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We developed novel CRISPR epigenetic editing tools (enCRISPRa/i) to precisely target enhancers. These systems effectively study enhancer function in situ and in vivo, advancing our understanding of gene regulation.

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Area of Science:

  • Molecular Biology
  • Epigenetics
  • Gene Regulation
  • CRISPR Technology

Background:

  • Tissue-specific gene expression relies on cis-regulatory elements (CREs), including enhancers.
  • Functional analysis of distal CREs like enhancers presents significant challenges.
  • Existing methods for enhancer perturbation often lack robustness and specificity.

Purpose of the Study:

  • To develop and validate CRISPR/dCas9-based epigenetic editing systems for enhancer targeting.
  • To enable efficient in situ and in vivo analysis of enhancer function.
  • To investigate the role of enhancers in gene transcription and biological processes.

Main Methods:

  • Development of enCRISPRa and enCRISPRi systems utilizing dual effectors for epigenetic editing at enhancers.
  • Application of CRISPR/dCas9 with single-guide RNA (sgRNA) to target specific enhancers.
  • In situ and in vivo validation in cell models, cancer xenotransplants, and a CRISPRi knock-in mouse model.

Main Results:

  • enCRISPRa and enCRISPRi successfully modulated gene transcription by remodeling epigenetic landscapes at targeted enhancers.
  • The systems demonstrated robust enhancer activity and gene transcription perturbations with minimal off-target effects compared to existing methods.
  • Allele-specific targeting of the TAL1 super-enhancer with enCRISPRa affected cancer progression; enCRISPRi in mice revealed essential roles of developmental enhancers in hematopoiesis.

Conclusions:

  • Enhancer-targeting CRISPR epigenetic editing (enCRISPRa/i) provides a powerful and efficient tool for interrogating enhancer function.
  • These systems allow for precise manipulation of enhancer activity in native biological contexts, both in vitro and in vivo.
  • The study establishes the utility of enCRISPR systems for understanding gene regulation, developmental processes, and disease mechanisms.