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A study of putative genetically determined defects of de novo purine synthesis using CRISPR‐Cas9 genome‐edited HeLa cells
Author(s) -
Skopova Vaclava,
Baresova Veronika,
Souckova Olga,
Krijt Matyas,
Kmoch Stanislav,
Zikanova Marie
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb139
Subject(s) - enzyme , crispr , hela , nuclease , dna , purine , gene , biology , purine metabolism , genome , biochemistry , chemistry , microbiology and biotechnology , cell
Objectives The diagnosis of patients with neurological impairment can be set down only in 2% of studied cases. The reason might be the inability to capture new, to date unknown, diseases. One of the metabolic pathways whose malfunctions could lead to neurological impairment is de novo purine synthesis (DNPS). Currently, two genetically determined defects of DNPS enzymes, ADSL deficiency and AICA‐ribosiduria, are known. Therefore, the existence of other defects manifested by neurological symptoms and by accumulating DNPS intermediates in body fluids, is highly presumable. Methods We knocked out the expression of DNPS enzyme‐coding genes in HeLa cells using GeneArt® CRISPR Nuclease Vector with OFP Reporter Kit. We tested the cells for presence of mutations by DNA sequencing, for the presence of knocked‐out proteins by Western blot, and we analyzed DNPS substrates accumulation in cell lysates and growth media by LC‐MS/MS. We enzymatically prepared the substrates of DNPS enzymes for use as standards. We also analyzed the ability of the knockout cells to form purinosome by immunofluorescence. Results We prepared CRISPR‐Cas9 genome edited HeLa cells deficient for particular steps of DNPS to model possible genetically determined defects of the DNPS enzymes. We developed an analytical method for screening of these defects based on LC‐MS/MS analysis of the accumulated enzyme substrates. The precise detection of the metabolites was accomplished using the prepared DNPS intermediates. In all prepared model cells except of one we detected an accumulation of the substrate(s) for knocked‐out enzyme. The purinosome formation was disrupted in cell lines where the DNPS enzymes GART, ADSL and ATIC were knocked‐out. In cell lines with knock‐out of the other proteins of DNPS was the purinosome formation reduced. Conclusion We conclude that, similar to ADSL deficiency and AICA‐ribosiduria, the deficiency of other DNPS enzymes will manifest with accumulation of substrates for these enzymes in the body fluids of patients. Our data allow the detection of PAICS and PFAS deficiency in a population of patients with severe neurological impairment that have not yet been diagnosed. Support or Funding Information This work was supported by grants AZV 15‐28979A from the Ministry of Health, CR, PRVOUK‐P24/LF1/3 programs of Charles University in Prague, CR, and NPU from the Ministry of Education, Youth and Sports, CR.