Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9

Area of Science:

• Molecular Biology
• Genetics
• Gene Regulation

Background:

• Transcription factor binding sites control gene expression and phenotype.
• Determining the function of individual binding sites in complex networks is challenging.

Purpose of the Study:

• To develop and validate a method (CRISPR interference) to precisely disrupt transcription factor binding sites.
• To investigate the role of specific binding sites in gene regulation and autoregulation.

Main Methods:

• Utilized deactivated Cas9 (dCas9) fused with guide RNAs to target and disrupt specific transcription factor binding sites.
• Applied CRISPR interference (CRISPRi) to Oct4 and Nanog binding sites within the Nanog and Pax6 gene promoters.
• Investigated effects on gene expression, protein binding, and cellular division.

Main Results:

• Disruption of an Oct4 binding site in the Nanog promoter reduced Nanog expression and slowed cell division.
• Disruption of an Oct4 binding site near Pax6 upregulated Pax6 transcription.
• Disruption of a Nanog binding site in its own promoter increased Nanog transcription, demonstrating autoregulation.

Conclusions:

• CRISPR interference provides a facile method to distinguish between activating and repressing transcription factor binding sites.
• This technique allows for the detailed examination of gene networks, autoregulation, and the functional impact of specific binding sites.
• The system allows for multiplexing and rapid reversibility, enhancing its utility in gene regulation studies.