CRISPR-mediated screens for Phosphoinositide signaling in T cells

The Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that control diverse signalling pathways affecting gene-transcription, cellular adhesion and trafficking, autophagy and metabolism via the generation of PIP3. While some of these readouts are controlled by the evolutionarily conserved PI3K-AKT-FOXO, PI3K-AKT-mTOR axes, there is a diverse network of PI3K effectors that are less well studied, especially in lymphocytes, but which nonetheless can have profound effects on lymphocyte biology. We have recently used CRISPR/Cas9 to perform a targeted screen of PI3K effectors by generating a library that specifically targets PIP3-binding proteins. Screening for genes that affect T cell adhesion, we identified RASA3 as a key protein linking PI3K to the activation of the integrin LFA-1 and found that RASA3 is critical for T cell migration, homeostasis and responses to immunization (Johansen et al Sci Signaling 2022; Trend Immunol 2023; Front Immunol. 2021). We have now generated extended CRISPR/Cas9 libraries that target the entire PI3K-ome (including the kinases, phosphatases and all known effector proteins). Potential projects include designing and implementing new screens for downstream readouts of PI function, including autophagy, endocytosis, regulation of humoral immunity in vivo or other readouts, and/or understanding how RASA3 and Kindlin3 regulate T cell function and the signaling pathways. Interestingly, while RASA3 and Kindlin3 are both regulated by PIP3, they have opposite effects on LFA1 activity.  We will use advanced imaging technologies to determine the differential effects of PI3K signalling on these two proteins.