P3a site-specific and cassette mutagenesis for seamless protein and plasmid engineering

Site-directed mutagenesis is a basic molecular tool required for protein and plasmid engineering. We have recently adopted an innovative strategy using primer pairs with 3-prime overhangs and developed an optimized protocol, referred to as P3 site-directed mutagenesis, with 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 systematically evaluated this improved method 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 (typically up to a dozen nucleotides), raising the technical problem how to carry out cassette mutagenesis for replacement, deletion or insertion of large DNA fragments. Accordingly, the high efficiency of P3a mutagenesis and the handshaking 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 and cassette mutagenesis method is highly efficient, likely resulting in its wide use for biomedical research.