In vitro Argonaute cleavage-mediated quantitative PCR facilitates versatile CRISPR/Cas-induced mutant analysis

CRISPR/Cas9 and associated base editing systems are used to precisely modify target genes in diverse cell types and organisms. However, CRISPR/Cas-induced mutants often contain small insertions and deletions (indels) or single nucleotide variations (SNVs) at edited sites with different genotypes, and gene editing frequency (GEF) typically varies between editing sites and editing systems. Therefore, methods are needed to identify mutants and their genotypes, and evaluate GEF. Herein, we describe a novel SMART approach for simultaneous analysis of CRISPR/Cas-induced mutants, genotypes and GEF, using DNA-guided Thermus thermophilus Argonaute (TtAgo) in vitro cleavage-mediated quantitative real-time PCR (qPCR) or digital PCR. SMART proved versatile for induced mutant identification, genotyping, and GEF evaluation using CRISPR/Cas9 gene-editing offspring lines harbouring the rice RNA-directed DNA methylation 3-like (RdDM3l) gene. SMART is higher specific, sensitive, and accurate than previously methods. SMART detected all types of mutations, including small indels, large indels and nucleotide substitutions, with good sensitivity regardless of mutation type. TtAgo has few strict requirements or restrictions for target sequences, such as restriction sites, GC content or protospacer-adjacent motifs (PAMs). SMART facilitates versatile CRISPR/Cas-induced mutant screening, genotyping, and GEF quantification, and it could be developed for clinical detection of rare mutations involving SNVs and indels.