Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools

Martina Celotti; Lucca L.M. Derks; Johan van Es; Ruben van Boxtel; Hans Clevers; Maarten H. Geurts

doi:10.1016/j.xpro.2024.103189

Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools

Martina Celotti^*, Lucca L.M. Derks, Johan van Es, Ruben van Boxtel, Hans Clevers, Maarten H. Geurts^*

^*Corresponding author for this work

Cancer

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Abstract

Isogenic disease models, such as genetically engineered organoids, provide insight into the impact of genetic variants on organ function. Here, we present a protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools. We describe steps for single guide RNA (sgRNA) design and cloning, electroporation, and selecting electroporated cells. We then detail procedures for clonal line generation. Next-generation CRISPR tools do not require double-stranded break (DSB) induction for their function, thus simplifying in vitro disease model generation. For complete details on the use and execution of this protocol, please refer to Geurts et al.¹^,²

Original language	English
Article number	103189
Number of pages	37
Journal	STAR protocols
Volume	5
Issue number	3
DOIs	https://doi.org/10.1016/j.xpro.2024.103189
Publication status	Published - 20 Sept 2024

Keywords

Cancer
CRISPR
Genetics
Stem Cells

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10.1016/j.xpro.2024.103189Licence: CC BY

1-s2.0-S266616672400354X-mainFinal published version, 8.77 MBLicence: CC BY

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abstract = "Isogenic disease models, such as genetically engineered organoids, provide insight into the impact of genetic variants on organ function. Here, we present a protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools. We describe steps for single guide RNA (sgRNA) design and cloning, electroporation, and selecting electroporated cells. We then detail procedures for clonal line generation. Next-generation CRISPR tools do not require double-stranded break (DSB) induction for their function, thus simplifying in vitro disease model generation. For complete details on the use and execution of this protocol, please refer to Geurts et al.1,2",

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AU - Celotti, Martina

AU - Derks, Lucca L.M.

AU - van Es, Johan

AU - van Boxtel, Ruben

AU - Clevers, Hans

AU - Geurts, Maarten H.

PY - 2024/9/20

Y1 - 2024/9/20

N2 - Isogenic disease models, such as genetically engineered organoids, provide insight into the impact of genetic variants on organ function. Here, we present a protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools. We describe steps for single guide RNA (sgRNA) design and cloning, electroporation, and selecting electroporated cells. We then detail procedures for clonal line generation. Next-generation CRISPR tools do not require double-stranded break (DSB) induction for their function, thus simplifying in vitro disease model generation. For complete details on the use and execution of this protocol, please refer to Geurts et al.1,2

AB - Isogenic disease models, such as genetically engineered organoids, provide insight into the impact of genetic variants on organ function. Here, we present a protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools. We describe steps for single guide RNA (sgRNA) design and cloning, electroporation, and selecting electroporated cells. We then detail procedures for clonal line generation. Next-generation CRISPR tools do not require double-stranded break (DSB) induction for their function, thus simplifying in vitro disease model generation. For complete details on the use and execution of this protocol, please refer to Geurts et al.1,2

KW - Cancer

KW - CRISPR

KW - Genetics

KW - Stem Cells

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DO - 10.1016/j.xpro.2024.103189

M3 - Article

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SN - 2666-1667

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JO - STAR protocols

JF - STAR protocols

IS - 3

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ER -

Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools

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