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EP-3429635-B1 - ANTI-CRISPR COMPOUNDS AND METHODS OF USE

EP3429635B1EP 3429635 B1EP3429635 B1EP 3429635B1EP-3429635-B1

Inventors

  • SONTHEIMER, Erik, J.
  • DAVIDSON, ALAN
  • MAXWELL, Karen
  • PAWLUK, APRIL
  • LEE, JOOYOUNG
  • AMRANI, NADIA
  • ZHANG, YAN

Dates

Publication Date
20260506
Application Date
20170313

Claims (15)

  1. A composition comprising a fusion protein having a bacterial Type II-C anti-CRISPR (Acr) protein and a C-terminal or N-terminal adduct, wherein said Acr protein: comprises at least a portion of an amino acid sequence selected from the group consisting of: an AcrIIC1Boe amino acid sequence (SEQ ID NO:2); an AcrIIC1Nme amino acid sequence (SEQ ID NO:4); an AcrIIC2Nme amino acid sequence (SEQ ID NO:6); an AcrIIC3Nme amino acid sequence (SEQ ID NO:8); an AcrIIC4Hpa amino acid sequence (SEQ ID NO: 10); an AcrIIC5Smu amino acid sequence (SEQ ID NO: 12); and a mutated Acr protein thereof comprising one to three non-wild type amino acid residues; and said Acr protein specifically binds a Type II-C Cas9 protein.
  2. The composition of Claim 1, wherein said Acr protein is a dimer Acr protein.
  3. The composition of Claim 1, wherein said adduct is selected from the group consisting of a nuclear localization sequence, an affinity tag, an epitope sequence tag, and a DYKDDDDK.
  4. The composition of Claim 1, wherein said Acr protein specifically binds to said Type II-C Cas9 protein binding site selected from the group consisting of a Type II-C Cas9 protein and a Type II-C dCas9 protein.
  5. The composition of Claim 4, wherein said Type II-C Cas9 protein or said Type II-C dCas9 protein is selected from the group consisting of a Brackiella oedipodis Cas9 protein, a Neisseria meningitidis Cas9 protein, a Haemophilus influenzae Cas9 protein, a Simonsiella muelleri Cas9 protein, and a Ralstonia solanacearum Cas9 protein.
  6. A composition according to any preceding claim for use in therapy.
  7. An in vitro method of reducing Cas9 gene editing, the method comprising: a) providing; i) a biological cell comprising at least one suspect gene; ii) a Type II-C Cas9/sgRNA complex or a Type II-C dCas9/RNA complex wherein said sgRNA is capable of hybridizing to a target site of at least one suspect gene and said Type II-C Cas9 protein or said Type II-C dCas9 protein comprises a binding site; and iii) a fusion protein comprising a bacterial Type II-C anti-CRISPR (Acr) protein and an adduct wherein said Acr protein: comprises at least a portion of an amino acid sequence selected from the group consisting of: an AcrIIC1Boe amino acid sequence (SEQ ID NO:2); an AcrIIC1Nme amino acid sequence (SEQ ID NO:4); an AcrIIC2Nme amino acid sequence (SEQ ID NO:6); an AcrIIC3Nme amino acid sequence (SEQ ID NO:8); an AcrIIC4Hpa amino acid sequence (SEQ ID NO: 10); an AcrIIC5Smu amino acid sequence (SEQ ID NO: 12); and a mutated Acr protein thereof comprising one to three non-wild type amino acid residues; and said Acr protein specifically binds to said Type II-C Cas9 binding site or said Type II-C dCas9 binding site; b) contacting said Type II-C Cas9/sgRNA complex or said Type II-C dCas9/sgRNA complex with said biological cell such that said Type II-C Cas9/sgRNA complex or said Type II-C dCas9/sgRNA complex hybridizes to said target site; c) editing said suspect gene with said Type II-C Cas9/sgRNA complex or said Type II-C dCas9/sgRNA complex; and d) contacting said binding site with said fusion protein such that said editing is reduced.
  8. The method of Claim 7, wherein said adduct is selected from the group consisting of a nuclear localization sequence, an affinity tag, an epitope sequence tag, and a DYKDDDDK protein.
  9. The method of Claim 7, wherein said Type II-C Cas9 or said Type II-C dCas9 is selected from the group consisting of a Brackiella oedipodis Cas9 protein, a Neisseria meningitidis Cas9 protein, a Haemophilus influenzae Cas9 protein, a Simonsiella muelleri Cas9 protein, and a Ralstonia solanacearum Cas9 protein.
  10. The method of Claim 7, wherein said type II-C dCas9/sgRNA complex is a dNmeCas9/sgRNA complex.
  11. The method of Claim 7, wherein said reduced editing of said suspect gene: i) occurs during the G1 phase of the cell cycle; or ii) prevents mosaic genotypes; or iii) inhibits a gene drive or iv) prevents at least one off-target event of Cas9 binding selected from the group consisting of DNA cleavage and DNA mutation.
  12. The method of Claim 7, wherein said contacting said binding site reduces said editing of said suspect gene by at least 70%, 75%, 90% or 100%.
  13. The method of Claim 7, wherein said contacting said binding site precisely and/or predictably controls the activity of said editing of said suspect gene.
  14. The method of Claim 7, wherein said at least one suspect gene is selected from the group consisting of a dysfunctional suspect gene, a dysregulated gene, a mutated gene and a damaged gene.
  15. A kit, comprising: a) a first container comprising at least one fusion protein comprising a bacterial Type II-C anti-CRISPR (Acr) protein and an adduct wherein said Acr protein: comprises at least a portion of an amino acid sequence selected from the group consisting of: an AcrIIC1Boe amino acid sequence (SEQ ID NO:2); an AcrIIC1Nme amino acid sequence (SEQ ID NO:4); an AcrIIC2Nme amino acid sequence (SEQ ID NO:6); an AcrIIC3Nme amino acid sequence (SEQ ID NO:8); an AcrIIC4Hpa amino acid sequence (SEQ ID NO:10); an AcrIIC5Smu amino acid sequence (SEQ ID NO:12); and a mutated Acr protein thereof comprising one to three non-wild type amino acid residues; and said Acr protein specifically binds a Cas9 protein binding site; b) a second container comprising said Cas9 protein selected from the group consisting of a Type II-C Cas9 protein, a Type II-C dCas9 protein and a Type II-C dNmeCas9 protein; c) a third container comprising an sgRNA; and wherein said Cas9 binding site is selected from the group consisting of a Brackiella oedipodis Cas9 protein binding site, a Neisseria meningitidis Cas9 protein binding site, a Haemophilus influenzae Cas9 protein binding site, a Simonsiella muelleri Cas9 protein binding site, and a Ralstonia solanacearum Cas9 protein binding site.

Description

Field Of The Invention The present invention is related to Type II-C Cas9 anti-CRISPR (Acr) inhibitors that control Cas9 gene editing activity, methods of use and a kit comprising the same. Co-administration of such Acr inhibitors may provide an advantageous adjunct in permitting safe and practical biological therapeutics through spatial or temporal control of Cas9 activity; controlling Cas9-based gene drives in wild populations to reduce the ecological consequences of such forced inheritance schemes; and contributing to general research into various biotechnological, agricultural, and medical applications of gene editing technologies. Background CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats; CRISPR-associated system) comprises a bacterial immune system that recognizes and destroys foreign nucleic acids. The development of Type II CRISPR-Cas9 systems as programmable nucleases for genome engineering has been beneficial in the biomedical sciences. For example, a Cas9 platform has enabled gene editing in a large variety of biological systems, where both gene knockouts and tailor-made alterations are possible within complex genomes with unprecedented accuracy and efficiency. The CRISPR-Cas9 system has the potential for application to gene therapy approaches for disease treatment, whether for the creation of custom, genome-edited cell-based therapies or for direct correction or ablation of aberrant genomic loci within patients. Mutant versions of Cas9 in which the DNA cleavage activity has been inactivated ["dead" Cas9 (dCas9)] have also been developed for RNA-guided genome binding, enabling further applications in gene expression control and genome structure visualization. The safe application of CRISPR-Cas9 in gene therapy requires an ability to control the gene editing activity of a Cas9/sgRNA complex once the intended use has been realized. While several engineered systems allow for controlled activation of CRISPR-Cas9 to increase precision, all of these systems still lack the ability to provide predictable control and robust inhibition. What is needed in the art is the ability to predictably control gene editing (Cas9) or genome binding (dCas9) activity to prevent unintended Cas9 cleavage or DNA binding activity once a specific goal has been attained. Additionally, the ability to restrict Cas9 cleavage activity to a particular site, tissue, or cell cycle stage would greatly improve the efficacy and safety of Cas9-based clinical treatments and research applications. Summary Of The Invention The present invention is related to the field of CRISPR-Cas9 gene editing platforms. In particular, the present invention has identified Type II-C Cas9 anti-CRISPR (Acr) inhibitors that control Cas9 gene editing activity. Co-administration of such Acr inhibitors may provide an advantageous adjunct in permitting safe and practical biological therapeutics through spatial or temporal control of Cas9 activity; controlling Cas9-based gene drives in wild populations to reduce the ecological consequences of such forced inheritance schemes; and contributing to general research into various biotechnological, agricultural, and medical applications of gene editing technologies. The present invention is set out in the appended set of claims. In one example, the present disclosure contemplates a Type II-C anti-CRISPR (Acr) protein. The Acr protein of the present disclosure may be a truncated protein. The truncated protein disclosed herein is derived from an amino acid sequence including, but not limited to, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and/or SEQ ID NO: 16. In one embodiment, the Acr protein is a fusion protein. In one embodiment, the Acr fusion protein comprises a C-terminal adduct. In one embodiment, the Acr fusion protein comprises an N-terminal adduct. In one embodiment, the adduct is a nuclear localization sequence. In one embodiment, the adduct is an affinity tag. In one embodiment, the affinity tag is FLAG. In one embodiment, the Acr protein is a dimer protein. In one example, the Acr dimer protein may be an Acr homodimer protein. In one example, the Acr dimer protein may be an Acr heterodimer protein. In one embodiment, the Acr protein is a mutated protein. In one embodiment, said Acr protein comprises at least a portion of an amino acid sequence selected from the group consisting of AcrIIC1Boe, AcrIIC1Nme, AcrIIC2Nme, AcrIIC3Nme, AcrIIC4Hpa, and AcrIIC5Smu. In one example, said protein may be less than approximately 14 kDa. In one example, the present disclosure contemplates a composition comprising a Type II-C Cas9 protein comprising a binding site and a Type II-C anti-CRISPR (Acr) protein, wherein said Acr protein binds with specific affinity to said binding site. In one example, the Type II-C Cas9 protein is a Brackiella oedipodis Cas9 protein. In one example, the Type II-C Cas9 protein is a Neisseria meningitidis Cas9 protein.