C. elegans Gene Regulatory Alleles and Reporter Bashing Studies.

Gene regulation has been studied in C. elegans for over 30 years. In this analysis of 102 publications, we find that most transcriptional cis-regulatory elements are located within 5,000 bp of the transcription start site. Over 75% of studies conclude that transcriptional elements and 5'UTRs activate-, while 3'UTRs repress gene expression. While gene regulatory mutations make up less than 0.8% of alleles in forward genetics screens, recent CRISPR-Cas approaches are increasing the number of tested mutations. This work provides a resource of known gene regulatory sequences in C.elegans .

Estimation of C. elegans cell- and tissue volumes.

Although C. elegans is one of the best-studied model organisms, an estimate of its cell sizes and tissues is missing. Here we used the Virtual Worm that is based on electron microscopy images to calculate a zeroth-order approximation of cell and tissue sizes of C. elegans. We conclude that the intestine is the largest tissue, followed by the hypodermis, gonads, body wall muscles, pharynx, and neurons. Thus, we provide an approximation of tissue volumes of young adult C. elegans.

Parallel genetics of regulatory sequences using scalable genome editing in vivo.

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.