CRISPR/dCas9在细菌转录调控中的应用及展望
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摘要
CRISPR/dCas9是以CRISPR/Cas9系统为基础发展而来的转录调控工具.本文综述了CRISPR/dCas9作为转录抑制工具(CRISPRi)和转录激活工具(CRISPRa)的发展及应用,总结了在细菌中应用CRISPR/dCas9系统时转录调控系统的选择、构建、靶位点的选择以及gRNA的设计等方面存在的问题,并对相关解决方法进行了展望.
CRISPR/dCas9 is a transcriptional regulation tool developed from the CRISPR/Cas9 system. The development and application of CRISPR/dCas9 as a transcriptional interference(CRISPRi)and transcription activator(CRISPRa)were summarized in this paper. Problems in the application of CRISPR/dCas9 system in bacteria,such as target sites,target selection and construction of the transcriptional regulatory system,the design of gRNA,and possible solutions were also discussed in the paper.
CRISPR/dCas9 is a transcriptional regulation tool developed from the CRISPR/Cas9 system. The development and application of CRISPR/dCas9 as a transcriptional interference(CRISPRi)and transcription activator(CRISPRa)were summarized in this paper. Problems in the application of CRISPR/dCas9 system in bacteria,such as target sites,target selection and construction of the transcriptional regulatory system,the design of gRNA,and possible solutions were also discussed in the paper.
引文
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[2]Wiedenheft B,Sternberg S H,Doudna J A.RNA-guided genetic silencing systems in bacteria and archaea[J].Nature,2012,482(7385):331-338.
[3]Chylinski K,Makarova K S,Charpentier E,et al.Classification and evolution of type II CRISPR-Cas systems[J].Nucleic Acids Research,2014,42(10):6091-6105.
[4]Jinek M,Chylinski K,Fonfara I,et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science,2012,337(6096):816-821.
[5]Lanct?t C,Cheutin T,Cremer M,et al.Dynamic genome architecture in the nuclear space:Regulation of gene expression in three dimensions[J].Nature Reviews Genetics,2007,8(2):104-115.
[6]Nishimasu H,Ran F A,Hsu P D,et al.Crystal structure of Cas9 in complex with guide RNA and target DNA[J].Cell,2014,156(5):935-949.
[7]Mali P,Aach J,Stranges P B,et al.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering[J].Nature Biotechnology,2013,31(9):833-838.
[8]Jiang F,Doudna J A.CRISPR-Cas9 structures and mechanisms[J].Annual Review of Biophysics,2017,46(1):505-529.
[9]Sternberg S H,Redding S,Jinek M,et al.DNA interrogation by the CRISPR RNA-guided endonuclease Cas9[J].Biophysical Journal,2014,106(2):695a.
[10]Qi L S,Larson M,Gilbert L,et al.Repurposing CRISPRas an RNA-guided platform for sequence-specific control of gene expression[J].Cell,2013,152(5):1173-1183.
[11]Bikard D,Jiang W,Samai P,et al.Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system[J].Nucleic Acids Research,2013,41(15):7429-7437.
[12]Wang H,Russa M L,Qi L S.CRISPR/Cas9 in genome editing and beyond[J].Annual Review of Biochemistry,2016,85(1):227-264.
[13]Shalem O,Sanjana N E,Zhang F.High-throughput functional genomics using CRISPR-Cas9[J].Nature Reviews Genetics,2015,16(5):299-311.
[14]Doudna J A,Charpentier E.The new frontier of genome engineering with CRISPR-Cas9[J].Science,2014,346(6213):1258096.
[15]Harrison M M,Jenkins B V,O'Connorgiles K M,et al.ACRISPR view of development[J].Genes&Development,2014,28(17):1859-1872.
[16]Hsu P,Lander E,Zhang F.Development and applications of CRISPR-Cas9 for genome engineering[J].Cell,2014,157(6):1262-1278.
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[20]Choudhary E,Thakur P,Pareek M,et al.Gene silencing by CRISPR interference in mycobacteria[J].Nature Communications,2015,6:6267.
[21]Chavez A,Scheiman J,Vora S,et al.Highly-efficient Cas9-mediated transcriptional programming[J].Nature Methods,2015,12(4):326-328.
[22]Tong Y,Charusanti P,Zhang L,et al.CRISPR-Cas9based engineering of actinomycetal genomes[J].ACSSynthetic Biology,2015,4(9):1020-1029.
[23]Gilbert L A,Larson M H,Morsut L,et al.CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[J].Cell,2013,154(2):442-451.
[24]Konermann S,Brigham M D,Trevino A E,et al.Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex[J].Nature,2015,517(7536):583-588.
[25]Tanenbaum M E,Gilbert L A,Qi L S,et al.A protein tagging system for signal amplification in gene expression and fluorescence imaging[J].Cell,2014,159(3):635-646.
[26]Konermann S,Brigham M D,Trevino A,et al.Optical control of mammalian endogenous transcription and epigenetic states[J].Nature,2013,500(7463):472-476.
[27]Cleary M A,Kilian K,Wang Y,et al.Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis[J].Nature Methods,2004,1(3):241-248.
[28]Leproust E M,Peck B J,Spirin K,et al.Synthesis of high-quality libraries of long(150mer)oligonucleotides by a novel depurination controlled process[J].Nucleic Acids Research,2010,38(8):2522-2540.
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[38]Larson M H,Gilbert L A,Wang X,et al.CRISPR interference(CRISPRi)for sequence-specific control of geneexpression[J].Nature Protocols,2013,8(11):2180-2196.
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