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G3 (Bethesda). 2016 Apr 7;6(4):905-15. doi: 10.1534/g3.116.027029.

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

Author information

  • 1Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, Texas 77843.
  • 2Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
  • 3Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
  • 4Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, Texas 77843 cmerlin@bio.tamu.edu.

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.

KEYWORDS:

CRISPR; TALENs; clock genes; germline targeting; insect

PMID:
26837953
PMCID:
PMC4825660
DOI:
10.1534/g3.116.027029
[PubMed - in process]
Free PMC Article
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