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Genome-scale CRISPR screening for modifiers of cellular LDL uptake.
Emmer, Brian T; Sherman, Emily J; Lascuna, Paul J; Graham, Sarah E; Willer, Cristen J; Ginsburg, David.
Afiliação
  • Emmer BT; Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.
  • Sherman EJ; Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America.
  • Lascuna PJ; Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America.
  • Graham SE; Chemical Biology Program, University of Michigan, Ann Arbor, Michigan, United States of America.
  • Willer CJ; Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America.
  • Ginsburg D; Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.
PLoS Genet ; 17(1): e1009285, 2021 01.
Article em En | MEDLINE | ID: mdl-33513160
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.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Receptores de LDL / Colesterol / Aterosclerose / Lipoproteínas LDL Tipo de estudo: Diagnostic_studies / Guideline / Prognostic_studies / Risk_factors_studies / Screening_studies Limite: Humans Idioma: En Ano de publicação: 2021 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Receptores de LDL / Colesterol / Aterosclerose / Lipoproteínas LDL Tipo de estudo: Diagnostic_studies / Guideline / Prognostic_studies / Risk_factors_studies / Screening_studies Limite: Humans Idioma: En Ano de publicação: 2021 Tipo de documento: Article