1.CRISPR-assisted transcription activation by phase-separation proteins.
Jiaqi LIU ; Yuxi CHEN ; Baoting NONG ; Xiao LUO ; Kaixin CUI ; Zhan LI ; Pengfei ZHANG ; Wenqiong TAN ; Yue YANG ; Wenbin MA ; Puping LIANG ; Zhou SONGYANG
Protein & Cell 2023;14(12):874-887
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.
Humans
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Transcriptional Activation
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RNA, Guide, CRISPR-Cas Systems
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Gene Expression Regulation
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CRISPR-Cas Systems/genetics*
Result Analysis
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