1. Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases
- Author
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Christopher M. Waters, Elisa Galli, François Xavier Barre, Ankur B. Dalia, Soo Hun Yoon, Triana N. Dalia, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Evolution et maintenance des chromosomes circulaires (EMC2), Département Biologie des Génomes (DBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,Exonuclease ,DNA, Bacterial ,Exonucleases ,[SDV]Life Sciences [q-bio] ,Mutant ,DNA, Single-Stranded ,Computational biology ,Genome ,DNA Mismatch Repair ,03 medical and health sciences ,chemistry.chemical_compound ,Genome editing ,Bacterial Proteins ,Genetics ,Nucleoid ,Homologous Recombination ,Vibrio cholerae ,030304 developmental biology ,Whole genome sequencing ,Gene Editing ,0303 health sciences ,biology ,Acinetobacter ,030306 microbiology ,Oligonucleotide ,Escherichia coli Proteins ,030104 developmental biology ,chemistry ,RNA splicing ,Mutation ,biology.protein ,Transformation, Bacterial ,Phosphorus-Oxygen Lyases ,Homologous recombination ,Synthetic Biology and Bioengineering ,Multiplex Polymerase Chain Reaction ,DNA ,Genome, Bacterial - 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 laboriousin vitroDNA splicing. InVibriocholerae, 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 selectionin cis. Consequently, editing constructs are generated in a single PCR 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 inV. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation inAcinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.
- Published
- 2017
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