Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing

• US 20150259684A1
• Filed: 03/31/2015
• Published: 09/17/2015
• Est. Priority Date: 07/10/2013
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: CHURCH, George M., ESVELT, Kevin, MALI, Prashant
• Granted Patent: US 9,587,252 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07K 2/00 Peptides of undefined number of amino acids; Derivatives thereof
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid
• CPC Class Codes: C12N 2320/33 Alteration of splicing
• CPC Class Codes: C12N 2320/50 Methods for regulating/modulating their activity
• CPC Class Codes: C12N 2800/107 for mammalian
• CPC Class Codes: C12N 2800/40 Systems of functionally co-operating vectors
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 2810/10 Vectors comprising a non-peptidic targeting moiety
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/34 being a transcription initiation element
• CPC Class Codes: C12N 2999/007 Technological advancements, e.g. new system for producing known virus, cre-lox system for production of transgenic animals
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)

Abstract

Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes.

Claims

1. A method of modulating expression of two or more target nucleic acids in a cell comprising

introducing into the cell a first foreign nucleic acid encoding two or more guide RNAs complementary to the two or more target nucleic acids,

introducing into the cell a second foreign nucleic acid encoding two or more orthogonal RNA guided nuclease null DNA binding proteins that respectively bind to the two or more target nucleic acids and are guided by the two or more guide RNAs,

introducing into the cell a third foreign nucleic acid encoding two or more transcriptional regulator proteins or domains,

wherein the RNAs, the orthogonal RNA guided nuclease-null DNA binding proteins, and the transcriptional regulator proteins or domains are expressed,

wherein two or more co-localization complexes form between an RNA, an orthogonal RNA guided nuclease null DNA binding protein, a transcriptional regulator protein or domain and a target nucleic acid and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

2. The method of claim 1 wherein the foreign nucleic acid encoding an orthogonal RNA guided nuclease null DNA binding protein further encodes the transcriptional regulator protein or domain fused to the orthogonal RNA guided nuclease null DNA binding protein.

3. The method of claim 1 wherein the foreign nucleic acid encoding two or more RNAs further encodes a target of an RNA-binding domain and the foreign nucleic acid encoding the transcriptional regulator protein or domain further encodes an RNA-binding domain fused to the transcriptional regulator protein or domain.

4. The method of claim 1 wherein the cell is a eukaryotic cell.

5. The method of claim 1 wherein the cell is a yeast cell, a plant cell or an animal cell.

6. The method of claim 1 wherein the RNA is between about 10 to about 500 nucleotides.

7. The method of claim 1 wherein the RNA is between about 20 to about 100 nucleotides.

8. The method of claim 1 wherein the transcriptional regulator protein or domain is a transcriptional activator.

11. The method of claim 1 wherein the transcriptional regulator protein or domain is a transcriptional repressor.

16. The method of claim 1 wherein the one or more RNAs is a tracrRNA-crRNA fusion.

17. The method of claim 1 wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

18. The method of claim 1 wherein a first target nucleic acid is activated and a second target nucleic acid is repressed.

19. The method of claim 1 wherein a first plurality of target nucleic acids are activated and a second plurality of target nucleic acids are repressed.

20. The method of claim 1 wherein a first one or more target nucleic acids are activated and a second one or more target nucleic acids are repressed.

37. The method of claim 1 wherein the RNA guided nuclease null DNA binding protein is a DNA binding protein of a Type II CRISPR system.

38. The method of claim 1 wherein the RNA guided nuclease null DNA binding protein is an orthogonal nuclease-null Cas9 protein.

9-10. -10. (canceled)

12-15. -15. (canceled)

21. A method of altering one or more target nucleic acids in a cell simultaneous with regulating expression of one or more target nucleic acids in a cell comprising

introducing into the cell a first foreign nucleic acid encoding two or more guide RNAs with at least one guide RNA being complementary to the one or more target nucleic acids to be altered and at least one guide RNA being complementary to the one or more target nucleic acids to be regulated,

introducing into the cell a second foreign nucleic acid encoding 1) one orthogonal RNA-guided protein nickase having one inactive nuclease domain and being guided by the one or more guide RNAs complementary to the one or more target nucleic acids to be altered, and 2) one or more orthogonal RNA guided nuclease null DNA binding proteins that bind to and are guided by the one or more guide RNAs complementary to the target nucleic acids to be regulated,

introducing into the cell a third foreign nucleic acid encoding one or more transcriptional regulator proteins or domains,

wherein the guide RNAs, the orthogonal RNA guided protein nickase, the orthogonal RNA guided nuclease-null DNA binding proteins, and the transcriptional regulator proteins or domains are expressed,

wherein at least one co-localization complex forms between a guide RNA, an orthogonal RNA guided protein nickase, and a target nucleic acid to be altered and wherein the protein nickase nicks the target nucleic acid to be altered,

wherein at least one co-localization complex forms between a guide RNA, an orthogonal RNA guided nuclease null DNA binding protein, a transcriptional regulator protein or domain and a target nucleic acid to be regulated and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid to be regulated.

22. A method of altering one or more target nucleic acids in a cell simultaneous with regulating expression of one or more target nucleic acids in a cell comprising

introducing into the cell a first foreign nucleic acid encoding two or more RNAs with at least one RNA being complementary to the one or more target nucleic acids to be altered and at least one RNA being complementary to the one or more target nucleic acids to be regulated,

introducing into the cell a second foreign nucleic acid encoding 1) one orthogonal RNA-guided protein nuclease and being guided by the one or more RNAs complementary to the one or more target nucleic acids to be altered, and 2) one or more orthogonal RNA guided nuclease null DNA binding proteins that bind to and are guided by the one or more RNAs complementary to the target nucleic acids to be regulated,

introducing into the cell a third foreign nucleic acid encoding one or more transcriptional regulator proteins or domains,

wherein the RNAs, the orthogonal RNA guided protein nuclease, the orthogonal RNA guided nuclease-null DNA binding proteins, and the transcriptional regulator proteins or domains are expressed,

wherein at least one co-localization complex forms between an RNA, an orthogonal RNA guided protein nuclease, and a target nucleic acid to be altered and wherein the nuclease cuts the target nucleic acid to be altered,

wherein at least one co-localization complex forms between an RNA, an orthogonal RNA guided nuclease null DNA binding protein, a transcriptional regulator protein or domain and a target nucleic acid to be regulated and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid to be regulated.

23. A cell comprising

a first foreign nucleic acid encoding two or more guide RNAs complementary to two or more respective target nucleic acids,

a second foreign nucleic acid encoding two or more RNA guided nuclease null DNA binding proteins, and

a third foreign nucleic acid encoding two or more transcriptional regulator proteins or domains wherein each guide RNA, orthogonal RNA guided nuclease null DNA binding protein and transcriptional regulator protein or domain is a member of a co-localization complex for a respective target nucleic acid.

24. The cell of claim 23 wherein the foreign nucleic acid encoding an orthogonal RNA guided nuclease null DNA binding protein further encodes the transcriptional regulator protein or domain fused to the orthogonal RNA guided nuclease null DNA binding protein.

25. The cell of claim 23 wherein the foreign nucleic acid encoding one or more guide RNAs further encodes a target of an RNA-binding domain and the foreign nucleic acid encoding the transcriptional regulator protein or domain further encodes an RNA-binding domain fused to the transcriptional regulator protein or domain.

26. The cell of claim 23 wherein the cell is a eukaryotic cell.

27. The cell of claim 23 wherein the cell is a yeast cell, a plant cell or an animal cell.

28. The cell of claim 23 wherein the RNA includes between about 10 to about 500 nucleotides.

29. The cell of claim 23 wherein the RNA includes between about 20 to about 100 nucleotides.

30. The cell of claim 23 wherein the transcriptional regulator protein or domain is a transcriptional activator.

35. The cell of claim 23 wherein the one or more guide RNAs is a tracrRNA-crRNA fusion.

36. The cell of claim 23 wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

39. The cell of claim 23 wherein the RNA guided nuclease null DNA binding protein is a DNA binding protein of a Type II CRISPR system.

40. The cell of claim 23 wherein the RNA guided nuclease null DNA binding protein is an orthogonal nuclease-null Cas9 protein.

31-34. -34. (canceled)