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成簇规律间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)系统作为一种高效、精确的基因编辑工具，已被广泛应用于基因功能研究、疾病治疗和生物技术开发。近年来，通过改造CRISPR系统，尤其是dCas9蛋白和单链向导RNA(single guide RNA,sgRNA),研究人员开发出多种基因表达调控策略。其中可诱导的CRISPR基因表达调控系统通过整合外部刺激响应元件(如光、化学小分子、温度等),实现了对基因表达的时间和空间精准控制。本文综述了基于CRISPR系统的基因表达调控策略与方法的最新进展，重点介绍了基于蓝光、红光/远红光、化学小分子以及热触发的基因表达调控系统的原理、设计策略。这些策略不仅为功能基因组学研究提供了强有力的工具，也为新一代基因治疗技术的发展奠定了基础。
Abstract：As an efficient and accurate gene editing tool, the clustered regularly interspaced short palindromic repeats(CRISPR) system has been widely used in gene function research, disease treatment, and biotechnology development. In recent years, by modifying the CRISPR system, particularly the dCas9 protein and single guide RNA(sgRNA), researchers have developed various strategies for regulating gene expression. The inducible CRISPR gene expression regulation system enables temporal and spatial control of gene expression by integrating external stimulus response elements(such as light, chemical small molecules, temperature, etc.). This review highlights recent advancements in CRISPR-based gene expression regulation strategies and methods, focusing on the principles and design strategies of systems triggered by blue light, red/far-red light, chemical small molecules, and heat. These strategies not only provide powerful tools for functional genomics research, but also lay the foundation for the development of next-generation of gene therapy technologies.
ALTINBAY M,WANG J,CHEN J,et al.,2024.Chem-CRISPR/dCas9FCPF:a platform for chemically induced epigenome editing.Nucleic Acids Res.,52(19):11587-11601.
BANANI S F,LEE H O,HYMAN A A,et al.,2017.Biomolecular condensates:organizers of cellular biochemistry.Nat.Rev.Mol.Cell Biol.,18(5):285-298.
BAO Z H,JAIN S,JAROENPUNTARUK V,et al.,2017.Orthogonal genetic regulation in human cells using chemically induced CRISPR/Cas9 activators.ACS Synth.Biol.,6(4):686-693.
CHAVEZ A,SCHEIMAN J,VORA S,et al.,2015.Highly efficient Cas9-mediated transcriptional programming.Nat.Methods,12(4):326-328.
CHEN T J,GAO D,ZHANG R S,et al.,2017.Chemically controlled epigenome editing through an inducible dCas9 system.J.Am.Chem.Soc.,139(33):11337-11340.
CHOI J,CHEN J,SCHREIBER S L,et al.,1996.Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP.Science,273(5272):239-242.
DOMINGUEZ A A,LIM W A,QI L S,2016.Beyond editing:repurposing CRISPR-Cas9 for precision genome regulation and interrogation.Nat.Rev.Mol.Cell Biol.,17(1):5-15.
FLORES-IBARRA A,MAIA R N A,OLASZ B,et al.,2024.Light-oxygen-voltage (LOV)-sensing domains:activation mechanism and optogenetic stimulation.J.Mol.Biol.,436(5):168356.
GAMBOA L,PHUNG E V,LI H X,et al.,2020.Heat-triggered remote control of CRISPR-dCas9 for tunable transcriptional modulation.ACS Chem.Biol.,15(2):533-542.
GAO Y C,XIONG X,WONG S,et al.,2016.Complex transcriptional modulation with orthogonal and inducible dCas9 regulators.Nat.Methods,13(12):1043-1049.
GELLER S H,ANTWI E B,DI VENTURA B,et al.,2019.Optogenetic repressors of gene expression in yeasts using light-controlled nuclear localization.Cell.Mol.Bioeng.,12(5):511-528.
GILBERT L A,LARSON M H,MORSUT L,et al.,2013.CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.Cell,154(2):442-451.
GRIFFITHS J,MURASE K,RIEU I,et al.,2006.Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis.Plant Cell,18(12):3399-3414.
HARPER S M,CHRISTIE J M,GARDNER K H,2004.Disruption of the LOV-Jalpha helix interaction activates phototropin kinase activity.Biochemistry,43(51):16184-16192.
HILTBRUNNER A,TSCHEUSCHLER A,VICZIáN A,et al.,2006.FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A photoreceptor.Plant Cell Physiol.,47(8):1023-1034.
HO S N,BIGGAR S R,SPENCER D M,et al.,1996.Dimeric ligands define a role for transcriptional activation domains in reinitiation.Nature,382(6594):822-826.
HSU P D,LANDER E S,ZHANG F,2014.Development and applications of CRISPR-Cas9 for genome engineering.Cell,157(6):1262-1278.
JINEK M,CHYLINSKI K,FONFARA I,et al.,2012.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science,337(6096):816-821.
KAWANO F,SUZUKI H,FURUYA A,et al.,2015.Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins.Nat.Commun.,6:6256.
KIM C,YUN S R,LEE S J,et al.,2024.Structural dynamics of protein-protein association involved in the light-induced transition of Avena sativa LOV2 protein.Nat.Commun.,15(1):6991.
KIM Y J,LEE M Jr,LEE Y T,et al.,2023.Light-activated macromolecular phase separation modulates transcription by reconfiguring chromatin interactions.Sci.Adv.,9(13):eadg1123.
KONERMANN S,BRIGHAM M D,TREVINO A E,et al.,2015.Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.Nature,517(7536):583-588.
KONG D Q,ZHOU Y,WEI Y,et al.,2024.Exploring plant-derived phytochrome chaperone proteins for light-switchable transcriptional regulation in mammals.Nat.Commun.,15(1):4894.
KUWASAKI Y,SUZUKI K,YU G G,et al.,2022.A red light-responsive photoswitch for deep tissue optogenetics.Nat.Biotechnol.,40(11):1672-1679.
LI R,XIA X Y,WANG X,et al.,2020.Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model.PLoS Biol.,18(11):e3000749.
LI Z X,ZHANG D D,XIONG X Y,et al.,2017.A potent Cas9-derived gene activator for plant and mammalian cells.Nat.Plants,3(12):930-936.
LIU H T,YU X H,LI K W,et al.,2008.Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis.Science,322(5907):1535-1539.
LIU J Q,CHEN Y X,NONG B T,et al.,2023.CRISPR-assisted transcription activation by phase-separation proteins.Protein Cell,14(12):874-887.
LIU X,CHEN Y,ZHANG Y Y,et al.,2020.Multiplexed capture of spatial configuration and temporal dynamics of locus-specific 3D chromatin by biotinylated dCas9.Genome Biol.,21(1):59.
LIU X,ZHANG Y Y,CHEN Y,et al.,2017.In situ capture of chromatin interactions by biotinylated dCas9.Cell,170(5):1028-1043.e19.
LIU Y W,LI X,LI K W,et al.,2013.Multiple bHLH proteins form heterodimers to mediate CRY2-dependent regulation of flowering-time in Arabidopsis.PLoS Genet.,9(10):e1003861.
MA S F,LIAO K T,LI M R,et al.,2023.Phase-separated DropCRISPRa platform for efficient gene activation in mammalian cells and mice.Nucleic Acids Res.,51(10):5271-5284.
MA Y,SZOSTKIEWICZ I,KORTE A,et al.,2009.Regulators of PP2C phosphatase activity function as abscisic acid sensors.Science,324(5930):1064-1068.
MACHENS F,RAN G Y,RUEHMKORFF C,et al.,2023.PhiReX 2.0:a programmable and red light-regulated CRISPR-dCas9 system for the activation of endogenous genes in Saccharomyces cerevisiae.ACS Synth.Biol.,12(4):1046-1057.
MAEDER M L,LINDER S J,CASCIO V M,et al.,2013.CRISPR RNA-guided activation of endogenous human genes.Nat.Methods,10(10):977-979.
MCCARTY N S,GRAHAM A E,STUDENá L,et al.,2020.Multiplexed CRISPR technologies for gene editing and transcriptional regulation.Nat.Commun.,11(1):1281.
MELCHER K,NG L M,ZHOU X E,et al.,2009.A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors.Nature,462(7273):602-608.
MORGAN S L,MARIANO N C,BERMUDEZ A,et al.,2017.Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping.Nat.Commun.,8:15993.
MORITA S,HORII T,KIMURA M,et al.,2020.Synergistic upregulation of target genes by TET1 and VP64 in the dCas9-SunTag platform.Int.J.Mol.Sci.,21(5):1574.
MUENCH P,FIUMARA M,SOUTHERN N,et al.,2025.A modular toolbox for the optogenetic deactivation of transcription.Nucleic Acids Res.,53(3):gkae1237.
NIHONGAKI Y,FURUHATA Y,OTABE T,et al.,2017.CRISPR-Cas9-based photoactivatable transcription systems to induce neuronal differentiation.Nat.Methods,14(10):963-966.
NIHONGAKI Y,YAMAMOTO S,KAWANO F,et al.,2015a.CRISPR-Cas9-based photoactivatable transcription system.Chem.Biol.,22(2):169-174.
NIHONGAKI Y,KAWANO F,NAKAJIMA T,et al.,2015b.Photoactivatable CRISPR-Cas9 for optogenetic genome editing.Nat.Biotechnol.,33(7):755-760.
PAN C T,WU X C,MARKEL K,et al.,2021.CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants.Nat.Plants,7(7):942-953.
POLSTEIN L R,GERSBACH C A,2015.A light-inducible CRISPR-Cas9 system for control of endogenous gene activation.Nat.Chem.Biol.,11(3):198-200.
QI L S,LARSON M H,GILBERT L A,et al.,2013.Repurpo-sing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.Cell,152(5):1173-1183.
QIAO L L,NIU L X,WANG M Y,et al.,2024.A sensitive red/far-red photoswitch for controllable gene therapy in mouse models of metabolic diseases.Nat.Commun.,15(1):10310.
QU G P,JIANG B C,LIN C T,2024.The dual-action mechanism of Arabidopsis cryptochromes.J.Integr.Plant Biol.,66(5):883-896.
SABARI B R,DALL′AGNESE A,BOIJA A,et al.,2018.Coactivator condensation at super-enhancers links phase se-paration and gene control.Science,361(6400):eaar3958.
SANDER J D,JOUNG J K,2014.CRISPR-Cas systems for editing,regulating and targeting genomes.Nat.Biotechnol.,32(4):347-355.
SHAO J W,WANG M Y,YU G L,et al.,2018.Synthetic far-red light-mediated CRISPR-dCas9 device for inducing functional neuronal differentiation.Proc.Natl.Acad.Sci.USA,115(29):E6722-E6730.
TAGUE N,CORIANO-ORTIZ C,SHEETS M B,et al.,2024.Light-inducible protein degradation in E.coli with the LOVdeg tag.eLife,12:RP87303.
TANENBAUM M E,GILBERT L A,QI L S,et al.,2014.A protein-tagging system for signal amplification in gene expression and fluorescence imaging.Cell,159(3):635-646.
TAYLOR R J,AGUILAR RANGEL M,GEESON M B,et al.,2022.π-clamp-mediated Homo- and heterodimerization of single-domain antibodies via site-specific homobifunctional conjugation.J.Am.Chem.Soc.,144(29):13026-13031.
THAKORE P I,D′IPPOLITO A M,SONG L Y,et al.,2015.Highly specific epigenome editing by CRISPR-Cas9 repre-ssors for silencing of distal regulatory elements.Nat.Methods,12(12):1143-1149.
TROJANOWSKI J,FRANK L,RADEMACHER A,et al.,2022.Transcription activation is enhanced by multivalent interactions independent of phase separation.Mol.Cell,82(10):1878-1893.e10.
UEGUCHI-TANAKA M,ASHIKARI M,NAKAJIMA M,et al.,2005.GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin.Nature,437(7059):693-698.
VAN ESSEN D,ENGIST B,NATOLI G,et al.,2009.Two modes of transcriptional activation at native promoters by NF-kappaB p65.PLoS Biol.,7(3):e73.
WU H,WANG F L,JIANG J H,2021.Inducible CRISPR-dCas9 transcriptional systems for sensing and genome regulation.Chembiochem,22(11):1894-1900.
XUE Y N,YAN X J,LI D,et al.,2024.Proinflammatory polarization of engineered heat-inducible macrophages reprogram the tumor immune microenvironment during cancer immunotherapy.Nat.Commun.,15(1):2270.
ZALATAN J G,LEE M E,ALMEIDA R,et al.,2015.Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds.Cell,160(1-2):339-350.
ZETSCHE B,VOLZ S E,ZHANG F,2015.A split-Cas9 architecture for inducible genome editing and transcription modulation.Nat.Biotechnol.,33(2):139-142.
ZHANG C,WELBORN M,ZHU T Y,et al.,2016.π-clamp-mediated cysteine conjugation.Nat.Chem.,8(2):120-128.
ZHANG C M,TANG Y J,TANG S J,et al.,2024.An induci-ble CRISPR activation tool for accelerating plant regeneration.Plant Commun.,5(5):100823.
ZHANG H,JI X,LI P L,et al.,2020.Liquid-liquid phase separation in biology:mechanisms,physiological functions and human diseases.Sci.China Life Sci.,63(7):953-985.
ZHOU Y,KONG D Q,WANG X Y,et al.,2022.A small and highly sensitive red/far-red optogenetic switch for applications in mammals.Nat.Biotechnol.,40(2):262-272.
ZHOU Z,WANG Y Q,ZHENG X N,et al.,2024.Optimizing ABA-based chemically induced proximity for enhanced intracellular transcriptional activation and modification response to ABA.Sci.China Life Sci.,67(12):2650-2663.
ZUO J,NIU Q W,CHUA N H,2000.Technical advance:an estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants.Plant J.,24(2):265-273.
基本信息:
DOI：10.13417/j.gab.044.000750
中图分类号:Q78
引用信息:
[1]王清灏,曹浩博,吴雨璇,等.可诱导的CRISPR-dCas9基因表达调控策略与方法[J].基因组学与应用生物学,2025,44(07):750-763.DOI:10.13417/j.gab.044.000750.
基金信息:
国家自然科学基金项目(32300572)资助
2025-05-30
2025-05-30
2025-05-30