Bacteriophage genome engineering with CRISPR-Cas13a.
Jingwen GuanAgnès Oromí-BoschSenén D MendozaShweta KarambelkarJoel D BerryJoseph Bondy-DenomyPublished in: Nature microbiology (2022)
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.
Keyphrases
- crispr cas
- genome wide
- genome editing
- pseudomonas aeruginosa
- circulating tumor
- dna methylation
- genome wide identification
- cell free
- nucleic acid
- single molecule
- cystic fibrosis
- copy number
- cancer therapy
- biofilm formation
- acinetobacter baumannii
- bioinformatics analysis
- dna damage
- quantum dots
- genome wide analysis
- escherichia coli
- oxidative stress
- living cells
- risk assessment
- transcription factor
- high frequency
- staphylococcus aureus
- climate change
- label free
- big data