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. 2022:2454:559-574.
doi: 10.1007/7651_2020_337.

CRISPR/Cas9-Mediated Gene Knockout and Knockin Human iPSCs

Paolo Petazzi  1 , Pablo Menéndez  1   2   3 , Ana Sevilla  4
Affiliations

CRISPR/Cas9-Mediated Gene Knockout and Knockin Human iPSCs

Paolo Petazzi et al. Methods Mol Biol. 2022.

Abstract

The realization of the full potential of human pluripotent stem cells (hPSCs), including human induced PSCs (iPSC), relies on the ability to precisely edit their genome in a locus-specific and multiplex manner. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) serve as a guide for the endonuclease Cas9 (CRISPR-associated protein 9) to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. CRISPR/Cas9-mediated editing has become the gold standard for precise genome manipulation as it offers a unique, versatile, and limitless tool for fast, robust, and efficient genome editing. Here, we provide a protocol to successfully generate gene knockout and/or knockin iPSCs. We include detailed information on the design of guide RNAs (gRNAs), T7 endonuclease assay to detect on-target CRISPR/Cas9 editing events, DNA electroporation of the iPSCs with a ribonucleoprotein complex, and single-cell cloning steps for the selection of the genome-edited iPSC clones.

Keywords: CRISPR/Cas9; Donor ssODNs sequence; Genome editing; PAM sequence; T7 endonuclease; iPSCs.

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References

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