Using indCAPS to Detect CRISPR/Cas9 Induced Mutations

Background

Detecting mutations in biological samples is a common task in biological research. This is often performed using polymerase chain reaction (PCR) assays, where a sample’s genotype at one locus is determined by examining the size or sequence of amplified DNA. This protocol is a practical guide to using the indCAPS tool (Hodgens et al., 2017) available at http://indcaps.kieber.cloudapps.unc.edu/ to generate primers for PCR-based genotyping tasks. The indCAPS tool relies on a pair of techniques known as Cleaved Amplified Polymorphic Sequences (CAPS) and derived CAPS (dCAPS) (Neff et al., 1998), in which the genotype of a biological sample is determined by amplifying a region of DNA and assaying a diagnostic restriction site with a restriction enzyme. In a CAPS assay, the mutation of interest disrupts a known restriction site or creates a restriction site where one did not exist. In a dCAPS assay, a diagnostic site is created by introducing an intentional mismatch to one of the primers used to amplify the DNA, creating a restriction site in one genotype but not the other. Tools for generating primers for CAPS and dCAPS assays have been developed (Neff et al., 1998 and 2002) but are not compatible with insertion/deletion (indel) alleles such as those commonly acquired through CRISPR/Cas9 mutagenesis (Hodgens et al., 2017). With the recent rise of indel alleles in the literature due to CRISPR/Cas9 mutagenesis, a tool capable of generating dCAPS primers for indel alleles would make genotyping tasks easier. The indCAPS tool was developed to provide a tool for generating CAPS and dCAPS primers compatible with indels (Hodgens et al., 2017) and is intended to be easy to use and interpret for biologists designing genotyping assays. This protocol will describe the use of the indCAPS website for generating primers for genotyping a known polymorphism as well as detecting a novel mutation in a CRISPR/Cas9 mutagenesis experiment.