CRISPR/Cas9 system is a technology of RNA-guided Cas9 nuclease which can edit target genes. Combining Cas9 nuclease or genetically modified Cas9 nuclease with genome-wide gRNA library can achieve genome-wide gene knockout or gene transcription regulation, which can be used for various high-throughput screening.
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At SBS Genetech, we can construct shRNA library and gRNA library according to customers' needs, including whole-genome gene silencing, gene knockout, gene transcription activation, and transcription inhibition. At the same time, we provide high-throughput gene-editing services by using shRNA library or gRNA library and screening services for drug target genes related to diseases by using high-throughput gene-editing technology.
Forward screening: Screen which gene mutations cause cell tolerance to different treatments.
e.g. After the Cas9-gRNA library is transfected into cells, treat the cells with the target reagent (drugs, toxins, or pathogens). The surviving cells are resistant to the reagent due to gene mutation. High-throughput sequencing is used to analyze the changes of gRNA abundance before and after the treatment, and the mutate genes can be identified.
Negative screening: Screen which genes are more sensitive to different treatments.
e.g. After the Cas9-gRNA library is transfected into cells, compare the abundance of gRNA in the cells with different survival times. In the cells with a short survival time, the gene mutation caused by Cas9-gRNA is probably related to cell proliferation.
After predicting the target gene and designing the gRNA sequences, synthesize the gRNA sequences by microarray. The lentiviral vector is used to transfect all the gRNAs into the target cells (low MOI value). High-throughput sequencing technology is used to detect the changes of gRNA abundance in cells before and after treatment, and to infer the corresponding relationship between gene function and cell phenotype.