MultiSite Gateway Technology Is Useful for Donor DNA Plasmid Construction in CRISPR/Cas9-Mediated Knock-In System

Abstract

The clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 method is a powerful tool for genome editing, by introducing a DNA double-strand break (DSB) at the specific site. The gene knock-out can be achieved by the deletion or inser - tion at the CRISPR/Cas9-mediated DSB site by error-prone nonhomologous end joining repair in targeted cells. However, the gene knock-in is still difficult as compared to the knock-out, because of the low efficiency of homology directed repair with donor DNA in cells. Therefore, to efficiently select the knock-in cells, we developed a complicated donor DNA plasmid containing an antibiotic-resistance gene, in addition to the knock-in sequence and the two homology arms. MultiSite Gateway technology is a useful tool for constructing this complicated plasmid. We describe the MultiSite Gateway technology and provide an overview of the DSB repair pathways to clarify the knock-out and knock- in methods by the CRISPR/Cas9 system.