Transcript-specific induction of stop codon readthrough using a CRISPR-dCas13 system.

Area of Science:

• Molecular Biology
• Gene Expression Regulation
• CRISPR Technology

Background:

• Stop codon readthrough (SCR) allows translation to continue past a termination signal on mRNA.
• Natural SCR occurs in specific genes like AGO1 and VEGFA, influencing protein levels.
• Premature termination codons (PTCs) can lead to non-functional proteins and disease.

Purpose of the Study:

• To develop a transcript-selective method to enhance or induce stop codon readthrough.
• To investigate the mechanism of CRISPR-dCas13-mediated SCR, including ribosomal pausing.
• To demonstrate the therapeutic potential of inducing SCR across disease-causing PTCs.

Main Methods:

• Utilized a CRISPR-dCas13 system with specific guide RNAs to target regions downstream of stop codons.
• Employed readthrough assays to quantify SCR enhancement and induction in mammalian cell lines.
• Analyzed ribosomal pausing associated with dCas13-mediated SCR.
• Applied the system to correct PTCs in disease-relevant genes (HBB, SPTA1).

Main Results:

• CRISPR-dCas13 complexes significantly enhanced SCR of AGO1 and VEGFA mRNAs.
• The system successfully induced SCR across PTCs in reporter genes (GFP, TP53).
• Demonstrated readthrough of thalassemia- and spherocytosis-causing PTCs in HBB and SPTA1 mRNAs, respectively.
• Observed ribosomal pausing correlated with enhanced SCR.

Conclusions:

• CRISPR-dCas13 offers a programmable, transcript-selective strategy for controlling stop codon readthrough.
• This technology can enhance natural SCR and induce readthrough at PTCs.
• The approach holds potential for therapeutic applications by correcting genetic defects caused by PTCs.