[1]朱丽颖,郑月萍,徐雪珍,等.一种准确、简便测定CRISPR/Cas9基因编辑效率的方法[J].江苏农业学报,2020,(02):299-305.[doi:doi:10.3969/j.issn.1000-4440.2020.02.007]
　ZHU Li-ying,ZHENG Yue-ping,XU Xue-zhen,et al.A convenient and accurate method for determining the efficiency of CRISPR/Cas9-based gene editing[J].,2020,(02):299-305.[doi:doi:10.3969/j.issn.1000-4440.2020.02.007]
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一种准确、简便测定CRISPR/Cas9基因编辑效率的方法()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2020年02期
页码:
299-305
栏目:
遗传育种·生理生化
出版日期:
2020-04-30

文章信息/Info

Title:
A convenient and accurate method for determining the efficiency of CRISPR/Cas9-based gene editing
作者:
朱丽颖1; 郑月萍1; 徐雪珍1; 段芊芊1; 韩妮莎2
（1.浙江农林大学农业与食品科学学院，浙江杭州311300；2.浙江农林大学林业与生物技术学院，浙江杭州311300）
Author(s):
ZHU Li-ying1; ZHENG Yue-ping1; XU Xue-zhen1; DUAN Qian-qian1; HAN Ni-sha2
(1.School of Agricultural and Food Sciences， Zhejiang A&F University， Hangzhou 311300, China;2.School of Forestry and Biotechnology， Zhejiang A&F University， Hangzhou 311300, China）
关键词:
CRISPR/Cas9; 基因编辑; 突变体筛选; 脂肪酸组分分析
Keywords:
CRISPR/Cas9; gene editing; mutant screening; fatty acid composition analysis
分类号:
Q784
DOI:
doi:10.3969/j.issn.1000-4440.2020.02.007
文献标志码:
A
摘要:
CRISPR/Cas9基因编辑技术是近年来发展迅速的一项基因定点编辑技术，但对特定CRISPR/Cas9基因编辑技术的编辑效率进行有效评估的方法仍十分匮乏，本研究的目的是建立一种准确、简便测定CRISPR/Cas9基因编辑效率的方法。在我们的方法中，将与种子专一表达启动子（2S3）连接的mCherry荧光蛋白报告基因作为选择基因，并以编码脂肪酸脱氢酶FAD2的基因为靶序列；然后对经CRISPR/Cas9编辑的拟南芥种子进行脂肪酸组分分析，根据其组分变化计算该方法的编辑效率。结果显示，利用mCherry荧光蛋白报告基因的种子专一表达特性，可简便快速地筛选阳性的初级转化子和后代的无转基因突变子；而种子油脂分析法能快速、准确地鉴定CRISPR/Cas9编辑的突变体，可以精确地检测出核酸长度小至单个碱基对的突变，用该方法获得的CRISPR/Cas9基因编辑效率（54.5%～74.1%）远高于基于聚丙烯酰氨凝胶电泳法获得的编辑效率（3.7%～15.4%）。说明，该方法检测CRISPR/Cas9基因编辑效率的高效性与可行性。
Abstract:
In recent years, CRISPR/Cas9 gene editing technology is a fast-growing gene targeted editing technology, but there are few effective methods the evaluate its editing efficiency. The purpose of this study was to establish an accurate and convenient method for determining the gene editing efficiency. In our assay, the mCherry fluorescent protein reporter gene under the control of a seed-specific promoter (2S3) was employed as a selection gene, and a gene encoding the fatty acid desaturase FAD2 was used as the target sequence for editing. The editing efficiency of the CRISPR/Cas9 method was subsequently determined through monitoring the alteration in the seed fatty acid composition of Arabidopsis plants subjected to the gene editing. The results showed that the seed-specific expression of the mCherry fluorescent protein reporter gene allowed us to quickly and easily screen for the positive primary transformants and the transgene-free progeny. Moreover, seed oil analysis enabled us to quickly and accurately identify the mutants edited by CRISPR/Cas9, including those with a single-base insertion or deletion. The efficiency of CRISPR/Cas9 based gene editing measured based on seed oil analysis (54.5%-74.1%) was much higher than that measured based on polyacrylamide gel electrophoresis analysis (3.7%-15.4%). The above results demonstrate the effectiveness and feasibility of the present method for determining the efficiency of CRISPR/Cas9 gene editing.

参考文献/References:

[1]MALI P, YANG L, ESVELT K M, et al. RNA-guided human genome engineering via Cas9[J]. Science, 2013, 339: 823-826.
[2]CONG L, RAN F A, COX D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339: 819-823.
[3]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]. Proc Natl Acad Sci USA, 2014, 111: 4632-4637.
[4]LI J F, ZHANG D, SHEEN J. Cas9-based genome editing in Arabidopsis and tobacco[J]. Methods Enzymol, 2014, 546: 459-472.
[5]MA X, ZHANG Q, ZHU Q, et al. A Robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants[J]. Mol. Plant, 2015, 8: 1274-1284.
[6]XIE K, MINKENBERG B, YANG Y, et al.CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system[J]. Proc Natl Acad Sci USA, 2015, 112: 3570-3575.
[7]GAO X, CHEN J, DAI X, et al. An effective strategy for reliably isolating heritable and Cas9-Free Arabidopsis mutants generated by crispr/cas9-mediated genome editing[J]. Plant Physiol, 2016, 171: 1794-1800.
[8]PAN C, YE L, QIN L, et al. CRISPR/Cas9 mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations[J]. Sci Rep, 2016, 6: 24765.
[9]DING D, CHEN K, CHEN Y, et al. Engineering introns to express RNA guides for Cas9- and Cpf1-mediated multiplex genome editing[J]. Mol Plant, 2018, 11: 542-552.
[10]VANLEUVEN A J, PARK S, MENKE D B, et al. A PAGE screening approach for identifying CRISPR-Cas9-induced mutations in zebrafish[J]. Biotechniques, 2018, 64(6): 275-278.
[11]XING H L, DONG L, WANG Z P, et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants[J]. BMC Plant Biol, 2014, 14(1): 327.
[12]ZHOU Y, ZHU S, CAI C, et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells[J]. Nature, 2014, 509(7501): 487-491.
[13]唐国，黄安群，孙志鹏，等. 三种CRISPR/Cas9基因敲除变异检测方法的比较分析[J]. 淡水渔业，2019，49(2)：20-26.
[14]SHAN Q, WANG Y, LI J, et al. Genome editing in rice and wheat using the CRISPR/Cas system[J]. Nat Protoc, 2014, 9(10): 2395-2410.
[15]DAHLEM T J, HOSHIJIMA K, JURYNEC M J, et al. 2012. Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome[J]. PLoS genetics, 8(8): e1002861.
[16]THOMAS H R, PERCIVAL S M, YODER B K, et al. High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis[J]. PLoS One, 2014, 9(12): e114632.
[17]兰博，谢秋巧，明建军，等. 比较T7E1和Surveyor核酸内切酶对于点突变的检测效率[J]. 广西医科大学学报，2015，32(6)：887-890.
[18]ZHU X, XU Y, YU S, et al. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system[J]. Sci Rep, 2014, 4: 6420.
[19]WANG Z P, XING H L, DONG L, et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation[J]. Genome Biol, 2015, 16: 144.
[20]郭勇，王玉成，王智博. 一种基于农杆菌介导的拟南芥瞬时转化技术优化[J]. 东北林业大学学报，2016，44(6)：41-44，83.
[21]王芳，李晓静，周延清. 聚丙烯酰胺凝胶电泳检测大豆SRAP标记[J]. 中州大学学报，2014，31(6)：125-128.
[22]徐明明，史卓维，郑璐侠，等. 蓖麻油、氢化蓖麻油及其聚氧乙烯衍生物中的主要脂肪酸的鉴别[J]. 药物分析杂志，2015，35(10)：1862-1865.
[23]马斯霜，白海波，惠建，等．CRISPR/Cas9技术及其在水稻和小麦遗传改良中的应用综述[J]．江苏农业科学，2019，47(20)：29-33.
[24]黄娟，邓国富，高利军，等. CRISPR/Cas9系统及其在作物育种中的应用[J]. 南方农业学报，2018, 49(1): 14-21.
[25]沈明晨，薛超，乔中英，等. CRISPR/Cas9系统在水稻中的发展和利用[J].江苏农业科学，2019,47(10):5-10.
[26]SPRUNCK S, RADEMACHER S, VOGLER F, et al. Egg cell-secreted EC1 triggers sperm cell activation during double fertilization[J]. Science, 2012, 338: 1093-1097.
[27]AL AMIN N, AHMAD N, WU N, et al. CRISPR-Cas9 mediated targeted disruption of FAD2-2 microsomal omega-6 desaturase in soybean (Glycine max.L)[J]. BMC Biotechnol, 2019, 19(1):9.
[28]LOU Y, SCHWENDER J, SHANKLIN J. FAD2 and FAD3 desaturases form heterodimers that facilitate metabolic channeling in vivo[J]. J Biol Chem, 2014, 289(26): 17996-18007.

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备注/Memo

备注/Memo:
收稿日期：2019-12-25基金项目：科技部转基因重大专项（2016ZX08010003-003）作者简介：朱丽颖（1994-），女，河南郑州人，硕士研究生，主要从事植物生物技术研究。(E-mail)zhuly_6545@163.com通讯作者：郑月萍，(E-mail)zyp860819@126.com
更新日期/Last Update: 2020-05-18