Granted Patent
B2
US 12,202,861 · App. 18/304,187 · Granted Jan 21, 2025
Inducible, tunable, and multiplex human gene regulation using CRISPR-Cpf1
Inventors:
Y. Esther Tak (Charlestown, MA); Benjamin Kleinstiver (Boston, MA); J. Keith Joung (Winchester, MA)
Assignee:
The General Hospital Corporation
C07K14/005C12N9/22C12N9/52C12N15/111C12N15/62A61K38/00C07K2319/03C12N2310/153C12N2310/20C12N2710/16222C12N2800/30
View Patent ↗
Loading inventors, assignments & file history…
Abstract
Drug-inducible, tunable, and multiplexable Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (Cpf1)-based activators, and methods of use thereof.
Claims (8)
1. A method of increasing expression of a target gene in a cell, the method comprising contacting the cell with, or expressing in the cell: (a) a first fusion protein comprising a catalytically inactive Lachnospiraceae bacterium ND2006 Cpf1 (dLbCpf1) fused to more than one first dimerization domains, and (b) a second fusion protein comprising at least one activation domain fused to a second dimerization domain that dimerizes with at least one of the first dimerization domains in the first fusion protein in the presence of a dimerizing agent, wherein the first and second dimerization domains are rapamycin analog A/C heterodimerizer DmrA or DmrC, and at least one crRNA that directs the second fusion protein to a regulatory region of the target gene.
2. The method of claim 1 , wherein (i) the first dimerization domain in the first fusion protein is DmrA, and the second dimerization domain in the second fusion protein is DmrC, or (ii) the first dimerization domain in the first fusion protein is DmrC, and the second dimerization domain in the second fusion protein is DmrA.
3. The method of claim 1 , wherein the activation domain is a transcriptional activation domain from herpes simplex viral protein 16 (VP64), Epstein-Barr virus R transactivator (Rta), p65 domain from cellular transcription factor NF-κB (NF-κB p65), or a tripartite effector composed of VP64, p65, and Rta (VPR).
4. The method of claim 1 , further comprising an intervening linker between each of the activation domain(s) or the second dimerization domain.
5. The method of claim 1 , wherein the first fusion protein is fused to two, three or four first dimerization domains.
6. The method of claim 1 , wherein the cell is a mammalian cell.
7. The method of claim 6 , wherein the mammalian cell is a human cell.
8. The method of claim 1 , wherein the regulatory region is a promoter region.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jun 22, 2023
From: TAK, YU GYOUNG; KLEINSTIVER, BENJAMIN; JOUNG, J. KEITH
To: THE GENERAL HOSPITAL CORPORATION
Reel/Frame 064022/0076 →
Continuity (3)
Division
16606680
Provisional Application
62488585
· Apr 21, 2017
References Cited (110)
US 20030017149A1
· Hoeffler et al.
· 2003
[cited by applicant]
US 20070020627A1
· Barbas, III
· 2007
[cited by applicant]
US 20110294873A1
· Mermod et al.
· 2011
[cited by applicant]
US 20150315252A1
· Haugwitz et al.
· 2015
[cited by applicant]
WO WO2003072788
· 2003
[cited by applicant]
WO WO2012047726
· 2012
[cited by applicant]
WO WO2013176772
· 2013
[cited by applicant]
WO WO2014152432
· 2014
[cited by applicant]
WO WO2015139139
· 2015
[cited by applicant]
WO WO2016115355
· 2016
[cited by applicant]
WO WO2016191684
· 2016
[cited by applicant]
WO WO2016205711
· 2016
[cited by applicant]
WO WO2017015015
· 2017
[cited by applicant]
WO WO2017031370
· 2017
[cited by applicant]
WO WO2017141173
· 2017
[cited by applicant]
WO WO2018071892
· 2018
[cited by applicant]
WO WO2019222670
· 2019
[cited by applicant]
WO WO2021108501
· 2021
[cited by applicant]
WO WO2021243289
· 2021
[cited by applicant]
Tak et al., Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors. bioRxiv preprint doi: https://doi.org/10.1101/150656; this version posted Jun. 15, 2017: 21 pages. (Year:…
[cited by examiner]
Notice of Allowance in Japanese Appln. No. 2019-556605, dated Oct. 3, 2023, 6 pages (with English translation).
[cited by applicant]
Office Action in Chinese Appln. No. 201880041218.8, dated Sep. 2, 2023, 20 pages (with English translation).
[cited by applicant]
Office Action in Chinese Appln. No. 201880041218.8, dated Dec. 1, 2023, 20 pages (with English translation).
[cited by applicant]
Adamson et al., “A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response,” Cell, Dec. 2016, 167(7):1867-1882.
[cited by applicant]
Andersson et al., “A Unified Architecture of Transcriptional Regulatory Elements,” Trends in Genetics, Aug. 2015, 31(8):426-433, 8 pages.
[cited by applicant]
Bao et al., “Orthogonal Genetic Regulation in Human Cells Using Chemically Induced CRISPR/Cas9 Activators,” ACS Synthetic Biology, Apr. 2017, 6(4):686-693, 8 pages.
[cited by applicant]
Baron-Benhamou et al, “Using the LambdaN Peptide to Tether Proteins to RNAs,” Methods in Molecular Biology, Jan. 2004, 257:135-153.
[cited by applicant]
Bikard et al., “Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system,” Nucleic Acids Research, Jun. 2013, 41(15):7429-7437.
[cited by applicant]
Bird et al., “A dual role for zinc fingers in both DNA binding and zinc sensing by the Zap1 transcriptional activator,” EMBO J., Jul. 2000, 19(14):3704-3713.
[cited by applicant]
Chavez et al., “Comparison of Cas9 activators in multiple species,” Nature Methods, Jul. 2016, 13(7):563-567, 7 pages.
[cited by applicant]
Chavez et al., “Highly efficient Cas9-mediated transcriptional programming,” Nature Methods, Apr. 2015, 12(4):326-328.
[cited by applicant]
Cong et al., “Multiplex genome engineering using CRISPR/Cas systems,” Science, Feb. 2013, 339(6121):819-823.
[cited by applicant]
Deltcheva et al., “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III,” Nature, Mar. 2011, 471(7340):602-607.
[cited by applicant]
Dong et al., “The crystal structure of Cpf1 in complex with CRISPR RNA,” Nature, Apr. 2016, 532(7600):522-526.
[cited by applicant]
Doudna & Charpentier., “Genome editing. The new frontier of genome engineering with CRISPR-Cas9,” Science, Nov. 2014, 346(6213):1258096, 12 pages.
[cited by applicant]
EP Extended European Search Report in European Appln. No. 18787309.6, dated Jan. 11, 2021, 11 pages.
[cited by applicant]
Fagerlund et al., “The Cpf1 CRISPR-Cas protein expands genome-editing tools,” Genome Biol., Dec. 2015, 16(1):251, 3 pages.
[cited by applicant]
Fonfara et al., “The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA,” Nature, Apr. 2016, 532(7600):517-521, 19 pages.
[cited by applicant]
Frock et al., “Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases,” Nat. Biotechnol., Feb. 2015, 33(2):179-186 , 10 pages.
[cited by applicant]
Gao et al., “Complex transcriptional modulation with orthogonal and inducible dCas9 regulators,” Nature Methods, Dec. 2016, 13(12):1043-1049.
[cited by applicant]
Gilbert et al., “Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation,” Cell, Oct. 2014, 159(3):647-661.
[cited by applicant]
Guo et al., “An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells,” Protein & Cell, May 2017, 8(5):379-393.
[cited by applicant]
Han al., “Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions,” Nature Biotechnology, May 2017, 35(5):463, 15 pages.
[cited by applicant]
Hsu et al., “Development and applications of CRISPR-Cas9 for genome engineering,” Cell, Jun. 2014, 157(6):1262-1278.
[cited by applicant]
International Preliminary Report on Patentability in International Appln. No. PCT/US18/28898, dated Oct. 22, 2019, 9 pages.
[cited by applicant]
International Search Report and Written Opinion in International Appln. No. PCT/US18/28898, dated Jul. 23, 2018, 12 pages.
[cited by applicant]
International Search Report and Written Opinion in International Appln. No. PCT/US2014/027335, dated Jul. 16, 2014, 13 pages.
[cited by applicant]
International Search Report and Written Opinion in International Appln. No. PCT/US2017/056738, dated Mar. 6, 2018, 16 pages.
[cited by applicant]
International Search Report and Written Opinion in International Appln. No. PCT/US2019/032937, dated Oct. 17, 2019, 17 pages.
[cited by applicant]
International Search Report and Written Opinion in International Appln. No. PCT/US2020/062166, dated May 4, 2021, 13 pages.
[cited by applicant]
International Search Report and Written Opinion in International Appln. No. PCT/US2021/034996, dated Sep. 16, 2021, 12 pages.
[cited by applicant]
Jinek et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, Aug. 2012, 337(6096):816-821.
[cited by applicant]
Jinek et al., “RNA-programmed genome editing in human cells,” Elife 2, Jan. 2013, 2:e00471, 9 pages.
[cited by applicant]
Kabadi et al., “Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector,” Nucleic Acids Res., 2014, 42(19):e147.
[cited by applicant]
Khalil et al., “A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions,” Cell, Aug. 2012, 150(3):647-658.
[cited by applicant]
Kim et al., “Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells,” Nat. Methods, Mar. 2015, 12(3):237-243.
[cited by applicant]
Kim et al., “Efficient Transcriptional Gene Repression by Type V-A CRISPR-Cpf1 from Eubacterium eligens,” ACS Synthetic Biology, Jul. 2017, 6(7):1273-1282.
[cited by applicant]
Kim et al., “Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells,” Nature Biotechnology, Aug. 2016, 34(8):863-868.
[cited by applicant]
Kleinstiver et al., “Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells,” Nature Biotechnology, Aug. 2016, 34(8):869-874.
[cited by applicant]
Kleinstiver et al., “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects,” Nature, Jan. 2016, 529(7587):490-495, 17 pages.
[cited by applicant]
Konermann et al., “Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex,” Nature, Jan. 2015, 517(7536):583-588.
[cited by applicant]
Li et al., “Identification of critical base pairs required for CTCF binding in motif M1 and M2,” Protein Cell, Mar. 2017, 8(7):544-549, 6 pages.
[cited by applicant]
Lin et al., “A CRISPR Approach for Reactivating Latent HIV-1,” Molecular Therapy, Mar. 2016, 24(3):416-418.
[cited by applicant]
Lin et al., “Cellular toxicity induced by SRF-mediated transcriptional squelching,” Toxicological Sciences, Mar. 2007, 96(1):83-91.
[cited by applicant]
Maeder et al., “CRISPR RNA-guided activation of endogenous human genes,” Nat. Methods, Oct. 2013, 10(10):977-979.
[cited by applicant]
Maeder et al., “Genome-editing Technologies for Gene and Cell Therapy,” Mol. Ther., Mar. 2016, 24(3):430-446.
[cited by applicant]
Maji et al., “Multidimensional chemical control of CRISPR-Cas9,” Nature Chemical Biology, Jan. 2017, 13(1):9-11.
[cited by applicant]
Makarova et al., “An updated evolutionary classification of CRISPR-Cas systems,” Nat. Rev. Microbiol., Nov. 2015, 13(11):722-736.
[cited by applicant]
Mali et al., “RNA-guided human genome engineering via Cas9,” Science, Feb. 2013, 339(6121):823-826, 5 pages.
[cited by applicant]
Matis et al., “Differential and opposed transcriptional effects of protein fusions containing the VP16 activation domain,” FEBS Letters, Jun. 2001, 499(1-2):92-96.
[cited by applicant]
Nissim et al., “Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells,” Molecular Cell, May 2014, 54(4):698-710.
[cited by applicant]
Notice of Acceptance in Australian Appln. No. 2018254616, dated Jul. 13, 2022, 4 pages.
[cited by applicant]
Office Action in Australian Appln. No. 2018254616, dated Oct. 11, 2021, 5 pages.
[cited by applicant]
Office Action in Chinese Appln. No. 201880041218.8, dated Feb. 8, 2023, 27 pages (with English translation).
[cited by applicant]
Office Action in Japanese Appln. No. 2019-556605, dated Mar. 29, 2022, 8 pages (with English translation).
[cited by applicant]
Office Action in Japanese Appln. No. 2019-556605, dated Oct. 18, 2022, 6 pages (with English translation).
[cited by applicant]
Perez-Pinera et al., “RNA-guided gene activation by CRISPR-Cas9-based transcription factors,” Nat. Methods, Oct. 2013, 10(10):973-976.
[cited by applicant]
Polstein & Gersbach, “A light-inducible CRISPR-Cas9 system for control of endogenous gene activation,” Nature Chemical Biology, Mar. 2015, 11(3):198-200.
[cited by applicant]
Qi et al, “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression,” Cell, Feb. 2013, 152:1173-1183.
[cited by applicant]
Rivera et al., “Dimerizer-mediated regulation of gene expression in vivo,” Cold Spring Harbor Protocols, Jul. 2012, 2012(7):821-824.
[cited by applicant]
Rojano et al., “Regulatory variants: from detection to predicting impact,” Briefings in Bioinformatics, Sep. 2019, 20(5):1639-1654.
[cited by applicant]
Sander & Joung, “CRISPR-Cas systems for editing, regulating and targeting genomes,” Nat. Biotechnol., Apr. 2014, 32(4):347-355.
[cited by applicant]
Schunder et al., “First indication for a functional CRISPR/Cas system in Francisella tularensis,” Int. J. Med. Microbiol., Mar. 2013, 303(2):51-60.
[cited by applicant]
Shen et al., “Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions,” Nature Methods, Jun. 2017, 14(6):573, 9 pages.
[cited by applicant]
Slaymaker et al., “Rationally engineered Cas9 nucleases with improved specificity,” Science, Jan. 2016, 351(6268):84-88.
[cited by applicant]
Tak et al.: “Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors,” Nature Methods, Oct. 2017, 14(12):1163-1166.
[cited by applicant]
Tang et al., “A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants,” Nature Plants, Feb. 2017, 3:17018, 5 pages.
[cited by applicant]
Tsai et al., “Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing,” Nat. Biotechnol., Jun. 2014, 32(6):569-576.
[cited by applicant]
Tsai et al., “GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases,” Nat. Biotechnol., Feb. 2015, 33(2):187-197.
[cited by applicant]
Wang et al., “Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors,” Nat. Biotechnol., Feb. 2015, 33(2):175-178.
[cited by applicant]
Wong et al., “Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM,” Proc. Natl. Acad. Sci. USA., Mar. 2016, 113(9):2544-2549.
[cited by applicant]
Wright et al., “Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering,” Cell, Jan. 2016, 164(1-2):29-44.
[cited by applicant]
Xie et al., “Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system,” Proc. Natl. Acad. Sci. USA., Mar. 2015, 112(11):3570-3575.
[cited by applicant]
Xu et al., “Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA,” Nucleic Acids Res., Mar. 2017, 45(5):e28, 9 pages.
[cited by applicant]
Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA,” Cell, May 2016, 165(4):949-962.
[cited by applicant]
Zetsche et al., “A split-Cas9 architecture for inducible genome editing and transcription modulation,” Nature Biotechnology, Feb. 2015, 33(2):139-142.
[cited by applicant]
Zetsche et al., “Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system,” Cell, Oct. 2015, 163(3):759-771.
[cited by applicant]
Zetsche et al., “Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array,” Nature Biotechnology, Jan. 2017, 35(1):31-34.
[cited by applicant]
Zhang et al., “Multiplex gene regulation by CRISPR-ddCpf1,” Cell Discovery, Jun. 2017, 3(6):17018, 9 pages.
[cited by applicant]
Office Action in Canadian Appln. No. 3,059,208, dated May 9, 2024, 4 pages.
[cited by applicant]