Cargando…

Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori

We report the establishment of an efficient and heritable gene mutagenesis method in the silkworm Bombyx mori using modified type II clustered regularly interspaced short palindromic repeats (CRISPR) with an associated protein (Cas9) system. Using four loci Bm-ok, BmKMO, BmTH, and Bmtan as candidate...

Descripción completa

Detalles Bibliográficos
Autores principales: Wei, Wei, Xin, Huhu, Roy, Bhaskar, Dai, Junbiao, Miao, Yungen, Gao, Guanjun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094479/
https://www.ncbi.nlm.nih.gov/pubmed/25013902
http://dx.doi.org/10.1371/journal.pone.0101210
_version_ 1782325835565367296
author Wei, Wei
Xin, Huhu
Roy, Bhaskar
Dai, Junbiao
Miao, Yungen
Gao, Guanjun
author_facet Wei, Wei
Xin, Huhu
Roy, Bhaskar
Dai, Junbiao
Miao, Yungen
Gao, Guanjun
author_sort Wei, Wei
collection PubMed
description We report the establishment of an efficient and heritable gene mutagenesis method in the silkworm Bombyx mori using modified type II clustered regularly interspaced short palindromic repeats (CRISPR) with an associated protein (Cas9) system. Using four loci Bm-ok, BmKMO, BmTH, and Bmtan as candidates, we proved that genome alterations at specific sites could be induced by direct microinjection of specific guide RNA and Cas9-mRNA into silkworm embryos. Mutation frequencies of 16.7–35.0% were observed in the injected generation, and DNA fragments deletions were also noted. Bm-ok mosaic mutants were used to test for mutant heritability due to the easily determined translucent epidermal phenotype of Bm-ok-disrupted cells. Two crossing strategies were used. In the first, injected Bm-ok moths were crossed with wild-type moths, and a 28.6% frequency of germline mutation transmission was observed. In the second strategy, two Bm-ok mosaic mutant moths were crossed with each other, and 93.6% of the offsprings appeared mutations in both alleles of Bm-ok gene (compound heterozygous). In summary, the CRISPR/Cas9 system can act as a highly specific and heritable gene-editing tool in Bombyx mori.
format Online
Article
Text
id pubmed-4094479
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-40944792014-07-15 Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori Wei, Wei Xin, Huhu Roy, Bhaskar Dai, Junbiao Miao, Yungen Gao, Guanjun PLoS One Research Article We report the establishment of an efficient and heritable gene mutagenesis method in the silkworm Bombyx mori using modified type II clustered regularly interspaced short palindromic repeats (CRISPR) with an associated protein (Cas9) system. Using four loci Bm-ok, BmKMO, BmTH, and Bmtan as candidates, we proved that genome alterations at specific sites could be induced by direct microinjection of specific guide RNA and Cas9-mRNA into silkworm embryos. Mutation frequencies of 16.7–35.0% were observed in the injected generation, and DNA fragments deletions were also noted. Bm-ok mosaic mutants were used to test for mutant heritability due to the easily determined translucent epidermal phenotype of Bm-ok-disrupted cells. Two crossing strategies were used. In the first, injected Bm-ok moths were crossed with wild-type moths, and a 28.6% frequency of germline mutation transmission was observed. In the second strategy, two Bm-ok mosaic mutant moths were crossed with each other, and 93.6% of the offsprings appeared mutations in both alleles of Bm-ok gene (compound heterozygous). In summary, the CRISPR/Cas9 system can act as a highly specific and heritable gene-editing tool in Bombyx mori. Public Library of Science 2014-07-11 /pmc/articles/PMC4094479/ /pubmed/25013902 http://dx.doi.org/10.1371/journal.pone.0101210 Text en © 2014 Wei et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Wei, Wei
Xin, Huhu
Roy, Bhaskar
Dai, Junbiao
Miao, Yungen
Gao, Guanjun
Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori
title Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori
title_full Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori
title_fullStr Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori
title_full_unstemmed Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori
title_short Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori
title_sort heritable genome editing with crispr/cas9 in the silkworm, bombyx mori
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094479/
https://www.ncbi.nlm.nih.gov/pubmed/25013902
http://dx.doi.org/10.1371/journal.pone.0101210
work_keys_str_mv AT weiwei heritablegenomeeditingwithcrisprcas9inthesilkwormbombyxmori
AT xinhuhu heritablegenomeeditingwithcrisprcas9inthesilkwormbombyxmori
AT roybhaskar heritablegenomeeditingwithcrisprcas9inthesilkwormbombyxmori
AT daijunbiao heritablegenomeeditingwithcrisprcas9inthesilkwormbombyxmori
AT miaoyungen heritablegenomeeditingwithcrisprcas9inthesilkwormbombyxmori
AT gaoguanjun heritablegenomeeditingwithcrisprcas9inthesilkwormbombyxmori