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dc.contributor.authorMarkert, Matthew J.
dc.contributor.authorZhang, Ying
dc.contributor.authorEnuameh, Metewo Selase
dc.contributor.authorReppert, Steven M.
dc.contributor.authorWolfe, Scot A.
dc.contributor.authorMerlin, Christine
dc.date2022-08-11T08:09:44.000
dc.date.accessioned2022-08-23T16:41:44Z
dc.date.available2022-08-23T16:41:44Z
dc.date.issued2016-04-07
dc.date.submitted2016-05-18
dc.identifier.citation<p>G3 (Bethesda). 2016 Apr 7;6(4):905-15. doi: 10.1534/g3.116.027029. <a href="http://dx.doi.org/10.1534/g3.116.027029">Link to article on publisher's site</a></p>
dc.identifier.issn2160-1836 (Linking)
dc.identifier.doi10.1534/g3.116.027029
dc.identifier.pmid26837953
dc.identifier.urihttp://hdl.handle.net/20.500.14038/39945
dc.description.abstractThe 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.
dc.language.isoen_US
dc.relation<p><a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=26837953&dopt=Abstract">Link to Article in PubMed</a></p>
dc.rightsCopyright © 2016 Markert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectCRISPR
dc.subjectTALENs
dc.subjectclock genes
dc.subjectgermline targeting
dc.subjectinsect
dc.subjectComputational Biology
dc.subjectGenetics
dc.subjectGenomics
dc.subjectMolecular Genetics
dc.subjectNeuroscience and Neurobiology
dc.titleGenomic Access to Monarch Migration Using TALEN and CRISPR/Cas9-Mediated Targeted Mutagenesis
dc.typeJournal Article
dc.source.journaltitleG3 (Bethesda, Md.)
dc.source.volume6
dc.source.issue4
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=3759&amp;context=oapubs&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/2754
dc.identifier.contextkey8614706
refterms.dateFOA2022-08-23T16:41:44Z
html.description.abstract<p>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.</p>
dc.identifier.submissionpathoapubs/2754
dc.contributor.departmentReppert Lab
dc.contributor.departmentNeurobiology
dc.contributor.departmentBiochemistry and Molecular Pharmacology
dc.source.pages905-15


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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 (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Except where otherwise noted, this item's license is described as Copyright © 2016 Markert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.