Dna gel extraction kit

Class I integron-targeting primers were designed based on previously described sequences (28 (link)). After DNA gel extraction kit (Vazyme Biotech, Nanjing, China) purification, the class I integron-positive DNA samples were sequenced (Sangon Biotech, Shanghai, China). The DNA sequences obtained were compared with those in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) using the Basic Local Alignment Search Tool to determine the gene cassettes within the variable region of the class I integrons.

The DNA template used for the in vitro synthesis of biotinylated circGlis3 was generated by PCR. The forward primer contained the T7 RNA polymerase promoter sequence to allow for subsequent in vitro transcription. The PCR products were purified using the DNA Gel Extraction Kit (Vazyme), and in vitro transcription was performed using the Transcript Aid T7 High Yield Transcription Kit (Thermo) and RNA 3’ End Biotinylation Kit (Thermo) according to the manufacturer’s instructions. RNA was subsequently purified through phenol‒chloroform extraction. The primer sequences used in this study are listed in Supplementary Table 1.

For the in vitro experiments, genomic DNA was isolated from HEI-OC1 cells after transfection with the CRISPR/Cas9 systems, and genomic DNA from cultured cochlear tissues was isolated 8 days after transduction with the Anc80L65–SpCas9 system using the QuickExtract DNA Extraction Solution (Epicentre, #QE09050) following the manufacturer’s instructions. For the in vivo experiments, genomic DNA was extracted from the whole cochlear tissue of the inner ear that was injected with the SpCas9 or SaCas9 system at P1, dissected at P7, and cultured for another 7 days in vitro. GFP-positive cells were sorted by FACS. Genomic DNA from non-transfected HEI-OC1 cells, non-transduced cultured cochlear tissue, and non-injected cochlear tissue were used as controls for each experiment. The target sites were PCR-amplified by nested PCR amplification and purified with a Gel DNA Extraction Kit (Vazyme, # DC301) for deep sequencing.

Several known Bacillus laccase sequences were obtained from NCBI, and multiple alignments were carried out with sequences showing high similarities using ClustalW (https://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 15 June 2021) and illustrated with ESPript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/, accessed on 15 June 2021). Degenerate primers were designed according to conserved regions of several known laccase sequences. The forward B.SCotA/F and reverse B.SCotA/R primer sequences are shown in Table S1.
The genomic DNA of 12 Bacillus strains were used as DNA templates, and potential laccase genes were amplified by PCR. The PCR reaction system (50 μL) included 20 μL ddH₂O, 25 μL 2× Taq Master Mix, 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), and 1 μL DNA template. The PCR procedure was 95 °C for 5 min; 35 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min; and 72 °C for 10 min. The PCR product(s) was verified by 1.0% agarose gel electrophoresis, and the target band was excised using a gel DNA extraction kit (Vazyme). The PCR fragment and the pMD-19 T vector were ligated using the TA clone kit (Takara). Positive clones were confirmed by DNA sequencing by General Biosystems Co., Ltd. (Chuzhou, China).

The strains, plasmids and PCR primers used in this study are listed in Supplementary Table 11 and Supplementary Table 12. Primer synthesis and DNA sequencing were performed by Tsingke Biotechnology Co. (Beijing, China). Fast digest restriction endonucleases and T4 DNA ligase were purchased from Thermo Fisher (Shanghai, China). The Super-Fidelity DNA Polymerase, Gel DNA Extraction Kit, Plasmid Isolation Mini Kit, Bacterial DNA Isolation Mini Kit and ClonExpress MultiS / One Step Cloning Kit were purchased from Vazyme Biotech Co. (Nanjing, China). All these kits were used according to the manufacturer’s procedures. Tylosin, NADPH and NADP⁺ were purchased from Macklin Co. (Shanghai, China). Other chemicals and media components were obtained from standard commercial sources and used directly.