CRISPR-Cas9-mediated functional dissection of 3'-UTRs.

Area of Science:

• Molecular Biology
• Genetics
• Gene Regulation

Background:

• 3' untranslated regions (3'-UTRs) are known regulators of gene expression, primarily through mRNA stability and translation.
• Reporter assays have limitations in reflecting the native cellular environment for studying 3'-UTR functions.

Purpose of the Study:

• To develop and apply a gene editing approach using CRISPR-Cas9 to investigate 3'-UTR regulatory activities in their native genomic context.
• To explore the complex roles of 3'-UTRs beyond mRNA stability and translation.

Main Methods:

• Utilized dual-CRISPR-Cas9 targeting to delete specific chemokine 3'-UTRs.
• Employed metabolic labeling assays to assess mRNA stability and transcription rates.
• Investigated gene expression modulation and functional annotation of 3'-UTRs in situ.

Main Results:

• CRISPR-Cas9 deletion of six chemokine 3'-UTRs increased mRNA levels as anticipated.
• Deletion of CXCL1, CXCL6, and CXCL8 3'-UTRs unexpectedly decreased mRNA levels.
• These decreases were attributed to increased mRNA stability coupled with significantly reduced transcription, revealing a novel role in transcriptional regulation.

Conclusions:

• 3'-UTR sequences exhibit a complex, dual regulatory role impacting both mRNA stability and transcription.
• CRISPR-Cas9 gene editing provides a powerful tool for functional annotation and modulation of 3'-UTRs in their native context.
• This study expands the understanding of gene expression regulation by non-coding sequences.