ESTABLISHMENT OF A RAPID SCREENING METHOD FOR IN VITRO SITE-DIRECTED MUTANTS OR CRISPR/Cas9-MEDIA-TED KNOCK-IN MUTANTS

Abstract

Objective To establish a rapid screening method for in vitro site-directed mutants or CRISPR/Cas9-mediated knock-in mutants, and to improve mutant screening efficiency and reduce experimental costs. Methods Polymerase chain reactions (PCRs) were carried out with primers designed with the last one, two, and three nucleotides in the 3' end matching the mutated DNA sequences for the constructed in vitro site-directed mutants or CRISPR/Cas9-mediated knock-in mutants, and agarose gel electrophoresis was used to detect specific amplification bands. Results PCR using specific mutation screening primers effectively amplified DNA bands in correct mutants but failed in unsuccessfully mutated samples. The presence of DNA amplification bands revealed by agarose gel electrophoresis confirmed the successful screening for desired mutants. Conclusion The rapid screening method for mutants introduced in this study can easily and quickly identify correct mutants, which significantly simplifies the identification process of mutations, and thereby reduces experimental costs and improves work efficiency.

Keywords

• polymerase chain reaction|mutagenesis, site-directed|mutation|crispr/cas9|gene knock-in techniques|mutagenicity tests