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A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obt...

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Autores principales: Merenda, Alessandra, Andersson-Rolf, Amanda, Mustata, Roxana C., Li, Taibo, Kim, Hyunki, Koo, Bon-Kyoung
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612278/
https://www.ncbi.nlm.nih.gov/pubmed/28745625
http://dx.doi.org/10.3791/55916
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author Merenda, Alessandra
Andersson-Rolf, Amanda
Mustata, Roxana C.
Li, Taibo
Kim, Hyunki
Koo, Bon-Kyoung
author_facet Merenda, Alessandra
Andersson-Rolf, Amanda
Mustata, Roxana C.
Li, Taibo
Kim, Hyunki
Koo, Bon-Kyoung
author_sort Merenda, Alessandra
collection PubMed
description CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.
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spelling pubmed-56122782017-10-10 A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer Merenda, Alessandra Andersson-Rolf, Amanda Mustata, Roxana C. Li, Taibo Kim, Hyunki Koo, Bon-Kyoung J Vis Exp Bioengineering CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors. MyJove Corporation 2017-07-12 /pmc/articles/PMC5612278/ /pubmed/28745625 http://dx.doi.org/10.3791/55916 Text en Copyright © 2017, Journal of Visualized Experiments http://creativecommons.org/licenses/by/3.0/us/ This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License. To view a copy of this license, visithttp://creativecommons.org/licenses/by/3.0/us/
spellingShingle Bioengineering
Merenda, Alessandra
Andersson-Rolf, Amanda
Mustata, Roxana C.
Li, Taibo
Kim, Hyunki
Koo, Bon-Kyoung
A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
title A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
title_full A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
title_fullStr A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
title_full_unstemmed A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
title_short A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
title_sort protocol for multiple gene knockout in mouse small intestinal organoids using a crispr-concatemer
topic Bioengineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612278/
https://www.ncbi.nlm.nih.gov/pubmed/28745625
http://dx.doi.org/10.3791/55916
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