Genetic variants that affect mRNA splicing are a major cause of hereditary disorders, but the spliceogenicity of variants is challenging to predict. RNA diagnostics of clinically accessible tissues enable rapid functional characterization of splice-altering variants within their natural genetic context. However, this analysis cannot be offered to all individuals as one in five human disease genes are not expressed in easily accessible cell types. To overcome this problem, we have used CRISPR activation (CRISPRa) based on a dCas9-VPR mRNA-based delivery platform to induce expression of the gene of interest in skin fibroblasts from individuals with suspected monogenic disorders. Using this ex vivo splicing assay, we characterized the splicing patterns associated with germline variants in the myelin protein zero gene (MPZ), which is exclusively expressed in Schwann cells of the peripheral nerves, and the spastin gene (SPAST), which is predominantly expressed in the central nervous system. After overnight incubation, CRISPRa strongly upregulated MPZ and SPAST transcription in skin fibroblasts, which enabled splice variant profiling using reverse transcription polymerase chain reaction, next-generation sequencing, and long-read sequencing. Our investigations show proof of principle of a promising genetic diagnostic tool that involves CRISPRa to activate gene expression in easily accessible cells to study the functional impact of genetic variants. The procedure is easy to perform in a diagnostic laboratory with equipment and reagents all readily available.

影响 mRNA 剪接的遗传变异是遗传性疾病的主要原因，但变异的剪接性很难预测。对临床可及的组织进行 RNA 诊断能够在其自然遗传背景下快速表征剪接改变变异体的功能。然而，这种分析不能提供给所有个体，因为五分之一的人类疾病基因不能在容易获得的细胞类型中表达。为了克服这个问题，我们使用基于 dCas9-VPR mRNA 的递送平台的 CRISPR 激活 (CRISPRa) 来诱导疑似单基因疾病个体的皮肤成纤维细胞中目的基因的表达。使用这种离体剪接测定，我们表征了与髓鞘蛋白零基因（MPZ）和 spastin 基因（SPAST）中种系变异相关的剪接模式，该基因仅在周围神经的雪旺细胞中表达。在中枢神经系统中。经过过夜孵育后，CRISPRa 强烈上调皮肤成纤维细胞中的MPZ和SPAST转录，从而能够使用逆转录聚合酶链式反应、下一代测序和长读长测序进行剪接变异分析。我们的研究证明了一种有前途的遗传诊断工具的原理，该工具涉及 CRISPRa 来激活易于接近的细胞中的基因表达，以研究遗传变异的功能影响。该程序在诊断实验室中很容易执行，设备和试剂都很容易获得。