Abstract
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 invivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds ofmutations 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.
Highlights
Understanding gene regulation is fundamental for understanding development and tissue function in health and disease
How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease
Cas9 induction for targeted and parallel mutagenesis in C. elegans populations To introduce many different targeted mutations in vivo, we developed a scalable approach in C. elegans using inducible expression of Cas9 and several multiplexed single guide RNAs
Summary
Understanding gene regulation is fundamental for understanding development and tissue function in health and disease. Many generegulatory sequences encode multiple functions that can cooperate, compensate, and compete (Davidson, 2010; Levo and Segal, 2014; Long et al, 2016). The interaction between effectors and regulatory elements can be modulated by sequence structure, co-factors, chemical modifications, and the temporal order of binding, and sequence activity is dependent on native sequence context, cell type, development, and the environment (Davidson, 2010; Dominguez et al, 2018; Jankowsky and Harris, 2015; Levo and Segal, 2014; Long et al, 2016). To understand biological functions and mechanisms in animals, scalable approaches to target regulatory sequences with many different mutations are required
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