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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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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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 |
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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
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title_full | Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori
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title_fullStr | Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori
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title_full_unstemmed | Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori
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title_short | Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori
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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 |
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