Genome-scale CRISPR screening for modifiers of cellular LDL uptake
Autoři:
Brian T. Emmer aff001; Emily J. Sherman aff002; Paul J. Lascuna aff002; Sarah E. Graham aff001; Cristen J. Willer aff001; David Ginsburg aff001
Působiště autorů:
Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America
aff001; Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America
aff002; Chemical Biology Program, University of Michigan, Ann Arbor, Michigan, United States of America
aff003; Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, United States of America
aff004; Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, United States of America
aff005; Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, United States of America
aff006; Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan, United States of America
aff007
Vyšlo v časopise:
Genome-scale CRISPR screening for modifiers of cellular LDL uptake. PLoS Genet 17(1): e1009285. doi:10.1371/journal.pgen.1009285
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.pgen.1009285
Souhrn
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.
Klíčová slova:
CRISPR – Gene regulation – Genetic screens – Genome-wide association studies – Guide RNA – Cholesterol – Library screening – Regulator genes
Zdroje
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