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Large-Scale CRISPR Screen of LDLR Pathogenic Variants

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 diagnose...

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Autores principales: Li, Mengjing, Ma, Lerong, Chen, Yiwu, Li, Jianing, Wang, Yanbing, You, Wenni, Yuan, Hongming, Tang, Xiaochun, Ouyang, Hongsheng, Pang, Daxin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: AAAS 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10368174/
https://www.ncbi.nlm.nih.gov/pubmed/37496633
http://dx.doi.org/10.34133/research.0203
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author Li, Mengjing
Ma, Lerong
Chen, Yiwu
Li, Jianing
Wang, Yanbing
You, Wenni
Yuan, Hongming
Tang, Xiaochun
Ouyang, Hongsheng
Pang, Daxin
author_facet Li, Mengjing
Ma, Lerong
Chen, Yiwu
Li, Jianing
Wang, Yanbing
You, Wenni
Yuan, Hongming
Tang, Xiaochun
Ouyang, Hongsheng
Pang, Daxin
author_sort Li, Mengjing
collection PubMed
description 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.
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spelling pubmed-103681742023-07-26 Large-Scale CRISPR Screen of LDLR Pathogenic Variants Li, Mengjing Ma, Lerong Chen, Yiwu Li, Jianing Wang, Yanbing You, Wenni Yuan, Hongming Tang, Xiaochun Ouyang, Hongsheng Pang, Daxin Research (Wash D C) Research Article 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. AAAS 2023-07-25 /pmc/articles/PMC10368174/ /pubmed/37496633 http://dx.doi.org/10.34133/research.0203 Text en Copyright © 2023 Mengjing Li et al. https://creativecommons.org/licenses/by/4.0/Exclusive licensee Science and Technology Review Publishing House. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY 4.0) (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Li, Mengjing
Ma, Lerong
Chen, Yiwu
Li, Jianing
Wang, Yanbing
You, Wenni
Yuan, Hongming
Tang, Xiaochun
Ouyang, Hongsheng
Pang, Daxin
Large-Scale CRISPR Screen of LDLR Pathogenic Variants
title Large-Scale CRISPR Screen of LDLR Pathogenic Variants
title_full Large-Scale CRISPR Screen of LDLR Pathogenic Variants
title_fullStr Large-Scale CRISPR Screen of LDLR Pathogenic Variants
title_full_unstemmed Large-Scale CRISPR Screen of LDLR Pathogenic Variants
title_short Large-Scale CRISPR Screen of LDLR Pathogenic Variants
title_sort large-scale crispr screen of ldlr pathogenic variants
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10368174/
https://www.ncbi.nlm.nih.gov/pubmed/37496633
http://dx.doi.org/10.34133/research.0203
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