An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

Abstract

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

iKA‐CRISPR hESCs for inducible and multiplex orthogonal gene knockout and activation

“…In this study, we generated an iKA‐CRISPR hESC line, in which simultaneous gene knockout and gene activation could be achieved. Compared with previously reported gene knockout (iCRISPR) and gene activation (CRISPR‐ON) hESC lines , the new platform has obvious advantages, especially in terms of the construction method and application range.…”

“…The approach to construct the iKA‐CRISPR hESCs is simple. Two donors were used for the iCRISPR system (Puro‐TRE‐Cas9 and Neo‐CAG‐M2rtTA ) and the CRISPR‐ON system (Puro‐TRE‐dCas9‐VPR and Neo‐CAG‐M2rtTA ). After puromycin and neomycin double selection, the clones with the two donors inserted into the two AAVS1 alleles were screened out.…”

“…CRISPR/Cas9 system modifies the genome of hESCs with high efficiency . And platforms for inducible gene knockout, gene activation, and gene repression in hESCs have been established . However, a platform for simultaneous gene knockout and gene activation has not been reported yet.…”

Genome and epigenome engineering CRISPR toolkit for in vivo modulation of cis-regulatory interactions and gene expression in the chicken embryo

“…VP64 is a well-established transcriptional activator domain consisting of a tetrameric repeat of the minimal activation domain found in herpes simplex protein VP16 (Seipel et al, 1992). dCas9-VP64 fusion has been successfully used in CRISPR-ON experiments to activate gene expression ectopically (Cheng et al, 2013;Guo et al, 2017). Here, we use a dCas9-VP64 fusion in conjunction with the synergistic activation mediator (SAM) system (Konermann et al, 2015).…”

CRISPR/Cas9 Genome-Editing System in Human Stem Cells: Current Status and Future Prospects

“…Guo et al. 20 designed a doxycycline-inducible dCas9-VPR cassette to activate gene expression in human PSCs. These CRISPRi/a platforms should provide a more convenient strategy to explore gene functions and signaling pathways in human stem cell research, which are faster, more convenient, and more economical than other techniques such as RNAi and gene overexpression.…”