Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, Germany.
Bioinformatics and Omics Data Science Platform, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, Germany.
Cell Rep. 2021 Apr 13;35(2):108988. doi: 10.1016/j.celrep.2021.108988.
How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. Regulatory elements must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3' UTRs of 16 genes in C. elegans. Our software crispr-DART analyzes indel mutations in targeted DNA sequencing. We quantify the impact of mutations on expression and fitness by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3' UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression independently of each other. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of regulatory sequences directly in animals.
调控序列如何控制基因表达对于解释健康和疾病中的表型至关重要。调控元件最终必须在其基因组环境和发育或组织特异性背景中得到理解。由于这在技术上具有挑战性，因此很少有调控元件在体内得到了表征。在这里，我们使用诱导型 Cas9 和多重向导 RNA 在秀丽隐杆线虫的 16 个基因的增强子/启动子和 3'UTR 中创建了数百个突变。我们的软件 crispr-DART 分析靶向 DNA 测序中的插入缺失突变。我们通过靶向 RNA 测序和 DNA 采样来量化突变对表达和适应性的影响。当我们将这种方法应用于 lin-41 3'UTR 并生成数百个突变体时，我们发现 miRNA let-7 的两个相邻结合位点可以独立地调节 lin-41 的表达。最后，我们为几个基因将调控基因型映射到表型特征上。我们的方法能够在动物体内直接对调控序列进行平行分析。
