Dna loading buffer

Oligo pairs of 21 nt sgRNA sequences targeting the RHO-T17M mutation were designed based on the Benchling website (https://www.benchling.com/). Based on the RHO gene sequence (ENSG00000198947), we identified all the NNGRR PAMs for SaCas9. Oligonucleotides corresponding to the sgRNA of interest were purchased from Beijing Ruibio Biotech Co., Ltd. (Beijing, China).
An in vitro cutting assay using the SaCas9/SgRNA Cutting Efficiency Detection Kit (VK012, Viewsolid Biotech, Beijing, China) was performed to ensure the relative cutting efficiency and specificity of sgRNAs. The sgRNAs of interest were directly transcribed in vitro with T7 polymerase from double-stranded DNA templates. Primer pairs RHO17-F and R (Supplementary file 1i) were used to amplify the targeted region with or without c.50C>T mutation, yielding a 678 bp PCR fragment, respectively. Afterward, 1 μL of purified PCR amplicon (50 ng/μL), 1 μL of in vitro-transcribed sgRNA (50 ng/μL), 1 μL of SaCas9 protein (1 U/μL), 2 μL of 10× SaCas9 buffer, and 15 μL of ddH₂O were added into a tube and incubated for 30 min at 37°C. Subsequently, 2 μL of DNA loading buffer (9157, TAKARA, Kusatsu, Japan) was added to the reaction and incubated at 65°C for 5 min. The full reaction was run on 2% agarose gel (111860, Biowest).

The constructed plasmid pET21a-CaCDC42 containing the cdc42 gene of Candida albicans, which served as the template DNA, was recombined and prepared in the College of Life Science, Beijing Normal University, China. SYBR® Premix Ex Taq™ II containing Taq DNA polymerase catalyzing the elongation step of DNA replication, dNTPs, ROX Reference Dye II, DNA loading buffer, and SYBR Green II was purchased from Takara Bio Inc. (Dalian, China). The synthetic primers targeting cdc42 were provided by Sangon Biotech Co. Ltd. (Beijing, China) as follows: 5′-GTGATGGTGCCGTTGGTA-3′ (forward) and 5′-CCCTGTTCCTGGGTGATT-3′ (reverse), which produced a piece of DNA containing 397 bp. Thirteen heavy metal salts, ZnCl₂, Pb(NO₃)₂, CdCl₂·2.5H₂O, Ni(NO₃)₂·6H₂O, HgCl₂, CrCl₃, MnCl₂, CuSO₄, CoCl₂, Ce(NO₃)₃, Sr(NO₃)₂, Bi(NO₃)₃ and K₃Fe(CN)₆ were obtained from Beijing Chemical Reagent Company (Beijing, China). Ethylene diamine tetraacetic acid (EDTA), Ethidium bromide (EB), and agarose was from Sigma-Aldrich (St. Louis, USA). The DNA replication reactions in vitro were fulfilled and monitored using the Real-time PCR System ABI7500 (Applied Biosystems™, USA).

Restriction enzymes, T4 DNA ligase, DL5000 DNA markers, and DNA loading buffer were from Takara, plasmid extraction kits from Tiangen Biotech Co. Ltd. (Beijing, China), and gel extraction kits were bought from Axygen Biosciences (Union City, CA, USA. Protein markers from Cell Signaling Technology (Danvers, MA, USA), Trans 2xnEasy Taq PCR Super Mix, 4S Green Plus Nucleic acid stain, protein loading buffer from Yeasen Biotechnology Co. Ltd. (Shanghai, China), SDS gel stain wash buffer from Beyotime Biotechnology Co. Ltd. (Shanghai, China), R250 from Ourchem, Sinopharm Chemical Reagent Co. Ltd. (shanghai, China), Ni-NTA from TransGen Biotech Co. Ltd., (Shanghai, China). pET28a from Novagen (Foster City, CA, USA), PMD18T, Takara Co. Ltd. (Dalian, China).