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CRISPR-Mediated Gene Targeting of Human Induced Pluripotent Stem Cells


Susan M. Byrne, George M. Church

CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem cells and induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, an optimized protocol is described for genome engineering of human iPSCs using simple transient transfection of plasmids and/or single-stranded oligonucleotides without any further selection or enrichment steps. This protocol achieves transfection efficiencies >60%, with gene disruption efficiencies of 1-25% and gene insertion/replacement efficiencies of 0.5-10%. Details are also provided for designing optimal sgRNA target sites and donor targeting vectors, cloning individual iPSCs by single-cell FACS sorting, and genotyping successfully edited cells.

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Antibody

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Published
November, 2015

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  • CRISPR/Cas9 Antibody
    C15200203-100
    CRISPR/Cas9 Antibody – clone 7A9
  • CRISPR/Cas9 Antibody
    C15200229-100
    CRISPR/Cas9 Antibody
  • CRISPR/Cas9 Antibody
    C15200216-100
    CRISPR/Cas9 Antibody 4G10

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