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
Prime editing is a revolutionary gene-editing method that is capable of introducing insertions,deletions and base substitutions into the genome.However,the editing efficiency of Prime Editor(PE)is limited by the DNA r...Prime editing is a revolutionary gene-editing method that is capable of introducing insertions,deletions and base substitutions into the genome.However,the editing efficiency of Prime Editor(PE)is limited by the DNA repair process.Here,we show that overexpression of the flap structure-specific endonuclease 1(FEN1)and the DNA ligase 1(LIG1)increases the efficiency of prime editing,which is similar to the dominant negative mutL homolog 1(MLH1dn).In addition,MLH1 is still the dominant factor over FEN1 and LIG1 in prime editing.Our results help to further understand the relationship of proteins involved in prime editing and envisage future directions for the development of PE.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.
基金National Natural Science Foundation of China(32171413,61721003)the Basic Research Program of Tsinghua National Lab for Information Science and Technology and the Science,Technology and Innovation Commission of Shenzhen Municipality(KCXFZ202002011006448).
文摘Prime editing is a revolutionary gene-editing method that is capable of introducing insertions,deletions and base substitutions into the genome.However,the editing efficiency of Prime Editor(PE)is limited by the DNA repair process.Here,we show that overexpression of the flap structure-specific endonuclease 1(FEN1)and the DNA ligase 1(LIG1)increases the efficiency of prime editing,which is similar to the dominant negative mutL homolog 1(MLH1dn).In addition,MLH1 is still the dominant factor over FEN1 and LIG1 in prime editing.Our results help to further understand the relationship of proteins involved in prime editing and envisage future directions for the development of PE.
