A PCR-Induced Mutagenesis-Restriction Fragment Length Polymorphism Method for the Detection of CRISPR-Induced Indels.

Abstract

As CRISPR-based technologies are widely used for knocking out genes in cell lines and organisms, there is a need for the development of reliable, cost-effective, and fast methods that identify fully mutated clones. In this context, we present a novel strategy named PCR-induced mutagenesis-restriction fragment length polymorphism (PIM-RFLP), which is based on the well-documented robustness and simplicity of the classical PCR-RFLP approach. PIM-RFLP allows the assessment of the editing efficiency in pools of edited cells and the effective identification of fully mutated single-cell clones. It is based on the creation by mutagenic PCR of a restriction enzyme degenerate cleavage site in the PCR product of the wild-type allele, which can then be distinguished from the indel-containing alleles following the standard RFLP procedure. PIM-RFLP is highly accessible, can be executed in a single day, and appears to outperform Sanger sequencing deconvolution algorithms in the detection of fully mutated clones.