• 主题
高级搜索  >
历史搜索 清空历史
首页> 美国卫生研究院文献>Genome Research >High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9
【2h】

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

机译：使用CRISPR / Cas9在斑马鱼中进行高通量基因靶向和表型分析

代理获取
本网站仅为用户提供外文OA文献查询和代理获取服务，本网站没有原文。下单后我们将采用程序或人工为您竭诚获取高质量的原文，但由于OA文献来源多样且变更频繁，仍可能出现获取不到、文献不完整或与标题不符等情况，如果获取不到我们将提供退款服务。请知悉。
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published “rules” for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5′ end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.
机译：CRISPR / Cas9在各种模型生物中作为基因组编辑工具的使用已从根本上改变了靶向诱变。在这里，我们介绍了使用CRISPR / Cas9技术在斑马鱼中的高通量靶向诱变管线，这将使基因组的饱和诱变和大规模表型化成为可能。我们描述了无克隆的单向导RNA（sgRNA）的合成，再加上利用荧光PCR和多重，高通量测序的简化的突变体识别方法。我们报告了来自针对斑马鱼基因组中83个基因的162个基因座的种系传递数据，其中我们获得了99％的成功产生突变率和28％的平均种系传递率。我们通过高通量测序验证了来自58个基因的678个独特等位基因。我们证明了我们的方法可用于有效的多重基因靶向。我们还证明了可以通过近交两个注入的始祖鱼来在F1代中完成表型化，从而显着减少了畜牧业和时间。这项研究将CRISPR / Cas9的种系传播数据与TALEN和ZFN的种系传递数据进行了比较，结果表明CRISPR / Cas9的效率是其他技术的六倍。我们显示，除sgRNA的5'端的GG或GA二核苷酸基因组匹配外，大多数已发表的有效sgRNA设计“规则”都无法有效预测斑马鱼的种系传播速率。最后，我们表明预测的脱靶诱变是体内遗传研究中的低关注。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
代理获取

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