Blasticidin

A lentiviral vector for DYRK2 overexpression was prepared by Chongqing Genemine Biotechnology Co., LTD. Briefly, primers with restriction sites that were specific for the human DYRK2 mRNA sequences were constructed and used for PCR amplification, yielding the full-length human DYRK2 cDNA. This PC product and the pLenti-CMV vector were then digested with appropriate restriction enzymes and the DYRK2 cDNA was inserted into this pLenti-CMV vector to yield the pLenti-CMV-DYRK2 plasmid, which was subsequently transformed into E. coli DH5α that were then screened using ampicillin-containing plates. Positive clones were selected, plasmids were extracted, and restriction enzyme digestion and DNA sequencing were then performed to confirm the sequence identity.
Lentiviral transduction was conducted as reported previously^{41 (link)}. Briefly, cells (5 × 10⁴/well) were transduced with the DYRK2 or empty control lentiviruses at an appropriate multiplicity of infection in media containing 10% FBS. At 48 h post-transduction, cells were selected using 4 µg/mL blasticidin (Solarbio, China).

We first infect EKVX cells with virus for encoding Cas9, and selected with blasticidin (3513–03-9, Solarbio) for two weeks. Monoclone was picked to generate a stable cell line (EKVX-Cas9). EKVX-Cas9 was further infected with the sgRNA library (addgene, #73178). After 24 h infection, a portion of the cells were collected as a control. The cells were selected with puromycin (1 μg/mL) for a week to eliminate uninfected cells. The infected cells were inoculated at 2 × 10⁶ cells subcutaneously into nude mice. After 14 days, the tumor was harvested to prepare genomic DNA. DNA fragment containing CRISPR sequence were PCR amplified and sent for sequencing.

The generation of lentiviruses was conducted according to the previous reports^{51 (link),52 (link)}. Briefly, synthetic target sequences containing pathogenic point mutations together with psPAX2 and pMD2.G, were co-transfected into the packaging cell line HEK293T at a weight ratio of 3:2:1. Viral supernatants were collected 48 h later, clarified by filtration, and concentrated by ultracentrifugation. Then the concentrated viruses were used to infect 5–10⁵ cells (20–30% confluence) in a 60-mm dish with 5 mg/mL polybrene. Infected cells were selected by 4 μg ml⁻¹ blasticidin (Solarbio) to the culture medium. The target sequence-transduced HEK293T cells were then transfected with a mixture of plasmid encoding GGBE1.0 and targeted gRNA. After 5 days treatment with puromycin, cells were collected and the genomic DNA was subjected to deep sequencing to measure the editing efficiency of base editing.

To construct the pLVX-mU6-Tubb4b-SgRNA-EGFP vector, the cleavage-active target sequence and the empty vector (pLVX-EGFP) were digested by Nhe I (1241A, Takara, Guangzhou, China) and Xho I (1094A, Takara, Guangzhou, China), and connected together using T4 DNA Ligase (2011A, Takara, Guangzhou, China). Then Nhe I and Xho I digestion was used to confirm the vector’s assembly. After that, lentivirus packaging was performed as antecedently described [18 (link)] to obtain Tubb4b-SgRNA virus, and Polybrene (H9268, Sigma, Shanghai, China) at a final concentration of 6 μg/mL was added to the culture medium to assist infect the mouse spermatogonia.
After 12 h, the lentivirus was removed and mouse spermatogonia were selected with 10 µg·mL-1 blasticidin (B9300, Solarbio, Beijing, China) for 5 days to 7 days to gain mouse spermatogonia stably expressing Tubb4b-SgRNA. The fluorescence of mouse spermatogonia following infection may then be seen with a fluorescence microscope (TH4-200, Olympus).

A stable CRISPRa cell line was generated using lentiMPH v2 plasmid (Addgene). 293 T cells were transfected with pMDG.2, psPAX2 and lentiMPH v2 plasmid (Addgene). Lentiviral particles were then collected and used to transduce U251 cells, which were selected in 200 μg/ml hygromycin (Solarbio). sgRNAs were designed using an online tool (https://portals.broadinstitute.org/gppx/crispick/public) and ordered from Sangon Biotech, and their sequences are shown in Table S6. sgRNAs were cloned into lentiSAM v2 plasmid (Addgene) using BsmBI (New England Biolabs). Lentiviral particles of pMDG.2, psPAX2 and lentiSAM v2 plasmid were generated and used to transduce U251 cells, which were selected in 6 μg/ml blasticidin (Solarbio) for about 7 days.

The stable cell line 293-miR-HER2, expressing miRNA targeting HER2, was generated by transfection of the miR-HER2-E1 plasmid into HEK-293 cells. Forty-eight hours after transfection, cells were selected by the addition of blasticidin (Solarbio Life Sciences) to a final concentration of 6 μg/ml. A cell colony with green fluorescent protein (GFP) expression was selected and cultured in complete medium with 6 μg/ml blasticidin. The cell line was monitored for the expression of GFP and miR-HER2-E1.
To generate a cell line producing exosomes that adhere to the surface of HER2-positive cells, 293-miR-HER2 cells were either infected with lentivector XSTP724PA-1 (XStamp HER2 ligand exosome HER2 receptor targeting lentivector) or infected with control lentivector XSTP710PA-1 according to the manufacturer's instructions (XStamp Technology, SBI: XSTP724PA-1/XSTP710PA-1). The two cell lines were renamed 293-miR-XS-HER2 and 293-miR-XS (control), respectively. The lentivector XSTP724PA-1 contains two copies of the HER2 ligand fused to the 5′ N-terminal signal sequence leader and fused in frame to the 3′ C-terminal C1C2 XStamp domain that directs the entire fusion protein to be displayed on the surface of secreted exosomes [28 (link), 29 (link)]. The HER2 binding ability of the exosomes purified from 293-miR-XS-HER2 cells was confirmed by ELISA.