• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Molecular Biology >Multiple Parameters Drive the Efficiency of CRISPR/Cas9-Induced Gene Modifications in Yarrowia lipolytica
【24h】

Multiple Parameters Drive the Efficiency of CRISPR/Cas9-Induced Gene Modifications in Yarrowia lipolytica

机译：多个参数推动Yarrowia Lipolytica中CRISPR / CAS9诱导的基因修饰的效率

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Yarrowia lipolyticais an oleaginous yeast of growing industrial interest for biotechnological applications. In the last few years, genome edition has become an easier and more accessible prospect with the world wild spread development of CRISPR/Cas9 technology. In this study, we focused our attention on the production of the two key elements of the CRISPR–Cas9 ribonucleic acid protein complex in this non-conventional yeast. The efficiency of NHEJ-induced knockout was measured by time-course monitoring using multiple parameters flow cytometry, as well as phenotypic and genotypic observations, and linked to nuclease production levels showing that its strong overexpression is unnecessary. Thus, the limiting factor for the generation of a functional ribonucleic acid protein complex clearly resides in guide expression, which was probed by testing different linker lengths between the transfer RNA promoter and the sgRNA. The results highlight a clear deleterious effect of mismatching bases at the 5′ end of the target sequence. For the first time in yeast, an investigation of its maturation from the primary transcript was undertaken by sequencing multiple sgRNAs extracted from the host. These data provide insights into of the yeast small RNA processing, from synthesis to maturation, and suggests a pathway for their degradation inY. lipolytica. Subsequently, a whole-genome sequencing of a modified strain detected no abnormal modification due to off-target effects, confirming CRISPR/Cas9 as a safe strategy for editingY. lipolyticagenome. Finally, the optimized system was used to promotein vivodirected mutagenesisviahomology-directed repair with a ssDNA oligonucleotide.
机译：Yarrowia Lipolyticais一种含有生物技术应用营业利益的含油酵母。在过去的几年里，Genome Edition已经成为CRISPR / CAS9技术的世界狂野传播开发的更容易和更容易获得的前景。在这项研究中，我们将注意力集中在该非常规酵母中的CRISPR-CAS9核糖核酸蛋白复合物的两个关键要素的生产。使用多个参数流式细胞术以及表型和基因型观察，并与核酸酶产生水平有关的时间过程测量NHEJ诱导的敲除，表明其强烈过度表达是不必要的。因此，通过在转移RNA启动子和SGRNA之间测试不同的接头长度来探测，产生功能性核糖核酸蛋白复合物的产生的限制因素清楚地存在于引导表达中。结果突出了目标序列5'末端不匹配碱的明显有害效果。首次在酵母中，通过测序从宿主提取的多个SGRNA来进行对初级转录物的成熟的调查。这些数据提供了对酵母小RNA处理的洞察，从合成到成熟，并表明其降解Iny的途径。 Lipolytica。随后，由于偏离目标效果，改性菌株的全基因组测序检测到没有异常修改，确认CRISPR / CAS9作为编辑的安全策略。脂肪变性。最后，优化的系统用于通过SSDNA寡核苷酸引起vivodiowected诱变viahomologyology学。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号