The use of CRISPR-Cas and RNA-guided endonucleases has drastically changed research strategies for understanding and exploiting gene function, particularly for the generation of gene-edited animal models. This has resulted in an explosion in the number of gene-edited species, including highly biomedically relevant pig models. However, even with error-free DNA insertion or deletion, edited genes are occasionally not expressed and/or translated as expected. Therefore, there is a need to validate the expression outcomes gene modifications in vitro before investing in the costly generation of a gene-edited animal. Unfortunately, many gene targets are tissue specific and/or not expressed in cultured primary cells, making validation difficult without generating an animal. In this study, using pigs as a proof of concept, we show that CRISPR-dCas9 transcriptional activators can be used to validate functional transgene insertion in nonexpressing easily cultured cells such as fibroblasts. This is a tool that can be used across disciplines and animal species to save time and resources by verifying expected outcomes of gene edits before generating live animals.

中文翻译：

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使用 CRISPR 转录激活剂对基因编辑猪的转基因表达进行体外验证

CRISPR-Cas 和 RNA 引导的核酸内切酶的使用极大地改变了理解和利用基因功能的研究策略，尤其是基因编辑动物模型的生成。这导致基因编辑物种的数量激增，包括与生物医学高度相关的猪模型。然而，即使 DNA 插入或删除无错误，编辑后的基因有时也不会按预期表达和/或翻译。因此，需要在体外验证基因修饰的表达结果在投资昂贵的基因编辑动物一代之前。不幸的是，许多基因靶标是组织特异性的和/或在培养的原代细胞中不表达，这使得在不产生动物的情况下难以进行验证。在这项研究中，我们使用猪作为概念证明，表明 CRISPR-dCas9 转录激活剂可用于验证非表达的易培养细胞（如成纤维细胞）中的功能性转基因插入。这是一种可以跨学科和跨动物物种使用的工具，通过在生成活体动物之前验证基因编辑的预期结果来节省时间和资源。