Enhancing Multiplex Genome Editing by Natural Transformation (MuGENT) via inactivation of ssDNA exonucleases

Abstract

Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ∼50% without selection in cis. Consequently, editing constructs are generated in a single polymerase chain reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.

A bifunctional ATPase drives tad pilus extension and retraction

“…V. cholerae mutants were constructed by MuGENT and natural transformation as previously described 17,38,39 and derived from the El Tor isolate E7946 40 . In short, transforming DNA was constructed using splicing-by-overlap extension PCR and transformed or co-transformed into strains 38,39 .…”

Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-Mediated Counter-Selection

“…The recombineering system described here is the first effective and simple strategy for targeted and markerless genome-editing in S. oneidensis, yielding a high efficiency of recombinant formation that permits screening for desired mutations in the absence of selection. Our system was developed without strain engineering, however rational removal of single-strand exonucleases could potentially enhance efficiency as shown in V. cholera (Dalia et al 2017) and E. coli (Mosberg et al 2012;Costantino & Court 2003;Li et al 2013). In chapter IV, we show that coupling the recombineering system developed here with the cutting edge CRISPR/Cas9-technology (Ronda et al 2016;Yan et al 2017), enables facile isolation of recombinants.…”

“…This difference may be due to the inefficiency in co-transformation of multiple different DNA molecules versus a single molecule electroporation, but possibly other factors such as the difference in promoter type and plasmid copy number and/or stability. Although our system was developed without strain engineering, rational removal of single-and double-strand exonucleases could potentially enhance the frequency of recombineering as shown in V. cholera (Dalia et al 2017) and E. coli (Mosberg et al 2012;Costantino & Court 2003;Li et al 2013).…”

Horizontal gene transfer by natural transformation promotes both genetic and epigenetic inheritance of traits

“…All V. cholerae and S. pneumoniae mutant strains were generated by SOE PCR and natural transformation, cotransformation, or MuGENT exactly as previously described (Dalia, 2018; Dalia et al, 2014; Dalia et al, 2017). For a list of all primers used in strain construction see Table S2.…”