Fine-mapping of human genetic regulatory variants

The majority of GWAS (Genome-Wide Association Study) identified common genetic variants map to regulatory regions of gene, and are likely to influence disease risk by affecting gene expression. One of the most important challenges is to experimentally fine-map causal regulatory variants that typically lie in credible intervals of 100 or more variants. Another large proportion of genetic variants, rare variants, are expected to have large effects causing disease in individual, but are not detectable in GWAS. Herein, I provide both experimental and computational approaches for fine-mapping common and rare genetic variants accounting for medium and large effect on population or individual. First, I describe a single cell clone-based strategy for targeted single-nucleotide polymorphism (SNP) evaluation wherein microindels are introduced by CRISPR/Cas9. Multiple constraints, including the variability in mutability, clonal genotype and expense, render this approach infeasible for fine-mapping 10%-20% moderate effect size expression SNPs (eSNPs), which is also validated in a simulation study. Subsequently, I switch to a moderate-throughput parallel screening tool that characterizes multiplexed CRISPR/Cas9 perturbed transcriptomes by single-cell RNA-seq, called “expression CROP-seq”. Two causal SNPs, rs2251039 and rs35675666, are identified that significantly alter the expression of CISD1 and PARK7, respectively. The sites overlap with chromatin accessibility peaks and are risk loci of inflammatory bowel disease. Expression CROP-seq reduces the variability identified in previous method and is powerful to screen genetic regulatory variants within credible intervals. Finally, to extend its application to rare variants, I develop a novel gene categorization system according to gene intolerance to promoter polymorphism and depletion of rare regulatory variants with GTEx v8 data. 49 GTEx tissues are clustered into functional groups with gene features. It supports the use of tissue-gene genomic annotation for prioritization of GWAS tagged risk loci. In summary, this work comprehensively describes and evaluates two CRISPR/Cas9-based eSNP screening systems. The use of rare regulatory variants in gene classification with tissue information demonstrates its potential in rare disease diagnoses. Both researches inevitably contribute to the genetic interpretation of human complex disease and personalized medicine in post-GWAS era.Ph.D