Seamless and Highly Efficient Site‐directed Mutagenesis for Protein, RNA, and Plasmid Engineering

Abstract Site‐directed mutagenesis is indispensable for protein, RNA, and plasmid engineering. It is ideal to carry out such mutagenesis at an efficiency close to 100%, but many current methods fail to reach this goal and thus require extensive screening efforts. We have recently optimized an innovative site‐specific mutagenesis approach based on PCR with primer pairs possessing 3’‐overhangs, thereby reaching the ideal efficiency of ∼100%. Such high efficiency and the incorporation of the “handshaking” feature of primer pairs with 3’‐overhangs have led us to adapt this method for seamless cassette mutagenesis, thereby conferring highly efficient deletion (up to 5 kb), insertion (up to 0.4 kb) or replacement of DNA fragments. Conceptually, deletion and insertion are special cases of replacement mutations, where the respective sequences to be inserted and deleted are 0 bp. This is because replacement mutagenesis converts fragment A to fragment B; for deletion, fragment B is 0 bp in size, whereas for insertion, fragment A is 0 bp. Thus, this new method makes site‐directed and cassette mutagenesis a highly efficient and reliable tool for protein, RNA and plasmid engineering in different types of biomedical research. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : P3a site‐directed mutagenesis to introduce point mutations, deletions, insertions or replacements Basic Protocol 2 : Transformation of chemically competent DH5α cells to obtain bacterial colonies Basic Protocol 3 : Isolation of plasmids from bacterial colonies for sequence analysis to identify clones with designed mutations Alternate Protocol : P3 site‐directed mutagenesis to introduce deletions, insertions, or replacements