The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA.
Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, CT, 06030, USA.
Nat Commun. 2020 Jun 12;11(1):2973. doi: 10.1038/s41467-020-16806-4.
Alternative splicing allows expression of mRNA isoforms from a single gene, expanding the diversity of the proteome. Its prevalence in normal biological and disease processes warrant precise tools for modulation. Here we report the engineering of CRISPR Artificial Splicing Factors (CASFx) based on RNA-targeting CRISPR-Cas systems. We show that simultaneous exon inclusion and exclusion can be induced at distinct targets by differential positioning of CASFx. We also create inducible CASFx (iCASFx) using the FKBP-FRB chemical-inducible dimerization domain, allowing small molecule control of alternative splicing. Finally, we demonstrate the activation of SMN2 exon 7 splicing in spinal muscular atrophy (SMA) patient fibroblasts, suggesting a potential application of the CASFx system.
选择性剪接允许从单个基因表达 mRNA 异构体，从而扩大了蛋白质组的多样性。其在正常生物和疾病过程中的普遍性需要精确的调控工具。在这里，我们报告了基于 RNA 靶向 CRISPR-Cas 系统的 CRISPR 人工剪接因子 (CASFx) 的工程改造。我们表明，通过 CASFx 的不同定位，可以在不同的靶标上同时诱导外显子的包含和排除。我们还使用 FKBP-FRB 化学诱导二聚化结构域创建了诱导型 CASFx(iCASFx)，允许通过小分子控制选择性剪接。最后，我们证明了在脊髓性肌萎缩症 (SMA) 患者成纤维细胞中 SMN2 外显子 7 剪接的激活，这表明 CASFx 系统具有潜在的应用前景。
