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The Protein phosphatase 1 catalytic subunit alpha (PPP1CA) is a alpha subunit of the PP1 complex, which known to be involved in the regulation of a variety of cellular processes, such as cell division, glycogen metabolism, muscle contractility, protein synthesis, and HIV-1 viral transcription. Increased PP1 activity has been observed in the end stage of heart failure. Here, a PPP1CA knockout human embryonic stem cell line, WAe009-A-B, was generated using the CRISPR/Cas9 system to further study the function of PPP1CA. The cell line confirmed with pluripotency, normal karyotype and differentiation potential.