The non-viral delivery of the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) nuclease system provides promising solutions for gene therapy. However, traditional chemical and physical delivery approaches for gene knock-in are confronted by significant challenges to overcome the drawbacks of low efficiency and high toxicity. An alternative method for directly delivering CRISPR components into single cells is microinjection. Here, we present the high-throughput robotic microinjection of CRISPR machinery plasmids to produce gene insertions. We demonstrate that the microinjection of CRISPR/Cas9 with an enhanced green fluorescent protein (eGFP) donor template into single HepG2 cells can achieve reporter gene knock-in targeting the adeno-associated virus site 1 locus. Homology-directed repair-mediated knock-in can be observed with an efficiency of 41%. Assessment via T7E1 assay indicates that the eGFP knock-in cells exhibit no detectable changes at potential off-target sites. A case study of injecting the eGFP knock-in cells into zebrafish (Danio rerio) embryos to form an in vivo tumor model is conducted. Results demonstrate the efficiency of combining microinjection with the CRISPR/Cas9 system in achieving gene knock-in.