title: 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.
authors.affiliation: Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, Texas 77843.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Markert
authors.affiliation: Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, Texas 77843.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
authors.forename: Metewo S
authors.initials: MS
authors.lastname: Enuameh
authors.affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Reppert
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
authors.forename: Scot A
authors.initials: SA
authors.lastname: Wolfe
authors.affiliation: Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, Texas 77843 cmerlin@bio.tamu.edu.
authors.forename: Christine
authors.initials: C
authors.lastname: Merlin
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002460:Cell Line; D002645:Chickens; D023061:Gene Order; D018390:Gene Targeting / Q000379:methods; D017930:Genes, Reporter; D005818:Genetic Engineering; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods; D016296:Mutagenesis; D000069896:Transcription Activator-Like Effector Nucleases / Q000378:metabolism*
chemical_list: D000069896:Transcription Activator-Like Effector Nucleases
keywords: CRISPR; TALENs; clock genes; germline targeting; insect
references.citation: PLoS One. 2014;9(7):e101210
references.pmid: 25013902
references.citation: Proc Natl Acad Sci U S A. 2002 Jul 23;99(15):10162-6
references.pmid: 12107283
references.citation: Nature. 2014 Oct 16;514(7522):317-21
references.pmid: 25274300
references.citation: PLoS One. 2015;10(3):e0122353
references.pmid: 25815482
references.citation: Science. 2003 May 23;300(5623):1303-5
references.pmid: 12764200
references.citation: Cell. 1998 May 29;93(5):791-804
references.pmid: 9630223
references.citation: Neuron. 2006 May 4;50(3):465-77
references.pmid: 16675400
references.citation: PLoS Biol. 2008 Jan;6(1):e4
references.pmid: 18184036
references.citation: Genome Res. 2009 Jul;19(7):1279-88
references.pmid: 19470664
references.citation: Science. 2009 Sep 25;325(5948):1700-4
references.pmid: 19779201
references.citation: Trends Neurosci. 2010 Sep;33(9):399-406
references.pmid: 20627420
references.citation: Nucleic Acids Res. 2011 Jul;39(12):e82
references.pmid: 21493687
references.citation: Methods Cell Biol. 2011;104:51-8
references.pmid: 21924156
references.citation: Cell. 2011 Nov 23;147(5):1171-85
references.pmid: 22118469
references.citation: Genome Res. 2012 May;22(5):939-46
references.pmid: 22267522
references.citation: Nat Commun. 2012;3:958
references.pmid: 22805565
references.citation: Science. 2012 Aug 17;337(6096):816-21
references.pmid: 22745249
references.citation: Nat Commun. 2012;3:1017
references.pmid: 22910363
references.citation: PLoS One. 2012;7(9):e45035
references.pmid: 23028749
references.citation: Insect Biochem Mol Biol. 2013 Jan;43(1):17-23
references.pmid: 23142190
references.citation: Genome Res. 2013 Jan;23(1):159-68
references.pmid: 23009861
references.citation: Nat Biotechnol. 2013 Mar;31(3):227-9
references.pmid: 23360964
references.citation: Genome Res. 2013 Jun;23(6):1008-17
references.pmid: 23478401
references.citation: Nat Commun. 2014;5:3157
references.pmid: 24445736
references.citation: Sci Rep. 2014;4:4489
references.pmid: 24671069
references.citation: Insect Biochem Mol Biol. 2014 Jun;49:35-42
references.pmid: 24698835
references.citation: Annu Rev Biochem. 2014;83:409-39
references.pmid: 24606144
references.citation: Proc Natl Acad Sci U S A. 2015 Mar 31;112(13):4038-43
references.pmid: 25775608
references.citation: Cell Rep. 2015 Apr 7;11(1):51-60
references.pmid: 25818303
references.citation: Genetics. 2013 Aug;194(4):1029-35
references.pmid: 23709638
references.citation: Trends Biotechnol. 2013 Jul;31(7):397-405
references.pmid: 23664777
references.citation: Development. 2015 Aug 15;142(16):2832-9
references.pmid: 26160901
references.citation: Proc Natl Acad Sci U S A. 2013 Aug 20;110(34):13904-9
references.pmid: 23918387
references.citation: Mol Genet Genomics. 2013 Dec;288(12):683-90
references.pmid: 24077893
references.citation: Nat Commun. 2015;6:8212
references.pmid: 26354079
references.citation: Genome Biol Evol. 2015 Sep;7(9):2545-59
references.pmid: 26338190
references.citation: Sci Rep. 2015;5:15885
references.pmid: 26521965
references.citation: Annu Rev Entomol. 2016 Mar 11;61:25-42
references.pmid: 26473314
references.citation: Neuron. 1999 Dec;24(4):953-65
references.pmid: 10624958
references.citation: J Biol Chem. 2014 Jul 11;289(28):19681-93
references.pmid: 24872414