Fig. 1: Targeted DNA ADP-ribosylation drives template-mediated homologous recombination in E. coli.
a, Role of the bacterial DarT2 toxin in antiphage immunity. NAM, niacinamide. b, Conceptualized impact and resolution of DNA ADP-ribosylation on DNA replication in E. coli17. c, Experimental setup for the in vitro polymerase-blocking assay. EPEC DarT2 recognizes the 5′-TCTC-3′ but not the 5′-ACTC-3′ motif. d, Impact of DNA ADP-ribosylation by DarT2 on DNA polymerase extension in vitro. Gel images are representative of two independent experiments (additional controls in Extended Data Fig. 1). e, Configuration of the append editor using DarT2. The editor combines ScCas9 mutated to nick the target DNA strand and a fused DarT2 that ADP-ribosylates the nontarget DNA strand displaced as part of R-loop formation. This combination is predicted to drive homologous recombination with a provided repair template (RT). HR, homologous recombination. f, Experimental setup for reverting a prematurely terminated kanamycin resistance gene (kanR*) in E. coli. The chromosomally integrated gene contains a premature stop codon that is reverted as part of homologous recombination, thus conferring kanamycin resistance. Cm, chloramphenicol; Carb, carbenicillin; Kan, kanamycin. g, Impact of programmable DNA ADP-ribosylation on cell viability and kanamycin resistance frequency. Bars and error bars represent the geometric mean and geometric s.d. of three independent experiments started from separate transformations. Dots represent individual measurements. CFU, colony-forming units. Bottom, cartoons designate whether a given DNA strand is unaltered, nicked or ADP-ribosylated. h, Amplicon sequencing of the kanR* target site from batch cultures. Bars and error bars represent the mean and s.d. of three independent experiments starting from separate transformations. Dots represent individual measurements. i, Genome-wide profiling of off-target edits. The indicated editor was expressed with an NT sgRNA in the absence of an RT. More information on the identified edits can be found in Supplementary Table 1. Whole-genome sequencing was performed on genomic DNA extracted from cultures beginning with an individual colony. Both strands are considered for a given edit (for example, T > A and A > T are combined). sgRNA, single-guide RNA; SNP, single-nucleotide polymorphism.
