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Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines.

Julia B Carleton1, Kristofer C Berrett1, Jason Gertz2

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Summary
This summary is machine-generated.

Enhancer-i is a new CRISPR interference method for studying multiple enhancers simultaneously. This tool helps determine enhancer function and their combinatorial effects on gene expression at endogenous loci.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Gene Regulation

Background:

  • Multiple enhancers regulate gene expression, but their individual contributions and combinatorial effects are often unclear.
  • Genome-wide enhancer identification necessitates high-throughput functional analysis tools.
  • Current methods like Cas9 deletions are inefficient for studying multiple enhancer interactions.

Purpose of the Study:

  • To develop a high-throughput CRISPR interference (CRISPRi) method for simultaneous functional interrogation of multiple enhancers.
  • To enable the study of enhancer function and combinatorial effects at endogenous genomic loci.
  • To provide a tool for understanding enhancer contributions to gene expression, especially in response to stimuli.

Main Methods:

  • Enhancer-i utilizes nuclease-deficient Cas9 fused to repressive domains (SID and KRAB).
  • CRISPR interference targets enhancers for deactivation via histone deacetylation.
  • Transient transfection of guide RNAs allows for temporary inactivation of targeted regulatory regions.
  • The method is validated for specificity in genomic targeting and global gene expression.

Main Results:

  • Enhancer-i successfully enables simultaneous functional interrogation of multiple enhancers.
  • The method effectively blocks inducible transcriptional responses in tissue culture.
  • Enhancer-i demonstrates high specificity in genomic targeting and minimal off-target effects on global gene expression.
  • The protocol facilitates the assessment of individual enhancer contributions and their interplay.

Conclusions:

  • Enhancer-i is a powerful CRISPRi-based tool for high-throughput enhancer function analysis.
  • This method overcomes limitations of previous techniques for studying combinatorial enhancer effects.
  • Enhancer-i provides insights into the necessity and magnitude of enhancer contributions to gene expression.