pyogenes Cas9 protein (SpCas9) characterized in that the manipulated SpCas9 comprises one to three amino acid substitutions in a linking groove between the RuvC and HNH domains, with reference to the amino acid position numbering of the wild-type SpCas9 having the amino acid sequence presented in SEQ ID NO: 520, with the amino acid substitutions selected from: K775A; or R780A; or K810A; or R832A; or K848A; or K855A; or K862A; or K961A; or K968A; or R976A; or K1000A; or K1014A; or K1047A; or R1060A; or K1003A; or K1289A; or K1296A; or H1297A; or K1300A; or H1311A, or K1325A; or R780A and K810A; or R780A and K855A; or R780A and R976A; or K848A and R976A; or K855A and R976A; or R780A and K848A; or K810A and K848A; or K848A and K855A; or K810A and K855A; or H982A and R1060A; or H982A and R1003A; or K1003A and R1060A; or R780A and H982A; or K810A and H982A; or K848A and H982A; or K855A and H982A; or R780A and K1003A; or K810A and R1003A; or K848A and K1003A; or R780A and R1060A; or K810A and R1060A; or K848A and R1060A; or R780A and R1114A; or K848A and R1114A; or K810A and K1003A; or R780A, K1003A and R1060A; or K810A, K1003A and R1060A; or K848A, K1003A and R1060A; or K855A, K1003A and R1060A; or H982A, K1003A and R1060A; and in which the manipulated SpCas9 protein has enhanced specificity compared to wild-type SpCas9, with this specificity being activity on the target rather than off-target activity.
  2. 2. A manipulated SpCas9 protein, according to claim 1, characterized in that the SpCas9 protein comprises at least two modifications, each being an amino acid substitution of a positively charged residue with an uncharged residue in a linking groove between the RuvC and HNH domains.
  3. 3. A modified SpCas9 protein, according to any one of claims 1 to 2, characterized in that the modification comprises K775A, R780A, K810A, R832A, K848A, K855A, K862A, K961A, K968A, R976A, K1000A, K1014A, K1047A, R1060A, K1003A, K1289A, K1296A, H1297A, K1300A, H1311A or K1325A.
  4. 4. A modified SpCas9 protein, according to any one of claims 1 to 2, characterized in that the modification comprises R780A and K810A, or R780A and K855A, or R780A and R976A, or K848A and R976A, or K855A and R976A, or R780A and K848A, or K810A and K848A, or K848A and K855A, or K810A and K855A, or H982A and R1060A, or H982A and R1003A, or K1003A and R1060A, or R780A and H982A, or K810A and H982A, or K848A and H982A, or K855A and H982A, or R780A and K1003A, or K810A and R1003A, or K848A and K1003A, or R780A and R1060A, or K810A and R1060A, or K848A and R1060A, or R780A and R1114A, or K848A and R1114A, or K810A and K1003A.
  5. 5. A modified SpCas9 protein, according to any one of claims 1 to 2, characterized in that the modification comprises R780A, K1003A and R1060A; or K810A, K1003A and R1060A; or K848A, K1003A and R1060A; or K855A, K1003A and R1060A; or H982A, K1003A and R1060A.
  6. 6. A manipulated SpCas9 protein, according to any one of claims 1 to 5, characterized in that the manipulated SpCas9 protein is fused with one or more nuclear localization signaling (NLS) domains, preferably two or more NLS domains.
  7. 7. A manipulated SpCas9 protein, according to any one of claims 1 to 6, characterized in that the manipulated SpCas9 protein is fused to a transcription activation domain, preferably in that the manipulated SpCas9 protein is fused to a VP64 domain.
  8. 8. A manipulated SpCas9 protein, according to any one of claims 1 to 6, characterized in that the manipulated SpCas9 protein is fused to a transcription repression domain, preferably wherein the manipulated SpCas9 protein is fused to a KRAB domain or a SID domain.
  9. 9. A manipulated SpCas9 protein, according to any one of claims 1 to 6, characterized in that the manipulated SpCas9 protein is fused to a nuclease domain, preferably in that the manipulated SpCas9 protein is fused to a Fok1 domain.
  10. 10. A manipulated SpCas9 protein, according to any one of claims 1 to 6, characterized in that the manipulated SpCas9 protein is fused to either a methylase domain or a demethylase domain.
  11. 11. Composition characterized in that it comprises the SpCas9 protein manipulated as defined in any one of claims 1 to 10 and an RNA of the CRISPR-Cas system.
  12. 12. CRISPR-Cas system characterized in that it comprises the SpCas9 protein manipulated as defined in any one of claims 1 to 10 in a complex with an RNA of the CRISPR-Cas system.

Description

CROSS-REFERENCE / INCORPORATION BY REFERENCE [0001] This application is a continuation of U.S. Patent Application No. 16/697,018 filed November 26, 2019, issued as U.S. Patent No. 12,123,032, which is a continuation of U.S. Patent Application No. 16/158,295 filed October 11, 2018, issued as U.S. Patent No. 10,494,621, which is a continuation of U.S. Patent Application No. 15/844528 filed on December 16, 2017, issued as U.S. Patent No. 10,876,100, which is a continuation application in part of international patent application No. PCT/US2016/038034 filed on June 17, 2016, published as PCT Publication No. WO2016/205613 on December 22, 2016, which claims the benefit and priority of U.S. Provisional Application Serial No. 62/181,453, filed on June 18, 2015, U.S. Provisional Patent Application Serial No. 62/207,312, filed on August 19, 2015, U.S. Provisional Patent Application Serial No. 62/237,360, filed on October 5, 2015, U.S. Provisional Patent Application Serial No. U.S. Provisional Patent Application No. 62/255,256, filed November 13, 2015 and U.S. Provisional Patent Application Serial No. 62/269,876, December 18, 2015. [0002] The prior patent application(s), and all documents cited or referred to herein (“documents cited herein”), and all documents cited or referred to in the documents cited herein, together with any manufacturer’s instructions, descriptions, product specifications, and product specifications for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated as being incorporated by reference. STATEMENT REGARDING FEDERALLY SPONSORED INVESTIGATION [0003] This invention was made with government support under grants number NIH/MH100706 and MH110049 awarded by the National Institutes of Health. The government retains certain rights to the invention. SEQUENCE LISTING [0003.1] This application contains a Sequence Listing that was submitted electronically in XML format and is incorporated by reference in its entirety. The said XML copy, created on November 19, 2024, is named 114203-5901_SL.xml and is 791,987 bytes in size. FIELD OF THE INVENTION [0004] The present invention generally relates to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR enzyme (e.g., Cas or Cas9), CRISPR-Cas system or CRISPR or CRISPR-Cas complex, their components, nucleic acid molecules, e.g., vectors, involving them and the uses of all the foregoing, among other aspects. BACKGROUND OF THE INVENTION [0005] The first publication of a disclosure possibility of how to make and use a CRISPR-Cas system in eukaryotic cells is Cong et al., Science 2013; 339:819-823 (published online January 3, 2013). The first patent filing of a disclosure possibility of how to make and use a CRISPR-Cas system in eukaryotic cells is Zhang et al., U.S. Provisional Application Serial No. 61/736,527, filed December 12, 2012, to which many patent applications claim priority, including those that have won in the reference U.S. Patents Nos. 8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616, 8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945 and 8,697,359. SUMMARY OF THE INVENTION [0006] Consistent with providing groundbreaking advances that enabled the use of the CRISPR-Cas system in eukaryotic cells, the laboratory of Zhang et al. at the Broad Institute recognized that there continues to be a need for improved CRISPR enzymes to be used in making modifications at target loci, but which reduce or eliminate off-target activity. There is an urgent need for alternative and robust systems and techniques to reduce the off-target activity of CRISPR enzymes when complexed with guide RNAs. There is also an urgent need for alternative and robust systems and techniques to increase the activity of CRISPR enzymes when complexed with guide RNAs. [0007] Several strategies to improve Cas9 specificity have been developed, including reducing the amount of Cas9 in the cell, using Cas9 nickase mutants to create a pair of juxtaposed single-stranded DNA cuts, truncating the guide sequence at the 5’ end, and using a pair of catalytically inactive Cas9 nucleases, each fused into a FokI nuclease domain. [0008] The inventors have surprisingly determined that modifications can be made to CRISPR enzymes that confer reduced off-target activity compared with unmodified CRISPR enzymes and/or increased on-target activity compared with unmodified CRISPR enzymes. Thus, improved CRISPR enzymes are provided here that may be useful in a wide range of gene-modifying applications. Also provided here are CRISPR complexes, compositions and systems, as well as methods and uses, all comprising the modified CRISPR enzymes disclosed herein. CRISPR-Cas9 is preferred, i