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Genome-scale CRISPR screening for modifiers of cellular LDL uptake
Brian T Emmer1,2, Emily J Sherman2,3, Paul J Lascuna2
1Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.
Plos Genetics
|January 29, 2021
Summary
This study used CRISPR screening to identify genes regulating cellular cholesterol uptake via the LDL receptor. Findings reveal new genes and mechanisms influencing low-density lipoprotein (LDL) homeostasis.
Area of Science:
- Genetics
- Molecular Biology
- Cardiovascular Disease Research
Background:
- Hypercholesterolemia is a key risk factor for atherosclerotic cardiovascular disease.
- Cellular cholesterol homeostasis relies on low-density lipoprotein (LDL) receptor-mediated endocytosis into hepatocytes.
Purpose of the Study:
- To identify genetic regulators of cellular LDL uptake using genome-scale CRISPR screening.
- To validate and characterize candidate LDL uptake regulators through secondary screening and orthogonal assays.
Main Methods:
- Genome-scale CRISPR screening in HuH7 cells under varying lipoprotein conditions.
- Secondary screening with a customized gRNA library for enhanced validation.
- Orthogonal screens assessing specificity, epistasis with LDLR deletion, and effects on LDLR expression/trafficking.
Main Results:
- A secondary screen with a customized library significantly improved performance over the primary genome-wide screen.
- Identified previously unrecognized genes involved in LDL uptake.
- Characterized candidate regulators for specificity, LDLR interaction, and cross-cell type generalizability.
Conclusions:
- The study successfully identified novel genetic regulators of LDL uptake.
- These findings provide insights into mechanisms of cholesterol homeostasis and potential therapeutic targets for hypercholesterolemia.
