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

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.