Systems, methods and compositions for sequence manipulation with optimized functional CRISPR-Cas systems

Patent metadata

Field	Value
Publication number	US-10550372-B2
Application number	US-201615179912-A
Country	US
Kind code	B2
Filing date	Jun 10, 2016
Priority date	Dec 12, 2013
Publication date	Feb 4, 2020
Grant date	Feb 4, 2020

Abstract

The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are structural information on the Cas protein of the CRISPR-Cas system, use of this information in generating modified components of the CRISPR complex, vectors and vector systems which encode one or more components or modified components of a CRISPR complex, as well as methods for the design and use of such vectors and components. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system. In particular the present invention comprehends optimized functional CRISPR-Cas enzyme systems.

First claim

What is claimed is:

1. A method of screening for gain of function (GOF) or loss of function (LOF), comprising:
(a) introducing a guide RNA (sgRNA) into cells of a mammalian cell line containing or expressing a CRISPR enzyme having at least one mutation, such that the CRISPR enzyme has no more than 5% of the nuclease activity of the CRISPR enzyme not having the at least one mutation, wherein the CRISPR enzyme is Cas9,
wherein the sgRNA comprises
(i) a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in the cells of the mammalian cell line, and
(ii) a modified loop comprising an insertion of a distinct RNA sequence(s) that binds to one or more adaptor proteins, wherein tetraloop and/or stem-loop 2 of the sgRNA is modified, wherein the one or more adaptor proteins is associated with or comprises one or more functional domains, and

(b) monitoring for GOF or LOF.

2. The method of claim 1 , wherein the CRISPR enzyme has a diminished nuclease activity of at least 97% as compared with the CRISPR enzyme not having the at least one mutation.

3. The method of claim 1 , wherein the CRISPR enzyme comprises two or more mutations in a residue selected from D10, E762, H840, N854, N863, or D986 according to a Streptococcus pyogenes Cas9 (SpCas9) protein or any corresponding ortholog or N580 according to a Staphylococcus aureus Cas9 (SaCas9) protein are mutated.

4. The method of claim 1 , wherein the at least one mutation is an H840A mutation according to a Streptococcus pyogenes Cas9 protein.

5. The method of claim 1 , wherein the CRISPR enzyme comprises a mutation selected from H840A, or D10A and H840A, or D10A and N863A, according to a Streptococcus pyogenes Cas9 (SpCas9) protein or any corresponding ortholog.

6. The method of claim 1 , wherein the CRISPR enzyme comprises: N580A according to a Staphylococcus aureus Cas9 (SaCas9) protein or any corresponding ortholog; or D10A according to a Streptococcus pyogenes Cas9 (SpCas9) protein, or any corresponding ortholog, and N580A according to a SaCas9 protein.

7. The method of claim 1 , wherein the CRISPR enzyme is associated with one or more functional domains.

8. The method of claim 1 , wherein the one or more functional domains is a heterologous functional domain.

9. The method of claim 7 , wherein the one or more functional domains associated with the CRISPR enzyme is a heterologous functional domain.

10. The method of claim 1 , wherein the adaptor protein is a fusion protein comprising the one or more functional domains and a region that binds to the distinct RNA sequence(s) of the sgRNA, wherein the fusion protein optionally comprises a linker between the one or more functional domains and the region, and wherein the linker optionally comprises a GlySer linker.

11. The method of claim 1 , wherein the sgRNA comprises at least one unmodified loop that does not comprise an insertion of the distinct RNA sequence(s).

12. The method of claim 11 , wherein the unmodified loop comprises a tetraloop or a stem-loop 2.

13. The method of claim 1 , wherein the one or more functional domains is a transcriptional activation domain.

14. The method of claim 7 , wherein the one or more functional domains associated with the CRISPR enzyme is a transcriptional activation domain.

15. The method of claim 13 , wherein the transcriptional activation domain comprises a transcriptional activation domain of VP64, p65, MyoD1, HSF1, RTA, or SET7/9.

16. The method of claim 14 , wherein the transcriptional activation domain comprises a transcriptional activation domain of VP64, p65, MyoD1, HSF1, RTA, or SET7/9.

17. The method of claim 1 , wherein the one or more functional domains is a transcriptional repressor domain.

18. The method of claim 7 , wherein the one or more functional domains associated with the CRISPR enzyme is a transcriptional repressor domain.

19. The method of claim 17 , wherein the transcriptional repressor domain is a KRAB domain, NuE domain, NcoR domain, SID domain or a SID4X domain.

20. The method of claim 18 , wherein the transcriptional repressor domain is a KRAB domain, NuE domain, NcoR domain, SID domain or a SID4X domain.

21. The method of claim 1 , wherein the one or more functional domains has one or more activities comprising methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity or nucleic acid binding activity.

22. The method of claim 7 , wherein the one or more functional domains associated with the CRISPR enzyme has one or more activities comprising methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity, nucleic acid binding activity, or molecular switch activity or chemical inducibility or light inducibility.

23. The method of claim 21 , wherein the DNA cleavage activity is due to a nuclease.

24. The method of claim 23 , wherein the nuclease comprises a Fok1 nuclease.

25. The method of claim 1 , further comprising introducing the one or more adaptor proteins into the cells of the mammalian cell line.

26. The method of claim 1 , further comprising introducing the one or more functional domains into the cells of the mammalian cell line.

27. The method of claim 7 , further comprising introducing the one or more functional domains associated with the CRISPR enzyme into the cells of the mammalian cell line.

28. The method of claim 1 , wherein the sgRNA comprises at least two modified loops, wherein each modified loop is modified by insertion of a distinct RNA sequence that binds to one or more adaptor proteins.

29. The method of claim 28 , wherein the two modified loops are a tetraloop and a stem-loop 2.

30. The method of claim 28 , wherein the two modified loops are modified by insertion of the same distinct RNA sequence.

31. The method of claim 28 , wherein the two modified loops are modified by insertion of different distinct RNA sequences.

32. The method of claim 30 , wherein the distinct RNA sequence inserted into the two modified loops are bound by adaptor proteins associated with or comprising the same functional domain.

33. The method of claim 30 , wherein the distinct RNA sequence inserted into the two modified loops are bound by adaptor proteins associated with or comprising different functional domains.

34. The method of claim 31 , wherein the distinct RNA sequences inserted into the two modified loops are bound by adaptor proteins associated with or comprising the functional domain.

35. The method of claim 31 , wherein the distinct RNA sequences inserted into the two modified loops are bound by adaptor proteins associated with or comprising different functional domains.

36. The method of claim 1 , wherein the CRISPR enzyme comprises at least one or more nuclear localization sequences.

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Univ Tokyo
• Harvard College

Inventors

• Konermann Silvana
• Trevino Alexandro
• Brigham Mark
• Ran Fei
• Hsu Patrick
• Lin Chie-Yu
• Nureki Osamu
• Nishimasu Hiroshi
• Ishitani Ryuichiro
• Zhang Feng

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61K48/005

  characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• A61K38/465

  acting on ester bonds (3.1), e.g. lipases, ribonucleases · CPC title

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title