Programmable transcriptional condensates for enhanced CRISPR-based gene regulation

Abstract

Rationale Efficient gene activation or repression through programmable CRISPR-Cas9 has revolutionized molecular biology and drug development. Nonetheless, the currently available CRISPRa/i approaches are modestly potent and require multi-component delivery, which hampers the wide use of the technology in both research and therapy. Methods We developed a modular CRISPR-condensate platform by appending a multivalent RNA nanostar to the 3’ end of a single-guide RNA, producing a sgRNA-nanostar chimera that mediates phase separation at Cas9-bound genomic loci. The nanostar scaffold also contains MS2 stem-loops, which recruit MCP-tagged transcriptional effectors (VP64 for activation, KRAB for repression) to the condensate microenvironment at high local concentration. We examined condensate formation, genome targeting, and transcriptional output by using live-cell imaging, RT-qPCR, ChIP-seq, RNA-seq and CUT&Tag in HEK293T, HeLa, U-2 OS, MDA-MB-231, as well as human iPSC cell lines. Results The CRISPR-condensate design resulted in up to 50–100-fold target-gene activation, compared with 5–10-fold activation by direct VP64 fusion, and 20–30-fold transcriptional repression, compared with 3–5-fold repression by direct KRAB fusion, with high target specificity (12 versus 28 non-target differentially expressed genes assessed by RNA-seq). Orthogonal kissing-loop (KL) pairings enabled independent condensate systems for simultaneous activation and repression of multiplexed targets. Janus condensates containing both activating and repressive domains enabled bidirectional regulation at a single locus. The system requires delivery of only three independently expressible components—dCas9-NLS, an sgRNA-nanostar chimera bearing MS2 stem-loops (MS2SLs), and an MCP-fused effector (VP64-MCP for activation or KRAB-MCP for repression)—and showed minimal innate immune response and high cell viability. Conclusions The CRISPR-condensate system merges the dramatically enhanced transcriptional efficacy with the reduced complexity of components, providing a modular system for fine-tuned gene expression regulation. This strategy makes biomolecular condensation a general principle for enhancing CRISPR gene regulation, opening up possibilities for functional genomics, cell engineering, and therapy development.