CRISPR-Cas9 Precision Editing in Multi-Drug Resistant Bacterial Strains

Our team has developed a novel Cas9 variant that reduces off‑target effects by 73%, paving the way for safer therapeutic applications. The study, published in Nature Biotechnology, demonstrates efficient editing in clinical isolates of multi‑drug resistant Mycobacterium tuberculosis and Staphylococcus aureus.

Overcoming the off‑target challenge

Conventional CRISPR‑Cas9 can introduce unintended mutations. By engineering the REC3 domain of Cas9, we enhanced specificity while maintaining on‑target activity. In vitro assays showed 94% editing efficiency in the target gene with undetectable off‑target effects in human‑off targets predicted by computational models.

“This is a significant step toward using CRISPR directly in patients to treat infections caused by resistant bacteria, without risking damage to the patient’s own genome.”

Validation in multi‑drug resistant strains

We tested the new variant on 24 clinical isolates of MRSA and MDR‑TB. The system successfully disrupted resistance genes (mecA in MRSA, katG in MDR‑TB) in 22 strains, restoring susceptibility to methicillin and isoniazid respectively. Whole‑genome sequencing of edited clones confirmed the absence of off‑target mutations.

Delivery and future directions

Using lipid nanoparticle formulations, we achieved efficient delivery to bacterial cells in a mouse model of wound infection. Treated mice showed a 4‑log reduction in bacterial load compared to controls. Work is underway to optimize for systemic delivery and advance toward clinical trials.