Continuous target-specific mutagenesis and rapid gene evolution by diversity-generating retroelements in Escherichia coli

Continuous site-directed mutagenesis and selection in Escherichia coli

The programmed diversification of genes and other encoded genetic elements, through site directed- and site saturation-mutagenesis, underpins approaches to learning the relationship between DNA sequence and function, and forms a foundation for creating new function through directed evolution. However, current approaches for generating large genetic libraries commonly generate diversity in vitro and then transform the resulting library into cells; this multistep process is inefficient and this paradigm places limits on the scale of diversity that can be achieved and the size of diversified genetic elements that can be introduced into cells. Here we demonstrate a distinct paradigm for library generation through: 1) efficient transfer of genetic elements, as cargos in F plasmids, to recipient cells bearing libraries of retron editors, 2) efficient editing of cargo genes in recipient cells, and 3) continuous iteration of the conjugation-editing cycles with selection for recipients in sequential cycles using three selection markers in series. In this paradigm, the library diversity emerges multiplicatively through the iteration of conjugation-editing cycles. Using this paradigm, we generated substantial libraries that enabled the selection of new phenotypes, with library members containing up to six distinct edits and edits arising from several conjugation-editing cycles.

Exploring the Evolutionary Landscape with Targeted In Vivo Hypermutations

In this review, a review of recent in vivo hypermutation tools that enable rapid sampling of the vast evolutionary landscape, all while supporting simultaneous selection of the best proteins within living organisms are discussed.

CRISPR/Cas9-Mediated Site-Directed Mutagenesis of Genes in Klebsiella pneumoniae.

This protocol minimizes off-target editing, shortens experimental timelines, reduces screening workload, and provides a reliable platform for investigating resistance mechanisms, validating candidate genes, and supporting functional genomics studies in clinically relevant bacterial pathogens.

Programmable enzymes for targeted gene insertion.

Canonically minimal RNA-guided insertion sequences expand into large elements that disseminate antimicrobial resistance

Using high-throughput junction mapping together with large-scale comparative genomics, this work redefined the in vivo structural boundaries, growth, and mobilization of IS110 elements and uncovered a previously unrecognized size continuum extending to ∼100 kb, driven by progressive additions.

Advances in understanding of plasmid biology through the application of transposon insertion sequencing

Plasmids are extra-chromosomal DNA molecules capable of autonomous replication, stable inheritance in a bacterial population, and horizontal transfer to other bacteria. Plasmids can harbour auxiliary genetic material that contributes to host bacterial fitness, the most prominent example being antimicrobial resistance (AMR) determinants, which remain the greatest threat to modern medicine. Since their discovery in the early 1950s, plasmids have been extensively studied due to their diversity, their capacity to spread between bacterial hosts, and their ability to carry and disseminate multiple AMR genes simultaneously. Recent advances in sequencing technology have transformed plasmid research, with transposon–insertion sequencing (TIS) enabling simultaneous analysis of millions of mutants and providing unprecedented scale, speed and resolution for studying plasmid biology. Here, we briefly outline a recommended methodology for generating plasmid transposon mutant libraries, which can be combined with TIS to investigate plasmid replication, maintenance and conjugation. We further summarise data from nine comprehensive plasmid TIS studies to date on five distinct plasmids, discuss alternative uses for plasmid libraries, challenges and future perspectives.