Table 1 Comparison among Cas12a crRNA engineering strategies
From: Tailoring Cas12a functionality with a user-friendly and versatile crRNA variant toolbox
crRNA engineering strategy | Comprehensive direct repeat sequence mutations (This work) | Selective nucleotide substitutions in the loop region53 | Circular free gRNAs21 | 5’ RNA G-quadruplex (RG4) engineering54 | 2-aminoadenine incorporation17 | Integration of a 3’-terminal HDV ribozyme55 | 7-mer DNA extension16 | Split/truncated crRNAs or ortholog-derived crRNAs18 |
|---|---|---|---|---|---|---|---|---|
Function | Tunable regulation of Cas12a substrate binding and nuclease activity in vitro and in vivo | Enhancement of genome-editing efficiency and fidelity | Improved stability and availability of crRNA | Modulation of Cas12a cleavage activity via RG4-stabilizing compounds | Enhanced DNA target-binding capability of Cas12a, leading to improved editing efficiency | Facilitating crRNA maturation | Improved trans-cleavage activity and target specificity | Improved cis- and trans-cleavage efficiency of Cas12a |
Economic cost | Low | Low | Low | Mediumb | High | Low | High | Low |
Technical complexity | Low | Low | Mediuma | Low | High | Low | High | Low |
Application Spectrum | Expression regulation, base editing, homologous recombination-mediated gene editing, and CRISPR-Dx | Introducing indels | Activating gene expression and cleaving genomic DNA | CRISPR-Dx | HDR-mediated knock-in and multiplex editing | Introducing indels and gene activation | CRISPR-Dx | Introducing indels and CRISPR-Dx |
