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

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

DOI:
10.3791/55916-v

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11:53 min

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July 12, 2017

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, , , , ,
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

Capítulos

  • 00:05Título
  • 00:50Cloning of gRNAs into the CRISPER-concatemer Vector
  • 03:40Assessing Success of Cloning by Restriction Digest
  • 05:28Preparing Intestinal Organoids for Electroporation
  • 08:08Electroporation
  • 10:36Results: Successful Generation of CRISPER-concatemers and Electroporation of Intestinal Organoids
  • 11:24Conlcusion

Summary

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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.

Tags

Keywords: CRISPR-concatemerMultiple Gene KnockoutMouse Small Intestinal OrganoidsCRISPRGuide RNAsElectroporationSimultaneous KnockoutLoss Of Function StudiesParallel CompensationCloningBbs1 Shuffling ReactionThree GRNA ConcatemerFour GRNA Concatemer

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