An Alternate Approach to Generate Induced Pluripotent Stem Cells with Precise CRISPR/Cas9 Tool.

Abstract

The induced pluripotent stem cells (iPSCs) are considered powerful tools in pharmacology, biomedicine, toxicology, and cell therapy. Multiple approaches have been used to generate iPSCs with the expression of reprogramming factors. Here, we generated iPSCs by integrating the reprogramming cassette into a genomic safe harbor, CASH-1, with the use of a precise genome editing tool, CRISPR/Cas9. The integration of cassette at CASH-1 into target cells did not alter the pattern of proliferation and interleukin-6 secretion as a response to ligands of multiple signaling pathways involving tumor necrosis factor-α receptor, interleukin-1 receptor, and toll-like receptors. Moreover, doxycycline-inducible expression of OCT4, SOX2, and KLF4 reprogrammed engineered human dermal fibroblasts and human embryonic kidney cell line into iPSCs. The generated iPSCs showed their potential to make embryoid bodies and differentiate into the derivatives of all three germ layers. Collectively, our data emphasize the exploitation of CASH-1 by CRISPR/Cas9 tool for therapeutic and biotechnological applications including but not limited to reprogramming of engineered cells into iPSCs.