Abstract LB201: ENIGMAC platform enables at-scale CRISPRi screenings for macrophage target discovery

Abstract RNA sequencing has improved our understanding of cellular heterogeneity by providing in-depth transcriptome information at the single cell level. However, this “dataset infodemic” did not translate into an abundance of new validated targets due to the difficulties in performing gene-to-function studies. The main goal of the ENIGMAC™ discovery platform is to support gene-to-function studies and to deliver new validated macrophage targets. ENIGMAC™ combines custom, disease relevant assays and unique genome editing methods supported by multi-omics bioinformatics analyses yielding selected gene sets for interrogation. We use a human Induced Pluripotent Stem Cell (iPSC) line that yields macrophages phenotypically and functionally similar to MDM. By employing this iPSC system, we can produce millions of macrophages per week, which allows multiple high throughput assays to support target validation and drug discovery. Furthermore, we developed several proprietary technologies allowing fast and reliable gene editing of macrophages, including gene Knock In (KI), Knock Out (KO) and Knock Down (KD) with high efficiency both at iPSC and macrophage level while maintaining expression/silencing during macrophage differentiation. Here we report preliminary results of the CRISPRi screen, aiming to identify novel regulators of INFg-mediated PD-L1 upregulation on macrophages, a surrogate readout for a suppressive phenotype. To this aim, we developed a proprietary human iPSC line that constitutively expresses dCAS9-KRAB protein, allowing for a highly gene specific guide RNA (sgRNA) -mediated method of silencing of gene transcription. Notably, our proprietary anti-silencing system allows to maintains dCAS9-KRAB expression throughout the entire macrophage differentiation process. During the screen, 100 million iPSC-derived microphages were transduced with a 6500 gRNAs library targeting 1300 genes selected from disease-relevant macrophage datasets. Macrophages were subsequently stimulated with INFg and sorted for loss of PD-L1 expression using flow cytometry. Sequencing of the PD-L1 low macrophage population successfully identified PD-L1 regulators such as IFNGR1, IFNGR2, JAK1, STAT1 and IRF1 among other hits. The novel hits are being now individually validated, and selected ones will be progressed to drug discovery. Importantly, this macrophage CRISPRi screen provides technical proof of concept for the ENIGMAC™ platform to perform screens at scale and to identify novel hits. In conclusion, the ENIGMAC™ platform represents a unique tool for gene to function studies using human macrophages. Furthermore, it is disease agnostic and can be integrated with a variety of disease-specific conditions and phenotypic readouts. Citation Format: Thomas W. Monteiro Crozier, Martha Lopez-Yrigoyen, Helena Engman, Moritz Haneklaus, Samuel Witham, Krzystof B. Wicher, Steven Myatt, Luca Cassetta, Carola Ries. ENIGMAC platform enables at-scale CRISPRi screenings for macrophage target discovery abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (7Suppl): Abstract nr LB201.