# US 12,215,326 B2 · RNA-targeting system > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,215,326 B2 - Document ID: US12215326B2 - Status: Granted - Kind: B2 - Application number: 15/632,067 - Filed: 2017-06-23 - Published: 2017-10-26 - Granted: 2025-02-04 - Earliest claimed: 2014-12-23 - Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology; President and Fellows of Harvard College - Applicants: The Broad Institute, Inc.; Massachusetts Institute of Technology; President and Fellows of Harvard College - Inventors: Feng Zhang; Patrick Hsu; Jonathan S. Gootenberg; Aaron Smargon - Web page: https://patentgrep.com/patent/US12215326B2 ## CPC - C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes} - 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} - C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids} - C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers} - C12N 15/86: Viral vectors - C12Y 301/21004: Type II site-specific deoxyribonuclease (3.1.21.4) - C07K 2319/09: containing a nuclear localisation signal - C12N 2310/10: Type of nucleic acid - C12N 2310/16: Aptamers - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12N 2330/51: Specially adapted vectors - C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## 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 ### Claim 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 Rec1 domain, Rec2 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) Rec1 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. ### Claim 2 (depends on 1) The vector system according to claim 1, wherein said FnCas protein is a type II Cas protein. ### Claim 3 (depends on 1) The vector system according to claim 1, wherein said scaRNA sequence is fused to said tracrRNA sequence. ### Claim 4 (depends on 1) The vector system according to claim 1, wherein said FnCas protein is codon optimized for expression in a eukaryotic cell. ### Claim 5 (depends on 1) 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. ### Claim 6 (depends on 1) The vector system according to claim 1, wherein said RNA-targeting guide RNA is designed to direct said FnCas protein to said target RNA. ### Claim 7 (depends on 1) A composition comprising a vector system according to claim 1. ### Claim 8 (depends on 1) 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. ### Claim 9 (depends on 8) 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. ### Claim 10 (depends on 9) The method according to claim 9, wherein the synthesis of said protein is suppressed by knock-down of said RNA molecule encoding said protein. ### Claim 11 (depends on 10) The method according to claim 10, wherein said RNA-targeting Cas protein is codon optimized for expression in a eukaryotic cell. ### Claim 12 (depends on 11) 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. ### Claim 13 (depends on 12) The method according to claim 12, wherein the cell is a eukaryotic cell. ### Claim 14 (depends on 11) The method according to claim 11, wherein the eukaryotic cell is a mammalian or human cell. ### Claim 15 (depends on 7) 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) Rec1 domain at which the one or more heterologous functional domains are positioned are modified. ### Claim 16 (depends on 15) 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. ### Claim 17 (depends on 16) 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. ### Claim 18 (depends on 17) 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). ### Claim 19 (depends on 18) 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. ### Claim 20 (depends on 1) 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. ### Claim 21 (depends on 20) The vector system according to claim 20, wherein the transcriptional activation domain comprises VP64. ### Claim 22 (depends on 20) The vector system according to claim 20, wherein the transcriptional repression domain comprises a KRAB domain or a SID domain. ### Claim 23 (depends on 20) The vector system according to claim 20, wherein the nuclease domain comprises Fok1. ### Claim 24 (depends on 1) 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. ### Claim 25 (depends on 7) The composition according to claim 7, wherein the tracrRNA or the scaRNA comprises one or more protein-binding RNA aptamers. ### Claim 26 (depends on 25) The composition according to claim 25, wherein the one or more aptamers is designed to bind a bacteriophage coat protein. ### Claim 27 (depends on 26) 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. ### Claim 28 (depends on 7) The composition according to claim 7, wherein the tracrRNA is 30 or more, 40 or more or 50 or more nucleotides in length. ### Claim 29 (depends on 7) 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. ### Claim 30 (depends on 29) 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. ### Claim 31 (depends on 7) 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. ### Claim 32 (depends on 7) A host cell or cell line comprising the composition according to claim 7, or progeny thereof. ### Claim 33 (depends on 32) 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. ### Claim 34 (depends on 1) The vector system according to claim 1, wherein the one or more functional domains are associated with an adaptor protein. ### Claim 35 (depends on 1) The vector system according to claim 1, wherein the FnCas protein comprises two or more functional domains. ### Claim 36 (depends on 1) 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.