Correction Of Glucose-6-Phosphate Dehydrogenase (G6PD) Viangchan Mutation In Monocytes Using CRISPR/CAS9
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Date
2018-07
Authors
Vengidasan, Lelamekala
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
G6PD deficiency is one of the most common enzymopathy in human with approximately 400 million people affected worldwide. Two hundred seventeen types of mutations have been described so far with some mutations are life threating. G6PD deficiency has also been linked to various types of cancers, tumours and metabolic diseases. This study aimed to correct G6PD deficiency by using CRISPR/Cas9 system. In this study, G6PD Viangchan as used; a type of mutation which is common in Malaysian Malays. G6PD Viangchan has cloned into pET26b (+) expression plasmid and expressed in BL21 (DE3) system. The recombinant G6PD Viangchan protein was verified by comparing its enzyme activity with G6PD wild type. Results showed a successful production of G6PD Viangchan protein by using heterologous expression system. The enzyme expression was reduced up to 67 % compared to G6PD WT. Subsequently, G6PD Viangchan was cloned into a lentiviral expression plasmid, pLJM-eGFP, to produce lentiviral particles. These particles were used to transduce THP-1 cell line, in order to generate stable G6PD Viangchan-expressing cells. The stable cell line was verified by using enzyme activity assay. THP-1/G6PD Viangchan cells produced a lower amount of G6PD enzyme (approximately 50 %) compared to untransduced THP-1 cells. Consecutively, the deficient was corrected by using CRISPR/Cas9 genome editing system. Guide RNAs flanking the mutation site were produced by using PCR approach and in vitro transcribed into RNA. G6PD deficient THP-1 cells were transfected with gRNA, Cas9 DNA and donor DNA by using Lipofectamine 3000. However, CRISPR-based gene editing only managed to increase approximately 8.8 % of G6PD enzyme compared to the untransfected control cells. Optimisations for CRISPR-based gene editing are much needed to obtain a higher percentage of gene correction. Nevertheless, this study forms a fundamental structure in providing a new opportunity in therapy for G6PD deficiency by using molecular approach.
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Keywords
Glucose-6-phosphate dehydrogenase viangchan , mutation in monocytes using CRISPR/CAS9