Froehlich, Jonathan et al. (2017) International Worm Meeting "Targeted, high-throughput interrogation of regulatory elements in C. elegans."

Rajewsky, Nikolaus, Herzog, Margareta, Froehlich, Jonathan

[International Worm Meeting, 2017]

Gene regulatory elements determine when, where and how much a protein is expressed. C. elegans allows tracking of cell-, tissue- or developmental stage specific expression in vivo. Furthermore it is possible to evaluate viability or other phenotypes and to observe several generations in a short time. Regulatory regions controlling gene expression have been studied in C. elegans by cloning different regions and injection of each construct ("bashing"). Few elements have been isolated in forward genetic screens and studied for effects on phenotype. Here we present our approach for unbiased interrogation of regulatory regions for a given locus, with CRISPR/Cas9 based systematic mutation. Using strains with inducible Cas9, staged embryos, and pools of sgRNAs, we are able to obtain thousands of independent mutant F1s in parallel, requiring very few injections. We have set up a targeted sequencing approach using large 3-6 kb amplicons to analyze indels with deep sequencing. As a proof of concept we applied this method to the his-72 locus. We focused on the 3' untranslated region, also targeting promoter-, coding-, and intron sequences at different developmental stages. In addition, we cover the lin-41 3'UTR with different sets of sgRNAs and analyze the depletion of genotypes over several generations. We will report about these ongoing experiments.

Froehlich, Jonathan et al. (2021) International Worm Meeting "Parallel genetics of regulatory sequences using induced genome editing"

Herzog, Margareta, Uyar, Bora, Rajewsky, Nikolaus, Akalin, Altuna, Theil, Kathrin, Froehlich, Jonathan, Glazar, Petar

[International Worm Meeting, 2021]

Understanding how regulatory sequences control gene expression is fundamental to explain how phenotypes arise in health and disease. The functions of regulatory elements must ultimately be understood within their genomic environment and developmental- or tissue-specific contexts. Here, we used induced Cas9 expression and multiplexed guide RNAs in C. elegans to create hundreds of mutations in enhancers, promoters and 3′ UTRs of 16 genes in parallel. We then analyzed the resulting complex populations by either selecting for phenotypic traits or reporter expression, or by DNA- or RNA amplicon sequencing of bulk samples. We developed a software pipeline, crispr-DART, to analyze targeted sequencing and describe the characteristics of >12,000 dsDNA break-induced indel mutations. We also analyzed the lin-41 3′ UTR and found that the two let-7 miRNA binding sites can function independently and that one of the sites is more important for mRNA repression and phenotype. In summary, our approach enables highly parallelized functional analysis of regulatory sequences in vivo.

Froehlich, Jonathan et al. (2019) International Worm Meeting "Conditional, targeted and multiplexed mutagenesis of regulatory sequences in animals."

Froehlich, Jonathan, Schilling, Marcel, Theil, Kathrin, Rajewsky, Nikolaus, Uyar, Bora, Herzog, Margareta, Akalin, Altuna

[International Worm Meeting, 2019]

High-throughput mutagenesis using CRISPR/Cas9 has been successfully applied in cell lines to interrogate regulatory elements or protein coding variants. However, regulation and function is often highly cell-type- and development specific and therefore only appears when tested in an animal. Here we use transient Cas9 induction and pooled guide RNAs in C. elegans to generate thousands of mutant animals in parallel. For example, when we target a 250 bp region of interest, indel mutations create hundreds of unique genotypes in the analyzed population. We apply this method to target regulatory regions, in particular 3'UTRs, of more than a dozen genes and isolate mutations which lead to very clear loss-of-function phenotypes. For one 3'UTR we found that a surprising number of insertions in different regions can have strong repressive effects and consequent phenotypes, while deletions are well tolerated. We also show that with targeted RNA sequencing we can directly measure the effect of different 3'UTR mutations on mRNA levels at particular developmental stages within a population. Altogether we created several thousand mutations which allow us to look at DNA repair patterns created by Cas9 in the germline and to compare efficiencies of different sgRNAs. Our method is useful to understand genotype to phenotype relations and to discover new functional elements directly in an animal.