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 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 did not only promote naïve pluripotent gene expression but also enhanced cell survival and clonogenicity, and it enabled integration of hESCs 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.

Genome and epigenome engineering CRISPR toolkit for probing in vivo cis-regulatory interactions 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 ectopically activate gene expression (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).…”

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. previously reported gene knockout (iCRISPR) and gene activation (CRISPR-ON) hESC lines [10,12], 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.…”

“…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 [10]) and the CRISPR-ON system (Puro-TRE-dCas9-VPR and Neo-CAG-M2rtTA [12]). 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 [8,9]. And platforms for inducible gene knockout, gene activation, and gene repression in hESCs have been established [10][11][12]. However, a platform for simultaneous gene knockout and gene activation has not been reported yet.…”

Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors

“…Catalytically inactive SpCas9 ("dead" SpCas9 or dSpCas9) have been fused to transcriptional activation or repression domains to alter the expression of individual genes 1,2 or to perform genome-wide library screens [3][4][5] in mammalian cells. Both small molecule-and light-inducible dSpCas9-based fusions have been developed [6][7][8][9][10] , enabling the ability to regulate the activity of this platform. Recently described CRISPR-Cpf1 nucleases offer additional capabilities beyond those of SpCas9 including shorter length CRISPR RNAs (crRNAs) for guiding Cpf1 to targets, the ability to target T-rich PAMs [11][12][13] , and RNase processing of multiple crRNAs from a single transcript by sequence-specific ribonuclease activity resident within Cpf1 itself 14,15 .…”