Dneasy1 blood tissue kit

The identification of the isolated bacteria was carried out using conventional methods, including Gram stain, morphological, biochemical tests, VITEK 2 C15 configuration Automated system (Biomérieux, Marcy-l'Étoile, France), and serology tests; then, the molecular method (PCR assay) was used to confirm this identification. After the DNA was extracted with a DNeasy1 Blood & Tissue Kit (Qiagen, Hilden, Germany), the 16S rRNA genes were targeted to confirm S. aureus. The commercially available master mix (2x HotStarTaq Plus Master Mix; Qiagen, Hilden, Germany) was used to set up the PCR reaction. The reaction mixture used in this study included: 5 μL of DNA, 20 μL of PCR mixture of 2x HotStarTaq Plus Master Mix (Qiagen, Hilden, Germany), and 1.5 μM of each forward and reverse primer. Target gene amplification was performed using PCR with a Thermal Cycler Corbett Research PCR Thermal Cycler. The thermal cycling conditions were optimized with an initial denaturation at 95 °C for 15 min, 35 cycles (denaturation, 94 °C, 1 min; annealing, 54 °C, 1 min; extension, 72 °C, 1 min), with a final extension at 72 °C for 10 min, and held at 4 °C.

To assess the efficiency of pgRNAs, the pY010 (AsCpf1) vector (Supplementary Fig. S8) containing different pgRNAs (3 µg each) were transfected into 2 × 10⁶ CHO-K1 and S cells. At day 5 post transfection, the genomic DNA (gDNA) was isolated with the DNeasy1 Blood & Tissue Kit (Qiagen, USA), according to the manufacturer’s protocol. The isolated gDNA was tested by deletion PCR with specific primers (Supplementary Table S5) using GoTaq^@ G2 DNA polymerase (Promega, USA). The amplified DNA products were visualized of by agarose gel electrophoresis using Midori Green Advance (Biozym, Germany) for DNA staining and documentation with the Gel Doc™ XR system (Bio-Rad, USA). The pgRNAs that showed the deleted PCR products were selected for further GS gene deletion experiments.