Inhibition of genes is a powerful approach to study their function. While RNA interference is a widely used method to achieve this goal, mounting evidence indicates that such an approach is prone to off-target effects. An alternative approach to gene function inhibition is genetic mutation, such as the CRISPR/cas9 method. A recent report, however, demonstrated that genetic mutation and inhibition of gene expression do not always give corresponding results. This can be explained by off-target effects, but it was recently shown, at least in one case, that these differences are the result of a compensatory mechanism induced only by genetic mutation. We present here a combination of RNA inhibition and CRISPR/cas9 methods to identify possible off targets as well as potential compensatory effects. This approach is demonstrated by testing a possible role for Sema4B in glioma biology, in which our results implicate Sema4B as having a critical function. In stark contrast, by using shRNA over CRISPR/cas9 combined methodology, we clearly demonstrate that the Sema4B targeted shRNA effects on cell proliferation is the result of off-target effects. Nevertheless, it also revealed that certain splice variants of Sema4B are important for the ability of glioma cells to grow as individual clones.

中文翻译：

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在CRISPR / cas9上结合shRNA，作为检测脱靶效应和潜在补偿机制的方法。

抑制基因是研究其功能的有力方法。尽管RNA干扰是实现此目标的一种广泛使用的方法，但越来越多的证据表明，这种方法易于产生脱靶效应。抑制基因功能的另一种方法是基因突变，例如CRISPR / cas9方法。然而，最近的一份报告表明，遗传突变和基因表达的抑制并不总是能给出相应的结果。这可以通过脱靶效应来解释，但最近至少在一种情况下显示，这些差异是仅由基因突变诱导的补偿机制的结果。我们在这里介绍了RNA抑制和CRISPR / cas9方法的组合，以识别可能的脱靶以及潜在的补偿作用。通过测试Sema4B在神经胶质瘤生物学中的可能作用证明了这种方法，其中我们的结果暗示Sema4B具有关键功能。与之形成鲜明对比的是，通过在CRISPR / cas9组合方法上使用shRNA，我们清楚地证明了Sema4B靶向shRNA对细胞增殖的作用是脱靶作用的结果。然而，它也揭示了Sema4B的某些剪接变体对于神经胶质瘤细胞作为单个克隆生长的能力很重要。