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.
Fabian B. H. Rehm, Martin Spinck, Jason W. Chin· bioRxiv· 0 citations
Sequence-programmable directed evolution systems have great potential to accelerate bioengineering. Diversity-generating retroelements (DGRs) are natural hypermutation systems widely distributed in prokaryotes and bacteriophages with the capacity to introduce diverse mutations at template-specified sites of target genes. Here, we show that DGRs can be installed in E. coli and reprogrammed for the continuous, iterative mutagenesis of user-defined target genes. We show that the DGR template RNA can be reprogrammed for gene- and residue-specific mutagenesis, leaving untargeted, adjacent residues unchanged. Furthermore, we establish continuous DGR-enabled mutagenesis with conjugation-mediated horizontal gene transfer of target genes (HGT-DGR) into a new host for the progressive accumulation of target-specific mutations. Iterative HGT-DGR mutagenesis over seven cycles yielded an average mutation load of approximately 6% across adenine positions in the target segment, generating a diverse library of variants comprising 40% mutant sequences, with a median pairwise Hamming distance of 4 among mutant variants. HGT-DGR enables iterative diversification of either the same or different user-specified segments of the target gene, as demonstrated with the directed evolution of the M. mazei pyrrolysyl-tRNA synthetase for non-canonical amino acid incorporation. HGT-DGR provides a simple, low-cost, sequence-programmable system that enables iterative, position-specific and tunable in vivo mutagenesis of any target sequence for applications in biotechnology and medicine.
Yang Liu, Yang-Qi Gu, Ganesh Agam et al.· bioRxiv· 0 citations
