The clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms.Current CRISPR-activation(CRISPR...The clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms.Current CRISPR-activation(CRISPRa)platforms often require multiple components because of inefficient transcriptional activation.Here,we fused different phase-separation proteins to dCas9-VPR(dCas9-VP64-P65-RTA)and observed robust increases in transcriptional activation efficiency.Notably,human NUP98(nucleoporin 98)and FUS(fused in sarcoma)IDR domains were best at enhancing dCas9-VPR activity,with dCas9-VPR-FUS IDR(VPRF)outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity.dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR.These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.展开更多
基金supported by the National Key Research and Development Program of China(2017YFA0102801)the National Natural Science Foundation of China(91640119,81330055,and 32001063)+2 种基金the Guangdong Special Support Program(2019BT02Y276)the Natural Science Foundation of Guangdong Province(2023A1515010176)the Guangzhou Science and Technology Program key projects(2023A04J1952)。
文摘The clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms.Current CRISPR-activation(CRISPRa)platforms often require multiple components because of inefficient transcriptional activation.Here,we fused different phase-separation proteins to dCas9-VPR(dCas9-VP64-P65-RTA)and observed robust increases in transcriptional activation efficiency.Notably,human NUP98(nucleoporin 98)and FUS(fused in sarcoma)IDR domains were best at enhancing dCas9-VPR activity,with dCas9-VPR-FUS IDR(VPRF)outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity.dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR.These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.
