IP Library › Granted Patent US 12,215,326
Granted Patent B2
US 12,215,326 · App. 15/632,067 · Granted Feb 4, 2025

RNA-targeting system

Inventors: Feng Zhang (Cambridge, MA); Patrick Hsu (Cambridge, MA); Jonathan S. Gootenberg (Cambridge, MA); Aaron Smargon (Cambridge, MA)
Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology; President and Fellows of Harvard College
C12N15/63C12N9/22C12N15/11C12N15/111C12N15/115C12N15/86C12Y301/21004C07K2319/09C12N2310/10C12N2310/16C12N2310/20C12N2330/51C12N2800/22
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Quick Facts
Patent No.
US 12,215,326
App. No.
15/632,067
Granted
Feb 4, 2025
Kind
B2
Abstract

The invention provides for systems, methods, and compositions for targeting RNA. In particular, the invention provides a non-naturally occurring or engineered RNA-targeting system comprising an RNA-targeting Cas protein and at least one RNA-targeting guide RNA, wherein said RNA-targeting guide RNA is capable of hybridizing with a target RNA in a cell.

Claims (47)

1. A non-naturally occurring or engineered vector system comprising one or more vectors comprising:

a) a first regulatory element operably linked to a first polynucleotide sequence encoding an RNA-targeting guide RNA (Rt-gRNA), wherein said Rt-gRNA is designed to hybridize with a target RNA, wherein said Rt-gRNA comprises:

(i) a small CRISPR/Cas system associated RNA (scaRNA) sequence, and

(ii) a trans-activating CRISPR/Cas system RNA (tracrRNA) sequence, wherein said scaRNA and said tracrRNA are designed to at least partially hybridize to each other, and

b) a second regulatory element operably linked to a second polynucleotide sequence encoding

(i) a Francisella novicida Cas protein (FnCas) or ortholog, homolog, or fragment thereof comprising a Rec 1 domain, Rec 2 domain, HNH domain, and PAM Interacting (PI) domain, and

(ii) one or more heterologous functional domains positioned at one or more sites in FnCas that are analogous to amino acid positions 534-676 corresponding to a wild type Streptococcus pyogenes Cas9 (SpCas9) Rec 1 domain

wherein the Rt-gRNA is capable of forming an RNA-targeting complex with the FnCas and of directing sequence-specific binding of the RNA-targeting complex to the target RNA,

wherein each of the one or more heterologous functional domains comprise one or more of the following activities: methylase activity, demethylase activity, translation activation activity, translation repression activity, histone modification activity, RNA cleavage activity, or DNA cleavage activity, and

wherein components (a) and (b) are located on the same or different vectors of the system and wherein the Rt-gRNA and said FnCas protein do not naturally occur together.

2. The vector system according to claim 1 , wherein said FnCas protein is a type II Cas protein.

3. The vector system according to claim 1 , wherein said scaRNA sequence is fused to said tracrRNA sequence.

4. The vector system according to claim 1 , wherein said FnCas protein is codon optimized for expression in a eukaryotic cell.

5. The vector system according to claim 1 , wherein said one or more vectors are viral vectors, or wherein said one or more vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

6. The vector system according to claim 1 , wherein said RNA-targeting guide RNA is designed to direct said FnCas protein to said target RNA.

7. A composition comprising a vector system according to claim 1 .

8. A method for modulating synthesis of a protein comprising introducing into a cell containing an RNA molecule encoding said protein, the vector system of claim 1 .

9. The method according to claim 8 , wherein the synthesis of said protein is suppressed; or the synthesis of said protein is modulated by editing of said RNA molecule encoding said protein or splicing of said RNA molecule encoding said protein.

10. The method according to claim 9 , wherein the synthesis of said protein is suppressed by knock-down of said RNA molecule encoding said protein.

11. The method according to claim 10 , wherein said RNA-targeting Cas protein is codon optimized for expression in a eukaryotic cell.

12. The method according to claim 11 , wherein said one or more vectors are viral vectors, or wherein said one or more vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

13. The method according to claim 12 , wherein the cell is a eukaryotic cell.

14. The method according to claim 11 , wherein the eukaryotic cell is a mammalian or human cell.

15. The composition according to claim 7 , wherein one or more amino acid residues of the FnCas protein, other than those amino acid positions in FnCas that are analogous to amino acid positions 534-676 corresponding to a Streptococcus pyogenes Cas9 (SpCas9) Rec 1 domain at which the one or more heterologous functional domains are positioned are modified.

16. The composition according to claim 15 , wherein the modification comprises mutation of one or more, or two or more, or three or more amino acid residues of the FnCas protein.

17. The composition according to claim 16 , wherein the one or more,

or two or more, or three or more mutations are in one or more catalytically active domains of the FnCas protein.

18. The composition according to claim 17 , wherein the FnCas protein has reduced or abolished nuclease activity compared with an FnCas protein lacking said mutation(s).

19. The composition according to claim 18 , wherein the one or more,

or two or more mutations are in a catalytically active domain of the FnCas protein comprising a RuvCI, RuvCII or RuvCIII domain.

20. The vector system according to claim 1 , wherein the FnCas further comprises one or more heterologous functional domains selected from the group consisting of transcriptional activation domain, transcriptional repression domain and nuclease domain.

21. The vector system according to claim 20 , wherein the transcriptional activation domain comprises VP64.

22. The vector system according to claim 20 , wherein the transcriptional repression domain comprises a KRAB domain or a SID domain.

23. The vector system according to claim 20 , wherein the nuclease domain comprises Fok1.

24. The vector system according to claim 1 , wherein the one or more functional domains further have one or more of the following activities: deaminase activity, transcription activation activity, transcription repression activity, transcription release factor activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity.

25. The composition according to claim 7 , wherein the tracrRNA or the scaRNA comprises one or more protein-binding RNA aptamers.

26. The composition according to claim 25 , wherein the one or more aptamers is designed to bind a bacteriophage coat protein.

27. The composition according to claim 26 , wherein the bacteriophage coat protein is selected from the group consisting of QB, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ¢Cb5, ¢Cb8r, ¢Cb12r, ¢Cb23r, 7s and PRR1.

28. The composition according to claim 7 , wherein the tracrRNA is 30 or more, 40 or more or 50 or more nucleotides in length.

29. The composition according to claim 7 , wherein the FnCas protein is modified to comprise a PAM sequence specificity which is different from the PAM sequence specificity of the FnCas protein without said further modification.

30. The composition according to claim 29 , wherein said modification comprises the introduction of one or more amino acid mutations into the FnCas protein, or by truncation of the FnCas protein, or by deletion and/or insertion of specific amino acids or amino acid sequences into the FnCas protein.

31. The composition according to claim 7 , wherein the one or more vectors comprise a plurality of polynucleotide sequences encoding multiple Rt-gRNAs, wherein each Rt-gRNAs is specific for a different target RNA whereby there is multiplexing.

32. A host cell or cell line comprising the composition according to claim 7 , or progeny thereof.

33. The host cell or cell line or progeny thereof according to claim 32 , which is ex vivo or in vitro, a stem cell or a stem cell line, or a plant cell or a plant cell line.

34. The vector system according to claim 1 , wherein the one or more functional domains are associated with an adaptor protein.

35. The vector system according to claim 1 , wherein the FnCas protein comprises two or more functional domains.

36. The vector system according to claim 1 , wherein the FnCas protein or ortholog, homolog, or fragment thereof, further comprises one or more heterologous thereof, further comprises one or more heterologous functional domains positioned at one or more sites in FnCas that are analogous to amino acid positions 175-306 of a wild type SpCas9 Recl domain.

Assignments (6)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 7, 2019
From: HSU, PATRICK
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 048266/0852 →
CORRECTIVE ASSIGNMENT TO CORRECT THE RECEIVING PARTY PREVIOUSLY RECORDED AT REEL: 043167 FRAME: 0557. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Recorded Sep 17, 2018
From: ZHANG, FENG
To: THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 047098/0929 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 27, 2018
From: GOOTENBERG, JONATHAN
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 046705/0568 →
CONFIRMATORY LICENSE Recorded Sep 25, 2017
From: BROAD INSTITUTE, INC.
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 043987/0100 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 2, 2017
From: ZHANG, FENG
To: THE BROAD INSTITUTE INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 043167/0557 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 13, 2017
From: SMARGON, AARON
To: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 043183/0015 →
Continuity (6)
Continuation In Part PCTUS2015067151 · Dec 21, 2015
Provisional Application 62096324 · Dec 23, 2014
Provisional Application 62098059 · Dec 30, 2014
Provisional Application 62181641 · Jun 18, 2015
Provisional Application 62181667 · Jun 18, 2015
Related Publication 20170306335A1 · Oct 26, 2017
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