Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases

Abstract

The development of reverse-genetic tools in “nonmodel” insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into “one nucleus” stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and “nonmodel” insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes.

Vertebrate-like CRYPTOCHROME 2 from monarch regulates circadian transcription via independent repression of CLOCK and BMAL1 activity

“…S2C). These results stand in sharp contrast with previous work showing no difference in the timing of eclosion between monarchs carrying either two or a single functional allele of dpCry2 in a wildtype dpBMAL1 background (18).…”

“…3A and Fig. S2C), as expected based on the previously reported circadian arrhythmicity of dpCry2 knockouts (18). However, to our surprise, dpBmal1ΔCter homozygous mutants carrying a single functional allele of dpCry2 (dpBmal1 m2; dpCry2…”

“…We first examined the timing of pupal eclosion (i.e., the emergence of the adult from its pupal case), a robust and quantifiable behavior under the control of the brain circadian clock (18,22,23), focusing our analysis on female dpCyc-like knockouts, heterozygous males, and wild-type siblings of both sexes (Materials and Methods). We found that the circadian timing of adult eclosion was abolished in dpCyc-like hemizygous butterflies, while heterozygous and wild-type siblings eclosed rhythmically with a similar peak of eclosion during the early subjective day (P > 0.05, one-way ANOVA followed by Tukey's post hoc test) ( Fig.…”

“…Not only does the monarch butterfly possess a single copy of a mammalian-like repressive CRY (designated "CRY2") (18)(19)(20) and an ortholog of mammalian Significance Daily rhythms in animal behavior, physiology, and metabolism are driven by cell-autonomous mechanisms that keep time and control overt rhythms via transcriptional feedback loops, making it fundamental to define the mechanisms driving rhythmic transcription. In mammals, PERIOD and CRYPTOCHROME (CRY) rhythmically repress CLOCK:BMAL1 transcriptional activity, but the mechanisms by which CRY represses CLOCK:BMAL1 activity are not fully understood.…”

A Practical Guide to CRISPR/Cas9 Genome Editing in Lepidoptera

“…Despite strong interest in this group, however, there has been a frustrating lack of progress in developing routine approaches for manipulative genetic work. While the last two decades have seen examples of transgenesis and targeted knockouts using methods like transposon insertion (Tamura et al 2000), zinc-finger nucleases (Takasu et al 2010;Merlin et al 2013), and TALENs (Takasu et al 2013;Markert et al 2016), especially in the silk moth Bombyx mori, these approaches have resisted widespread application due to their laborious nature. We see two other main reasons manipulative genetics has failed to become routine in Lepidoptera.…”