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Editing the Genome of Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 Ribonucleoprotein Complexes

  • Michaela Bruntraeger
  • Meg Byrne
  • Kathleen Long
  • Andrew R. BassettEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1961)

Abstract

Genome editing using the CRISPR/Cas9 system has rapidly established itself as an essential tool in the genetic manipulation of many organisms, including human cell lines. Its application to human induced pluripotent stem cells (hiPSCs) allows for the generation of isogenic cell pairs that differ in a single genetic lesion, and therefore the identification and characterization of causal genetic variants. We describe a simple, effective approach to perform delicate manipulations of the genome of hiPSCs through delivery of Cas9 RNPs along with ssDNA oligonucleotide repair templates that can generate mutations in up to 98% of single cell clones and introduce single nucleotide changes at an efficiency of up to 40%. We describe our use of a T7 endonuclease assay to identify active guide RNAs, and a high-throughput sequencing genotyping strategy that allows the identification of correctly edited clones. We also present our experiences of generating single nucleotide changes at 15 sites, which show considerable variability between both guides and target sites in the efficiency at which such changes can be introduced.

Key words

CRISPR/Cas9 Genome editing Human iPSC RNP Point mutation SNP 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Michaela Bruntraeger
    • 1
  • Meg Byrne
    • 1
  • Kathleen Long
    • 1
  • Andrew R. Bassett
    • 1
    Email author
  1. 1.Wellcome Sanger InstituteWellcome Genome CampusHinxtonUK

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