Phage-based delivery of CRISPR-associated transposases for targeted bacterial editing

• 成果类型: Article
• 署名作者: Roberts, Avery; Adler, Benjamin A.; Cress, Brady F.; Doudna, Jennifer A.; Barrangou, Rodolphe
• 署名单位: North Carolina State University; University of California System; University of California Berkeley
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-11232
• DOI: 10.1073/pnas.2504853122
• 发表日期: 2025-07-29
• 关键词: bacteriophage-lambda

摘要

Phage lambda, a well- characterized temperate phage, has been recently leveraged for bacterial genome editing by selectively delivering base editors into targeted bacterial species. We extend this concept by engineering phage lambda to deliver CRISPR- guided transposases, accomplishing large insertions and targeted gene disruptions. To achieve this, we engineered phage lambda using homologous recombination paired with Cas13a- based counterselection for precise phage modifications. Initially, we established the utility of Cas13a in phage lambda by conducting minimal recoding edits, deletions, and insertions. Subsequently, we scaled up the engineering to embed the comprehensive DNA- editing CRISPR- Cas modified lambda- DART phages were then employed to infect Escherichia coli, generating CRISPR RNA- guided transposition events in the host genome. Applying our engineered lambda- DART phages to monocultures and a mixed bacterial community comprising three genera led to efficient, precise, and specific gene knockouts and insertions in the targeted E. coli cells, achieving editing efficiencies surpassing 50% of the population. This research enhances phage- mediated genome editing by enabling efficient in situ gene integrations in bacteria, offering an avenue for further application in microbial in situ to manipulate the function and composition of diverse ecosystems.