Large-Scale CRISPR Screen of LDLR Pathogenic Variants  

作　　者：Mengjing Li Lerong Ma Yiwu Chen Jianing Li Yanbing Wang Wenni You Hongming Yuan Xiaochun Tang Hongsheng Ouyang Daxin Pang 

机构地区：[1]Key Lab for Zoonoses Research,Ministry of Education,Animal Genome Editing Technology Innovation Center,College of Animal Sciences,Jilin University,Changchun,Jilin Province 130062,China [2]The Institute of Translational Medicine,Tianjin Union Medical Center of Nankai University,Tianjin 300071,China [3]Chongqing ResearchInstitute,Jilin University,Chongqing 401123,China [4]Chongqing Jitang Biotechnology Research Institute Co.Ltd.,Chongqing,China

出　　处：《Research》2024年第2期213-225,共13页研究（英文）

基　　金：supported by the National Key R&D Program of China(2019YFA0110702 and 2021YFA0805901).

摘　　要：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.

关 键 词：LDLR SCREEN STRAIGHT 

分 类 号：R54[医药卫生—心血管疾病]