The CRISPR systems provide powerful genome-editing tools for wide applications in biological and medical research fields. However, the safety issue due to off-target effects of CRISPR has been one of the major hindrances of its application to regenerative medicine. The conventional CRISPR system has the intrinsic danger of inducing unpredictable mutations at non-targeted genomic loci via erroneous double-strand DNA breaks (DSBs). In this study, we demonstrate a safety-enhanced application of a recently discovered CRISPR-Cpf1 for targeted gene activation, without DNA double-strand break, to facilitate osteogenic differentiation of human umbilical-cord-derived mesenchymal stem cells (UC-MSCs). To this end, we developed a catalytically inactive AsCpf1 fused to tripartite transcription activator domain (dAsCpf1-VPR) that can induce upregulation of targeted gene expression in mammalian cells. We observed that the CRISPR-dAsCpf1-VPR activator can be applied to enhance the osteogenic differentiation of human UC-MSCs, via increasing the expression level of endogenous BMP4 gene. The results suggested that the CRISPR-Cpf1 activator provides versatile methods applicable for bone regeneration and regenerative medicine.

中文翻译：

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内源性BMP4基因的CRISPR-Cpf1激活用于脐带间充质干细胞成骨分化。

CRISPR系统为生物和医学研究领域的广泛应用提供了强大的基因组编辑工具。然而，由于CRISPR的脱靶作用而引起的安全性问题一直是其应用于再生医学的主要障碍之一。常规的CRISPR系统具有固有的危险，即通过错误的双链DNA断裂（DSB）在非靶向基因组位点诱导不可预测的突变。在这项研究中，我们证明了最近发现的CRISPR-Cpf1在不需DNA双链断裂的情况下靶向基因激活的安全性得到增强的应用，以促进人脐带来源的间充质干细胞（UC-MSCs）的成骨分化。为此，我们开发了一种与三方转录激活剂域（dAsCpf1-VPR）融合的无催化活性的AsCpf1，可诱导哺乳动物细胞中靶基因表达的上调。我们观察到CRISPR-dAsCpf1-VPR激活剂可通过增加内源性BMP4基因的表达水平来增强人UC-MSC的成骨分化。结果表明，CRISPR-Cpf1激活剂提供了适用于骨骼再生和再生医学的通用方法。