A Comparison of the Efficiency of the T7-Endonuclease I Enzyme and Polyacrylamide Gel Electrophoresis System in Evaluating the Cleavage Efficiency of the CRISPR/Cas9 System

Assessment of Cas9 cleavage in CRISPR (Clustered Regularly Interspersed Short Palindromic Repeat) system and screening of mutations is one of the challenges of this system. This study suggests a high-efficiency PCR-based method using non-denaturing PAGE that does not depend on the formation of heteroduplexes.

In this study, to investigate the CRISPR-Cas9 function, first, a proper guide RNA sequence with the target sequence was designed and synthesized. The BbsI digested pSpCas9(BB)-2A-GFP (PX458) vector and the guide RNA were cloned into the cleavage site. The recombinant plasmid was extracted, and after confirming the correctness of cloning using the sequencing technique, it was transfected into the HEK293T cell line. After confirming and determining the transfected percentage by flow cytometry, the genomic DNA of the cells carrying the recombinant vector was extracted. Two different PCR reactions were performed to check the function of the Cas9 enzyme in the desired region on the Beta-globin gene, and the cutting percentage was evaluated.

The enzymatic digestion of T7EI on the genome showed 32%, and the polyacrylamide gel electrophoresis system results showed a 66% function of the Cas9 enzyme. In this way, the PAGE system reliably detects changes as small as 5-10 base pairs in the length of the nucleic acid at the desired location.

The PAGE-based technique can replace the T7EI assay as a routine laboratory protocol for genotyping human cell lines produced by the CRISPR/Cas9 system.