Genetic Engineering & Biotechnology News

DEC 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Page 54 of 69 | DECEMBER 2017 | 21 zebrafish. "The zebrafish is a practical model organism for creating genetic mutations on the basis of human variants," says Alaa Koleilat, a Ph.D. student at the Mayo Clinic Rochester. "The zebrafish genome contains [a] high percentage of related orthologue human genes." Continues Koleilat, "Additionally, zebrafish have a series of mechanosen- sory receptors similar to human hair cells, easily accessible in the fish lateral line. Most importantly, deaf zebrafish display a characteristic circular swim- ming behavior, making it straightfor- ward to distinguish [them] from wild type." Dr. Schimmenti's team hopes to create model organisms for discovery of pharmacological treatments for hearing loss. Using a gene-editing technology based on transcription activator-like nucleases (TALENs), the team has modeled the most common human variant in GJB2, c.35delG. "Unlike the CRISPR system, the TALEN system is unrestricted in terms of the sequence types it can modify," notes Koleilat. "A TALEN is guided by its own DNA-binding domain, which can be precisely engineered for a target DNA sequence. TALENs can be developed in less than a week." The team created a mutant line with an 8-bp out-of-frame deletion that results in an early stop codon in the zebrafish orthologue of GJB2, cx30.3. Dr. Schimmenti explains that in situ detection of the protein is chal- lenging because the protein itself is small. "So far, we observed that homozy- gous recessive embryos have decreased expression of cx30.3 in the ear and no expression behind the heart," reports Dr. Schimmenti. "And yet these embryos display normal hair cell structure and also have normal mechanotransduction channel activ- ity." A subset of homozygous embryos demonstrates circling behavior linked to deafness. The team continues to perform functional validation studies in knock- out model of cx30.3. Going forward, the team hopes to extend its genetic- engineering methodology to model other genetic variants associated with hearing and vision loss in humans. Novel Vector Suite for Precise Integration Gene knockout and knockin are critical techniques in functional gene analysis, and they may become stan- dard approaches in gene therapy. These techniques have already demon- strated greater utility, given their in- corporation of custom CRISPR/Cas9 nuclease systems. CRISPR/Cas9-based knockout and knockin techniques are being devel- oped by Drena Dobbs, Ph.D., Jeffrey Essner, Ph.D., and Maura McGrail, Ph.D., Iowa State University scientists who specialize in bioinformatics, cel- lular biology, and genome dynamics, respectively. These scientists are col- laborating not just with each other, but also with Stephen C. Ekker, Ph.D., and Karl J. Clark, Ph.D.—Mayo Clinic researchers who apply genome engineering to zebrafish models. (These researchers also work on be- half of Lifengine Technologies, which provides gene-editing reagent kits.) "We built on an in-frame inte- gration method that was recently described in Scientific Reports, in a pa- per credited to Hisano et al., informs Jordan M. Welker, a graduate student at Iowa State University. "When this method is used, a reporter gene flanked by short regions of homol- ogy can be efficiently integrated at a DNA double-strand break using B E S T O F C R I S P R 2 017 ➜ Figure 2. A team of Iowa State University scientists is developing specialized vector technologies that enable precise knockins. This confocal image, provided by the team's Wesley Wierson, shows a zebrafish embryo for which a fluorescent reporter has been integrated (at the esama locus).

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