A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
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Peer-reviewed
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Abstract
This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.
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Keywords
Animals, CRISPR-Cas Systems, Clustered Regularly Interspaced Short Palindromic Repeats, Gene Knockout Techniques, Mice, Mice, Knockout, Organoids, Transfection
Journal Title
Journal of Visualized Experiments
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Journal ISSN
1940-087X
1940-087X
1940-087X
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Publisher
JoVE Corporation
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Sponsorship
Medical Research Council (MC_PC_12009)
Wellcome Trust (101241/Z/13/Z)
Wellcome Trust (101241/Z/13/Z)
A.M. is supported by Wntsapp (Marie Curie ITN), A.A-R. is supported by the Medical Research Council (MRC), and B-K.K. and R.M. are supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society [101241/Z/13/Z] and receive support through a core grant from the Wellcome Trust and MRC to the Wellcome Trust - MRC Cambridge Stem Cell Institute.