Knock-in of a Large Reporter Gene via the High-Throughput Microinjection of the CRISPR/Cas9 System

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 draw-backs 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 re-porter 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.