Department of Biomedical Molecular Biology, Ghent University, Technologiepark 71, 9052, Ghent (Zwijnaarde), Belgium.
Cancer Research Institute Ghent, Ghent, Belgium.
Sci Rep. 2020 Sep 4;10(1):14662. doi: 10.1038/s41598-020-71412-0.
CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F generation.
CRISPR/Cas9 基因组编辑技术在脊椎动物功能基因组学领域引发了革命。然而，由于双链断裂 (DSB) 修复后编辑结果的嵌合性质，CRISPR/Cas9 编辑的 F 代动物常常表现出可变的表型外显率。即使基因组编辑效率很高，表型也可能被比例存在的仍然产生功能蛋白的无义突变所掩盖。最近，细胞培养系统中的研究表明，CRISPR/Cas9 介导的突变的性质可能依赖于局部序列背景，并可以通过计算方法进行预测。在这里，我们证明了类似的方法可以用于预测 Xenopus tropicalis、Xenopus laevis 和斑马鱼中的 CRISPR/Cas9 基因编辑结果。我们表明，先前在小鼠胚胎干细胞培养中训练的一种公开可用的神经网络 (InDelphi-mESC) 能够准确预测早期脊椎动物胚胎中的 CRISPR/Cas9 基因编辑结果。我们的观察结果可能对实验设计具有直接影响，允许选择具有预测修复结果特征的向导 RNA，这些特征富集在移码突变上，从而最大限度地提高 F 代 CRISPR/Cas9 表型的外显率。
