Session Information
Session Type: Poster Session B
Session Time: 10:30AM-12:30PM
Background/Purpose: Genetic studies have identified thousands of individual disease-associated non-coding alleles, but identification of the causal alleles and their functions remain critical bottlenecks. Even though CRISPR-Cas editing has enabled targeted modification of DNA, inefficient editing leads to heterogeneous outcomes within individual cells, limiting the ability to detect functional consequences of disease alleles particularly in primary human cells.
Methods: To overcome these challenges, we have developed MINECRAFTseq: a multi-omic single cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome, and measures cell surface protein expression. We apply this novel assay to cell lines and primary human CD4 T cells from non-autoimmune donors genomically edited with CRISPR-Cas enzymes.
Results: In cell lines, we show that CRISPR induced deletion of a regulatory region specifically associated with HLA-DQB1 gene expression, knockout of FBXO11 by splicing disruption alters CD40 mediated signaling, and CRISPR Cas base-editing identifies the causal variant in an expression quantitative trait locus of RPL8. We then apply this technique to primary human CD4 T cells. We induce an early stop codon with base editors in PTPRC and identify significant changes in gene and protein expression associated with hyperactivation and lineage skewing. Finally, we identify the state-specific effects of an IL2RA autoimmune variant in primary human T cells polarized under Treg but not Th1 conditions.
Conclusion: Multimodal functional genomic single cell assays including DNA sequencing bridges a crucial gap in our understanding of complex human diseases by directly identifying causal variation in primary human cells.
To cite this abstract in AMA style:
Baglaenko Y, Curtis M, Al Suqri M, Agnew R, Nathan A, Mire H, Yoo Mah-Som A, Liu D, Newby G, Raychaudhuri S. Defining the Function of Disease Variants with CRISPR Editing and Multimodal Single Cell Sequencing [abstract]. Arthritis Rheumatol. 2024; 76 (suppl 9). https://acrabstracts.org/abstract/defining-the-function-of-disease-variants-with-crispr-editing-and-multimodal-single-cell-sequencing/. Accessed .« Back to ACR Convergence 2024
ACR Meeting Abstracts - https://acrabstracts.org/abstract/defining-the-function-of-disease-variants-with-crispr-editing-and-multimodal-single-cell-sequencing/