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 -ho-mologous 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 identifi-cation of the mutant allele and can be used for genotyping offspring during sub-sequent breeding for colony establishment.For complete details on the use and execution of this protocol, please refer to Vasu et al. (2021).