Nearly 40% of our genomes consist of regulatory elements that control when and where a gene is expressed. Although biomedical research has focused on protein-coding regions of genomes, understanding how regulatory regions control gene expression is central to understanding attributes of health and disease.

While progress has been made in understanding gene regulation in cell lines and yeast, few studies have been done in live animals or in large populations. One of the technical challenges of studying regulatory regions is that they must ultimately be understood in the context of their genomic and tissue environments and developmental timing.

Systems biologists at the Max Delbrück Center (MDC) for Molecular Medicine in Berlin report a new in vivo parallel genetics approach in their article “Parallel genetics of regulatory sequences using scalable genome editing in vivo” published in Cell Reports that introduce diverse mutations on a large scale in the genomes of thousands of microscopic worms called Caenorhabditis elegans and tracks their physiological effects. This offers a systematic method to link genotype to phenotype—a monumental task at the current frontiers of biology.

“With cell lines, you are missing development processes, many cell-types, as well as interaction between cell types that all affect gene regulation,” says Jonathan Froehlich, a PhD student in MDC’s Systems Biology of Gene Regulatory Elements Lab in the Berlin Institute for Medical Systems Biology (BIMSB) and co-first author on the article. “We can now really test these regulatory sequences in the environment where they are important and observe the consequences on the organism.”

The researchers use CRISPR-Cas9 to introduce large scale mutations in the form of genomic deletions or insertions (indels) in a parent generation of the hermaphrodite worms and studied the physiological effects in thousands of worms in subsequent generations. “One part is controlled, the part where we design the guide RNAs and tell the Cas9 nuclease where to go, but the outcome of this is semi-random,” says Froehlich. “You will have many different types of outcomes and we can see what the effect is on the animal.”

The authors link several mutations in regulatory regions to specific physiological effects using the new approach. One of their expected findings was the identification of two independently functioning let-7 microRNA binding sites in the downstream regulatory region of a gene called lin-41. If at least  one of the two sites was intact, the worms developed normally, else gene expression was mis-regulated and the worms developed abnormally and died.

“This demonstrates nicely how this system can be used to study gene regulation during development,” says Nikolaus Rajewsky, PhD, scientific director of MDC’s BIMSB, who oversaw the project.

Existing analytical tools proved inadequate in analyzing the large scale data-types from the diverse mutations and the interactive data visualizations required to make sense of it all. Bioinformatics scientist Bora Uyar, PhD, designed a new software package, called crispr-DART—short for Downstream Analysis and Reporting Tool to analyze the data generated using this parallel editing approach, which does not lead to mutations in the target areas with consistency.

“That’s why I call it crispr-DART, you are throwing some arrows in the genome and the tool tells you if you are actually successful or not,” Uyar says. The publicly available software can handle a variety of different sequencing data types—long read, short read, single reads, paired reads, DNA, RNA.

This large-scale screen for sequences that affect phenotypic traits doubles the regulatory alleles registered at Wormbase in the last 40 years. The authors are confident that this new approach will help decipher the logic underlying gene regulation and reveal mechanisms that affect morphological and physiological attributes in living organisms.

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