A Powerful CRISPR/Cas9‐Based Method for Targeted Transcriptional Activation

Abstract

Abstract:The targeted transcriptional activation of endogenous genes is an important tool for understanding physiological transcriptional networks, synthesizing genetic circuits, and inducing cellular phenotype changes. The CRISPR/Cas9 system has a great potential to achieve this purpose, however it has not yet succeeded to activate endogenous genes and induce cellular phenotype changes, efficiently. We herein show a powerful method for transcriptional activation by the CRISPR/Cas9-based system. Replacement of the methylated promoter with unmethylated one by the CRISPR/Cas9-based system was sufficient to activate the expression of the neural cell-specific gene OLIG2 and the embryonic stem cell-specific gene NANOG in HEK293T cells. Moreover, CRISPR/Cas9-based OLIG2 activation induced embryonic carcinoma cell line NTERA-2 to express neuronal marker βIII-tubulin. Thus, these data provide a novel method to handle genetic circuits and phenotype changes.Technologies for editing the epigenetic marks of targeted genes are required to understand complex transcriptional networks, accurately. Small molecules, such as DNA methylation inhibitors, alter the epigenetic state globally, but cannot target any specific loci. The CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat/CRISPR-associated protein 9) system has been shown to target specific genomic locus and induce the sitedirected DNA brake, when combined with a single-guide RNA (sgRNA) that contains the complementary 20 nucleotides of the target sequence and the protospacer-adjacent motif (PAM), NGG [1]-[7] . Recent studies have demonstrated that the nucleasenull Cas9 (dCas9), which was fused with transcription activation domains (e.g. VP16 or VP64), functioned as a transcriptional activator [8]-[18] , suggesting that the CRISPR/Cas9-based system can be used to control the expression of specific genes. Since the transcriptional activity of the dCas9 activators was not high [8], [16] , however, the efficacy of the cell-fate changes was still very low (~10%) [17] . In addition, it was concerned that the dCas9 activators may trigger non-targeted transcriptional activation with unknown transcription regulators and their transcriptional activity may not be a physiological level. It was, therefore, required to establish new system that activates the target gene expression at physiological level and induces cell-fate changes.In order to achieve this, we have focused on the microhomology-mediated end-joining (MMEJ)-dependent integration of donor DNA using CRISPR/Cas9 [19], [20] . MMEJ is a DNA double-stranded break (DSB) repair mechanism that utilizes microhomologous sequences (5-25 bp) for error-prone end-joining [21] . Using the MMEJ-combined CRISPR/Cas9 system, here we show a powerful method that activates the silenced gene by replacing its methylated promoter with unmethylated one. This system can be used for the analysis of transcriptional networks, cell-fates and genetic circuits in various types of cells.We designed a CRISPR/Cas9-based epigenome edit...

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