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Updated: Aug 2, 2026
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
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.
Insights
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:
- 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.
Abstract:
Familial hypercholesterolemia (FH) is a frequently occurring genetic disorder that is linked to early-onset cardiovascular disease. If left untreated, patients with this condition can develop severe cardiovascular complications. Unfortunately, many patients remain undiagnosed, and even when diagnosed, the treatment is often not optimal. Although mutations in the LDLR gene are the primary cause of FH, predicting whether novel variants are pathogenic is not a straightforward task. Understanding the functionality of LDLR variants is crucial in uncovering the genetic basis of FH. Our study utilized CRISPR/Cas9 cytosine base editors in pooled screens to establish a novel approach for functionally assessing tens of thousands of LDLR variants on a large scale. A total of more than 100 single guide RNAs (sgRNAs) targeting LDLR pathogenic mutations were successfully screened with relatively high accuracy. Out of these, 5 sgRNAs were further subjected to functional verification studies, including 1 in the promoter, 1 in the antisense RNA, 1 in the exon, and 2 in the intron. Except for the variant caused by the sgRNA located at intron 16, the functionalities of the other LDLR variants were all downregulated. The high similarity of LDLR intron sequences may lead to some false positives. Overall, these results confirm the reliability of the large-scale screening strategy for functional analysis of LDLR variants, and the screened candidate pathogenic mutations could be used as an auxiliary means of clinical gene detection to prevent FH-induced heart disease.
