Genetic Engineering by DNA Recombineering

Abstract

Recombineering inserts PCR products into DNA using homologous recombination. A pair of short homology arms (50 base pairs) on the ends of a PCR cassette target the cassette to its intended location. These homology arms can be easily introduced as 5 primer overhangs during the PCR reaction. The flexibility to choose almost any pair of homology arms enables the precise modification of virtually any DNA for purposes of sequence deletion, replacement, insertion, or point mutation. Recombineering often offers significant advantages relative to previous homologous recombination methods that require the construction of cassettes with large homology arms, and relative to traditional cloning methods that become intractable for large plasmids or DNA sequences. However, the tremendous number of variables, options, and pitfalls that can be encountered when designing and performing a recombineering protocol for the first time introduce barriers that can make recombineering a challenging technique for new users to adopt. This article focuses on three recombineering protocols we have found to be particularly robust, providing a detailed guide for choosing the simplest recombineering method for a given application and for performing and troubleshooting experiments. C 2019 by John Wiley & Sons, Inc.Keywords: Escherichia coli r genetic engineering r homologous recombination r recombineering r viral genome engineering

Expanded MutaT7 toolkit efficiently and simultaneously accesses all possible transition mutations in bacteria

“…Mutagenic E. coli strains were engineered using lambda red recombineering strategies for large seamless insertions or gene replacements, as described by Papa and Shoulders ( 33 ) and summarized in the Supplementary Data. The strains Δ ung and MutaT7 C→T (previously termed ‘MutaT7 strain’) were constructed and described by Moore et al.…”

“…The MP6 strain used in this work was derived by transforming the Δ ung strain with the MP6 plasmid ( 7 ). All other strains (TadA7.10 only, TadA8e only, MutaT7 A→G , eMutaT7 A→G , Dual7 and Dual8) were specifically constructed for this work via two-step lambda red recombineering ( 33–35 ). In the first step of this process, a kan-ccdB selection–counterselection cassette is integrated into the desired genomic locus.…”

ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes

“…The subsequent generation of transgenic cell lines based on these libraries has further been used to precisely determine the cellular localization and the quantitative interactome of more than 1,000 proteins ( Hubner et al, 2010 ; Hutchins et al, 2010 ; Hein et al, 2015 ). On the contrary, current techniques to introduce point mutations in BACs still require more intensive work, whether by a counterselection-based two-step procedure ( Bird et al, 2011 ; Wang et al, 2014 ; Näsvall, 2017 ; Papa and Shoulders, 2019 ), a lower efficiency one-step procedure requiring extensive PCR screening ( Lyozin et al, 2014 ), or CRISPR-guided methods ( Pyne et al, 2015 ). Here, we present a simple and efficient one-step procedure to introduce point mutations in BAC transgenes, harnessing introns to carry selectable markers, which reduces the time and cost of generating mutagenized constructs.…”