CRISPR/Cas基因编辑系统及其在植物中的研究进展

doi:10.11924/j.issn.1000-6850.casb14120051

摘要/Abstract

摘要： 靶向基因编辑系统是一种研究植物基因功能的工具。其中，CRISPR/Cas基因编辑系统的克隆策略相对简单，成本低廉，因而在生物学界获得了广泛的关注。为了更好地对其在植物中进行研究，笔者介绍了该系统的基本结构特征与作用机理，归纳了在拟南芥、烟草、水稻等植物中对该系统进行的报道，分析了该系统尚需改进的问题如脱靶效应，探讨了多种解决方法，如优化sgRNA序列长度、利用双Cas9切口酶、合成fCas9复合体以及应用RFNs二聚体等，以降低脱靶效应。

关键词: 生根率, 生根率

Abstract: Targeted gene editing system is a kind of tool to study the functions of plant genes. In this system, the cloning strategy of CRISPR/Cas gene editing system is relatively simple and the cost is low, thus it has drawn widespread attention in biology. In order to study the system in plant better, the author introduced the basic structure and the mechanism of the system, summarized research reports on the system in plants like Arabidopsis, tobacco, rice and so on, analyzed problems of the system that needs to be improved, such as off-target effects, and discussed several solutions, including optimizing sgRNA sequence length, using double Cas9 nickases, synthesizing fCas9 complex and applying RFNs dimers, to reduce off-target effects.

赵欣,胡军. CRISPR/Cas基因编辑系统及其在植物中的研究进展[J]. 中国农学通报, 2015, 31(12): 187-192.

Zhao Xin and Hu Jun. CRISPR/Cas Gene Editing System and Its Progress in Plants[J]. Chinese Agricultural Science Bulletin, 2015, 31(12): 187-192.

参考文献

[1] Mahfouz M M, Piatek A, Stewart C N. Genome engineering via TALENs and CRISPR/Cas9 systems: challenges and perspectives [J]. Plant biotechnology journal,2014,12(8):1006-1014.
[2] Lozano-Juste J, Cutler S R. Plant genome engineering in full bloom [J]. Trends in plant science,2014,19(5):284-287.
[3] Liu L, Fan X D. CRISPR–Cas system: a powerful tool for genome engineering [J]. Plant molecular biology,2014,85(3):209-218.
[4] Nakata A, Amemura M, Makino K. Unusual nucleotide arrangement with repeated sequences in the Escherichia coli K-12 chromosome [J]. Journal of bacteriology,1989,171(6):3553-3556.
[5] Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies[J]. Microbiology,2005,51(3):653-663.
[6] Mojica F J, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements [J]. Journal of Molecular evolution, 2005,60(2):174-182.
[7] Bolotin A, Quinquis B, Sorokin A, et al. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin [J]. Microbiology, 2005,151(8):2551-2561.
[8] Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes [J]. Science, 2007, 315 (5819): 1709-1712.
[9] Bland C, Ramsey T L, Sabree F, et al. CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats [J]. BMC bioinformatics, 2007,8(1):209.
[10] Klug A. The discovery of zinc fingers and their applications in gene regulation and genome manipulation [J]. Annual review of biochemistry, 2010,79:213-231.
[11] Wei C, Liu J, Yu Z, et al. TALEN or Cas9–rapid, efficient and specific choices for genome modifications [J]. Journal of Genetics and Genomics, 2013,40(6):281-289.
[12] Grissa I, Vergnaud G, Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats [J]. Bmc Bioinformatics, 2007,8(1):172.
[13] Richter H, Randau L, Plagens A. Exploiting CRISPR/Cas: Interference mechanisms and applications [J]. International journal of molecular sciences, 2013,14(7):14518-14531.
[14] Marraffini L A, Sontheimer E J. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea [J]. Nature Reviews Genetics, 2010,11(3):181-190.
[15] Mojica F, Diez-Villasenor C, Garcia-Martinez J, et al. Short motif sequences determine the targets of the prokaryotic CRISPR defence system [J]. Microbiology, 2009,155(3):733-740.
[16] Gasiunas G, Barrangou R, Horvath P, et al. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria [J]. Proceedings of the National Academy of Sciences, 2012,10(39): E2579-E2586.
[17] Xie K, Zhang J, Yang Y. Genome-Wide Prediction of Highly Specific Guide RNA Spacers for CRISPR–Cas9-Mediated Genome Editing in Model Plants and Major Crops [J]. Molecular plant, 2014,7(5):923-926.
[18] Jiang W, Yang B, Weeks D P. Efficient CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana and inheritance of modified genes in the T2 and T3 generations [J]. PloS one, 2014,9(6):e99225.
[19] Hyun Y, Kim J, Cho S W, et al. Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles [J]. Planta, 2014, doi:10.1007/s00425-014-2180-5.
[20] Feng Z, Mao Y, Xu N, et al. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis [J]. Proceedings of the National Academy of Sciences, 2014,111(12):4632-4637.
[21] Fauser F, Schiml S, Puchta H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana [J]. The Plant Journal, 2014,79(2):348-359.
[22] Zhang H, Zhang J, Wei P, et al. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation [J]. Plant biotechnology journal, 2014,12(6):797-807.
[23] Xu R, Li H, Qin R, et al. Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice [J]. Rice, 2014,7(1):5.
[24] Xie K,Yang Y. RNA-guided genome editing in plants using a CRISPR-Cas system [J]. Molecular plant, 2013,6(6):1975-1983.
[25] Jiang W, Zhou H, Bi H, et al. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice [J]. Nucleic acids research, 2013, doi: 10.1093/nar/gkt780.
[26] Shan Q, Wang Y, Li J, et al. Genome editing in rice and wheat using the CRISPR/Cas system [J]. Nature protocols, 2014,9(10):2395-2410.
[27] Ga J, Wang G, Ma S. et al. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum [J]. Plant molecular biology, 2014:1-12.
[28] Nekrasov V, Staskawicz B, Weigel D, et al. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease [J]. Nature biotechnology, 2013,31(8):691-693.
[29] Liang Z, Zhang K, Chen K, et al. Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System [J]. Journal of Genetics and Genomics, 2014,41(2):63-68.
[30] Jia H, Wang N. Targeted genome editing of sweet orange using Cas9/sgRNA [J]. PLoS One, 2014,9(4):e93806.
[31] Brooks C, Nekrasov V, Lippman Z B, et al. Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System [J]. Plant physiology, 2014,166(3):1292-1297.
[32] Sharma S, Upadhyay S K. Functional Characterization of Expressed Sequence Tags of Bread Wheat (Triticum aestivum) and Analysis of CRISPR Binding Sites for Targeted Genome Editing [J]. American Journal of Bioinformatics Research, 2014,4(1):11-22.
[33] Semenova E, Nagornykh M, Pyatnitskiy M, et al. Analysis of CRISPR system function in plant pathogen Xanthomonas oryzae [J]. FEMS microbiology letters, 2009,296(1):110-116.
[34] Zhou H, Liu B, Weeks D P, et al. Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice [J]. Nucleic acids research, 2015,42(17):10903-10914.
[35] Ryan O W, Skerker J M, Maurer M J, et al. Selection of chromosomal DNA libraries using a multiplex CRISPR system [J]. eLife, 2014,3:e03703.
[36] Feng Z, Zhang B, Ding W, et al. Efficient genome editing in plants using a CRISPR/Cas system [J]. Cell research, 2013,23(10):1229-1232.
[37] Upadhyay S K, Sharma S. SSFinder: high throughput CRISPR-Cas target sites prediction tool [J]. BioMed research international, 2014:742482.
[38] Güell M, Yang L, Church G M. Genome editing assessment using CRISPR Genome Analyzer (CRISPR-GA) [J]. Bioinformatics, 2014,30(20):2968-2970.
[39] Yan M, Zhou S R, Xue H W. CRISPR Primer Designer: Design primers for knockout & chromosome imaging CRISPR-Cas system [J]. Journal of integrative plant biology, 2014, doi: 10.1111/jipb.12295.
[40] O’Connell M R, Oakes B L, Sternberg S H, et al. Programmable RNA recognition and cleavage by CRISPR/Cas9 [J]. Nature, 2014, doi:10.1038/nature13769.
[41] Fu Y, Sander J D, Reyon D, et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs [J]. Nature biotechnology, 2014,32(3):279-284.
[42] Ran F A, Hsu P D, Lin C-Y, et al. Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity [J]. Cell, 2013,155(2):479-480.
[43] Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification [J]. Nature biotechnology, 2014,32:577-582.
[44] Tsai S Q, Wyvekens N, Khayter C, et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing [J]. Nature biotechnology, 2014,32(6):569-576.
[45] Ranganathan V, Wahlin K, Maruotti J, et al. Expansion of the CRISPR–Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs [J]. Nature communications, 2014, doi:10.1038/ncomms5516.
[46] Gogleva A A, Gelfand M S, Artamonova I I. Comparative analysis of CRISPR cassettes from the human gut metagenomic contigs [J]. BMC genomics, 2014,15(1):202.
[47] Small I, Puchta H. Emerging tools for synthetic biology in plants [J]. The Plant Journal, 2014,78(5):725-726.
[48] Kumar V, Jain M. The CRISPR–Cas system for plant genome editing: advances and opportunities [J]. Journal of Experimental Botany, 2014, doi: 10.1093/jxb/eru429.
[49] Hartung F, Schiemann J. Precise plant breeding using new genome editing techniques: opportunities, safety and regulation in the EU [J]. The Plant Journal, 2014,78(5):742-752.
[50] Webber P. Does CRISPR-Cas open new possibilities for patents or present a moral maze? [J]. Nature biotechnology, 2014,32(4):331-333.