Human pluripotent reprogramming with CRISPR activators

Jere Weltner,Timo Otonkoski,Maxim Bespalov,Shintaro Katayama,Ras Trokovic,Eeva-Mari Jouhilahti,Kaarel Krjutškov,Juha Kere,Diego Balboa

doi:10.1038/s41467-018-05067-x

Abstract

CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSCs) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC, and LIN28A promoters. The low basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif). This effect is mediated in part by more efficient activation of NANOG and REX1. These data demonstrate that human somatic cells can be reprogrammed into iPSCs using only CRISPRa. Furthermore, the results unravel the involvement of EEA-motif-associated mechanisms in cellular reprogramming.

Highlights

CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci
CRISPRa system relies on sequence specific recruitment of a catalytically inactivated version of Cas[9] protein to genomic sequences defined by short guide RNA molecules[1,2,3]
CRISPRa-mediated POU5F1 (OCT4) activation has been used to replace transgenic OCT4 in human fibroblast reprogramming, while the transgenic expression of only OCT4 has been shown to be sufficient for the reprogramming of neuroepithelial stem cells (NSCs) into iPSCs12,22

Summary

Introduction

CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. The low basal reprogramming efficiency can be improved by an order of magnitude by targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif) This effect is mediated in part by more efficient activation of NANOG and REX1. A conserved Alu-motif was recently reported to be enriched in the promoter areas of the first genes expressed during human embryo genome activation (EGA)[19] This sequence is likely to be involved in the control of early embryonic transcriptional networks. Efficiency of the method depends on the targeting of the EEA-motif, which results in improved activation of a subset of endogenous genes that work as reprogramming factors, including NANOG and ZFP42 (REX1) These results exemplify the potential in targeting cell type enriched regulatory elements for controlling cell fate

Methods

Results

Conclusion