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【专题讨论】希望和大家一起学习下CRISPR在植物上的应用

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先总结下最近应用CRISPR在植物上应用的文献：
1. Belhaj, K., Chaparro-Garcia, A., Kamoun,S., and Nekrasov, V. 2013. Plant genome editing made easy: targeted mutagenesisin model and crop plants using the CRISPR/Cas system. Plant Methods. 9:39.
2. Bos,J. I. B., Kanneganti, T.-D., Young, C., Cakir, C., Huitema, E., Win, J., et al.2006. The C-terminal half of Phytophthora infestans RXLR effector AVR3a issufficient to trigger R3a-mediated hypersensitivity and suppress INF1-inducedcell death in Nicotiana benthamiana. The Plant Journal. 48:165–176.
3. Carroll,D. 2013. Staying on target with CRISPR-Cas. Nat Biotech. 31:807–809.
4. D’Halluin,K., Vanderstraeten, C., Van Hulle, J., Rosolowska, J., Van Den Brande, I.,Pennewaert, A., et al. 2013. Targeted molecular trait stacking in cottonthrough targeted double-strand break induction. Plant Biotechnology Journal.11:933–941.
5. Deltcheva,E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., etal. 2011. CRISPR RNA maturation by trans-encoded small RNA and host factorRNase III. Nature. 471:602–607.
6. Fauser,F., Roth, N., Pacher, M., Ilg, G., Sánchez-Fernández, R., Biesgen, C., et al.2012. In planta gene targeting. PNAS. 109:7535–7540.
7. Feng,Z., Zhang, B., Ding, W., Liu, X., Yang, D.-L., Wei, P., et al. 2013. Efficientgenome editing in plants using a CRISPR/Cas system. Cell Res. 23:1229–1232.
8. Vander Hoorn, R. A. L., Laurent, F., Roth, R., and De Wit, P. J. G. M. 2000.Agroinfiltration Is a Versatile Tool That Facilitates Comparative Analyses of Avr9/ Cf-9 -Induced and Avr 4/ Cf-4 -Induced Necrosis.Molecular Plant-Microbe Interactions. 13:439–446.
9. Hsu,P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V.,et al. 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. NatBiotech. 31:827–832.
10. Jiang, W., Zhou, H., Bi, H., Fromm, M.,Yang, B., and Weeks, D. P. 2013. Demonstration of CRISPR/Cas9/sgRNA-mediatedtargeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucl.Acids Res. 41:e188–e188.
11. Kuzma, J., and Kokotovich, A. 2011.Renegotiating GM crop regulation. EMBO reports. 12:883–888.
12. Li, J.-F., Norville, J. E., Aach, J.,McCormack, M., Zhang, D., Bush, J., et al. 2013. Multiplex and homologousrecombination-mediated genome editing in Arabidopsis and Nicotiana benthamianausing guide RNA and Cas9. Nat Biotech. 31:688–691.
13. Lusser, M., and Davies, H. V. 2013. Comparativeregulatory approaches for groups of new plant breeding techniques. NewBiotechnology. 30:437–446.
14. Lusser, M., Parisi, C., Plan, D., andRodríguez-Cerezo, E. 2012. Deployment of new biotechnologies in plant breeding.Nat Biotech. 30:231–239.
15. Mali, P., Aach, J., Stranges, P. B.,Esvelt, K. M., Moosburner, M., Kosuri, S., et al. 2013. CAS9 transcriptionalactivators for target specificity screening and paired nickases for cooperativegenome engineering. Nat Biotech. 31:833–838.
16. Mao, Y., Zhang, H., Xu, N., Zhang, B.,Gou, F., and Zhu, J.-K. 2013. Application of the CRISPR-Cas System forEfficient Genome Engineering in Plants. Molecular Plant. 6:2008–2011.
17. Miao, J., Guo, D., Zhang, J., Huang, Q.,Qin, G., Zhang, X., et al. 2013. Targeted mutagenesis in rice using CRISPR-Cassystem. Cell Res. 23:1233–1236.
18. Mussolino, C., and Cathomen, T. 2013. RNAguides genome engineering. Nat Biotech. 31:208–209.
19. Nekrasov, V., Staskawicz, B., Weigel, D.,Jones, J. D. G., and Kamoun, S. 2013. Targeted mutagenesis in the model plantNicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotech. 31:691–693.
20. Pattanayak, V., Lin, S., Guilinger, J.P., Ma, E., Doudna, J. A., and Liu, D. R. 2013. High-throughput profiling ofoff-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. NatBiotech. 31:839–843.
21. Qi, Y., Zhang, Y., Zhang, F., Baller, J.A., Cleland, S. C., Ryu, Y., et al. 2013. Increasing frequencies ofsite-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNArepair pathways. Genome Res. 23:547–554.
22. Shan, Q., Wang, Y., Li, J., Zhang, Y.,Chen, K., Liang, Z., et al. 2013. Targeted genome modification of crop plantsusing a CRISPR-Cas system. Nat Biotech. 31:686–688.
23. Voytas, D. F. 2013. Plant GenomeEngineering with Sequence-Specific Nucleases. Annual Review of Plant Biology.64:327–350.
24. Weber, E., Engler, C., Gruetzner, R.,Werner, S., and Marillonnet, S. 2011. A Modular Cloning System for StandardizedAssembly of Multigene Constructs. PLoS ONE. 6:e16765.

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donggua 编辑于 2014-03-06 16:26

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第一篇文献，是关于crispr在植物上应用的一个小综述。
摘要：
Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.
与zfns 和talens 的设计复杂和各种实验室试验相比，crispr或许相对简单点。crispr／cas系统允许个性化的小的非编吗rna来介导靶标基因组dna的切割，从而使基因能够通过非同源末端结合和同源接到的修复机制来修饰基因。
本文主要汇总和讨论最近该技术在植物上的应用。
植物内编辑基因组的模式图。

The Cas9 nuclease and the sgRNA matching the gene of interest are co-expressed using Agrobacterium tumefaciens as a vector inN. benthamiana leaves or transfected into protoplasts from Arabidopsis, wheat or rice. Then, the genomic DNA is extracted from the leaf tissues or protoplasts and subject to PCR-amplification with primers flanking the target site. The presence of Cas9/sgRNA-induced mutations can be easily detected using the restriction enzyme (RE) site loss method. The RE-resistant band (lane 3) can be cloned. The exact nature of the mutations is then revealed by sequencing individual clones.
cas9核酶与感兴趣的sgrna利用农杆菌作为载体在本生烟叶子上共表达，或转染拟南芥，小麦，水稻的原生质体。随后，从叶片上或原生质体提取基因组dna，pcr扩增包含靶标的基因。cas9/sgrna诱导的突变是否成功可以利用限制性酶酶切pcr片段，限制性酶丢失与否来检测是否成功，克隆限制性酶抗性片段。通过测序来验证突变效果。
编辑植物基因组cas9的各种变异体。

he Cas9 nuclease was expressed as a fusion protein with a tag (FLAG or GFP as indicated) under various constitutive promoters. Diagonal lines indicate an intron inserted into the Cas9 gene. NLS, nuclear localization signal.

sgrna基因组结构及识别机制

sgRNA is expressed under the U6 promoter inA. thaliana, N. benthamiana, O. sativa, T. aestivum and S. bicolor, and under the U3 promoter in O. sativa. The transcript initiation nucleotide in the case of U6p and U3p is “G” and “A”, respectively.
sgrna在u6启动子下表达，转录起始的核苷酸在u6p 和u3p分别是g和a
通过靶标pds中两个相邻序列来产生染色体缺失

A. Cartoon explaining setup of the experiment. B. Detection of deletion mutations using the AFLP analysis. Agarose gel shows PCR bands amplified across targets 1 and 2 using genomic DNA extracted from respective leaf samples. Cas9, sgRNA1 and 2 were expressed in N. benthamiana leaf tissue using the standard agroinfiltration protocol. In lane 2, Cas9/sgRNA1/sgRNA2 were expressed from three separate plasmids, while in lane 4 they were expressed from a single plasmid. C. Types of deletion mutations identified. Bottom PCR bands from lanes 2 and 4 were cloned into a high copy vector and 15 individual clones were sequenced. All clones contained deletions that can be grouped in three different types (m1-3).
a：实验设计，b：通过aflp分析来检测突变体

2014-01-24 16:43