An in vitro site-specific cleavage assay of CRISPR-Cas9 using a personal glucose meter
The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system, as a significant genome editing tool, has been applied in many research fields. The evaluation for site-specific cleavage of CRISPR-Cas9 is of great importance before in vivo application of CRISPR-Cas9. However, current methods for assessing CRISPR-Cas9 cleavage efficiency usually rely on expensive equipment and experienced trainers. Herein, we developed a sensitive and simple assay based on a personal glucose meter to detect the site-specific cleavage of CRISPR-Cas9 in vitro. Specially, the target DNA was designed to hybridize with the sequences modified on invertase and magnetic beads. In the presence of target DNA, invertase could be linked to the surface of magnetic beads. The magnetic beads with invertase could be collected via magnetic separation and then catalyzed sucrose into glucose. After the target DNA was cleaved by CRISPR-Cas9, reduced glucose signal would be read out by personal glucose meter and thus site cleavage efficiency evaluation for CRISPR-Cas9 was realized. The strategy avoids the expensive analysis equipment and any labels for target DNA. The detection limit is as low as 1.1 nM. The developed assay provides a sensitive and simple strategy to investigate the cleavage efficiency of CRISPR-Cas9 in vitro and can be further explored to other applications of CRISPR-Cas9.