Project description:Innate immunity is a non-specific host immune response against pathogen invasion. It exists widely in most of cells and is considered as the first line of defense against pathogen infection. Upon virus infection, the natural immune system is activated to generate immune response, thereby exerting the antiviral effect and effectively inhibiting the spread of virus in the body. With recent decades of research, people have found and identified the key regulatory factors of the natural immune response and the signal transduction. However the regulatory mechanisms of the factors are still unclear and need to be further explored. Our work used the CRISPR/Cas9 library to perform large-scale genome-wide, unbiased screening in immune cells, and discovered some novel genes involved in the regulation of type I IFN responses.
Project description:Here we developed a massively parallel in-library ligation methodology to simultaneously perturb four pre-designed targets in CRISPR/Cas9 screening. Thousands of pairs of sequences precisely ligated with their counterparts in library, which enabled simultaneous expression of four gRNAs from each single vector. We demonstrated this novel method with 6,236 4-gene combinations targeting 1,599 immune response related genes, and generated a plasmid library with 1,400x coverage. The library performance was evaluated in a canonical T cell activation experiment, and combinations involved in TCR signaling pathway or TCR complex were successfully identified as positive regulators. Novel combination that is reflecting a potential pathway crosstalk was also verified. This new methodology expands the capacity of the perturbation in CRISPR screening and provided a powerful tool for researches in broad fields to study the combinatorial outcomes from coordinated gene behaviors.