CRISPR/Cas9系统中引导RNA的研究进展
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摘要
CRISPR/Cas9系统作为重要的一种基因编辑工具,自产生以来广泛应用于各种生物体基因组序列的精确编辑,可在特定位点产生核苷酸的删除、插入或替换,从而改变编码基因的功能。以CRISPR/Cas9为代表的基因编辑技术将应用于包括生物育种在内的各项生物技术领域,产生新一代生物技术产品。目前关于CRISPR/Cas9的研究报道多集中于Cas9蛋白的结构功能,以及CRISPR/Cas9系统的工作原理。引导RNA(Guide RNA)作为CRISPR/Cas9系统的重要组分之一,也是基因编辑技术领域的研究热点,近来有多篇文章报道通过改良引导RNA从而提高CRISPR/Cas9的编辑效率和精确性。对CRISPR/Cas9系统中的引导RNA研究进展进行了综述,围绕引导RNA的序列组成、结构特征以及转录产生方式这3方面内容,有助于全面了解CRISPR/Cas9系统的结构特征,讨论了引导RNA对CRISPR/Cas9基因编辑效率的影响,有利于CRISPR/Cas9系统的使用。最后,展望引导RNA今后的研究发展趋势,旨在解决CRISPR/Cas9目前存在的相关问题,优化出效率更高、精度更高的基因编辑技术。
The CRISPR/Cas9 system,as a critical gene editing tool,has been used widely for the precise editing of genome sequences in a variety of organisms since its emergence,and it may generate the nucleotides deletion,insertion or replacement at specific targeted sites,thus changing the functions of encoding genes. Gene-editing technique based on CRISPR/Cas9 system will be applied into various biotechnology fields including biological breeding for producing a new generation of biotechnology products. Current research reports on CRISPR/Cas9 focus on the Cas9 protein structure and function,as well as the working principle of CRISPR/Cas9 system. As one of the critical components of CRISPR/Cas9 system,guide RNA is also the research hotspot in gene-editing technology fields,there are many article reporting that optimization of guide RNA resulted in the increase of editing efficiency and precision of CRISPR/Cas9. Therefore here we reviewed the research progress on guide RNA in CRISPR/Cas9 system,focusing on the sequence composition,structural characteristics and transcriptional ways of guide RNA,which is conducive to completely understand the characteristics of CRISPR/Cas9 system. Moreover,we discussed the effects of guided RNA on the gene editing efficiency of CRISPR/Cas9,which is beneficial in using CRISPR/Cas9 system. Finally,we prospected the future research trend of guided RNA,aiming at solving the current issues of using CRISPR/Cas9 and optimizing the gene editing technology with higher efficiency and accuracy.
The CRISPR/Cas9 system,as a critical gene editing tool,has been used widely for the precise editing of genome sequences in a variety of organisms since its emergence,and it may generate the nucleotides deletion,insertion or replacement at specific targeted sites,thus changing the functions of encoding genes. Gene-editing technique based on CRISPR/Cas9 system will be applied into various biotechnology fields including biological breeding for producing a new generation of biotechnology products. Current research reports on CRISPR/Cas9 focus on the Cas9 protein structure and function,as well as the working principle of CRISPR/Cas9 system. As one of the critical components of CRISPR/Cas9 system,guide RNA is also the research hotspot in gene-editing technology fields,there are many article reporting that optimization of guide RNA resulted in the increase of editing efficiency and precision of CRISPR/Cas9. Therefore here we reviewed the research progress on guide RNA in CRISPR/Cas9 system,focusing on the sequence composition,structural characteristics and transcriptional ways of guide RNA,which is conducive to completely understand the characteristics of CRISPR/Cas9 system. Moreover,we discussed the effects of guided RNA on the gene editing efficiency of CRISPR/Cas9,which is beneficial in using CRISPR/Cas9 system. Finally,we prospected the future research trend of guided RNA,aiming at solving the current issues of using CRISPR/Cas9 and optimizing the gene editing technology with higher efficiency and accuracy.
引文
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[30]Hamada H,Liu YL,Nagira Y,et al.Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat[J].Scientific Reports,2018,8:1-7.
[31]Kato-Inui T,Takahashi G,Hsu S,et al.Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair[J].Nucleic Acids Research,2018,46:4677-4688.
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[33]Nielsen S,Yuzenkova Y,Zenkin N.Mechanism of eukaryotic RNApolymerase III transcription termination[J].Science,2013,340:1577-1580.
[34]Mekler V,Minakhin L,Semenova E,et al.Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3'-terminal segment of guide RNA[J].Nucleic Acids Research,2016,44:2837-2845.
[35]Xu JY,Lian W,Jia YN,et al.Optimized guide RNA structure for genome editing via Cas9[J].Oncotarget,2017,8:94166-94171.
[36]Nahar S,Sehgal P,Azha M,et al.A G-quadruplex motif at the 3'end of sgRNAs improves CRISPR-Cas9 based genome editing efficiency[J].Chemical Communications,2018,54:2377-2380.
[37]Zhang F,LeBlanc C,Irish VF,et al.Rapid and efficient CRISPR/Cas9 gene editing in Citrus using the YAO promoter[J].Plant Cell Rep,2017,36:1883-1887.
[38]Canver MC,Lessard S,Pinello L,et al.Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci[J].Nature Genetics,2017,49:625-634.
[39]Tycko J,Myer VE and Hsu PD.Methods for optimizing CRISPR-Cas9 genome editing specificity[J].Molecular Cell,2016,63:355-370.
[40]Butt H,Eid A,Ali Z,et al.Efficient CRISPR/Cas9-mediated genome editing using a chimeric single-guide rna molecule[J].Frontiers in Plant Science,2017,8:1-8.
[41]Burstein D,Harrington LB,Strutt SC,et al.New CRISPR-Cas systems from uncultivated microbes[J].Nat,2016,542:237-243.
[2]Cong L,Ran FA,Cox D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339:819-823.
[3]Mali P,Yang LH,Esvelt KM,et al.RNA-guided human genome engineering via Cas9[J].Science,2013,339:823-826.
[4]Barrangou R,Doudna JA.Applications of CRISPR technologies in research and beyond[J].Nat Biotech,2016,34:933-941.
[5]Jinek M,Chylinski K,Fonfara I,et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science,2012,337:816-821.
[6]Miao J,Guo D,Zhang JZ,et al.Targeted mutagenesis in rice using CRISPR-Cas system[J].Cell Res,2013,23:1233-1236.
[7]Zhou HB,Liu B,Weeks DP,et al.Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice[J].Nucleic Acids Research,2014,42:10903-10914.
[8]Hsu PD,Scott DA,Weinsteinet JA,al.DNA targeting specificity of RNA-guided Cas9 nucleases[J].Nature Biotechnology,2013,31:827-832.
[9]Dang Y,Jia GX,Choi J,et al.Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency[J].Genome Biology,2015,16:280-290.
[10]Briner AE,Donohoue PD,Gomaa AA,et al.Guide RNA functional modules direct Cas9 activity and orthogonality[J].Molecular Cell,2014,56:333-339.
[11]Nishimasu H,Ran FA,Hsu PD,et al.Crystal structure of Cas9 in complex with guide rna and target DNA[J].Cell,2014,156:935-949.
[12]Jiang FG,Zhou KH,Ma LL,et al.A Cas9-guide RNA complex reorganized for target DNA recognition[J].Science,2015,348:1477-1481.
[13]Anders C,Niewoehner O,Duerst A,et al.Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease[J].Nat,2014,513:569-573.
[14]Hu XX,Meng XB,Liu Q,et al.Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice[J].Plant Biotechnol J,2018,16:292-297.
[15]Weeks DP,Yang B.Progress in molecular biology and translational science[M].New York:Academic Press,2017.
[16]Lowder LG,Zhang DW,Baltes N,et al.A CRISPR/Cas9toolbox for multiplexed plant genome editing and transcriptional regulation[J].Plant Physiol,2015,169:971-985.
[17]Fu YF,Sander JD,Reyon D,et al.Improving CRISPR-Cas nuclease specificity using truncated guide RNAs[J].Nat Biotech,2014,32:279-284.
[18]Durr J,Papareddy R,Nakajima K,et al.Highly efficient heritable targeted deletions of gene clusters and non-coding regulatory regions in Arabidopsis using CRISPR/Cas9[J].Scientific Reports,2018,8:1-11.
[19]Mikami M,Toki S,Endo M.In planta processing of the SpCas9-gRNA Complex[J].Plant Cell Physiol,2017,58:1857-1867.
[20]Wang M,Mao YF,Lu YM,et al.Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems[J].Journal of Integrative Plant Biology,2018,60:626-631.
[21]Ma XL,Zhang QY,Zhu QL,et al.A robust CRISPR/Cas9 system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants[J].Molecular Plant,2015,8:1274-1284.
[22]Tsai SQ,Wyvekens N,Khayter C,et al.Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing[J].Nat Biotech,2014,32:569-576.
[23]?ermák T,Curtin SJ,Gil-Humanes J,et al.A multipurpose toolkit to enable advanced genome engineering in plants[J].Plant Cell,2017,29:1196-1217.
[24]Qi WW,Zhu T,Tian ZR,et al.High-efficiency CRISPR/Cas9multiplex gene editing using the glycine tRNA-processing systembased strategy in maize[J].BMC Biotechnology,2016,16:58-64.
[25]Xie KB,Minkenberg B,Yang Y.Boosting CRISPR/Cas9multiplex editing capability with the endogenous tRNA-processing system[J].Proceedings of the National Academy of Sciences,2015,112:3570-3575.
[26]Gao YB and Zhao YD.Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing[J].Journal of Integrative Plant Biology,2014,56:343-349.
[27]Kim SJ,Kin D,Cho SW,et al.Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9ribonucleoproteins[J].Genome Research,2014,24:1012-1019.
[28]Svitashev S,Schwartz C,Lenderts B,et al.Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes[J].Nat Commun,2016,7:1-7.
[29]Kelley ML,Strezoska Z,He KZ,et al.Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing[J].Journal of Biotechnology,2016,233:74-83.
[30]Hamada H,Liu YL,Nagira Y,et al.Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat[J].Scientific Reports,2018,8:1-7.
[31]Kato-Inui T,Takahashi G,Hsu S,et al.Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair[J].Nucleic Acids Research,2018,46:4677-4688.
[32]Zhang JH,Adikaram P,Pandey M,et al.Optimization of genome editing through CRISPR-Cas9 engineering[J].Bioengineered,2016,7:166-174.
[33]Nielsen S,Yuzenkova Y,Zenkin N.Mechanism of eukaryotic RNApolymerase III transcription termination[J].Science,2013,340:1577-1580.
[34]Mekler V,Minakhin L,Semenova E,et al.Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3'-terminal segment of guide RNA[J].Nucleic Acids Research,2016,44:2837-2845.
[35]Xu JY,Lian W,Jia YN,et al.Optimized guide RNA structure for genome editing via Cas9[J].Oncotarget,2017,8:94166-94171.
[36]Nahar S,Sehgal P,Azha M,et al.A G-quadruplex motif at the 3'end of sgRNAs improves CRISPR-Cas9 based genome editing efficiency[J].Chemical Communications,2018,54:2377-2380.
[37]Zhang F,LeBlanc C,Irish VF,et al.Rapid and efficient CRISPR/Cas9 gene editing in Citrus using the YAO promoter[J].Plant Cell Rep,2017,36:1883-1887.
[38]Canver MC,Lessard S,Pinello L,et al.Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci[J].Nature Genetics,2017,49:625-634.
[39]Tycko J,Myer VE and Hsu PD.Methods for optimizing CRISPR-Cas9 genome editing specificity[J].Molecular Cell,2016,63:355-370.
[40]Butt H,Eid A,Ali Z,et al.Efficient CRISPR/Cas9-mediated genome editing using a chimeric single-guide rna molecule[J].Frontiers in Plant Science,2017,8:1-8.
[41]Burstein D,Harrington LB,Strutt SC,et al.New CRISPR-Cas systems from uncultivated microbes[J].Nat,2016,542:237-243.
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