CRISPR_Cas___scRNAseq_in_human_iPSC

Multiple core questions in human genetics could be addressed by measuring transcriptomes of single cells in response to various genetic perturbations. Building on two rounds of successful pilot single cell CRISPR screen projects executed 2019-2021. Here we apply our approach at scale on multiple iPSC lines from healthy donors of the HIPSCI cohort with different genetic backgrounds, targeting large sets of genes with a range of fitness effects. We aim to 1) optimize experimental design of genome-wide knockdown screens in iPSCs; 2) establish the baseline measurement of magnitude and modifiability of gene perturbation effects on the transcriptome; 3) identify how genetic background influences the effect of gene knockdown on gene expression across individuals. To do so, we will perform CRISPR inhibition screens in multiple iPSC lines (~64 lines) for 3-6 days using several CRISPRi gRNA libraries. Then single cells are sorted for dCas9 and gRNA presence after CRISPR/Cas9 perturbation, followed directly by 10x loading. We will construct single cell RNA library preparation using the 10x Genomics 5' v2 kit. This will allow direct capture of guide RNAs and transcriptome simultaneously from each single cell of these donors. Then the gene expression and guide RNA libraries will be deep sequenced (total 72 lanes of NovaSeq S4) to reveal the effect of the different genetic perturbations on gene expression. These screens are part of an effort to establish gene regulatory networks in cell fitness and differentiation, and their differences across different individuals.