Targeting cellular mRNAs translation by CRISPR-Cas9

Area of Science:

• Molecular Biology
• Gene Regulation
• CRISPR Technology

Background:

• The CRISPR-Cas9 system is known for targeting viral RNA.
• Its potential influence on cellular messenger RNA (mRNA) translation remains largely unexplored.

Purpose of the Study:

• To investigate whether the CRISPR-Cas9 system can repress the translation of cellular mRNAs.
• To elucidate the mechanism and key components involved in this translational repression.

Main Methods:

• Utilized both natural and catalytically dead Cas9 variants.
• Assessed the role of the 5' end of the single-guide RNA (sgRNA) in repression.
• Employed RNA aptamer-ligand complexes to probe the translation machinery.
• Monitored protein expression and phenotypic changes.

Main Results:

• CRISPR-Cas9, including catalytically dead Cas9, represses cellular mRNA translation.
• The first 14 nucleotides of the sgRNA's 5' end are critical for this repression.
• CRISPR-Cas9 suppresses unintended target gene protein expression without DNA sequence alteration, leading to unexpected phenotypic changes.
• A roadblock mechanism involving physical obstruction of the translation machinery was identified.

Conclusions:

• CRISPR-Cas9 possesses the capability to inhibit cellular mRNA translation, independent of DNA modification.
• The findings necessitate comprehensive assessment of CRISPR-Cas9 off-target effects at both DNA and protein levels.
• Understanding this translational repression mechanism is crucial for accurate interpretation of CRISPR-Cas9 experimental outcomes.