standard Nasonia Genome Editing

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Deanna Arsala is a Ph.D. student in the laboratory of Dr. Jeremy Lynch, Biological Sciences University of Illinois at Chicago  MORE ABOUT THE AUTHOR

Scientists in the Ferree and Akbari laboratories developed a CRISPR/Cas9 protocol for germline editing in Nasonia vitripennis, adding an essential tool for the study of this important insect.  They report their results in bioArxiv (Li et. al 2017).

With its haplodiploid genetics and tractability in the laboratory, the parasitoid jewel wasp Nasonia is a powerful model system which continues to make substantial contributions to our understanding of ecology, behavior, evolutionary and developmental biology. Unlike the popular model organism, Drosophila, Nasonia’s haplodiploid sex determination system makes it significantly easier to screen for recessive mutations. Its fully sequenced genome has allowed for several transcriptome and methylome studies. While RNAi has been successful in Nasonia, there were no published protocols to generate targeted mutations.

cinnabar

Ommochrome (brown eye pigment) biosynthetic pathway beginning with tryptophan. The cinnabar gene encodes for kynurenine 3-monooxygenase.

The authors targeted the conserved eye pigmentation gene cinnabar, which results in a mutant phenotype of red eye-color when silenced through larval RNAi, making it the ideal gene to test and develop a CRISPR/Cas9 protocol for Nasonia.  cinnabar encodes the enzyme kynurenine monoxygenase that converts kynureninc to hydroxykinurenine, an essential step in the biosynthesis of brown eye color pigments, ommochromes.

Li et al’s protocol resulted in 32-36% of adult wasps developing from injected embryos (G0) displaying the mutant cinnabar phenotype (red eyes). These mutations were also efficiently transmitted into the germ-line, producing stable, homozygous mutant lines.

cinnabar mutation in Drosophila melanogaster

Nasonia presents a number of technical challenges that make the work of Li et al. notable.  Nasonia female lay eggs in the pupae of other insects.  In the laboratory Sarchophaga bullata is a common host whose pupae are commercially available and can be stored in cool temperatures for prolonged periods.  When needed the pupae are presented to gravid females in a way the results in Nasonia eggs only being laid at one end of the pupae.  To access the freshly laid eggs the pupal cuticle at the end containing the eggs is removed, exposing a cluster of eggs.

The eggs have a very thin, transparent chorion and while small, the eggs are readily injected using technologies and procedures available for the injection of other insect embryos.

The injected eggs are then placed back into a pupa and allowed to develop and emerge.  Li et al (2017) have successfully solved these various technical challenges and have successfully created mutations using Cas9.

Nasonia

Nasonia vitripennis life cycle. Image Credit: Here

Much of the current work in Nasonia to data has relied on RNAi, which produces inconsistent gene knockdowns. As the CRISPR/Cas9 protocol can be used to target other Nasonia genes, a fully functional reverse genetics toolkit would allow Nasonia geneticists to expand their experimental repertoire while continuing to utilize the wasp’s unique advantages as an emerging model system.

On the horizon, CRISPR/Cas9 can also be utilized for the introduction of novel genetic elements and tools for more sophisticated genetic analyses. Although homology-directed repair (HDR) has yet to be demonstrated in the wasp, heritable site-specific transgenesis would further expand the genetic toolbox of Nasonia, bringing forth its preeminence as a model system across biological fields.

 

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