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Related Experiment Video

Updated: Jul 21, 2025

Pooled CRISPR-Based Genetic Screens in Mammalian Cells
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Large-Scale CRISPR Screen of LDLR Pathogenic Variants.

Mengjing Li1,2, Lerong Ma1,3,4, Yiwu Chen1,3,4

  • 1Key Lab for Zoonoses Research, Ministry of Education, Animal Genome Editing Technology Innovation Center, College of Animal Sciences, Jilin University, Changchun, Jilin Province 130062, China.

Research (Washington, D.C.)
|July 27, 2023
PubMed
Summary

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This study introduces a new CRISPR/Cas9 base editing method for large-scale functional assessment of LDLR variants. This approach aids in identifying pathogenic mutations for Familial Hypercholesterolemia (FH) and improving clinical gene detection.

Area of Science:

  • Genetics
  • Molecular Biology
  • Cardiovascular Disease Research

Background:

  • Familial hypercholesterolemia (FH) is a common genetic disorder causing early-onset cardiovascular disease.
  • Many FH patients are undiagnosed or undertreated, highlighting a need for better diagnostic tools.
  • Identifying pathogenic LDLR gene variants is crucial for understanding FH, but challenging for novel mutations.

Purpose of the Study:

  • To develop and validate a high-throughput CRISPR/Cas9 cytosine base editing screening method for functional assessment of LDLR variants.
  • To identify and functionally verify candidate pathogenic mutations in the LDLR gene associated with FH.
  • To establish a reliable strategy for large-scale functional analysis of genetic variants impacting LDLR function.

Main Methods:

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Last Updated: Jul 21, 2025

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  • Utilized CRISPR/Cas9 cytosine base editors in pooled screens to assess tens of thousands of LDLR variants.
  • Screened over 100 sgRNAs targeting known pathogenic LDLR mutations.
  • Performed functional verification studies on 5 selected sgRNAs in different genomic regions (promoter, antisense RNA, exon, introns).

    • Main Results:

    • Successfully screened over 100 sgRNAs targeting LDLR mutations with high accuracy.
    • Functional verification showed downregulation of LDLR variant activity for most tested sgRNAs, except for one in intron 16.
    • The study confirmed the reliability of the large-scale screening strategy for LDLR variant functional analysis.

    Conclusions:

    • The developed CRISPR/Cas9 base editing screen is a reliable method for large-scale functional analysis of LDLR variants.
    • Screened pathogenic mutations can serve as auxiliary tools for clinical gene detection in FH.
    • This approach can aid in preventing cardiovascular complications associated with FH.