RT Journal Article SR Electronic T1 ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes JF Life Science Alliance JO Life Sci. Alliance FD Life Science Alliance LLC SP e202000836 DO 10.26508/lsa.202000836 VO 4 IS 2 A1 Arnaud Rondelet A1 Andrei Pozniakovsky A1 Devika Namboodiri A1 Richard Cardoso da Silva A1 Divya Singh A1 Marit Leuschner A1 Ina Poser A1 Andrea Ssykor A1 Julian Berlitz A1 Nadine Schmidt A1 Lea Röhder A1 Gerben Vader A1 Anthony A Hyman A1 Alexander W Bird YR 2021 UL https://www.life-science-alliance.org/content/4/2/e202000836.abstract AB Bacterial artificial chromosome (BAC)–based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. Although homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here, we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call exogenous/synthetic intronization (ESI) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.