Monarch Reverse Genetics

Danaus plexippus By William Warby (https://flic.kr/p/dtKZF2) [CC BY 2.0 (http://creativecommons.org/licenses/by/2.0)], via Wikimedia Commons

Danaus plexippus By William Warby (https://flic.kr/p/dtKZF2) [CC BY 2.0 (http://creativecommons.org/licenses/by/2.0)], via Wikimedia Commons

Markert et al. just reported their effective use Cas9 and TALENs to knock out cryptochrome 2 and clock in Danaus plexippus using some strategies and approaches that are generally useful to insect scientists and much more efficient than earlier reports of gene editing in D. plexippus.

In 2013 Merlin et al reported the successful use of zinc finger nucleases (ZFNs) to knock out the gene cryptochrome 2 in D. plexippus and while this was an interesting application of engineered nuclease technology it required injection of thousands of D. plexippus embryos in order to recover only a few germline mutants.  Given the challenges of working with this species in the laboratory, this was a herculean effort not likely to be repeated by many similarly interested investigators.

Markert et al. have revisited this problem using TALENs and Cas9 in lieu of ZFNs along with some streamlining of the workflow so that now germline mutants can be obtained after injecting fewer than 100 embryos, making reverse genetic approaches to D. plexippus feasible.

Cas9 with functional domains highlighted in relation to target DNA and guideRNA

Cas9 with functional domains highlighted in relation to target DNA and guideRNA

Markert et al. targeted cryptochrome 2 as well as clock in this case.  The resulting knockouts confirmed the role of CLK in the monarchs circadian clock.

Also of interest are some of the technical aspects of this work that are likely to be of broader interest within the insect science community.

CLOCK (CLK) and CYCLE (CYC) join together in the nucleus and drive the transcription of the per, tim, and cry2 genes to make PERIOD (PER), TIMELESS (TIM), and CRYPTOCHROME 2 (CRY2). In the cytoplasm, PER, TIM and CRY2 form complexes and CRY2 is shuttled into the nucleus. There, it shuts down the transcription driven by CLK and CYC, completing the loop. Meanwhile, PER is progressively phosphorylated and likely helps translocate CRY2 into nucleus. CRYPTOCHROME 1 (CRY1) is a circadian photoreceptor which, upon light exposure (lightning bolt), causes TIM degradation to gain access to the central clock mechanism. The thick gray arrows represent output functions for CRY1 and for CRY2. Image from The Reppert Lab http://reppertlab.org/migration/the-circadian-clock/clockwork/

In some cases the authors chose target sites that included the presence of endogenous restriction endonuclease sites and by so doing gave themselves a simple and inexpensive means by which they could detect mutants.  After amplifying across the target site fragments from mutants would be resistant to digestion with the corresponding restriction enzyme.

To identify mutants in their Cas9 experiments they performed in vitro cleavage assays in which instead of restriction enzymes, T7 endonuclease or Cel1 endonuclease (Surveyor ®) to digest PCR products that included the target site following mutagenesis they use purified Cas9 and the gRNA specific for the target site.  They expressed Cas9 using pET28-Cas9 and purified it using nickel-based affinity chromatography, significantly reducing costs.

All embryos were injected with Cas9 mRNA and specific gRNA very soon after being deposited by females – within 20 minutes, and this probably maximized mosaicism within those embryos and increased the likelihood that the germline would have a mutagenized genome.

Danaus plexippus 5th instar larva Photo (c)2007 Derek Ramsey (Ram-Man) (Own work) [GFDL 1.2 (http://www.gnu.org/licenses/old-licenses/fdl-1.2.html)], via Wikimedia Commons

Danaus plexippus 5th instar larva By Photo (c)2007 Derek Ramsey (Ram-Man) (Own work) [GFDL 1.2 (http://www.gnu.org/licenses/old-licenses/fdl-1.2.html)], via Wikimedia Commons

Markert et al. also genotyped all G0 larvae by clipping their ‘horns’, which could be done without killing the larvae.  Larvae with the highest levels of mosaicism based on in vitro cleavage assays were of particular interest for obtaining G1 progeny.

From 24 G1 larvae in their TALEN mutagenesis experiment to create cry2 mutants, 1 was a mutant.  Their efforts to mutagenize clk were more efficient.  All G0s (17) were mosaics and 33 0f 68 G1s were mutants (52%), 12 of which were bi-allelic mutants (‘homozygotes’), a very workable efficiency.

Cas9 experiments were similarly efficient.

Compared to earlier work with ZFNs, it appears that Cas9 and TALENs are much less toxic to D. plexippus.

The workflow described by Markert et al. is likely to be applicable to many other insects and even those who not work on Lepidoptera will find this interesting and relevant.

Genomic Access to Monarch Migration Using TALEN and CRISPR/Cas9-Mediated Targeted Mutagenesis Matthew J. Markert, Ying Zhang, Metewo S. Enuameh, Steven M. Reppert, Scot A. Wolfe, and Christine Merlin G3 g3.116.027029; Early Online February 2, 2016, doi:10.1534/g3.116.027029

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