Outbred genome sequencing and CRISPR/Cas9 gene editing in butterflies

Abstract

Butterflies are exceptionally diverse but their potential as an experimental system has been limited by the difficulty of deciphering heterozygous genomes and a lack of genetic manipulation technology. Here we use a hybrid assembly approach to construct high quality reference genomes for Papilio xuthus (contig and scaffold N50: 492 Kb, 3.4 Mb) and P. machaon (contig and scaffold N50: 81 Kb, 1.15 Mb), highly heterozygous species that differ in host plant affiliations and adult and larval color patterns. Integrating comparative genomics and analyses of gene expression yields multiple insights into butterfly evolution, including potential roles of specific genes in recent diversification. To functionally test gene function, we develop an efficient (up to 92.5%) CRISPR/Cas9 gene editing method that yields obvious phenotypes with three genes, Abdominal-B, ebony, and frizzled. Our results provide valuable genomic and technological resources for butterflies, and unlock their potential as a genetic model system.

A Practical Guide to CRISPR/Cas9 Genome Editing in Lepidoptera

“…Although we have not rigorously quantified this effect, after extensive work with pigmentation genes in V. cardui, we found that injecting earlier (e.g., at 1 h) typically produces more and larger mutant clones compared to injection performed later (e.g., at 4 h). This is consistent with previous studies that have found a higher deletion frequency when embryos are injected at earlier versus later stages (Li et al 2015a). Thus, for most of our deletion experiments, we aim to inject~1-2 h after oviposition.…”

“…Unfortunately, to our knowledge, these remain the only two examples of lepidopteran knockins outside of the new data we present below. The first example of Cas9 genome editing in a species besides B. mori was described by Li et al (2015a), who produced deletions in three genes in the swallowtail butterfly Papilio xuthus. This was an important case study because it showed that the general approach used by Wang et al (2013) in B. mori could be transferred to other species and still retain the same level of high efficiency.…”

Genomic adaptation to polyphagy and insecticides in a major East Asian noctuid pest

“…We obtained peptides and genome sequences for B. mori 66 , Papilio xuthus 67 and H. melpomene 11 . If a gene had more than one transcript, only the first transcript in the annotation was used.…”

Molecular Mechanisms Underlying Color Vision and Color Formation in Dragonflies

“…Molecular bases underlying color pattern formation and its evolution in dragonflies are just as fascinating and challenging as in butterflies. Recently, effective RNAi-and genome-editing methods have been developed for gene functional analyses in butterflies (Ando and Fujiwara 2013;Nishikawa et al 2015;Li et al 2015;Perry et al 2016;Zhang and Reed 2016;Beldade and Peralta 2017). Applying these methods to dragonflies will be an important step toward future studies in this field (Okude et al 2017).…”