基于CRISPR-Cas9的筛选文库的类型与应用
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摘要
文库筛选技术广泛应用于生命科学研究各领域,加速了生物医药基础科研和临床实践的进展。本文对基于CRISPR-Cas9的文库类型和应用进行综述。CRISPR-Cas9文库包括敲除、活化和抑制文库。敲除文库通过Cas9/sgRNA靶向切割DNA序列,产生移码突变进行基因敲除。活化文库包括两种:一种是dCas9/sgRNA与转录活化蛋白质融合,例如dCas9-SAM,dCas9-SunTag和dCas9-VPR系统;另一种是dCas9与表观遗传修饰酶融合,例如d Cas9-Tet1和d Cas9-p300系统。CRISPR-Cas9抑制文库通过dCas9与表观遗传修饰蛋白质融合,抑制转录,例如d Cas9-KRAB和d Cas9-Dnmt3a系统。目前,CRISPR-Cas9文库广泛用于功能基因筛选、药物靶点和耐药靶点筛选、病毒靶点筛选和揭示信号通路,并在基因互作筛选及揭示顺式调节元件功能等方面初步展现其优势。CRISPR-Cas9文库优势在于其设计灵活、操作便捷、筛选高效。伴随基因编辑系统的研发,新的筛选文库靶向性和突变将更加精准,应用将更加拓展和深化。基于CRISPR-Cas9筛选文库不仅可以筛选病理和生理过程中的关键基因和非编码DNA,还可以揭示其发挥功能的分子机制,是剖析生命复杂调控网络的手术刀。
The library screening technology is widely used in various fields of life science research,and it greatly accelerates the progress of basic research and clinical practice in biomedicine. This article reviews types and applications of libraries based on CRISPR-Cas9. CRISPR-Cas9 based libraries include knockout libraries,activation libraries,and inhibition libraries. The knockout library identifies and cuts specific DNA sequences through the Cas9/sgRNA complex,resulting in a frame shift mutation for gene knockout. Activation libraries include two types: one is dCas9/sgRNA fused to a transcriptional activator protein,such as dCas9-SAM,dCas9-SunTag and dCas9-VPR systems; the other is dCas9 fused to an epigenetic modification enzyme,such as dCas9-Tet1 and dCas9-p300 systems. Inhibitory libraries are fused to epigenetic modified proteins by dCas9 to inhibit transcription,such as d Cas9-KRAB and dCas9-Dnmt3 a systems. The advantages of the CRISPR-Cas9 library are its flexible design,easy operation and efficient screening. Currently,CRISPR-Cas9 based libraries play an irreplaceable role in functional gene screening,drug targets and resistance targets screening,viral targets screening,identification of signaling pathways,gene interaction screening,and revealing the function of cis-regulator elements. With the development of gene editing systems,novel screening libraries may emerge in the future,which target and mutate more precisely,and their applications will be deeper and expanded. CRISPR-Cas9 based libraries can not only screen key genes and non-coding DNAs in pathological and physiological processes,but also reveal the molecular mechanisms of their function. It is a scalpel that dissects the complex regulatory network of life.
The library screening technology is widely used in various fields of life science research,and it greatly accelerates the progress of basic research and clinical practice in biomedicine. This article reviews types and applications of libraries based on CRISPR-Cas9. CRISPR-Cas9 based libraries include knockout libraries,activation libraries,and inhibition libraries. The knockout library identifies and cuts specific DNA sequences through the Cas9/sgRNA complex,resulting in a frame shift mutation for gene knockout. Activation libraries include two types: one is dCas9/sgRNA fused to a transcriptional activator protein,such as dCas9-SAM,dCas9-SunTag and dCas9-VPR systems; the other is dCas9 fused to an epigenetic modification enzyme,such as dCas9-Tet1 and dCas9-p300 systems. Inhibitory libraries are fused to epigenetic modified proteins by dCas9 to inhibit transcription,such as d Cas9-KRAB and dCas9-Dnmt3 a systems. The advantages of the CRISPR-Cas9 library are its flexible design,easy operation and efficient screening. Currently,CRISPR-Cas9 based libraries play an irreplaceable role in functional gene screening,drug targets and resistance targets screening,viral targets screening,identification of signaling pathways,gene interaction screening,and revealing the function of cis-regulator elements. With the development of gene editing systems,novel screening libraries may emerge in the future,which target and mutate more precisely,and their applications will be deeper and expanded. CRISPR-Cas9 based libraries can not only screen key genes and non-coding DNAs in pathological and physiological processes,but also reveal the molecular mechanisms of their function. It is a scalpel that dissects the complex regulatory network of life.
引文
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[12] Gilbert LA,Horlbeck MA,Adamson B,et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation[J]. Cell,2014,159(3):647-661
[13] Heaton BE, Kennedy EM, Dumm RE, et al. A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor[J]. Cell Rep,2017,20(7):1503-1512
[14] Liu XS,Wu H,Ji X,et al. Editing DNA Methylation in the Mammalian Genome[J]. Cell,2016,167(1):233-247.e217
[15] Hilton IB,D'Ippolito AM,Vockley CM,et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers[J]. Nat Biotechnol,2015,33(5):510-517
[16] Nihongaki Y,Furuhata Y,Otabe T,et al. CRISPR-Cas9-based photoactivatable transcription systems to induce neuronal differentiation[J]. Nat Methods,2017,14(10):963-966
[17] Sanjana NE. Genome-scale CRISPR pooled screens[J]. Anal Biochem,2017,532:95-99
[18] Phelps MP,Bailey JN,Vleeshouwer-Neumann T,et al. CRISPR screen identifies the NCOR/HDAC3 complex as a major suppressor of differentiation in rhabdomyosarcoma[J]. Proc Natl Acad Sci U S A,2016,113(52):15090-15095
[19] Wang C,Jin H,Gao D,et al. A CRISPR screen identifies CDK7as a therapeutic target in hepatocellular carcinoma[J]. Cell Res,2018,28(6):690-692
[20] Yau EH,Kummetha IR,Lichinchi G,et al. Genome-Wide CRISPR Screen for Essential Cell Growth Mediators in Mutant KRAS Colorectal Cancers[J]. Cancer Res,2017,77(22):6330-6339
[21] Song CQ,Li Y,Mou H,et al. Genome-Wide CRISPR Screen Identifies Regulators of Mitogen-Activated Protein Kinase as Suppressors of Liver Tumors in Mice[J]. Gastroenterology,2017,152(5):1161-1173.e1
[22] Chow RD,Guzman CD,Wang G,et al. AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma[J]. Nat Neurosci,2017,20(10):1329-1341
[23] Shi CX,Kortum KM,Zhu YX,et al. CRISPR Genome-Wide Screening Identifies Dependence on the Proteasome Subunit PSMC6 for Bortezomib Sensitivity in Multiple Myeloma[J]. Mol Cancer Ther,2017,16(12):2862-2870
[24] Ruiz S, Mayor-Ruiz C, Lafarga V, et al. A Genome-wide CRISPR Screen Identifies CDC25A as a Determinant of Sensitivity to ATR Inhibitors[J]. Mol Cell,2016,62(2):307-313
[25] Hou P,Wu C,Wang Y,et al. A Genome-Wide CRISPR Screen Identifies Genes Critical for Resistance to FLT3 Inhibitor AC220[J]. Cancer Res,2017,77(16):4402-4413
[26] Ishizuka JJ,Manguso RT,Cheruiyot CK,et al. Loss of ADAR1in tumours overcomes resistance to immune checkpoint blockade[J]. Nature,2019,565(7737):43-48
[27] Manguso RT,Pope HW,Zimmer MD,et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target[J].Nature,2017,547(7664):413-418
[28] Ma L,Boucher JI,Paulsen J,et al. CRISPR-Cas9-mediated saturated mutagenesis screen predicts clinical drug resistance with improved accuracy[J]. Proc Natl Acad Sci U S A,2017,114(44):11751-11756
[29] Hess GT,Fresard L,Han K,et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells[J].Nat Methods,2016,13(12):1036-1042
[30] Neggers JE,Kwanten B,Dierckx T,et al. Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes[J]. Nat Commun,2018,9(1):502
[31] Reczek CR, Birsoy K, Kong H, et al. A CRISPR screen identifies a pathway required for paraquat-induced cell death[J].Nat Chem Biol,2017,13(12):1274-1279
[32] Jost M,Chen Y,Gilbert LA,et al. Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent[J]. Mol Cell,2017,68(1):210-223.e6
[33] Hultquist JF, Schumann K, Woo JM, et al. A Cas9Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells[J]. Cell Rep,2016,17(5):1438-1452
[34] Flint M,Chatterjee P,Lin DL,et al. A genome-wide CRISPR screen identifies N-acetylglucosamine-1-phosphate transferase as a potential antiviral target for Ebola virus[J]. Nat Commun,2019,10(1):285
[35] Zhang R,Miner JJ,Gorman MJ,et al. A CRISPR screen defines a signal peptide processing pathway required by flaviviruses[J].Nature,2016,535(7610):164-168
[36] Potting C, Crochemore C, Moretti F, et al. Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy[J]. Proc Natl Acad Sci U S A,2018,115(2):E180-E189
[37] Kong X,Kuilman T,Shahrabi A,et al. Cancer drug addiction is relayed by an ERK2-dependent phenotype switch[J]. Nature,2017,550(7675):270-274
[38] Koirala P,Huang J,Ho TT,et al. LncRNA AK023948 is a positive regulator of AKT[J]. Nat Commun,2017,8:14422
[39] Zhao H,Choi K. A CRISPR screen identifies genes controlling Etv2 threshold expression in murine hemangiogenic fate commitment[J]. Nat Commun,2017,8(1):541
[40] Wu Y,Kang T. Protein stability regulators screening assay(ProSRSA):protein degradation meets the CRISPR-Cas9 library[J].Chin J Cancer,2016,35(1):60
[41] Shang W,Jiang Y,Boettcher M,et al. Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation[J]. Proc Natl Acad Sci U S A,2018,115(17):E4051-E4060
[42] Shen JP,Zhao D,Sasik R,et al. Combinatorial CRISPR-Cas9screens for de novo mapping of genetic interactions[J]. Nat Methods,2017,14(6):573-576
[43] Han K,Jeng EE,Hess GT,et al. Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions[J]. Nat Biotechnol,2017,35(5):463-474
[44] Horlbeck MA,Xu A,Wang M,et al. Mapping the Genetic Landscape of Human Cells[J]. Cell,2018,174(4):953-967.e22
[45] Boettcher M,Tian R,Blau JA,et al. Dual gene activation and knockout screen reveals directional dependencies in genetic networks[J]. Nat Biotechnol,2018,36(2):170-178
[46] Thurman RE,Rynes E,Humbert R,et al. The accessible chromatin landscape of the human genome[J]. Nature,2012,489(7414):75-82
[47] Gasperini M,Hill AJ,Mc Faline-Figueroa JL,et al. A Genomewide Framework for Mapping Gene Regulation via Cellular Genetic Screens[J]. Cell,2019,176(1-2):377-390.e319
[48] Rajagopal N,Srinivasan S,Kooshesh K,et al. High-throughput mapping of regulatory DNA[J]. Nat Biotechnol,2016,34(2):167-174
[49] Fulco CP,Munschauer M,Anyoha R,et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference[J]. Science,2016,354(6313):769-773
[50] Klann TS, Black JB, Chellappan M, et al. CRISPR-Cas9epigenome editing enables high-throughput screening for functional regulatory elements in the human genome[J]. Nat Biotechnol,2017,35(6):561-568
[2]李欢欢,黄承浩.基于CRISPR-Cas9的功能基因筛选研究进展[J].生物工程学报(Li HH,Huang CH. Functional genetic screening using CRISPR-Cas9 system[J]. Chin J Biotechnol),2018,34(4):461-472
[3] Qi X,Zhang J,Zhao Y,et al. The applications of CRISPR screen in functional genomics[J]. Brief Funct Genomics,2017,16(1):34-37
[4] Kweon J, Kim Y. High-throughput genetic screens using CRISPR-Cas9 system[J]. Arch Pharm Res,2018,41(9):875-884
[5] Thakore PI,D'Ippolito AM,Song L,et al. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements[J]. Nat Methods,2015,12(12):1143-1149
[6] Joung J, Engreitz JM, Konermann S, et al. Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood[J]. Nature,2017,548(7667):343-346
[7] Tanenbaum ME,Gilbert LA,Qi LS,et al. A protein-tagging system for signal amplification in gene expression and fluorescence imaging[J]. Cell,2014,159(3):635-646
[8]张雪梅,高旭,马宁.基于CRISPR/Cas9技术的基因敲入/敲除策略[J].中国生物化学与分子生物学报(Zhang XM,Gao X,Ma N. Strategy of gene knockin/knockout based on CRISPR/Cas9 technology[J]. Chin J Biochem Mol Biol),2019,35(1):1-6
[9] Chen S,Sanjana NE,Zheng K,et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis[J].Cell,2015,160(6):1246-1260
[10] Shalem O, Sanjana NE,Hartenian E, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells[J]. Science,2014,343(6166):84-87
[11] Zhu S,Li W,Liu J,et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library[J]. Nat Biotechnol,2016,34(12):1279-1286
[12] Gilbert LA,Horlbeck MA,Adamson B,et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation[J]. Cell,2014,159(3):647-661
[13] Heaton BE, Kennedy EM, Dumm RE, et al. A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor[J]. Cell Rep,2017,20(7):1503-1512
[14] Liu XS,Wu H,Ji X,et al. Editing DNA Methylation in the Mammalian Genome[J]. Cell,2016,167(1):233-247.e217
[15] Hilton IB,D'Ippolito AM,Vockley CM,et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers[J]. Nat Biotechnol,2015,33(5):510-517
[16] Nihongaki Y,Furuhata Y,Otabe T,et al. CRISPR-Cas9-based photoactivatable transcription systems to induce neuronal differentiation[J]. Nat Methods,2017,14(10):963-966
[17] Sanjana NE. Genome-scale CRISPR pooled screens[J]. Anal Biochem,2017,532:95-99
[18] Phelps MP,Bailey JN,Vleeshouwer-Neumann T,et al. CRISPR screen identifies the NCOR/HDAC3 complex as a major suppressor of differentiation in rhabdomyosarcoma[J]. Proc Natl Acad Sci U S A,2016,113(52):15090-15095
[19] Wang C,Jin H,Gao D,et al. A CRISPR screen identifies CDK7as a therapeutic target in hepatocellular carcinoma[J]. Cell Res,2018,28(6):690-692
[20] Yau EH,Kummetha IR,Lichinchi G,et al. Genome-Wide CRISPR Screen for Essential Cell Growth Mediators in Mutant KRAS Colorectal Cancers[J]. Cancer Res,2017,77(22):6330-6339
[21] Song CQ,Li Y,Mou H,et al. Genome-Wide CRISPR Screen Identifies Regulators of Mitogen-Activated Protein Kinase as Suppressors of Liver Tumors in Mice[J]. Gastroenterology,2017,152(5):1161-1173.e1
[22] Chow RD,Guzman CD,Wang G,et al. AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma[J]. Nat Neurosci,2017,20(10):1329-1341
[23] Shi CX,Kortum KM,Zhu YX,et al. CRISPR Genome-Wide Screening Identifies Dependence on the Proteasome Subunit PSMC6 for Bortezomib Sensitivity in Multiple Myeloma[J]. Mol Cancer Ther,2017,16(12):2862-2870
[24] Ruiz S, Mayor-Ruiz C, Lafarga V, et al. A Genome-wide CRISPR Screen Identifies CDC25A as a Determinant of Sensitivity to ATR Inhibitors[J]. Mol Cell,2016,62(2):307-313
[25] Hou P,Wu C,Wang Y,et al. A Genome-Wide CRISPR Screen Identifies Genes Critical for Resistance to FLT3 Inhibitor AC220[J]. Cancer Res,2017,77(16):4402-4413
[26] Ishizuka JJ,Manguso RT,Cheruiyot CK,et al. Loss of ADAR1in tumours overcomes resistance to immune checkpoint blockade[J]. Nature,2019,565(7737):43-48
[27] Manguso RT,Pope HW,Zimmer MD,et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target[J].Nature,2017,547(7664):413-418
[28] Ma L,Boucher JI,Paulsen J,et al. CRISPR-Cas9-mediated saturated mutagenesis screen predicts clinical drug resistance with improved accuracy[J]. Proc Natl Acad Sci U S A,2017,114(44):11751-11756
[29] Hess GT,Fresard L,Han K,et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells[J].Nat Methods,2016,13(12):1036-1042
[30] Neggers JE,Kwanten B,Dierckx T,et al. Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes[J]. Nat Commun,2018,9(1):502
[31] Reczek CR, Birsoy K, Kong H, et al. A CRISPR screen identifies a pathway required for paraquat-induced cell death[J].Nat Chem Biol,2017,13(12):1274-1279
[32] Jost M,Chen Y,Gilbert LA,et al. Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent[J]. Mol Cell,2017,68(1):210-223.e6
[33] Hultquist JF, Schumann K, Woo JM, et al. A Cas9Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells[J]. Cell Rep,2016,17(5):1438-1452
[34] Flint M,Chatterjee P,Lin DL,et al. A genome-wide CRISPR screen identifies N-acetylglucosamine-1-phosphate transferase as a potential antiviral target for Ebola virus[J]. Nat Commun,2019,10(1):285
[35] Zhang R,Miner JJ,Gorman MJ,et al. A CRISPR screen defines a signal peptide processing pathway required by flaviviruses[J].Nature,2016,535(7610):164-168
[36] Potting C, Crochemore C, Moretti F, et al. Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy[J]. Proc Natl Acad Sci U S A,2018,115(2):E180-E189
[37] Kong X,Kuilman T,Shahrabi A,et al. Cancer drug addiction is relayed by an ERK2-dependent phenotype switch[J]. Nature,2017,550(7675):270-274
[38] Koirala P,Huang J,Ho TT,et al. LncRNA AK023948 is a positive regulator of AKT[J]. Nat Commun,2017,8:14422
[39] Zhao H,Choi K. A CRISPR screen identifies genes controlling Etv2 threshold expression in murine hemangiogenic fate commitment[J]. Nat Commun,2017,8(1):541
[40] Wu Y,Kang T. Protein stability regulators screening assay(ProSRSA):protein degradation meets the CRISPR-Cas9 library[J].Chin J Cancer,2016,35(1):60
[41] Shang W,Jiang Y,Boettcher M,et al. Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation[J]. Proc Natl Acad Sci U S A,2018,115(17):E4051-E4060
[42] Shen JP,Zhao D,Sasik R,et al. Combinatorial CRISPR-Cas9screens for de novo mapping of genetic interactions[J]. Nat Methods,2017,14(6):573-576
[43] Han K,Jeng EE,Hess GT,et al. Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions[J]. Nat Biotechnol,2017,35(5):463-474
[44] Horlbeck MA,Xu A,Wang M,et al. Mapping the Genetic Landscape of Human Cells[J]. Cell,2018,174(4):953-967.e22
[45] Boettcher M,Tian R,Blau JA,et al. Dual gene activation and knockout screen reveals directional dependencies in genetic networks[J]. Nat Biotechnol,2018,36(2):170-178
[46] Thurman RE,Rynes E,Humbert R,et al. The accessible chromatin landscape of the human genome[J]. Nature,2012,489(7414):75-82
[47] Gasperini M,Hill AJ,Mc Faline-Figueroa JL,et al. A Genomewide Framework for Mapping Gene Regulation via Cellular Genetic Screens[J]. Cell,2019,176(1-2):377-390.e319
[48] Rajagopal N,Srinivasan S,Kooshesh K,et al. High-throughput mapping of regulatory DNA[J]. Nat Biotechnol,2016,34(2):167-174
[49] Fulco CP,Munschauer M,Anyoha R,et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference[J]. Science,2016,354(6313):769-773
[50] Klann TS, Black JB, Chellappan M, et al. CRISPR-Cas9epigenome editing enables high-throughput screening for functional regulatory elements in the human genome[J]. Nat Biotechnol,2017,35(6):561-568