P3 site-directed mutagenesis: An efficient method based on primer pairs with 3′-overhangs

Site-directed mutagenesis is a fundamental tool indispensable for protein and plasmid engineering. An important technological question is how to achieve the ideal efficiency of 100%. Based on complementary primer pairs, the QuickChange method has been widely used, but it requires significant improvements due to its low efficiency and frequent unwanted mutations. An alternative and innovative strategy is to utilize primer pairs with 3'-overhangs, but this approach has not been fully developed. As the first step toward reaching the efficiency of 100%, we have optimized this approach systematically (such as use of newly designed short primers, test of different Pfu DNA polymerases, and modification of PCR parameters) and evaluated the resulting method extensively with >100 mutations on 12 mammalian expression vectors, ranging from 7.0 to 13.4 kb in size and encoding ten epigenetic regulators linked to cancer and neurodevelopmental disorders. We have also tested the new method with two expression vectors for the SARS-CoV-2 spike protein. Compared to the QuickChange method, the success rate has increased substantially, with an average efficiency of ∼50%, with some at or close to 100%, and requiring much less time for engineering various mutations. Therefore, we have developed a new site-directed mutagenesis method for efficient and economical generation of various mutations. Notably, the method failed with a human KAT2B expression plasmid that possesses extremely GC-rich sequences. Thus, this study also sheds light on how to improve the method for developing ideal mutagenesis methods with the efficiency of ∼100% for a wide spectrum of plasmids.