High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9.
High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9.
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DOI：
10.1101/gr.186379.114
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发表时间：
2015-07
期刊：
影响因子：
7
通讯作者：
Burgess SM
中科院分区：
文献类型：
--
作者：
Varshney GK;Pei W;LaFave MC;Idol J;Xu L;Gallardo V;Carrington B;Bishop K;Jones M;Li M;Harper U;Huang SC;Prakash A;Chen W;Sood R;Ledin J;Burgess SM
在各种模式生物中使用CRISPR/Cas9作为基因组编辑工具，从根本上改变了靶向诱变。在这里，我们在斑马鱼中使用CRISPR/Cas9技术提出了一个高通量靶向诱变管道，这将使基因组的饱和诱变和大规模表型工作成为可能。我们描述了一种无克隆的单导RNA （sgRNA）合成，结合了利用荧光PCR和多路高通量测序的流线型突变体鉴定方法。我们报告了斑马鱼基因组中针对83个基因的162个位点的种系传播数据，我们获得了99%的成功率产生突变，平均种系传播率为28%。通过高通量测序，从58个基因中鉴定出678个独特等位基因。我们证明了我们的方法可以用于高效的多路基因靶向。我们还证明，通过近交两个注射奠基鱼，可以在F1代中进行表型分型，大大减少了畜牧业和时间。本研究比较了CRISPR/Cas9与TALENs和ZFNs的种系传播数据，发现CRISPR/Cas9的效率是其他技术的六倍。我们发现，除了sgRNA 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”并不能有效预测斑马鱼的种系传播率。最后，我们表明预测的脱靶突变在体内遗传研究中是不受关注的。
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.
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作者：
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