基于高分辨率熔解曲线技术的CRISPR/Cas9介导的细胞基因突变快速检测研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-1477

摘要/Abstract

摘要：

旨在建立一种通过高分辨率熔解曲线（high-resolution melting，HRM）分析技术快速检测CRISPR/Cas9介导的细胞基因突变的方法。采用野生型、纯合突变及杂合突变小鼠胚胎干细胞优化和完善HRM检测条件，然后应用建立的HRM方法对30个测序结果已知的CRISPR/Cas9技术编辑过的小鼠胚胎干细胞单克隆进行检测，根据熔解温度与熔解曲线的差异区分野生型、纯合突变型及杂合突变型，以验证HRM方法的可行性和准确性。结果表明，优化的HRM检测方法能够鉴别野生型、纯合突变型及杂合突变型小鼠胚胎干细胞。应用建立的HRM方法分析30个CRISPR/Cas9技术编辑过的小鼠胚胎干细胞单克隆，结果显示，14个单克隆为杂合突变型、2个单克隆为纯合突变型、14个单克隆为野生型，与测序结果一致，准确率为100%。本研究建立的高分辨率熔解曲线法能对CRISPR/Cas9介导的细胞基因突变进行快速筛选，是一种灵敏、准确、简便、高通量的方法。

关键词: 高分辨率熔解曲线, CRISPR/Cas9, 细胞基因突变, 检测

Abstract:

This study is aimed to establish a method for rapid detection of CRISPR/Cas9-mediated gene mutations in cells using high-resolution melting（HRM）assay. The condition of HRM analysis was optimized and perfected using wild-type mouse embryonic stem cells（mESCs），homozygous mutation mESCs and heterozygous mutation mESCs. In order to verify the feasibility and accuracy of HRM method，the established HRM analysis was used to detect 30 monoclones of CRISPR/Cas9-edited mESCs with known their sequencing results. According to the difference of melting temperature and melting curve，wild-type mESCs，homozygous mutation mESCs and heterozygous mutation mESCs were distinguished. The results showed that the optimized HRM assay identified wild-type mESCs，homozygous mutation mESCs and heterozygous mutation mESCs. Thirty monoclones of mESCs edited by CRISPR/Cas9 technique were analyzed using the established HRM method. The results indicated that 14 monoclones were heterozygous mutation mESCs，2 monoclones were homozygous mutation mESCs and 14 monoclones were wild-type mESCs. These results were consistent with the sequencing results at accuracy of 100%.The high-resolution melting assay established in this study is a sensitive，accurate，simple and high-throughput method，and may rapidly scan CRISPR/Cas9-mediated gene mutations in cells.

Key words: high-resolution melting, CRISPR/Cas9, gene mutation in cells, detection

陈黎, 卢茜, 杨红兰, 张鹏, 何志旭. 基于高分辨率熔解曲线技术的CRISPR/Cas9介导的细胞基因突变快速检测研究[J]. 生物技术通报, 2022, 38(10): 90-96.

CHEN Li, LU Xi, YANG Hong-lan, ZHANG Peng, HE Zhi-xu. Methodological Research on Rapid Detection of CRISPR/Cas9-Mediated Gene Mutations in Cells Based on High-resolution Melting Technique[J]. Biotechnology Bulletin, 2022, 38(10): 90-96.

图/表 7

图1 野生型PCR扩增产物为394 bp时，野生型、纯合突变型及杂合突变型高分辨率熔解曲线分析图

Fig.1 Analysis of high-resolution melting of wild-type mESCs，homozygous mutation mESCs and heterozygous mutation mESCs when the PCR product of wild-type mESCs is 394 bp

图2 野生型PCR扩增产物为91 bp时，野生型、纯合突变型及杂合突变型高分辨率熔解曲线分析图

Fig.2 Analysis of high-resolution melting of wild-type mE-SCs，homozygous mutation mESCs and heterozygous mutation mESCs when the PCR product of wild-type mESCs is 91 bp

图3 野生型、杂合突变型和纯合突变型的测序结果

Fig.3 Sequencing results of wild-type mESCs，heteroz-ygous mutation mESCs and homozygous mutation mESCs

图4 代表性野生型、杂合突变型和纯合突变型的测序结果

Fig. 4 Sequencing results of typical wild-type mESCs，he-terozygous mutation mESCs and homozygous mut-ation mESCs

图5 30例CRISPR/Cas9技术编辑过的小鼠胚胎干细胞单克隆熔解温度结果横坐标代表熔解温度，纵坐标代表30个不同的小鼠胚胎干细胞单克隆，其中C7-B7为野生型单克隆，B6-C8为杂合突变型单克隆，D7、C2为纯合突变型单克隆

Fig.5 Results of melting temperature of 30 monoclones of mESCs edited by CRISPR/Cas9 techniqe The abscissa refers to the melting temperature，and the vertical axis refers to 30 different monoclones of mESCs，among which monoclones C7-B7 are wild-type mESCs，monoclones B6-C8 are heterozygous mutation mESCs，and monoclones D7 and C2 are homozygous mutantion mESCs

图6 30例CRISPR/Cas9技术编辑过的小鼠胚胎干细胞单克隆高分辨率熔解曲线图

Fig.6 High-resolution melting curve of 30 monoclones of mESCs edited by CRISPR/Cas9 technique

表1 3种检测CRISPR/Cas9基因编辑产物方法比较

Table 1 Comparison of three methods of detecting gene products edited by CRISPR/Cas9 technique

方法 Method	检测SNP Detecting SNP	灵敏度 Sensitivity	检测周期 Detection period	检测成本 Detection cost	检测步骤 Detection steps	高通量方法 High-throughput method
酶错配切割方法 Mismatch cleavage assays	能Yes	低Low	较长 A little long	低Low	繁琐Complex	否No
DNA测序法Sanger sequencing	能Yes	高High	长Long	高High	繁琐Complex	否No
高分辨率熔解曲线法HRMA	能Yes	高High	短Short	低Low	简单Simple	是Yes

参考文献 23

[1]	Gasanov EV, Jędrychowska J, Pastor M, et al. An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique[J]. Mol Biol Rep, 2021, 48(2):1951-1957. doi: 10.1007/s11033-020-06125-8 pmid: 33481178
[2]	Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157(6):1262-1278. doi: S0092-8674(14)00604-7 pmid: 24906146
[3]	胡廷栋, 王蒙, 刘晓蕊, 等. 利用CRISPR/Cas9建立PIN1基因敲除的成体神经干细胞系[J]. 南京医科大学学报:自然科学版, 2021, 41(4):483-488, 521.
	Hu TD, Wang M, Liu XR, et al. Establishment of neural stem cells line with PIN1 gene knockout by CRISPR/Cas9[J]. J Nanjing Med Univ Nat Sci, 2021, 41(4):483-488, 521.
[4]	Druml B, Cichna-Markl M. High resolution melting(HRM)analysis of DNA—its role and potential in food analysis[J]. Food Chem, 2014, 158:245-254. doi: 10.1016/j.foodchem.2014.02.111 URL
[5]	温鹏强, 王国兵, 陈占玲, 等. 应用高分辨率熔解曲线分析快速筛查和诊断citrin缺陷导致的新生儿肝内胆汁淤积症[J]. 中华医学遗传学杂志, 2012, 29(2):167-171.
	Wen PQ, Wang GB, Chen ZL, et al. Utilization of high-resolution melting analysis to screen patients with neonatal intrahepatic cholestasis caused by citrin deficiency[J]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 2012, 29(2):167-171.
[6]	Tricarico R, Crucianelli F, Alvau A, et al. High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene[J]. BMC Cancer, 2011, 11:305. doi: 10.1186/1471-2407-11-305 pmid: 21777424
[7]	Marotta RV, Turri O, Morandi A, et al. High resolution melting analysis to genotype the most common variants in the HFE gene[J]. Clin Chem Lab Med, 2011, 49(9):1453-1457. doi: 10.1515/CCLM.2011.237 pmid: 21627541
[8]	Bodnar GC, Martins HM, de Oliveira CF, et al. Comparison of HRM analysis and three REP-PCR genomic fingerprint methods for rapid typing of MRSA at a Brazilian hospital[J]. J Infect Dev Ctries, 2016, 10(12):1306-1317. doi: 10.3855/jidc.7887 URL
[9]	Chambliss AB, Resnick M, Petrides AK, et al. Rapid screening for targeted genetic variants via high-resolution melting curve analysis[J]. Clin Chem Lab Med, 2017, 55(4):507-516. doi: 10.1515/cclm-2016-0603 pmid: 27732553
[10]	Reed GH, Wittwer CT. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis[J]. Clin Chem, 2004, 50(10):1748-1754. pmid: 15308590
[11]	Switzeny OJ, Christmann M, Renovanz M, et al. MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response[J]. Clin Epigenetics, 2016, 8:49. doi: 10.1186/s13148-016-0204-7 pmid: 27158275
[12]	Wang XM, Shi H, Zhou JJ, et al. Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation[J]. J Genet Genomics, 2020, 47(5):249-261. doi: S1673-8527(20)30086-2 pmid: 32703661
[13]	张鹏, 杨红兰, 刘含, 等. 甲基结合蛋白1对小鼠胚胎干细胞增殖及克隆形态的影响[J]. 贵州医科大学学报, 2019, 44(6):621-625.
	Zhang P, Yang HL, Liu H, et al. Effect of methyl-CpG binding domain protein 1 on the colony morphology and proliferation of mouse embryonic stem cells[J]. J Guizhou Med Univ, 2019, 44(6):621-625.
[14]	Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and Archaea[J]. Nature, 2012, 482(7385):331-338. doi: 10.1038/nature10886 URL
[15]	Nelles DA, Fang MY, O’Connell MR, et al. Programmable RNA tracking in live cells with CRISPR/Cas9[J]. Cell, 2016, 165(2):488-496. doi: 10.1016/j.cell.2016.02.054 pmid: 26997482
[16]	龚美玲, 张琳琳, 郑翠侠. 利用CRISPR/Cas9系统对人A549肺癌细胞NRF2基因的稳定敲除及其功能研究[J]. 中国癌症杂志, 2019, 29(11):855-861.
	Gong ML, Zhang LL, Zheng CX. Stable knockout of NRF2 gene in human A549 lung cancer cells by CRISPR/Cas9 system and its functional research[J]. China Oncol, 2019, 29(11):855-861.
[17]	Gupta D, Bhattacharjee O, Mandal D, et al. CRISPR-Cas9 system:a new-fangled dawn in gene editing[J]. Life Sci, 2019, 232:116636. doi: 10.1016/j.lfs.2019.116636 URL
[18]	Memi FN, Ntokou A, Papangeli I. CRISPR/Cas9 gene-editing:research technologies, clinical applications and ethical considerations[J]. Semin Perinatol, 2018, 42(8):487-500. doi: 10.1053/j.semperi.2018.09.003 URL
[19]	Janik E, Niemcewicz M, Ceremuga M, et al. Various aspects of a gene editing system-CRISPR-Cas9[J]. Int J Mol Sci, 2020, 21(24):9604. doi: 10.3390/ijms21249604 URL
[20]	Vouillot L, Thélie A, Pollet N. Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases[J]. G3(Bethesda), 2015, 5(3):407-415.
[21]	Zischewski J, Fischer R, Bortesi L. Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases[J]. Biotechnol Adv, 2017, 35(1):95-104. doi: S0734-9750(16)30158-6 pmid: 28011075
[22]	Martín-Núñez GM, Gómez-Zumaquero JM, Soriguer F, et al. High resolution melting curve analysis of DNA samples isolated by different DNA extraction methods[J]. Clin Chimica Acta, 2012, 413(1/2):331-333. doi: 10.1016/j.cca.2011.09.014 URL
[23]	Pham QT, Raad S, Mangahas CL, et al. High-throughput assessment of mutations generated by genome editing in induced pluripotent stem cells by high-resolution melting analysis[J]. Cytotherapy, 2020, 22(10):536-542. doi: S1465-3249(20)30754-4 pmid: 32768274