This project aimed at the development and implementation of techniques that would allow the concurrent interrogation of host and pathogens at the single cell level,
The scientific objectives and goals have been addressed via three specific work packages: (1) develop a dual smFISH protocol to detect both host and pathogen transcripts; (2) single-cell CRISPRi genetic screens in Salmonella; (3) and their implementation in infection models.
For the first package, a successful protocol has been calibrated and applied to Salmonella infection in macrophages to follow the expression of virulence genes across time.
The second package has been accomplished by achieving down-regulation of Salmonella genes during infection in macrophages, and has been implemented as part of the third package. Moreover, to transfer the CRISPRi technology to the single-cell level, the scaffold region of the sgRNAs has been successfully engineered to include a capture sequence, not affecting efficiency of knock-down (KD) and allowing for direct recovery of the identity of the perturbation. A collection of perturbations targeting two-component systems was generated and used for subsequent experiments. The fellow has set up a protocol compatible with mainstream commercial single-cell technologies for recovering, at the single-cell level, both the gene expression profile of the host and the identity of Salmonella’s perturbations with a very high recovery rate. The protocols for these methods will be open access on online platforms, after manuscript submission. The CRISPRi plasmids have already been sent to addgene, and will be distributed following manuscript submission. These results were also disseminated in the several conferences the fellow attended.
