CRISPR/Cas9-Mediated Gene Knockout and Knockin Human iPSCs.

Paolo Petazzi, Pablo Menéndez, Ana Sevilla

Methods in molecular biology (Clifton, N.J.) 2022

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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. © 2020. Springer Science+Business Media New York.

Citation

Paolo Petazzi, Pablo Menéndez, Ana Sevilla. CRISPR/Cas9-Mediated Gene Knockout and Knockin Human iPSCs. Methods in molecular biology (Clifton, N.J.). 2022;2454:559-574