Bacteria play a crucial role in human life, and modifying them can enhance their usefulness. Bacterial functions are encoded in the genome, which is composed of deoxyribonucleic acid (DNA). Gene editors enable targeted modifications to bacterial genomes by chemically altering DNA. These modifications typically involve cleaving molecular bonds, such as the phosphodiester bond between nucleotides (nucleases), the carbon-nitrogen bond in nitrogenous bases (deaminases), or the glycosidic bond between a nitrogenous base and a ribose (glycosylases). However, no gene editors to date function by adding chemical groups to DNA. DNA ADP-ribosyltransferase (DarT) is the toxin component of a bacterial defense system against invading phages. As its name suggests, DarT transfers adenosine diphosphate ribose (ADPr) from nicotinamide adenine dinucleotide (NAD⁺) to thymine at the motif 5′–TYT–3′. ADPr-thymine triggers skipping of DNA polymerase III, and the creation of post-replicative ssDNA gaps. Bacteria repair these gaps through RecA-mediated homologous recombination. This work introduces the Append Editor, a fusion of DarT to the N-terminus of nCas9, creating a novel gene editor that functions in E. coli by triggering homology-directed repair. A gain-of-function assay is developed to quantify gene editing efficiency based on the restoration of kanamycin resistance. This assay is then used to demonstrate that opposite-strand nicking enhances ADPr-mediated gene editing. Next, long-read ONT sequencing confirms high editing rates and the absence of base mutations at the target thymine. Furthermore, extended expression of the Append Editor does not generate off-target mutations across the genome, distinguishing it from cytidine base editors. To mitigate Append Editor cytotoxicity, rational design of DarT was undertaken. Three amino acid substitutions were found to greatly reduce cytotoxicity while only slightly decreasing gene editing efficiency. A growth-based assay in a hypersensitive ΔrecA strain highlights the degree to which ADPr-mediated cytotoxicity has been reduced. This attenuated DarT variant enabled exploration of the genetic basis by which the Append Editor functions, confirming its dependence on RecA and RecFOR. Finally, the improved Append Editor outperformed standard Cas9 in gene editing involving the conjugative transfer of a single editing plasmid between E. coli donor and recipient strains. These findings highlight the potential for deploying the Append Editor in situ for gut microbiome engineering.
Harris Bassett (Thu,) studied this question.