Paired CRISPR screens to map gene regulation incisandtrans

ABSTRACT

Recent massively-parallel approaches to decipher gene regulatory circuits have focused on the discovery of eithercis-regulatory elements (CREs) ortrans-acting factors. Here, we develop a scalable approach that pairscis- andtrans-regulatory CRISPR screens to systematically dissect how the key immune checkpointPD-L1is regulated. In human pancreatic ductal adenocarcinoma (PDAC) cells, we tile thePD-L1locus using ∼25,000 CRISPR perturbations in constitutive and IFNγ-stimulated conditions. We discover 67 enhancer- or repressor-like CREs and show that distal CREs tend to contact the promoter ofPD-L1and related genes. Next, we measure how loss of all ∼2,000 transcription factors (TFs) in the human genome impacts PD-L1 expression and, using this, we link specific TFs to individual CREs and reveal novel PD-L1 regulatory circuits. For one of these regulatory circuits, we confirm the binding of predictedtrans-factors (SRF and BPTF) using CUT&RUN and show that loss of either the CRE or TFs potentiates the anti-cancer activity of primary T cells engineered with a chimeric antigen receptor. Finally, we show that expression of these TFs correlates withPD-L1expressionin vivoin primary PDAC tumors and that somatic mutations in TFs can alter response and overall survival in immune checkpoint blockade-treated patients. Taken together, our approach establishes a generalizable toolkit for decoding the regulatory landscape of any gene or locus in the human genome, yielding insights into gene regulation and clinical impact.