Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension

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. Simple cloning by prolonged overlap extension utilizes overlapping PCR fragments to create large repetitive multimers, which can be used to directly transform bacteria. Internal circularization of these long concatemers generates the desired plasmid.