safe harbor locus-targeted, ChR2 knocked-in human pluripotent stem cells (hPSCs), followed by the differentiation of these genetically engineered hPSCs into forebrain organoids. The resulting ChR2-expressing human forebrain organoids showed homogeneous cellular expression of ChR2 throughout entire regions without any structural and functional perturbations and displayed consistent and robust neural activation upon light stimulation, allowing for the non-virus mediated, spatiotemporal optogenetic control of neural activities. Furthermore, in the hybrid platform in which brain organoids are connected with spinal cord organoids and skeletal muscle spheroids, ChR2 knocked-in forebrain organoids induced strong and consistent muscle contraction upon brain-specific optogenetic stimulation. Our study thus provides a novel, non-virus mediated, preclinical human organoid system for light-inducible, consistent control of neural activities to study neural circuits and dynamics in normal and disease-specific human brains as well as neural connections between brain and other peripheral tissues.