Screening of CRISPR-Cas9-generated point mutant mice using MiSeq and locked nucleic acid probe PCR

CRISPR-Cas9-mediated, site-directed mutagenesis in mice generates mosaic founder mice with varied efficiency of desired point mutation and other non-homologous end-joined variants. Here, we present a protocol for design, sample preparation, and analysis for identification of mice with the desired mutation. Deep sequencing provides the proportion of reads of a particular allele for each mouse line. Locked nucleic acid probe-based qPCR provides rapid identification of the mutant allele and can be used for genotyping offspring during subsequent breeding for colony establishment. For complete details on the use and execution of this protocol, please refer to Vasu et al. (2021).