Heritable genome editing with CRISPR/Cas9 in the silkworm, Bombyx mori.
Heritable genome editing with CRISPR/Cas9 in the silkworm, Bombyx mori.
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使用 CRISPR/Cas9 对家蚕进行遗传基因组编辑
DOI：
10.1371/journal.pone.0101210
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Gao G
中科院分区：
文献类型：
--
作者：
Wei W;Xin H;Roy B;Dai J;Miao Y;Gao G
我们报道了一种在家蚕（Bombyx mori）中建立的高效且可遗传的基因突变方法，该方法使用了改良的II型成簇规律间隔短回文重复序列（CRISPR）及其相关蛋白（Cas9）系统。以Bm - ok、BmKMO、BmTH和Bmtan这四个基因座作为候选位点，我们证明了通过将特异性引导RNA和Cas9 - mRNA直接显微注射到家蚕胚胎中，可以诱导特定位点的基因组改变。在注射的这一代中观察到16.7% - 35.0%的突变频率，并且还发现了DNA片段缺失。由于Bm - ok基因被破坏的细胞具有易于确定的半透明表皮表型，因此使用Bm - ok嵌合突变体来检测突变的遗传性。使用了两种杂交策略。第一种是将注射后的Bm - ok蛾与野生型蛾杂交，观察到生殖系突变传递频率为28.6%。在第二种策略中，将两只Bm - ok嵌合突变蛾相互杂交，93.6%的后代在Bm - ok基因的两个等位基因上都出现了突变（复合杂合子）。总之，CRISPR/Cas9系统可作为一种在家蚕中高度特异性且可遗传的基因编辑工具。
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.
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影响因子：
3.8
作者：
Takasu, Yoko;Kobayashi, Isao;Zurovec, Michal
通讯作者：
Zurovec, Michal
影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
3.3
作者：
Tzur, Yonatan B.;Friedland, Ari E.;Colaiacovo, Monica P.
通讯作者：
Colaiacovo, Monica P.
影响因子：
46.9
作者：
通讯作者：
--
DOI：
10.1126/science.1232033
发表时间：
2013-02-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Mali P;Yang L;Esvelt KM;Aach J;Guell M;DiCarlo JE;Norville JE;Church GM
通讯作者：
Church GM