Mousavi, N.; Zhou, E.; Razavi, A.; Ebrahimi, E.; Varela-Castillo, P.; Yang, X.-J.

Site-directed mutagenesis is a fundamental tool indispensable for protein and plasmid engineering. An important technological question is how to achieve the efficiency at the ideal level 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-prime overhangs, but this approach has not been fully developed. As the first step towards reaching the efficiency of 100%, we have optimized this pproach systematically and evaluated the resulting method extensively with >100 mutations on 12 mammalian expression vectors, ranging from 7.0-13.4 kb in size and encoding ten epigenetic regulators with links 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, in this study, we have developed a new site-directed mutagenesis method for efficient, versatile and economical generation of various mutations. Importantly, the extensive experience from this study also sheds light on how to develop ideal mutagenesis methods with the efficiency at or close to 100% for a wide spectrum of plasmids.