Tu106 - An Efficient Functional Assay Platform of Autoimmunity Risk Alleles Using CRISPR Genome Editing

Takahiro Arakawa; Masahiro Nakano – Laboratory for Autoimmune Diseases – RIKEN Center for Integrative Medical Sciences; Hiroaki Hatano; Kazuyoshi Ishigaki

Abstract Text: Large-scale genetic association studies have identified hundreds of variants associated with rheumatoid arthritis development. The risk variants are enriched in regulatory regions of activated CD4+ T cells more than resting CD4+ T cells, suggesting that they involve in context-specific cis-regulatory machinery. However, previous functional genetics studies failed to conduct functional assays with sufficient variations of immunological context and were unable to detect their allelic effects on gene expression regulations. The failure is partially because the previous studies relied on the natural genetic variants that we possess from our birth. The major draw-back in using natural variants is its low-efficiency; we usually need to recruite huge numbers of donors to test allelic effects, and this requirement restricted the search space of immunological contexts. To overcome this limitation, we have established a novel experimental platform consisted of two components: (i) artificial variants generated by CRIPSR prime editing in human CD4+ T cells, and (ii) pooled target-assay for transposase-accessible chromatin with sequencing (pooled target-ATAC-seq). CRIPSR prime editing enables us to generate human primary immune cells with any variants with high efficiency. Using pooled target-ATAC-seq, we selectively amplify only tagment DNAs harboring specific risk alleles with unique molecular indexes, and sequence multiple samples simultaneously with customized barcorded transposome. Our novel platform requires only a few donors to evaluate risk variant’s allelic effect on the chromatin accessibility, and with this high efficiency we can test allelic effects in variety of immunological contexts. Our platform contributes to better understanding of the genetic etiology of autoimmune disease.