Rapid Screening of CRISPR/Cas9-Induced Mutants Using the ACT-PCR Method.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system provides a technological breakthrough in targeted mutagenesis. However, a significant amount of time and cost is required to screen for the CRISPR/Cas9-induced mutants from a typically large number of initial samples. Here, we describe a cost-effective and sensitive screening technique based on conventional polymerase chain reaction (PCR), termed "annealing at critical temperature PCR" (ACT-PCR), for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants.