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

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CRISPR-assisted transcription activation by phase-separation proteins.

Author: Jiaqi LIU ¹ ; Yuxi CHEN ¹ ; Baoting NONG ¹ ; Xiao LUO ¹ ; Kaixin CUI ¹ ; Zhan LI ¹ ; Pengfei ZHANG ¹ ; Wenqiong TAN ² ; Yue YANG ¹ ; Wenbin MA ¹ ; Puping LIANG ¹ ; Zhou SONGYANG ¹
Author Information

1. State Key Laboratory of Biocontrol, MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
2. Lumiere Therapeutics, Suzhou 215000, China.
Publication Type:Journal Article
Keywords: CRISPR; phase-separation proteins; transcriptional activation
MeSH: Humans; Transcriptional Activation; RNA, Guide, CRISPR-Cas Systems; Gene Expression Regulation; CRISPR-Cas Systems/genetics*
From: Protein & Cell 2023;14(12):874-887
CountryChina
Language:English
Abstract: 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.