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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.

CRISPR-Cpf1 Activation of Endogenous BMP4 Gene for Osteogenic Differentiation of Umbilical-Cord-Derived Mesenchymal Stem Cells