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

Chapters

  • 00:05Title
  • 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

Automatic Translation

Automatic Translation

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

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

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