We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-mediated homologous repair.
Keywords: donor template generation; gene editing; multiple site-directed mutagenesis; prolonged overlap extension; seamless cloning; seamless plasmid assembly.