Fig. 1

Design and integration of the “all-in-one” Tet-On system plasmid for targeted gene knock-in. A. Schematic representation of the all-in-one Tet-On plasmid. The human EF1α promoter drives the expression of the reverse tetracycline transactivator (rtTA), along with the blasticidin resistance (BSR) gene and mScarlet (mScar) reporter gene. The TRE3G promoter is oriented in the opposite direction to control the expression of the target gene (Gene). Two sets of adjacent BsaI restriction sites (2x BsaI) flank the regulatory elements. B. Generation of homology arms (HAs) through PCR amplification. The 5’ and 3’ homology arms, each approximately 450 bp, are designed to match the target genomic site for precise knock-in through homologous recombination. C. One-pot Golden Gate Cloning is used to insert the homology arms into the all-in-one plasmid. The BsaI restriction enzyme is used to ligate the homology arms with the Tet-On gene circuit. D. The plasmid (All-in-one Tet-On with HAs plasmid) is linearized using KpnI and NheI restriction enzymes to prepare it for integration into the genome. The final construct includes the homology arms and the regulatory elements of Tet-On system. E. Schematic of the targeted circuit integration. The linearized plasmid and a Cas9-gRNA vector are co-electroporated into mammalian cells. Cas9 creates a double-stranded break at the target site, and the homology arms direct homologous recombination (HDR), enabling precise knock-in of the genetic circuit into the desired genomic location. F. Final structure of the genomic integration, with the All-in-one Tet-On gene circuit at the target site