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.

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.

Kolundzic, Ena et al. (2017) International Worm Meeting "FACT is a reprogramming barrier in C. elegans and human cells."

Tursun, Baris, Sommermeier, Anne, Diecke, Sebastian, Kolundzic, Ena, Lacadie, Scott, Baytek, Gulkiz, Ofenbauer, Andreas, Akalin, Altuna, Uyar, Bora, Seelk, Stefanie, He, Mei

[International Worm Meeting, 2017]

Direct reprogramming of cellular identities by mis-expressing transcription factors (TFs) is limited in different tissue contexts. We previously identified the histone chaperone LIN-53 as a reprogramming barrier in C. elegans preventing the direct conversion of germ cells to specific neuron types (Tursun et al., Science 2011). Analogously, a recent study showed that the mammalian LIN-53/CAF-1p48-containing histone chaperone CAF-1 is a barrier for cellular reprogramming in mouse cells (Cheloufi et al., Nature 2015) suggesting that more barrier factors might be conserved among C. elegans and higher organisms. Therefore, we conducted genetic screens using transgenic animals expressing CHE-1, the ASE neuron fate-inducing TF, under the heat shock promoter together with ASE-neuron-specific reporter gcy-5::gfp and revealed that the histone chaperone FACT (FAcilitates Chromatin Transcription) is a novel reprogramming barrier. FACT protects different tissues from being directly reprogrammed into neuron-like cells in C. elegans. Moreover, we demonstrate that FACT depletion also enhances the reprogramming of human fibroblasts. FACT is predominantly known as a positive effector that regulates gene expression by facilitating transcription. Nevertheless, FACT can also act as a repressor in certain instances such as at the mating locus in yeast. We performed transcriptome analysis as well as chromatin profiling using ATAC-seq and found that loss of FACT results in transcriptional de-repression at specific genomic loci rather than disturbing chromatin organization globally. Furthermore, FACT depletion-mediated reprogramming requires the presence of the histone variant H2A.Z and Histone H3 phosphorylation by the Aurora Kinase. Hence, aberrant genomic H2A.Z and H3ph localization might facilitate gene activation leading to enhanced reprogramming. Our findings demonstrate the versatility of C. elegans as a discovery tool for identifying and understanding the role of epigenetic regulators in the context of safeguarding cellular identities.