Protocol for CRISPR-Cas12a genome editing of protein tyrosine phosphatases in human pluripotent stem cells and functional β-like cell generation

STAR Protoc. 2024 Sep 20;5(3):103297. doi: 10.1016/j.xpro.2024.103297. Epub 2024 Sep 6.

Abstract

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.

Keywords: CRISPR; Cell Differentiation; Metabolism; Model Organisms; Molecular Biology; Protein Biochemistry; Stem Cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems* / genetics
  • Cell Differentiation / genetics
  • Gene Editing* / methods
  • Human Embryonic Stem Cells / cytology
  • Human Embryonic Stem Cells / metabolism
  • Humans
  • Insulin-Secreting Cells* / cytology
  • Insulin-Secreting Cells* / metabolism
  • Mice
  • Pluripotent Stem Cells* / cytology
  • Pluripotent Stem Cells* / metabolism
  • Protein Tyrosine Phosphatases* / genetics
  • Protein Tyrosine Phosphatases* / metabolism

Substances

  • Protein Tyrosine Phosphatases