CRISPR-assisted transcription activation by phase-separation proteins

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.

Expanding RNA editing toolkit using an IDR-based strategy

“…Using the 15-residue minimalistic LLPS peptides or the split-ADAR2 strategy mentioned above can potentially solve the problem. Last, IDRs can potentiate CRISPRa 31 , 32 but are unable to stimulate Cas13d, a programmable RNA-cutting enzyme (unpublished). Thus, we propose that the utility of IDR-based strategy in various editing platforms (including CRISPR editing and CRISPR interference) be carefully characterized on a case-by-case basis.…”

Liquid–Liquid Phase Separation Sheds New Light upon Cardiovascular Diseases

“…Furthermore, the feasibility of using phase-separating proteins to enhance dCas9-VPR activity has been demonstrated. Liu et al fused the human NUP98 and FUS IDR domains to dCas9-VPR, and observed a substantial increase in the efficiency of transcriptional activation [138].…”

Expanding RNA editing toolkit using an IDR-based strategy

“…Using the 15-residue minimalistic LLPS peptides or the split-ADAR2 strategy mentioned above can potentially solve the problem. Lastly, IDRs can potentiate CRISPRa (Liu et al, 2023; Ma et al, 2023) but unable to stimulate Cas13d, a programmable RNA-cutting enzyme (unpublished). Thus, we propose that the utility of IDR-based strategy in various editing platforms (including CRISPR editing and CRISPR interference) be carefully characterized on a case-by-case basis.…”