Screening Of Genes Involved In The Regulation Of Innate Immune Signaling By Genome-scale CRISPR-Cas9 Knockout

Innate immune response aganist pathogen invasion plays an important role for maintaining their normal physiological function.The cGAS-STING pathway is recently discovered to be a major immune regulatory pathway involved in recognition of immunogenic DNA.However,the regulatory mechnism of the cGAS-STING pathway,especially its upstream regulation is still not well known.Large-scale screening of genes is an effective way for finding the novel regulatory genes involved in the regulation of natural immune pathways.The expression system in combination with the IFN? promoter-induced Luciferase screening system has been previously used for identifying the immune-regulatory genes such as MITA and STING.However,using the Luciferase screening system has been constrained due to its high screening background and huge workload.Another way is to employ the method of RNAi-mediated genome-wide functional deletion screening.However,this method has not been widely used because it can not completely knockout the protein function and has the potential risk of off-target function.In order to find other important regulatory genes involved in the innate immune signaling pathway,especially the cGAS-STING pathway,we combined the genome-scale CRISPR-Cas9 knockout library technology with Fluorescence activated Cell Sorting technique and TK/DTA negative screening technology to establish a cost-effective screening approach.We used the CRISPR genome knockout library to infect BGC-823 cells and then stimulated the cells with herpes simplex virus type ?(HSV-1).We isolated the cell clones which were resistant to HSV-1 infection.By examing the sgRNA fragments via high-throughput sequencing,we successfully identified about 30 genes which deletion might be related to the resistance to the viral infection.One of these genes is HCFC1R1,a regulator of HCF-1 which has been reported to be involved in the regulation of HSV-1 replication.We also identified the other genes which functions have not been previously reported.Thus,we have successfully used the system for identifying the regulatory genes involved in the regulation of viral infection.In another set of studies,we performed a truncated analysis of the promoter region of the IFN? gene,an important gene involved in the innate immune response,to determine the optimal promoter region.Two selection vector systems were successfully constructed:one was IFN?-induceble EGFP reporter gene vector;another contained the suicide gene TK or DTA as negative reporters which expression could be induced by IFN? upon immune stimulation.We demonstrate that the system can work normally and can be used for high-throughput screening of regulatory genes involved in innate immune signaling.In summary,by using a genome-scale CRISPR-Cas9 knockout library,we establish the systems used for identifying the regulatory genes involved in the innate immune signaling pathway including the cGAS-STING pathway and in HSV-1 infection.We successfully identified several regulatory genes involved in the regulation of HSV-1 infection by using the system.We will use the system to find more genes involved in the innate immune signaling pathways that recognize different pathogen-related molecular patterns(LPS,RNA,DNA)in the future.