Methodological Research on Rapid Detection of CRISPR/Cas9-Mediated Gene Mutations in Cells Based on High-resolution Melting Technique

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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

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

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

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

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