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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Autores principales:	Wen-Hsin Liu, Kerstin Völse, Daniela Senft, Irmela Jeremias
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a377dc79c39245ce948428dc257d1833
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spelling	oai:doaj.org-article:a377dc79c39245ce948428dc257d18332021-12-02T17:23:26ZA reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models10.1038/s41598-021-91760-92045-2322https://doaj.org/article/a377dc79c39245ce948428dc257d18332021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91760-9https://doaj.org/toc/2045-2322Abstract 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.Wen-Hsin LiuKerstin VölseDaniela SenftIrmela JeremiasNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-8 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q 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
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.
format	article
author	Wen-Hsin Liu Kerstin Völse Daniela Senft Irmela Jeremias
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 Portfolio
publishDate	2021
url	https://doaj.org/article/a377dc79c39245ce948428dc257d1833
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A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models

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