Development of methods for effective identification of CRISPR/Cas9-induced indels in rice

Abstract

Two methods, PCR and amplicon labeling based, were developed and successfully applied to reliably detect CRISPR/Cas9 induced indels in rice. The use of CRISPR/Cas9 has emerged as a powerful nuclease-based genome editing tool in several model organisms including plants for mutagenesis by inducing precise gene editing through efficient double strand DNA breaks (DSBs) at the target site and subsequent error-prone non-homologous end joining (NHEJ) repair, leading to indel mutations. Different molecular methods including enzymatic mismatch cleavage (EMC), high-resolution melting curve analysis (HRMA) and conventional polymerase chain reaction (PCR) combined with ligation detection reaction (LDR) have been developed to quick identify CRISPR/Cas9 induced mutations. However, their intrinsic drawbacks limit their application in the identification of indel mutants in plants. Here we present two methods (one simple PCR based and the other amplicon labeling based) for effective and sensitive detection of CRISPR/Cas9 induced indels in rice. In PCR-based method, targets were amplified using two pairs of primers for each target locus and visualized on gel electrophoresis, while in amplicon labeling-based method, targets were amplified using tri-primers (with one a universal 6-FAM 5'-labelled) and detected by DNA capillary electrophoresis. Both methods can accurately define indel sizes down to ± 1 bp, and are amenable for high throughput analysis, therefore, will significantly facilitate the identification of indel mutants generated by CRISPR/Cas9 for further functional analysis and breeding in rice and other plants.

Keywords: Amplicon labeling; CRISPR/Cas9; Indel mutation; Inner/Outer primer pair; Tri-primers.