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Summary: Gene editing of human pluripotent stem cells is a promising approach for developing targeted gene therapies for metabolic diseases. Here, we present a protocol for generating a CRISPR-Cas12a gene knockout of protein tyrosine phosphatases in human embryonic stem cells. We describe steps for differentiating the edited clones into pancreatic islet-like spheroids rich in β-like cells. We then detail procedures for implanting these spheroids under the murine kidney capsule for in vivo maturation. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.