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.

Key words:
Genomic editing; mutation screening; OsLCT1; bell