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 ...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 dCasg-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 pro- moter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naive pluripotsnt 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.展开更多
Dear Editor,Animal models,most commonly mice,that lack a protein of interest play an important role in phenotypic and functional studies of a target gene,allowing researchers to answer various biological questions(Cha...Dear Editor,Animal models,most commonly mice,that lack a protein of interest play an important role in phenotypic and functional studies of a target gene,allowing researchers to answer various biological questions(Chaible et al,2010).At pre-sent,a variety of tools act at the DNA or RNA level to enable researchers to model gene function(and thus protein)deficiency,including nucleic acid based RNA interference(EI-bashir et al.,2001),antisense oligonucleotides(Schoch and Miller,2017),and genome editing-based CRISPR-Cas9(Doudna and Charpentier,2014)strategies.However,challenges remain.展开更多
文摘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 dCasg-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 pro- moter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naive pluripotsnt 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.
文摘Dear Editor,Animal models,most commonly mice,that lack a protein of interest play an important role in phenotypic and functional studies of a target gene,allowing researchers to answer various biological questions(Chaible et al,2010).At pre-sent,a variety of tools act at the DNA or RNA level to enable researchers to model gene function(and thus protein)deficiency,including nucleic acid based RNA interference(EI-bashir et al.,2001),antisense oligonucleotides(Schoch and Miller,2017),and genome editing-based CRISPR-Cas9(Doudna and Charpentier,2014)strategies.However,challenges remain.
