Multiplexed CRISPRa/i in primary immune cells to elucidate gene regulatory networks underpinning T cell development

Immune cells adopt distinct cell type identities and functional states to enable robust defense against diverse challenges. These distinct cell types and states are established by networks of interacting transcription factors, chromatin regulators and cis-regulatory elements. Many key molecular components of these gene regulatory networks are now identified; however, it remains unclear how they work together to orchestrate immune cell state transitions. This gap is in part due to the lack of strategies to concurrently turn on or off multiple components, and thereby probe their identities and modes of interaction. CRISPR/Cas9 technologies can facilitate recruitment of distinct transcription effectors to different loci, thus potentially enabling concurrent activation and repression of different genes in the same cell. However, current CRISPR activation (CRISPRa) or interference (CRISPRi) systems are challenging to implement in primary immune cells. Furthermore, current systems often still show weak and variable effects at target loci, precluding their systematic deployment in diverse immune contexts. These challenges will be overcome by developing a compact viral system for robust, concurrent CRISPR activation and interference (CRISPRa/i) in primary murine immune cells (Aim 1). This system will have the following innovations: (1) CRISPR RNA-scaffolds to recruit distinct transcriptional effectors to different loci in the same cell using a single Cas9 protein; (2) next-generation hypercompact effectors with high transcriptional efficacy; and (3) use of truncated (14-15 nt) scaffold RNAs that will render this system compatible with the widely- used Cas9 knock-in mouse strains. The system will be developed in primary mouse progenitors and optimized for system designs that show both high efficacy and reliability in the targeting of multiple genes. Next, this compact CRISPRa/i system will be used to map the landscape of interactions in the gene regulatory network controlling Bcl11b activation and T cell lineage commitment (Aim 2). The transcription factor Bcl11b is essential for T cell lineage identity; its irreversible all-or-none activation shuts down alternate fate options and commits progenitors to the T cell lineage. Here, a series of single and pairwise CRISPR activation and interference screens in multipotent progenitors will identify transcription factors, chromatin regulators and cis-regulatory elements controlling Bcl11b activation. These studies will identify the gene regulatory network components underlying Bcl11b activation and comprehensively map the genetic interactions between these components. More broadly, they will establish a widely-accessible viral system to concurrently turn on or off genes in primary mouse immune cells, with broad applications in a variety of basic and translational applications. Project Number: 1R21AI193387-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Hao Yuan Kueh (+1 co-PI) | Institution: UNIVERSITY OF WASHINGTON, SEATTLE, WA | Award Amount: $405,497 | Activity Code: R21 | Study Section: Adaptive Immunity Study Section[AI] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19338701