Dataset Information

Quality Control Strategy for CRISPR-Cas9-Based Gene Editing Complicated by a Pseudogene.

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.

SUBMITTER: Hanss Z

PROVIDER: S-EPMC6961559 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Quality Control Strategy for CRISPR-Cas9-Based Gene Editing Complicated by a Pseudogene.

Hanss Zoé Z Boussaad Ibrahim I Jarazo Javier J Schwamborn Jens C JC Krüger Rejko R

Frontiers in genetics 20200108

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, <i>GBA ...[more]

PMID: 31998363

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Project description:Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species. We describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis, and C. metapsilosis We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis We show that editing can easily be carried out in multiple C. tropicalis isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in C. metapsilosis and C. tropicalis IMPORTANCE Candida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis We have also constructed a separate system for gene editing in C. tropicalis.

Dataset Information

Quality Control Strategy for CRISPR-Cas9-Based Gene Editing Complicated by a Pseudogene.

Publications

Quality Control Strategy for CRISPR-Cas9-Based Gene Editing Complicated by a Pseudogene.

Similar Datasets

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