A PCR-RFLP method for the detection of CRISPR-induced indels

Abstract

CRISPR-based technologies have revolutionised genome editing and are widely used for knocking out genes in cell lines and organisms. From a practical perspective, a critical factor that largely influences the successful outcome of CRISPR gene knockout experiments is the reliable and fast identification of fully mutated cells carrying exclusively null alleles of the target gene. Here we describe a novel strategy based on the well-documented reliability and simplicity of the classical PCR-Restriction Fragment Length Polymorphism (RFLP), which allows the assessment of the editing efficiency in pools of edited cells and the effective identification of cell clones that carry exclusively mutated alleles. This fast and cost-effective method, named PIM-RFLP (PCR Induced Mutagenesis-RFLP), is executed in two steps. In the first step, the editing target is amplified by a set of mutagenic primers that create a restriction enzyme degenerate cleavage site in the amplification product of the wild type allele. As a proof of principle, we chose the XcmI restriction site because it is especially suitable since it has the particularity of containing nine centrally placed non-specific nucleotides. This gives great flexibility in the mutagenic primers design and allows for efficient execution of the mutagenic PCR. In the second step, the evaluation of the editing efficiency in pools of edited cells or the identification of fully mutated single-cell derived clones is achieved following the standard procedure for any PCR-RFLP assay: digestion of the PCR products and analysis of the restriction fragments in an agarose gel.