Journal Article

Bacteriophage genome engineering with CRISPR–Cas13a

Oromí-Bosch, Agnès, Bondy-Denomy, Joseph, Mendoza, Senén D., Karambelkar, Shweta, Berry, Joel D., Guan, Jingwen

2022

نظرة عامة

Jumbo phages such as Pseudomonas aeruginosa ФKZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited by a lack of genetic engineering tools due to a proteinaceous ‘phage nucleus’ structure that protects from DNA-targeting CRISPR–Cas tools. To provide reverse-genetics tools for DNA jumbo phages from this family, we combined homologous recombination with an RNA-targeting CRISPR–Cas13a enzyme and used an anti-CRISPR gene ( acrVIA1 ) as a selectable marker. We showed that this process can insert foreign genes, delete genes and add fluorescent tags to genes in the ФKZ genome. Fluorescent tagging of endogenous gp93 revealed that it is ejected with the phage DNA while deletion of the tubulin-like protein PhuZ surprisingly had only a modest impact on phage burst size. Editing of two other phages that resist DNA-targeting CRISPR–Cas systems was also achieved. RNA-targeting Cas13a holds great promise for becoming a universal genetic editing tool for intractable phages, enabling the systematic study of phage genes of unknown function. A phage genetic engineering platform will enable a better understanding of phage biology and engineering of phage therapies.

• الناشر: Nature Publishing Group UK,Nature Publishing Group
• موضوع: 14 / 14/63 / 631/208/464 / 631/326/1321 / Antimicrobial agents / Bacteriophages - genetics / Biomedical and Life Sciences / Burst size / CRISPR / CRISPR-Cas Systems / Deoxyribonucleic acid / DNA / DNA structure / Gene Editing / Genetic Engineering / Genomes / Homologous recombination / Infectious Diseases / Life Sciences / Medical Microbiology / Microbiology / Parasitology / Phages / RNA / Tubulin / Virology