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

BACKGROUND: The CRISPR/Cas12a and CRISPR/Cas13d systems are widely used for fundamental research and hold great potential for future clinical applications. However, the short half-life of guide RNAs (gRNAs), particularly free gRNAs without Cas nuclease binding, limits their editing efficiency and durability. RESULTS: Here, we engineer circular free gRNAs (cgRNAs) to increase their stability, and thus availability for Cas12a and Cas13d processing and loading, to boost editing. cgRNAs increases the efficiency of Cas12a-based transcription activators and genomic DNA cleavage by approximately 2.1- to 40.2-fold for single gene editing and 1.7- to 2.1-fold for multiplexed gene editing than their linear counterparts, without compromising specificity, across multiple sites and cell lines. Similarly, the RNA interference efficiency of Cas13d is increased by around 1.8-fold. In in vivo mouse liver, cgRNAs are more potent in activating gene expression and cleaving genomic DNA. CONCLUSIONS: CgRNAs enable more efficient programmable DNA and RNA editing for Cas12a and Cas13d with broad applicability for fundamental research and gene therapy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-023-02992-z.