Pmd2 g

Seventy to ninety percent confluent Lenti-X 293T HEK cells cultured in DMEM 10% FBS media were transfected with pMD2.G and pCMVR8.74 alongside appropriate delivery plasmids: pLV[Exp]-U6 > sgRNA-hPGK > mApple (Vectorbuilder) plasmids (for sgRNA), empty pLVX-EF1α-IRES-Puro (Clontech, Takara Bio, 631988), V5-KANSL1 pLVX-EF1α-IRES-Puro or V5-KAT8 pLV[Exp]-EF1α-IRES-Puro at a 1:1:2 molar mass ratio using Lipofectamine 3000 (Invitrogen). The next day, a full media change was performed with mTeSR1 (for sgRNA lentivirus) or DMEM 10% FBS media and cells cultured for 24 h. The lentivirus containing mTeSR1/DMEM 10% FBS was collected and diluted 1:2 with fresh mTeSR1 or 10% FBS before filtering through 0.44 µm PES filters. pMD2.G (Addgene plasmid no. 12259, RRID:Addgene_12259) and pCMVR8.74 (Addgene plasmid #22036, RRID:Addgene_22036) were gifts from Didier Trono. KANSL1 cDNA (ENST00000432791.7) with N-terminal V5 tag was cloned into the pLVX-EF1α-IRES-Puro plasmid using SpeI and NotI restriction sites (doi:10.5281/zenodo.6903553). See Supplementary Table 1 for sgRNA plasmids and V5-KAT8 pLV[Exp]-EF1α-IRES-Puro plasmids (Vectorbuilder).

Initially, the plasmids encoding Cas9 along with pMD2.G and psPAX2 lentiviral plasmids (VectorBuilder, Guangzhou, China) were transfected into 293T cells. After a 60 h incubation period, the supernatant containing the generated lentivirus was harvested and utilized to infect the target cells, C2C12. Subsequent to drug screening for positive cells, monoclonal cell sorting and expansion procedures were carried out, culminating in obtaining a C2C12 monoclonal cell line expressing Cas9. C2C12-Cas9 cells were seeded into T75 flasks and then infected with the library lentiviruses at a multiplicity of infection (MOI) of 0.3. Three days post-infection, GFP-positive cells were sorted using Fluorescence-Activated Cell Sorting (FACS), while puromycin-resistant cells were selected with 1.5 μg/mL puromycin. Subsequently, the sorted (1B) and selected cells (2B) were reseeded into T75 flasks. The cells would then be divided into two groups, one was collected after 1 day of proliferation and the other was collected when the cells were left for continuous proliferation in the growth medium for 5 days.