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...
Main Authors: | , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
MDPI AG
2021-07-01
|
Series: | Genes |
Subjects: | |
Online Access: | https://www.mdpi.com/2073-4425/12/7/1076 |
id |
doaj-a1fb5518a3ad44368f97afc5778db576 |
---|---|
record_format |
Article |
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 |
work_keys_str_mv |
AT anniebrown recapturelysosomalenzymedeficiencyviatargetedgenedisruptioninthehumannearhaploidcelllinehap1 AT jiayizhang recapturelysosomalenzymedeficiencyviatargetedgenedisruptioninthehumannearhaploidcelllinehap1 AT brendanlawler recapturelysosomalenzymedeficiencyviatargetedgenedisruptioninthehumannearhaploidcelllinehap1 AT biaolu recapturelysosomalenzymedeficiencyviatargetedgenedisruptioninthehumannearhaploidcelllinehap1 |
_version_ |
1721288270533361664 |