Genome Editing: A Comparative Study on the Efficiency of CRISPR/Cas9 Nuclease Versus Nickase Using HIV as a Model System
- Author:
Ravichantar Nithya1, Ishola Oluwaseun Ayodeji1,2, Tan Benjy Jek Yang1,3,4, Ong Jing Kai1,5, Theva Das Kumitaa1
1
,
2
,
3
Author Information
1. 1 Infectomics Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, 13200 Kepala Batas, Pulau Pinang. 2 Helmholtz Research Zentrum, Munich, Germany 3 Division of Genomics &
2. Transcriptomics, Joint Research Center for Human Retrovirus Infection, Kumamoto University. 4 Laboratory of Retroviral Genomics &
3. Transcriptomics, International Research Center for Medical Sciences (IRCMS), Kumamoto University. 5 Pusat PERMATApintar Negara, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan.
- Publication Type:Journal Article
- Keywords:
CRISPR/Cas9;
Gene editing;
Genome engineering;
HIV
- From:Malaysian Journal of Medicine and Health Sciences
2019;15(SUPPLEMENT 9):122-129
- CountryMalaysia
- Language:English
-
Abstract:
Introduction: CRISPR/Cas9 nuclease has gained popularity as a genome editing tool due to its straight-forward mechanism. However, there are concerns that CRISPR nuclease would cause off-target and toxicity. The CRISPR/ Cas9 D10A nickase was designed to enhance genome editing. Nevertheless, this raised the question of whether the efficiency of nickase is compromised compared to CRISPR/Cas9 nuclease. Targeting HIV genes, we investigated if CRISPR nuclease performed better than the nickase in efficacy and safety. Methods: CRISPR nucleases and nickases were designed to target Gag, Pol, Rev, Vif, Tat and LTR. HIV latently infected cell line, ACH-2, was transfected with the nucleases and nickases. Changes to viral load after CRISPR treatment was measured using p24 ELISA. Safety of nuclease and nickase was monitored using GFP expression with fluorescence microscopy and flow cytometry. Targeting two sites within the same gene, and targeting multiple genes concurrently were also studied to determine efficacy of CRISPR in reducing viral load. Results: A 44.9 to 68.1% and a 34.4 to 49.7% decrease in viral load was seen in CRISPR nuclease and nickase respectively. Microscopy and flow cytometry results showed that the nickase system was slightly toxic with a 0.31 to 0.7-fold cell death. There was a 34% decrease in viral load when two sites were targeted within a gene, and the largest decrease was seen when all the nucleases were combined, giving a 75.4% decrease in viral load at day 5. Conclusion: The knowledge gained from this study will be employed to improve genome editing in other disease models.
- Full text:11.2019my05471.pdf
