Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1

Background: Advancement in genome engineering enables rapid and targeted disruption of any coding sequences to study gene functions or establish human disease models. We explored whether this approach can be used to study Gaucher disease, one of the most common types of lysosomal storage diseases (L...

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Main Authors: Annie Brown, Jiayi Zhang, Brendan Lawler, Biao Lu
Format: Article
Language:English
Published: MDPI AG 2021-07-01
Series:Genes
Subjects:
Online Access:https://www.mdpi.com/2073-4425/12/7/1076
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spelling doaj-a1fb5518a3ad44368f97afc5778db5762021-07-23T13:42:02ZengMDPI AGGenes2073-44252021-07-01121076107610.3390/genes12071076Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1Annie Brown0Jiayi Zhang1Brendan Lawler2Biao Lu3Department of Bioengineering, School of Engineering, Santa Clara University, 500 El Camino Real, Santa Clara, CA 95053, USADepartment of Bioengineering, School of Engineering, Santa Clara University, 500 El Camino Real, Santa Clara, CA 95053, USADepartment of Bioengineering, School of Engineering, Santa Clara University, 500 El Camino Real, Santa Clara, CA 95053, USADepartment of Bioengineering, School of Engineering, Santa Clara University, 500 El Camino Real, Santa Clara, CA 95053, USABackground: Advancement in genome engineering enables rapid and targeted disruption of any coding sequences to study gene functions or establish human disease models. We explored whether this approach can be used to study Gaucher disease, one of the most common types of lysosomal storage diseases (LSDs) in a near-haploid human cell line (HAP1). Results: CRISPR-Cas9 targeting to coding sequences of β-glucocerebrosidase (GBA), the causative gene of Gaucher disease, resulted in an insertional mutation and premature termination of GBA. We confirmed the GBA knockout at both the gene and enzyme levels by genotyping and GBA enzymatic assay. Characterization of the knockout line showed no significant changes in cell morphology and growth. Lysosomal staining revealed more granular lysosomes in the cytosol of the GBA-knockout line compared to its parental control. Flow cytometry analysis further confirmed that more lysosomes accumulated in the cytosol of the knockout line, recapturing the disease phenotype. Finally, we showed that this knockout cell line could be used to evaluate a replacement therapy by recombinant human GBA. Conclusions: Targeted gene disruption in human HAP1 cells enables rapid establishment of the Gaucher model to capture the key pathology and to test replacement therapy. We expect that this streamlined method can be used to generate human disease models of other LSDs, most of which are still lacking both appropriate human disease models and specific treatments to date.https://www.mdpi.com/2073-4425/12/7/1076β-glucocerebrosidaseGaucher diseaselysosomal storage disorderdisease modelCRISPR-Cas9
collection DOAJ
language English
format Article
sources DOAJ
author Annie Brown
Jiayi Zhang
Brendan Lawler
Biao Lu
spellingShingle Annie Brown
Jiayi Zhang
Brendan Lawler
Biao Lu
Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1
Genes
β-glucocerebrosidase
Gaucher disease
lysosomal storage disorder
disease model
CRISPR-Cas9
author_facet Annie Brown
Jiayi Zhang
Brendan Lawler
Biao Lu
author_sort Annie Brown
title Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1
title_short Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1
title_full Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1
title_fullStr Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1
title_full_unstemmed Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1
title_sort recapture lysosomal enzyme deficiency via targeted gene disruption in the human near-haploid cell line hap1
publisher MDPI AG
series Genes
issn 2073-4425
publishDate 2021-07-01
description Background: Advancement in genome engineering enables rapid and targeted disruption of any coding sequences to study gene functions or establish human disease models. We explored whether this approach can be used to study Gaucher disease, one of the most common types of lysosomal storage diseases (LSDs) in a near-haploid human cell line (HAP1). Results: CRISPR-Cas9 targeting to coding sequences of β-glucocerebrosidase (GBA), the causative gene of Gaucher disease, resulted in an insertional mutation and premature termination of GBA. We confirmed the GBA knockout at both the gene and enzyme levels by genotyping and GBA enzymatic assay. Characterization of the knockout line showed no significant changes in cell morphology and growth. Lysosomal staining revealed more granular lysosomes in the cytosol of the GBA-knockout line compared to its parental control. Flow cytometry analysis further confirmed that more lysosomes accumulated in the cytosol of the knockout line, recapturing the disease phenotype. Finally, we showed that this knockout cell line could be used to evaluate a replacement therapy by recombinant human GBA. Conclusions: Targeted gene disruption in human HAP1 cells enables rapid establishment of the Gaucher model to capture the key pathology and to test replacement therapy. We expect that this streamlined method can be used to generate human disease models of other LSDs, most of which are still lacking both appropriate human disease models and specific treatments to date.
topic β-glucocerebrosidase
Gaucher disease
lysosomal storage disorder
disease model
CRISPR-Cas9
url https://www.mdpi.com/2073-4425/12/7/1076
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