UC San Diego

Astrocytes are one of the primary cells in the central nervous system that regulate the inflammatory response. However, master regulatory proteins that mediate the astrocyte inflammatory response remain to be elucidated. To identify these regulator genes, we made two embryonic stem cell lines (ES) with a doxycycline inducible Cas9 through transduction into the AAVS1 (Adeno-Associated Virus Integration Site 1) safe harbor locus. We also developed an 8-day protocol for a CRISPR-cas9 Kinome-wide, pooled screen to identify genes that when knocked out differentially modulate the inflammatory response. Many inflammatory response pathways such as the Tumor Necrosis Factor Receptor, Interleukin 1 Receptor type 1, and Toll Like Receptor 4 converge on the Nuclear Factor Kappa B (NF-κB) transcription factor; thus, we use this gene to identify genes which enhance or activate or repress the inflammatory response. The inflammatory cytokine, Interleukin 1-beta (IL1β) was used to stimulate the inflammatory response in our astrocytes. However, in the process of screen construction, we observed unstable Cas9 protein expression within our cells and explored why this would occur.