Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes
Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes
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DOI：
10.1242/dev.134809
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发表时间：
2016-06-01
期刊：
影响因子：
4.6
通讯作者：
Mosimann, Christian
中科院分区：
文献类型：
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作者：
Burger, Alexa;Lindsay, Helen;Mosimann, Christian
CRISPR-Cas9能够实现有效的序列特异性突变，以创建模式生物的体细胞或种系突变。体内的关键限制因素仍然是具有最低毒性的活性Cas9-sgRNA核糖核蛋白复合体(RNPs)的表达和递送，取决于靶向序列的可变诱变效率，以及高突变嵌合体。在这里，我们将体外组装的荧光Cas9-sgRNA RNPs应用于增溶盐溶液中，以实现斑马鱼胚胎的最大诱变效率。使用CrispRVariants对单个胚胎中的目标基因座进行基于MiSeq的序列分析，CrispRVariants是一种用于突变量化和可视化的定制软件工具，揭示了高效的双等位基因突变，在几个测试的基因座位上达到饱和。这种几乎完全的突变暴露了体细胞突变胚胎中候选基因的功能丧失表型，从而为下一代稳定的种系突变提供了依据。我们进一步证明，利用饱和突变在基因调控区域定位非编码元件，可以发现转基因报告中的功能控制元件和注射胚胎中的内源基因。我们的结果表明，优化溶解的体外组装的荧光Cas9-sgRNA RNPs为直接和可扩展的功能丧失研究和应用提供了一种可重复的试剂，而不仅仅是斑马鱼实验，这些实验需要体内最大的DNA切割效率。
CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.