Increasing Gene Editing Efficiency for CRISPR-Cas9 by Small RNAs in Pluripotent Stem Cells

Abstract

Gene manipulations of human induced pluripotent stem cells (iPSCs) by CRISPR-Cas9 genome engineering are widely used for disease modeling and regenerative medicine applications. There are two competing pathways, non-homologous end joining (NHEJ) and homology directed repair (HDR) that correct the double-strand break generated by CRISPR-Cas9. Here, we improved gene editing efficiency of gene knock-in (KI) in iPSCs with minimum components by manipulating the Cas9 expression vector. Either we inserted short hairpin RNA expression cassettes to downregulate <i>DNAPK</i> and <i>XRCC4</i>, two main players of the NHEJ pathway, or we increased cell survival by inserting an anti-apoptotic expression cassette of miRNA-21 into the Cas9 vector. For an easy readout, the pluripotency gene <i>SOX2</i> was targeted with a T2A-tdTomato reporter construct. <i>In vitro</i> downregulating <i>DNAPK</i> and <i>XRCC4</i> increased the targeting efficiency of <i>SOX2</i> KI by around twofold. Furthermore, co-expression of miRNA-21 and Cas9 improved the efficiency of <i>SOX2</i> KI by around threefold. Altogether, our strategies provide a simple and valuable approach for efficient CRISPR-Cas9 gene editing in iPSCs.