Genomic Access to Monarch Migration Using TALEN and CRISPR/Cas9-Mediated Targeted Mutagenesis

Abstract

The eastern North American monarch butterfly, Danaus plexippus, is an emerging model system to study the neural, molecular, and genetic basis of animal long-distance migration and animal clockwork mechanisms. While genomic studies have provided new insight into migration-associated and circadian clock genes, the general lack of simple and versatile reverse-genetic methods has limited in vivo functional analysis of candidate genes in this species. Here, we report the establishment of highly efficient and heritable gene mutagenesis methods in the monarch butterfly using transcriptional activator-like effector nucleases (TALENs) and CRISPR-associated RNA-guided nuclease Cas9 (CRISPR/Cas9). Using two clock gene loci, cryptochrome 2 and clock (clk), as candidates, we show that both TALENs and CRISPR/Cas9 generate high-frequency nonhomologous end-joining (NHEJ)-mediated mutations at targeted sites (up to 100%), and that injecting fewer than 100 eggs is sufficient to recover mutant progeny and generate monarch knockout lines in about 3 months. Our study also genetically defines monarch CLK as an essential component of the transcriptional activation complex of the circadian clock. The methods presented should not only greatly accelerate functional analyses of many aspects of monarch biology, but are also anticipated to facilitate the development of these tools in other nontraditional insect species as well as the development of homology-directed knock-ins.

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

“…S1C). We found sex-based segregation for the mutation in the corresponding adults, with females being hemizygous and males heterozygous, demonstrating that dpBmal1 is located on the Z sex chromosome along with monarch Clk (21) [in lepidopterans, females are heterogametic (ZW), and males are homogametic (ZZ)]. Importantly, the 13-bp deletion germline mutation resulted in a frameshift leading to the truncation of the dpBmal1 C terminus and was designated "dpCyc-like.…”

“…However, the relative importance of these two sites on CLOCK:BMAL1 for CRY1 repression has not been firmly established through in vivo experiments, because the only existing mouse mutant lacking the BMAL1 C-terminal TAD harbors compromised transcriptional activity (28). In this work, we leveraged the monarch butterfly as an alternative system to directly test in vivo the importance of the BMAL1 TAD for vertebrate-like CRY repressive function because it possesses mammalian-like clock components and is readily amenable to CRISPR-mediated targeted mutagenesis (21). Through the generation of a mutant lacking the BMAL1 TAD α-helix but retaining the most distal C-terminal residues sufficient to provide transcriptional activity, we present in vivo genetic evidence showing that, despite regulating the circadian phase or period, the BMAL1 TAD α-helix is not necessary for repression of CLOCK-BMAL1 transcriptional activity by insect CRY2.…”

“…BMAL1 with a highly conserved TAD in the last 30 amino acids of its C terminus, but its genome can also be targeted with CRISPR/Cas9 (21). Here, we generated domain-specific mutations in the monarch (dp)BMAL1 in vivo using CRISPR/Cas9.…”

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.…”

Genome Editing of Wnt-1, a Gene Associated with Segmentation, via CRISPR/Cas9 in the Pine Caterpillar Moth, Dendrolimus punctatus

“…These two RNA molecules can be fused artificially to form a chimeric RNA molecule called single guide RNA (sgRNA). CRISPR/Cas9 system has been used to produce heritable mutations in non-model organisms, including RNAi-recalcitrant Lepidoptera, such as Bombyx mori, Danaus plexippus, Spodoptera litura, Plutella xylostella, Spodoptera littoralis , and Helicoverpa armigera (Wang et al, 2013, 2016; Daimon et al, 2014; Huang et al, 2016; Koutroumpa et al, 2016; Markert et al, 2016; Zhu et al, 2016). …”