# NIH grant: Developing a novel technology for studying T cell differentiation in vivo (1R21AI199345-01) — retained record

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[original source ↗](https://reporter.nih.gov/project-details/1R21AI199345-01)

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NIH grant 1R21AI199345-01: Developing a novel technology for studying T cell differentiation in vivo
Institute: {'code': 'AI', 'abbreviation': 'NIAID', 'name': 'National Institute of Allergy and Infectious Diseases', 'admin_org_id': '12500003', 'admin_funding_url': ''}. PI: WEIGUO  CUI. Award: $154394.
Start: 2026-06-18T00:00:00.
Summary
CRISPR-based genetic screens have revolutionized our understanding of gene functions and molecular
mechanisms across various biological processes. In the field of T cell biology, CRISPR screens have played a
pivotal role in identifying genes that impact critical aspects, such as T cell development, differentiation, and
function. However, traditional screens have struggled to distinguish genes with diverse mechanisms of action,
necessitating further investigations. To address this challenge, researchers have harnessed the power of
CRISPR screens combined with single-cell sequencing (scCRISPR-seq), enabling the simultaneous
assessment of genetic perturbations and high-dimensional phenotypes at the single-cell level. While scCRISPR-
seq has predominantly been performed in vitro using immortalized cell lines, its physiological relevance is limited
due to oversimplified biological context and disparities compared to primary cells. This limitation highlights the
urgent need for large-scale in vivo scCRISPR-seq with primary T cells. However, various challenges have
discouraged its widespread adoption. The use of viral vectors for sgRNA delivery compromises physiological
relevance, as the in vitro activation conditions fail to faithfully represent the intricate T cell priming process in
vivo. Moreover, viral vector components and continuous Cas9 expression can trigger immunogenicity and
cytotoxicity, leading to cell depletion and hindering long-term studies. Additionally, current scCRISPR-seq
methods face technical limitations, including low editing efficiency and inadequate perturbation identity recovery
rates, which impede efficient large-scale in vivo applications. Fortunately, recent advances in ribonucleoprotein
complex (RNP) transfection have addressed many of these challenges. This cutting-edge technology enables
efficient gene editing in primary T cells without the need for in vitro activation or permanent Cas9 expression.
Leveraging the high editing efficiency of RNP transfection, the investigator’s team aims to develop a novel
strategy for in vivo T cell CRISPR screens. This innovative approach involves arrayed RNP transfection and co-
transfer of T cells that recognize the relevant antigens. Instead of traditional genetic barcodes, the strategy
utilizes congenic markers (CD45.1/45.2 and CD90.1/CD90.2) from donor TCR transgenic T cells as "external
barcodes." These markers facilitate the recovery of gene perturbation identity at the single-cell level through the
application of CITE-seq. Importantly, this RNP-based strategy seamlessly integrates with existing single-cell
sequencing protocols, enabling the comprehensive assessment of transcripts, epitopes, and chromatin
accessibility simultaneously. To demonstrate the efficacy of this strategy, the team plans to develop two
benchmarking approaches: RNP-CET-seq to investigate the role of TCR regulators in T cell exhaustion and
RNP-CATE-seq to map the gene regulatory atlas of exhausted CD8 T cells. In summary, the proposed RNP-
based scCRISPR-seq strategy overcomes the limitations of current approaches, enabling large-scale, multi-
module in vivo genetic screens within a physiologically relevant context across various disease models.
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