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  1. Ravichantar Nithya, Ishola Oluwaseun Ayodeji, Tan Benjy Jek Yang, Ong Jing Kai, Theva Das Kumitaa
    MyJurnal
    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 im- prove genome editing in other disease models.
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