­Engineered Bacteriophages for in Situ Delivery of CRISPR RNA-Guided DNA Transposases

Bacteriophages are promising vectors for the delivery of engineered DNA to bacteria in diverse microbial communities. Here, we repurposed phage lambda to encode and deliver the DNA-editing all-in-one RNA-guided CRISPR-Cas transposase (DART) system for CRISPR RNA-guided DNA transposition in E. coli without lysogeny. For phage engineering, we employed homologous recombination with CRISPR-Cas13a counterselection to replace non-essential regions of the lambda genome, particularly those crucial for lysogeny, with the substantial 10-kb DART system. Consequently, these engineered phages had approximately 25% of their original genome replaced by the DART system, and this modification remained stable across successive passages. The selected lambda phages possess amber mutations and thus rely on amber suppressor E. coli hosts for lytic activity and propagation. Therefore, these phages cannot lyse or, due to engineering, establish lysogeny within wild-type E. coli hosts. When wild-type E. coli strains were infected by DART-encoding engineered phages, site-specific CRISPR RNA-guided integration of the transposon occurred, resulting in simultaneous gene knockout and the introduction of the genes contained within the transposon. Integration events were confirmed via PCR, while whole-genome sequencing verified their specificity and the lack of an associated prophage. Our current efforts focus on broadening target sites and diversifying transposon content for various phenotypic outcomes. By incorporating tailored host-range specificity and ensuring phage infection occurs without lysis or lysogeny, we aim for advancements in delivering the DART system for targeted editing in microbial communities. This work contributes to the development of generalizable phage-mediated genome editing strategies, with future work targeting rhizosphere microbial communities.