Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• CRISPR/Cas12a and CRISPR/Cas13d systems are vital tools in research with clinical potential.
• Short half-life of free guide RNAs (gRNAs) limits the efficiency and durability of CRISPR editing.
• Current limitations hinder the full therapeutic application of CRISPR-based gene editing.

Purpose of the Study:

• To engineer circular guide RNAs (cgRNAs) to enhance the stability and availability of gRNAs.
• To improve the editing efficiency and durability of CRISPR/Cas12a and CRISPR/Cas13d systems.
• To assess the efficacy of cgRNAs in both in vitro and in vivo settings.

Main Methods:

• Engineering of circular free guide RNAs (cgRNAs).
• Testing cgRNA stability and Cas nuclease binding compared to linear gRNAs.
• Evaluating CRISPR/Cas12a gene activation and DNA cleavage efficiency with cgRNAs.
• Assessing CRISPR/Cas13d RNA interference efficiency using cgRNAs.
• In vivo studies in mouse liver models.

Main Results:

• Circular gRNAs (cgRNAs) significantly increased Cas12a editing efficiency (2.1- to 40.2-fold) and Cas13d RNA interference (1.8-fold).
• cgRNAs demonstrated enhanced performance in both single and multiplex gene editing applications.
• Specificity was maintained across multiple cell lines and genomic sites.
• In vivo studies confirmed cgRNAs' potency in activating gene expression and cleaving DNA in mouse liver.

Conclusions:

• Engineered cgRNAs offer a stable and efficient alternative to linear gRNAs for CRISPR/Cas12a and Cas13d systems.
• cgRNAs enhance programmable DNA and RNA editing, broadening CRISPR applications.
• This innovation holds significant promise for fundamental research and the development of gene therapies.