Genome-scale CRISPR screening for modifiers of cellular LDL uptake

Abstract

Hypercholesterolemia is a causal and modifiable risk factor for atherosclerotic cardiovascular disease. A critical pathway regulating cholesterol homeostasis involves the receptor-mediated endocytosis of low-density lipoproteins into hepatocytes, mediated by the LDL receptor. We applied genome-scale CRISPR screening to query the genetic determinants of cellular LDL uptake in HuH7 cells cultured under either lipoprotein-rich or lipoprotein-starved conditions. Candidate LDL uptake regulators were validated through the synthesis and secondary screening of a customized library of gRNA at greater depth of coverage. This secondary screen yielded significantly improved performance relative to the primary genome-wide screen, with better discrimination of internal positive controls, no identification of negative controls, and improved concordance between screen hits at both the gene and gRNA level. We then applied our customized gRNA library to orthogonal screens that tested for the specificity of each candidate regulator for LDL versus transferrin endocytosis, the presence or absence of genetic epistasis with LDLR deletion, the impact of each perturbation on LDLR expression and trafficking, and the generalizability of LDL uptake modifiers across multiple cell types. These findings identified several previously unrecognized genes with putative roles in LDL uptake and suggest mechanisms for their functional interaction with LDLR.

Identification of cell type specific ACE2 modifiers by CRISPR screening

“…Multiple CRISPR screens have implicated cholesterol regulation as an important mediator of host cell interactions with SARS-CoV-2 [ 30 , 31 , 35 , 36 ]. In addition to canonical SREBP regulators, we also noted the identification of several genes in these studies that we had recently identified in a screen for regulators of low-density lipoprotein (LDL) uptake [ 43 ]. These included RAB10 and multiple components of the exocyst complex [ 31 , 35 , 36 ].…”

“…A total of ~20 million cells of FACS input were also collected for comparison to the starting plasmid pool. Genomic DNA was extracted and gRNA sequences amplified and sequenced as previously described, with pooling of samples performed after barcoding of PCR amplicons [ 43 ].…”

ACE2 protein expression within isogenic cell lines is heterogeneous and associated with distinct transcriptomes

“…An ACE2 overexpression construct was generated by HiFi DNA assembly (New England Biolabs #E2621) of the human ACE2 coding sequence (Sino Biological, Beijing China, #HG10108-M) into LeGO-iC2 (Addgene #27345, gifted by Boris Fehse) with simultaneous replacement of mCherry with a blasticidin resistance cassette. Lentivirus was generated and used to genetically engineer cell lines as previously described 59 Immunoblotting. Cells were lysed in RIPA buffer (Thermo Fisher, #89900) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis MO, #11836170001).…”

ACE2 protein expression within isogenic cell lines is heterogeneous and associated with distinct transcriptomes

“…An ACE2 overexpression construct was generated by HiFi DNA assembly (New England Biolabs #E2621) of the human ACE2 coding sequence (Sino Biological, Beijing China, #HG10108-M) into LeGO-iC2 (Addgene #27345, gifted by Boris Fehse) with simultaneous replacement of mCherry with a blasticidin resistance cassette. Lentivirus was generated and used to genetically engineer cell lines as previously described 57 . Primers used for qRT-PCR were: ACE2 -fwd [5’ -AAACATACTGTGACCCCGCAT-3’], ACE2 -rev [5’ -CCAAGCCTCAGCATATTGAACA-3’], ACTB -fwd [5’ - CCCTGGACTTCGAGCAAGAG-3’], ACTB -rev [5’ -ACTCCATGCCCAGGAAGGAA].…”