CRISPRi Screening of Enhancers in Human Primary Astrocytes Identifies Regulatory Circuitry Disrupted in Alzheimer’s Disease [NanoString]

We used CROP-seq to perform a high-throughput, parallel screen of 979 candidate enhancer perturbations in normal human astrocytes (NHAs), a primary cell-line. We identified the target gene(s) for 145 of these candidates, which were enriched for transcription with eRNA, transcription factor footprinting, and superenhancer annotation. Most regulatory interactions were <50kb, targeting the nearest gene in ~50% of cases, but were not typically captured by eQTL or in silico predictions. These data elucidate the regulatory network of an understudied-but-crucial brain cell-type. To validate results from our CRISPRi scRNA-seq screen, we selected 19 enhancers linked to 14 genes to validate in singleton experiments. The top performing sgRNA for each enhancer was cloned in CROP-seq-opti-GFP and transduced into NHA cells that stably express dCas9-KRAB at an MOI of 1. We also generated 4 pooled lines, where sgRNA for 2-3 enhancers linked to the same gene were transduced into the NHA-dCas9-KRAB cells at an MOI of 1 for each sgRNA. After 4 days, cells were selected with 3 µg/mL of puromycin and 10 µg/mL blasticidine S hydrochloride, and RNA was extracted after 7 days. Gene expression was then measured using a Nanostring nCounter Elements TagSets.