HRM-facilitated rapid identification and genotyping of mutations induced by CRISPR/Cas9 mutagenesis in rice

Abstract The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) technology has recently emerged as a powerful genomic editing tool with great potential for crop breeding. However, commonly used protocols for screening of CRISPR/Cas9-induced mutations are laborious, time-consuming, and costly. In the present study we examined the applicability of high resolution melting (HRM) analysis fast screening CRISPR/Cas9-induced mutations in T0 plants and subsequent genotyping of T1 populations. Comparative analysis demonstrated that HRM analysis could identify mutant T0 plants carrying various types of mutation, including single nucleotide substitutions and short insertion/deletions, with false positive/negative rates of 0-2.78%. Furthermore, T1 plants derived from single T0 plants could be correctly genotyped by HRM analysis using WT parents and T0 plants as controls. We hence recommend the adoption of HRM analysis in CRISPR/Cas9 mediated genetic studies and breeding in rice and other crop species.

摘要 成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeat，CRISPR）/CRISPR相关蛋白（CRISPR-associated，Cas）技术近年来已成为一种强大的基因组编辑工具，在作物育种中展现出巨大应用潜力。然而，当前常用的CRISPR/Cas9诱导突变筛选流程往往费力、耗时且成本高昂。本研究探究了高分辨率熔解曲线分析（high resolution melting，HRM）在T0代植株中快速筛选CRISPR/Cas9诱导突变，并用于后续T1代群体基因分型的适用性。对比分析结果显示，HRM分析可鉴定出携带各类突变的T0代突变植株，包括单核苷酸替换与短插入/缺失变异，其假阳性/假阴性率为0~2.78%。此外，以野生型亲本及T0代植株作为对照，HRM分析可正确对单株T0代衍生的T1代植株进行基因分型。因此，我们建议在水稻及其他作物的CRISPR/Cas9介导遗传研究与育种工作中采用HRM分析技术。