|
|
|
| Study of Qualitative and Quantitative Detection Methods for Exogenous Gene Cas9 in Gene Editing |
| DING Lin1,*, WANG Hao-Qian2,*, ZHANG Min1, WANG Yu-Ling1, WANG Xiao-Fu1, CHEN Xiao-Yun1, XU Jun-Feng1, ZHANG Xiu-Jie2,**, PENG Cheng1,** |
1 Institute of Agro-product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China;
2 Development Center for Science and Technology, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Beijing 100176,China |
|
|
|
|
Abstract The prerequisites of bringing gene-edited products to market are effective supervision of them and eliminating public doubts in China. At present, Cas9 endonuclease is the most commonly used method of gene-editing technology, therefore, it is necessary to establish a qualitative and quantitative detection method for Cas9 endonuclease. In this study, the most commonly used Cas9 endonuclease exogenous sequence in gene-editing was used as the target site. The primers and probes of conventional PCR and qPCR were designed, and the most suitable primers and probes were selected through experiments. The system was optimized in the conventional PCR method to determine the optimal primer concentration and annealing temperature for subsequent experiments. By testing specificity and sensitivity, conventional PCR and qPCR detection methods were established for detecting the Cas9 in gene-editing. Meanwhile, the detection limits of qualitative and quantitative detection methods were determined. In the specificity experiment of detection method, the target band was amplified by conventional PCR method in the samples containing Cas9, but the samples without Cas9 endonuclease were negative results. The qPCR method obtained amplification curves in samples containing Cas9, while other samples without Cas9 endonuclease were not amplified; In the method sensitivity test, conventional PCR could detect 0.1% of Cas9 event, and limit of detection (LOD) of qPCR method could reach 16 copies of gene-edited rice genomic DNA; In the method stability experiment, the conventional PCR performed 67 repeated experiments by using the gene-edited samples with a mass fraction of 0.1%, and the experimental results all amplified the target bands. The qPCR method was repeated 60 times at the corresponding minimum detection limit concentration, and the experimental results all obtained the amplification curve. At the same time, this qualitative and quantitative method is applicable to different gene-edited crops, such as rice (Oryza sativa), soybean (Glycine max) and rape (Brassica napus). The qualitative and quantitative detection methods of Cas9 established in this study have good specificity, high sensitivity and strong stability. The establishment of this method will provide a certain technical support for the effective supervision of gene-edited products in the future.
|
|
Received: 28 September 2021
|
|
|
|
Corresponding Authors:
** zhxj7410@sina.com; pc_phm@163.com
|
| About author:: * These authors contributed equally to this work |
|
|
|
[1] 李铁民, 杜波. 2011. CRISPR-Cas系统与细菌和噬菌体的共进化[J]. 遗传, 33(03): 213-218.
(Li T M, Du B.2011. CRISPR-Cas system and coevolution of bacteria and phages[J]. Hereditas, 33(3): 213-218.)
[2] 刘晋冀, 杨洁鸿, 李莲军. 2017. 基因编辑技术的发展及在畜牧业中的应用[J]. 基因组学与应用生物学, 36(05): 1953-1956.
(Liu J J, Yang J H, Li L J.2017. The development of transgenic technology and its application in animal husbandry[J]. Genomics and Applied Biology, 36(05): 1953-1956.)
[3] 孟泽松, 王飞飞, 王光林, 等. 2016. CRISPR/Cas9基因编辑技术在肿瘤研究及治疗中的应用[J]. 肿瘤, 36(12): 1395-1401.
(Meng Z S, Wang F F, Wang G L, et al.2016. Application of CRISPR /Cas9 genome editing system in research and therapy of cancer[J]. Tumor, 36(12): 1395-1401.)
[4] 汪小福, 陈笑芸, 缪青梅, 等. 2016. 食品分析水平测试计划(FAPAS)转基因检测能力验证中的方法分析与质量控制[J]. 中国食品学报, 16(7): 224-230.
(Wang X F, Chen X Y, Miao Q M, et al.2016. Analysis and quality control for the detection of transgenic in FAPAS proficiency tests[J]. Journal of Chinese Institute of Food Science and Technology, 16(7): 224-230.)
[5] 汪秀秀, 杨捷琳, 宋青, 等. 2014. 转基因棉花GHB119品系特异性定量PCR检测方法的建立[J]. 农业生物技术学报, 22(03): 380-388.
(Wang X X, Yang J L, Song Q, et al.2014. Establishment of a novel event-specific quantitative PCR method for genetically modified cotton (Gossypium sp.) GHB119 detection[J]. Journal of Agricultural Biotechnology, 22(3): 380-388.)
[6] 徐俊锋, 王鹏飞, 李玥莹, 等. 2015. 转基因植物中CaMV35S和tNOS元件的4种定性PCR检测方法的比较[J]. 农业生物技术学报, 23(3): 397-407.
(Xu J F, Wang P F, Li Y Y, et al.2015. Comparison of four qualitative PCR- based detection methords of CaMV35S and tNOS elements in transgenic plants[J]. Journal of Agricultural Biotechnology, 23(3): 397-407.)
[7] 杨华, 彭城, 肖英平, 等. 2018. 转基因大豆SHZD32-1转化体普通PCR和qRT-PCR检测方法的研究[J]. 农业生物技术学报, 26(03): 492-501.
(Yang H, Peng C, Xiao Y P, et al.2018. Study of conventional PCR and qRT-PCR detection methods for genetically modified soybean (Glycine max) SHZD32-1[J]. Journal of Agricultural Biotechnology, 26(3): 492-501.)
[8] 姚祝平, 程远, 万红建, 等. 2017. CRISPR/Cas9基因编辑技术在植物基因工程育种中的应用[J]. 分子植物育种, 15(07): 2647-2655.
(Yao Z P, Cheng Y, Wang H J, et al.2017. Application of CRISPR/Cas9 genome editing technology in plant genetic engineering breeding[J]. Molecular Plant Breeding, 15(07): 2647-2655.)
[9] 曾秀英, 侯学文. 2015. CRISPR/Cas9基因组编辑技术在植物基因功能研究及植物改良中的应用[J]. 植物生理学报, 51(9): 1351-1358.
(Zeng X Y, Hou X W.2015. Application of CRISPR/Cas9 genome editing technology in functional genomics and improvement of plants[J]. Plant Physiology Journal Plant Physiology Journal, 51(9): 1351-1358.)
[10] 郑怀国, 赵静娟, 秦晓婧, 等. 2021. 全球作物种业发展概况及对我国种业发展的战略思考[J]. 中国工程科学, 23(04): 45-55.
(Zheng H G, Zhao J J, Qin X J, et al.2021. Overview of the global crop seed industry and strategic thinking on its development in China[J]. Strategic Study of CAE, 23(04): 45-55.)
[11] Bedell V M, Wang Y, Campbell J M, et al.2012. In vivo genome editing using a high efficiency TALENs[J]. Nature, 491(7422): 114-118.
[12] Deveau H, Garneau J E, Moineau S.2010. CRISPR/Cas system and its role in phage-bacteria interactions[J]. Annual Review of Microbiology, 64(1): 475-493.
[13] Ding Y D, Li H, Chen L L, et al.2016. Recent advances in genome editing using CRISPR/Cas9[J]. Frontiers in Plant Science, 7: 703.
[14] Emily Waltz.GABA-enriched tomato is frst CRISPR-edited food to enter market[J]. Nature Biotechnology,2022,40:9-11.
[15] Gupta R M, Musunuru K.2014. Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9[J]. The Journal of Clinical Investigation, 124(10): 4154-4161.
[16] Huang S W, Weigel D, Beachy R N, et al.2016. A proposed regulatory framework for genome-edited crops[J]. Nature Genetics, 48(2): 109-111.
[17] Ishino Y, Shinagawa H, Makino K, et al.1987. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product[J]. Journal of Bacteriology, 169(12): 5429-5433.
[18] Jansen R, Embden J D, Gaastra W, et al.2002. Identification of genes that are associated with DNA repeats in prokaryotes[J]. Molecular Microbiology, 43(6): 1565-1575.
[19] Jinek M, Chylinski K, Fonfara I, et al.2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 337(6096): 816-821.
[20] Kim Y G, Cha J, Chandrasegaran S, et al.1996. Hybrid restriction enzymes: Zinc finger fusions to Fok Ⅰ cleavage domain[J]. Proceedings of the National Academy of Sciences of the USA, 93(3): 1156-1160.
[21] Kleinstiver B P, Prew M S, Tsai S Q, et al.2015. Engineered CRISPR-Cas9 nucleases with altered PAM specificities[J]. Nature, 523(7561): 481-485.
[22] Makarova K S, Haft D H, Barrangou R, et al.2011. Evolution and classification of the CRISPR-Cas systems[J]. Nature Reviews Microbiology, 9(6): 467-477.
[23] Mali P, Esvelt K M, Church G M.2013. Cas9 as a versatile tool for engineering biology[J]. Nature Methods, 10(10): 957-963.
[24] Mojica F J, Diez-Villasenor C, Soria E, et al.2000. Biological significance of a family of regularly spaced repeats in the genomes of archaea, bacteria and mitochondria[J]. Molecular Microbiology, 36(1): 244-246.
[25] Mussolino C, Cathomen T.2013. RNA guides genome engineering[J]. Nature Biotechnology, 31(3): 208-209.
[26] Niu Y Y, Shen B, Cui Y Q, et al.2014. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos[J]. Cell, 156(4): 836-843.
[27] Pelletier S, Gingras S, Green D R.2015. Mouse genome engineering using CRISPR-Cas9 for study of immune function[J]. Immunity, 42(1): 18-27.
[28] Peng C, Wang H, Xu X, et al.2018. High-throughput detection and screening of plants modified by gene editing using quantitative real-time PCR[J]. The Plant Journal, 95(3):557-567.
[29] Peng C, Zheng M, Ding L, et al.2020. Accurate detection and evaluation of the gene-editing frequency in plants using droplet digital PCR[J]. Frontiers in Plant Science, 11:610790.
[30] Torres R, Martin M C, Garcia A, et al.2014. Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system[J]. Nature Communications, 5(1): 3964-3971.
[31] Waltz E.2016. Gene-edited CRISPR mushroom escapes US regulation[J]. Nature, 532(7599): 293-293.
[32] Yin K Q, Gao C X, Qiu J L.2017. Progress and prospects in plant genome editing[J]. Nature Plants, 3(8): 17107. |
|
|
|
