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Bioengineering

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

Article DOI: 10.3791/55916-v • 11:53 min • July 12th, 2017
July 12th, 2017 •
1,2, 1,2, 1, 1, 3, 1,2
1Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, 2Department of Genetics, University of Cambridge, 3Department of Pathology, Yonsei University College of Medicine

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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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Protocol Multiple Gene Knockout Mouse Small Intestinal Organoids CRISPR-concatemer Guide RNAs Cloning Electroporation Lost Of Function Studies Parallel Compensation CRISPR-concatemer Strategy Cloning Step Simultaneous Knockout Alessandra Merenda PhD Student Reaction Mixture Top Strand GRNAs Bottom Strand GRNAs T4 DNA Ligase Buffer T4 Polynucleotide Kinase
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