Activation of Split Prime Editor in Bacteria: High-Precision Genome Editing Demonstrated in Escherichia coli and MRSA
-
Size‑limited barrier in prokaryotes and bottleneck of genome engineering in non‑model strains Prime editing is a next‑generation precise genome‑correction modality that can perform desired base substitutions, insertions, and deletions without double‑strand breaks (DSBs) or exogenous donor DNA. However, the conventional prime editor (PE) molecule, in which the Cas9 nickase and reverse‑transcriptase are fused into a massive single backbone, creates a delivery‑capacity bottleneck for bacterial (prokaryotic) systems, severely limiting intracellular uptake and expression. This problem is especially acute when attempting precise editing in genetically intractable non‑model strains such as clinically important methicillin‑resistant Staphylococcus aureus (MRSA), representing a long‑standing technical barrier to establishing reversible antibiotic‑resistance‑blocking pipelines.
-
Deployment of Split Prime Editor (Split PE2) and DeepPrime in silico design In this work we eliminated the spatial and physical delivery barriers in bacterial cells by physically separating the large PE2 protein into a reverse‑transcriptase domain and a Cas9 nickase domain, each encoded on independent plasmid backbones—a ‘Split PE2’ architecture. The team integrated DeepPrime, a generative‑AI‑based guide‑RNA optimization engine, to retrospectively calculate the binding free energy of pegRNAs for each bacterial target site. The results demonstrated that the individually delivered enzyme fragments self‑assemble in the cytoplasm to reconstitute an intact native PE2 platform, achieving statistically comparable knock‑in and substitution efficiencies.
-
Strain‑specific exonuclease interference control and structural superiority of SpPE2 Omics‑level variability tracking revealed that prime‑editing efficiency in E. coli is critically modulated by target‑site sequence entropy, edit topology, and the kinetic degradation of pegRNA flaps by endogenous exonucleases.
- Exonuclease control in E. coli: Computational inhibition or evasion of endogenous exonuclease activity via pegRNA flap‑stabilizing designs reduced false‑positive indel rates below baseline.
- Transport optimization in MRSA: The Streptococcus pyogenes‑derived SpPE2 architecture, which is chemically more flexible than the Staphylococcus aureus‑derived SaPE2 system, displayed overwhelming thermodynamic advantage in binding‑free‑energy tensors.
- Establishment of microbial‑engineering standards and next‑generation synthetic‑biology IND guidelines The computational genetic‑engineering and microbial‑target control matrix redefines microbiome and infectious‑disease governance from a simple antibiotic‑administration model to a programmable bacterial‑correction infrastructure that “pinpoints” essential endogenous resistance genes for lethal reprogramming. By embedding this framework into synthetic‑biology R&D pipelines for diverse industrial microbes, we built a computational backbone that proactively predicts gene‑circuit integrity. The calibrated reverse‑transcriptase rotation constant of the split SpPE2 will serve as a safety‑metric backbone for multinational pharmaceutical pipelines developing next‑generation antimicrobial peptides and microbial modulators, dramatically shortening global regulatory‑approval timelines.
Prime editing is a precise and rapid genome-editing technique that modifies short DNA sequences using tailored guide RNAs. To implement this technique in bacteria, we used Prime Editor 2 (PE2) with the DeepPrime gRNA design tool and assessed its gene-editing efficiency in Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) cells. Our findings indicate that a split PE2, comprising a reverse transcriptase and two Cas9 nickase domains, exhibited gene-editing efficiency comparable to that of the intact PE2. The efficiency observed in E. coli was significantly affected by the target sites, edit type, and the presence of exonucleases. In MRSA, which serves as a model to evaluate the applicability in non-model bacterial species, Streptococcus pyogenes PE2 (SpPE2) exhibited superior performance relative to Staphylococcus aureus PE2 (SaPE2). Furthermore, the split SpPE2 lacking the reverse transcriptase successfully induced the intended mutation in MRSA. This study demonstrates the feasibility of prime editing within bacterial systems.
The molecular discoveries of this study go beyond a theoretical paradigm shift and directly impact global bio‑security supply chains and industrial microbiology business lines. First, in clinical settings where multidrug‑resistant viruses and super‑bacterial strains are encountered, plasmid cassettes and chromosomal loci that trigger resistance can be instantly scanned with Python algorithms, eliminating the temporal noise associated with antibiotic‑non‑responsive precursors and preserving a reversible public‑health containment barrier. Simultaneously, integration of the split PE2 platform’s open‑source genomic‑database matrix enables virtual simulation of false‑positive exogenous‑variant confounders during clinical‑trial design and real‑time back‑calculation of target microbial‑community penetration concentrations for therapeutic devices, realized through a companion‑diagnostic panel interface. Moreover, during scale‑up of industrial production batches of valuable compounds by multinational firms, linking epigenetic metabolic‑flux thresholds of the production strain as correction coefficients eliminates genetic‑toxicity score attrition across production strains and maximizes the probability of regulatory approval, functioning as a backbone infrastructure.