Precision Mutant Libraries

Multidimensional Approach to Decoding the Mysteries of Animal Development

In this webinar hosted by GEN (Genetic Engineering&Biotechnology News) and sponsored by GenScript, Dr. Justin Crocker talks about his work developing high-throughput methods to elucidate the role of transcriptional enhancers in regulating patterns of gene expression in animal development.

Dr. Crocker’s work aims to understand how the genome code’s linear sequences support specific and detailed expression patterns. To address this fundamental question, he utilizes the fly as a model system combined with mutant libraries. Specifically, he focuses on enhancer regions found upstream and downstream of developmental genes and how they regulate the striped patterns along the anterior-posterior axis of the Drosophila embryo in early development.

Why are these regulatory mechanisms important?

• Transcriptional switches are conserved across invertebrates and vertebrates.
• In mammals, mutations within these regulatory regions are associated with various disease states.
• In humans, over 90% of pathologies map to transcriptional enhancer regions.
• Transcriptional enhancers play a role in shaping animal evolution.

Understanding how transcriptional enhancers work is critical to support predictive capacity and enable their manipulation.

Some of the key questions that his group is addressing include:

• How many binding sites are involved in regulation?
• How robust are enhancers?
• How evolvable are enhancers?
• Can we predict patterns of evolution?

To address these critical questions, Dr. Crocker’s lab studies the pathway involved in the development of hook-like structures in the fly larva, a process regulated by Shavenbaby (svb) transcriptional enhancer. 

Shavenbaby (svb) integrates signaling from pathways such as Hox, Wingless, EGF-R, Hedgehog, and Notch, giving rise to bands along the embryo’s anterior-posterior axis. Shavenbaby transcriptional enhancer regulates these eight bands’ patterning, ultimately forming identical bands containing hook-like structures in the larva.

To uncover the basis of Shavenbaby transcriptional enhancer function, Dr. Crocker’s lab follows a large scale unbiased enhancer screening approach. He has leveraged GenScripts services to develop an enhancer library with a 2% mutation rate. Together, this enhancer mutant library, newly developed robotics for high-throughput processing of embryos, and an imaging analysis pipeline allow his lab to elucidate how specific changes within enhancer sequences impact the ultimate larval phenotype.