A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models

Abstract CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-re...

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Main Authors: Wen-Hsin Liu, Kerstin Völse, Daniela Senft, Irmela Jeremias
Format: Article
Language:English
Published: Nature Publishing Group 2021-06-01
Series:Scientific Reports
Online Access:https://doi.org/10.1038/s41598-021-91760-9
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spelling doaj-a377dc79c39245ce948428dc257d18332021-06-20T11:34:22ZengNature Publishing GroupScientific Reports2045-23222021-06-011111810.1038/s41598-021-91760-9A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient modelsWen-Hsin Liu0Kerstin Völse1Daniela Senft2Irmela Jeremias3Research Unit Apoptosis in Hematopoietic Stem Cells, German Research Center for Environmental Health (HMGU), Helmholtz Zentrum MünchenResearch Unit Apoptosis in Hematopoietic Stem Cells, German Research Center for Environmental Health (HMGU), Helmholtz Zentrum MünchenResearch Unit Apoptosis in Hematopoietic Stem Cells, German Research Center for Environmental Health (HMGU), Helmholtz Zentrum MünchenResearch Unit Apoptosis in Hematopoietic Stem Cells, German Research Center for Environmental Health (HMGU), Helmholtz Zentrum MünchenAbstract CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems.https://doi.org/10.1038/s41598-021-91760-9
collection DOAJ
language English
format Article
sources DOAJ
author Wen-Hsin Liu
Kerstin Völse
Daniela Senft
Irmela Jeremias
spellingShingle Wen-Hsin Liu
Kerstin Völse
Daniela Senft
Irmela Jeremias
A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models
Scientific Reports
author_facet Wen-Hsin Liu
Kerstin Völse
Daniela Senft
Irmela Jeremias
author_sort Wen-Hsin Liu
title A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models
title_short A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models
title_full A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models
title_fullStr A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models
title_full_unstemmed A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models
title_sort reporter system for enriching crispr/cas9 knockout cells in technically challenging settings like patient models
publisher Nature Publishing Group
series Scientific Reports
issn 2045-2322
publishDate 2021-06-01
description Abstract CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems.
url https://doi.org/10.1038/s41598-021-91760-9
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