Peng, J., Zhou, Y., Zhu, S., & Wei, W. (2015). High‐throughput screens in mammalian cells using the CRISPR‐Cas9 system. The FEBS journal, 282(11), 2089-2096.
- Aimed to use CRISPR-Cas9 functional screening tools to discover the genes related to the IFN-α-induced apoptosis pathway.
- Independently introduced and designed sgRNA sequences, and construct sgRNA plasmids for gene screening.
- Constructed GoldenGate point mutation on plasmids.
- Carried out cell culture, virus extraction, and titer determination.
Background
- CRISPR: Cas9 targets the DNA through sgRNA, causes DSBs.
- iBAR is a 6nt sequence designed at frequently changing positions.
- TNF-α tends to be pro-inflammatory and apoptotic.
Functional screening
Construction sgRNA library (4 different iBARs/sgRNA) →Infection by lentivirus→3 rounds of TNF-α treatment→PCR→Detect false positives by iBAR(count changes of the sgRNA should be consistent) →High score is probably related to TNF- induced apoptosis