Continuous site-directed mutagenesis and selection in Escherichia coli
TL;DR
This work demonstrates a distinct paradigm for library generation through efficient transfer of genetic elements to recipient cells bearing libraries of retron editors, efficient editing of cargo genes in recipient cells, and continuous iteration of the conjugation-editing cycles with selection for recipients in sequential cycles.
Abstract
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.