Generation of a homozygous knock-in human embryonic stem cell line expressing mEos4b-tagged CTR1

Abstract

Copper transporter 1 (CTR1) is the major membrane protein responsible for cellular copper (Cu) uptake and mediates cellular copper homeostasis. To elucidate CTR1′s behavior using imaging approaches, we generated a homozygous knock-in human embryonic stem cell (hESC) clone expressing photoconvertible fluorescence protein mEos4b-tagged endogenous CTR1 using CRISPR-Cas9 mediated homologous recombination. The engineered cells express functional CTR1-mEos4b fusion and have normal stem cell morphology. They remain pluripotent and can be differentiated into all three germ layers in vitro. This resource allows the study of CTR1 at an endogenous level in different cellular contexts using microscopy.