An Efficient and Cost-Effective Novel Strategy for Identifying CRISPR-Cas-Mediated Mutants in Plant Offspring

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system has revolutionized targeted mutagenesis, but screening for mutations in large sample pools can be time-consuming and costly. We present an efficient and cost-effective polymerase chain reaction (PCR)-based strategy for identifying edited mutants in the T₁ generation. Unlike previous methods, our approach addresses the challenges of large progeny populations by using T₀ generation sequencing results for genotype prediction. The T₁ generation plants were then divided into two scenarios: ≥4 bp indels and 1–2 bp indels. Specific primers are designed for these categories, employing dual-primers critical annealing temperature PCR for ≥4 bp indels and the derived cleaved amplified polymorphic sequences (dCAPS) method for 1–2 bp indels. This method is straightforward, cost-effective, and allows rapid and precise identification of T₁ editing outcomes, distinguishing between wild-type, heterozygous, and homozygous plants. This strategy accelerates gene functional analysis in plants and beyond.