Yang, X.-J.

Site-directed mutagenesis is a basic molecular tool required for protein and plasmid engineering. We have recently adopted an innovative strategy using mutagenic primer pairs with 3-prime overhangs and developed an optimized protocol, referred to as P3 site-directed mutagenesis, succeeding with many plasmids at an efficiency of ~50%. This raises the important question how to enhance the efficiency to the ideal level of 100%. Here we report P3a site-directed mutagenesis with an efficiency at or close to 100% and have evaluated this improved method systematically by engineering >100 point mutations and small deletions (or insertions) on >20 mammalian expression vectors encoding various epigenetic regulators and the spike protein of SARS-COV-2. Moreover, all known mutagenesis methods are limited to point mutations and small deletions/insertions (like several nucleotides), raising the technical problem how to carry out efficient and seamless cassette mutagenesis for replacement, deletion or insertion of DNA fragments. Accordingly, the high efficiency of P3a mutagenesis and the hand-shaking feature of primer pairs with 3-prime overhangs have inspired us to adapt this method for efficient and seamless cassette mutagenesis, including high efficiency in epitope tagging and untagging, deletion of small and large DNA fragments and insertion of oligonucleotide duplexes (such as LoxP sites and sgRNA-coding sequences). Thus, this new site-specific or cassette mutagenesis method is highly efficient, likely resulting in its wide use for biomedical research.