Awakening prime editing for precision engineering of probioticEscherichia coliNissle 1917

CRISPR-Cas systems are transforming precision medicine with engineered probiotics as next-generation diagnostics and therapeutics. To promote human health and treat disease, engineering probiotic bacteria demands maximal versatility to enable non-natural functionalities while minimizing undesired genomic interferences. Here, we present a streamlined prime editing approach tailored for probiotic Escherichia coli Nissle 1917 utilizing only essential genetic modules and an optimized workflow. This was realized by assembling a prime editor consisting of the CRISPR-Cas system from Streptococcus pyogenes with its native codons and a codon-optimized reverse transcriptase, and by orchestrating the induction levels. As a result, we achieved all types of prime editing in every individual round of experiments with efficiencies of 25.0%, 52.0% and 66.7% for DNA deletion, insertion, and substitution, respectively. A comprehensive evaluation of off-target effects revealed a significant reduction in unintended mutations, particularly in comparison to two different base editing methods. Leveraging the prime editing system, we developed a barcoding system for strain tracking and an antibiotic-resistance-gene-free platform to enable non-natural functionalities. Our prime editing strategy awakens back-to-basics CRISPR-Cas systems devoid of complex or extraneous designs, paving the way for future innovations in engineered probiotics.