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

G3 : Genes - Genomes - Genetics
Matthew J MarkertChristine Merlin

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 accelerat...Continue Reading

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Methods Mentioned

BETA
gene knockouts
genotyping
dissecting
PCR
electrophoresis
cleavage assay
restriction digest
targeted mutations
dissection

Software Mentioned

ImageJ
QI Macros
monarch

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