Objective:To study gene therapy for Wilson′s disease (WD) with adeno-associated virus (AAV)-clustered regularly interspaced short palindromic repeats (CRISPR) system in vitro. Methods:Three small-guide RNAs (sgRNAs) were designed and AAV particles containing CRISPR-sgRNA1, 2, 3 or W/O homologous template (HT) were transduced into Toxic milk (TX) mouse hepatocytes, respectively. Gene editing efficiency and HT repair efficiency were evaluated by sequencing, and repair template-specific primers were used for PCR to verify HT incorporation.Results:Sanger sequencing data showed that the editing efficiency of sgRNA1 reached (20.2±2.3)%, which was significantly higher than others ( P<0.05). When AAV-HT treatment was combined, the corrected ATP7B gene band could be amplified by PCR, and NGS showed that repair efficiency was (7.9±2.7)%. Conclusion:AAV transduction can achieve pretty high editing efficiency. We selected sgRNA1 and verified its editing efficiency and the repair efficiency of HT, providing a theoretical and experimental basis for further in vivo experiments.

Objective:To study gene therapy for Wilson’s disease with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system in vitro. Methods:Three crRNAs, W/O homologous template, the single-stranded oligo DNA (ssODN), were transfected into Toxic milk (TX) mouse hepatocytes using CRISPR/Cas9 ribonucleoprotein (RNP) method, respectively. Gene editing efficiency and ssODN repair efficiency were verified by sequencing, and repair template-specific primers were used for PCR to verify ssODN incorporation. Data are expressed as mean±standard deviation of independent samples. Comparisons between two groups were performed by unpaired Student’s t-test. Results:Sanger sequencing showed that the editing efficiency of crRNA001 reached (57.8±5.1)%. The corrected ATP7B gene band can be amplified by PCR using specific primers, and NGS sequencing results showed that the ATP7B gene repair efficiency reached (7.45±2.54)%.Conclusion:Pretty high editing efficiency can be achieved by RNP transfection. We selected crRNA001 and verified its editing efficiency and the repair efficiency of ssODN, providing a theoretical and experimental basis for further in vivo experiments.