UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology; Department of Neurobiology; Reppert Lab

Publication Date


Document Type



Computational Biology | Genetics | Genomics | Molecular Genetics | Neuroscience and Neurobiology


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.


CRISPR, TALENs, clock genes, germline targeting, insect

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Copyright © 2016 Markert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

DOI of Published Version



G3 (Bethesda). 2016 Apr 7;6(4):905-15. doi: 10.1534/g3.116.027029. Link to article on publisher's site

Journal/Book/Conference Title

G3 (Bethesda, Md.)

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Link to Article in PubMed

PubMed ID


Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.