Abstract
CRISPR-Cas9 mediated gene editing in induced pluripotent stem cells became an efficient tool to investigate biological mechanisms underlying genetic-driven diseases while accounting for the respective genetic background. This technique relies on the targeting of a specific nucleotide sequence present in the gene of interest. Therefore, the gene editing of some genes can be complicated by non-coding pseudogenes presenting a high homology of sequence with their respective genes. Among them, GBA is raising special interest because of its implication as the most common genetic risk factor for Parkinson’s disease. In this study, we present an easy-to-use CRISPR-Cas9 gene editing strategy allowing for specific editing of point mutations in a gene without genetic alteration of its pseudogene exemplified by the correction or insertion of the common N370S mutation in GBA. A quality control strategy by combined fluorescence and PCR-based screening allows the early identification of correctly edited clones with unambiguous identification of the status of its pseudogene, GBAP1. Successful gene editing was confirmed by functional validation. Our work presents the first CRISPR-Cas9 based editing of a point mutation in GBA and paves the way for technically demanding gene engineering due to the presence of pseudogenes.
Highlights
Pseudogenes are DNA sequences with high homology to functional genes
The induced pluripotent stem cells (iPSC) lines used in this study were reprogrammed from patient-derived fibroblasts using Simplicon RNA Reprogramming Kit (Merck) and the expression of the pluripotency markers Nanog, Oct4, and SOX2 was validated by immunocytochemistry (Figure S1). iPSCs were maintained on Geltrex (Gibco) coated plates in E8 medium supplemented with 10% mTesR®
To illustrate the possibility of targeting this exon, we selected the mutation rs76763715 resulting in an amino-acid exchange (p.N370S) in GCase. This mutation was either corrected or inserted at the heterozygous state in patient-derived iPSCs in order to model its implication in Parkinson’s disease (PD)
Summary
Pseudogenes are DNA sequences with high homology to functional genes. More than 17,000 pseudogenes have been identified in the human genome and up to 10% of them are transcribed (Kovalenko and Patrushev, 2018). Pseudogenes can act as competing endogenous RNA (ceRNA) toward their parental gene and compete for regulatory microRNA (miRNA) (Costa et al, 2012; Kovalenko and Patrushev, 2018). By these mechanisms, the ratio between the amount of gene and pseudogene transcripts modulates the expression of the gene. The ratio between the amount of gene and pseudogene transcripts modulates the expression of the gene Of these functionalities, pseudogenes have been implicated in the development of human diseases, in particular in cancer and neurodegenerative disorders (Costa et al, 2012)
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