GoKawiilSleeping Beauty transgene overexpression
Bidirectional Sleeping Beauty plasmids encoding codon-optimized cDNA of the gene of interest (KIT) or its mutated variants were cloned downstream of an EF1α promoter. The same construct included a reporter cassette constituted by a fluorescent protein (mTagBFP2) and puromycin-acetyltransferase (PAC) separated by a P2A peptide downstream of an RPBSA chimeric promoter. dsDNA inserts used for cloning were synthesized by IDT (IDT gBlocks). Human (K562, HEK-293T) or mouse (NIH-3T3 or Ba/F3) cell lines were electroporated using Lonza 4D-Nucleofector system according to the manufacturer’s instructions with 500 ng pSB100x transposase plasmid (Addgene #34879) and 100 ng of the transfer plasmid, unless stated otherwise. Cells underwent puromycin selection (1–2 μg ml−1) starting 5 days after electroporation and analysed by flow cytometry 7–14 days after the start of the selection.
KIT mAbs
The SR-1 mAb (mouse IgG2a) was produced by BioXcell. The BA7.3 C.9 hybridoma line was purchased through ATCC and sent to BioXcell for mAb production and purification. SR-1 later became available as BioXcell InVivoMAb anti-human KIT (BE0380). Fab-79D was produced as recombinant mAb by Genescript using the TurboCHO service as human IgG1. A briquilimab research analogue sharing the publicly available sequence of briquilimab (record UNII QWX84D0DRC, DrugBank accession DB18136) was produced as an aglycosylated (N297Q) recombinant mAb by Genescript.
KIT degenerate codon library
We cloned a bidirectional Sleeping Beauty plasmid encoding for a codon-optimized human KIT cDNA bearing unique restriction sites flanking each ECD and a reporter cassette composed of mTagBFP2-P2A-puromycin N-acetyl-transferase (PAC). To introduce random amino acids, we generated a library insert by PCR amplification of 350 bp long pooled ssDNA oligos (IDT oPools), encoding the KIT ECD2 bearing degenerate bases (NNN) at each amino acid position, flanked by homology arms for the backbone plasmid. The gel-purified insert was then cloned into the Sleeping Beauty transfer by HiFi cloning (NEB E2621L) and plated on 10× 15 cm agar dishes to estimate library complexity by counting bacterial colonies. Electroporation of HEK-293T cells with low plasmid doses (50–250 ng per 100 μl electroporation volume) allowed to select cells transduced with (~5–10%) efficiency and obtain approximately 1 integration per cell. Sorting of cells positive for KIT control antibody (104D2 clone) and negative for therapeutic SR-1 clone and cells positive for both antibodies was carried out using a BD Melody or BD FACS Aria sorter. Genomic DNA (gDNA) of sorted fractions was extracted (Qiagen DNAeasy Blood&Tissue Kit, 69504) and the library region was amplified by PCR with primers bearing Illumina partial adapters for NGS sequencing (Genewiz Amplicon EZ, Azenta Life Sciences).
Fluorescent ligand binding assay
hSCF (Peprotech 300-07) was resuspended at 1 mg ml−1 and conjugated with AlexaFluor-488 or AlexaFluor-647 (Invitrogen AF488 or AF647 Antibody Labeling Kit, A20181 and A20186) for 1 h at room temperature. The reaction was quenched with 1% 1 M Tris-HCl pH 8. Cell lines transduced with the respective KIT variant by Sleeping Beauty transposase were incubated in the presence of AF488-conjugated SCF for 15 min at room temperature, washed with PBS + 2% FBS and analysed by flow cytometry. The data obtained from the antibody and ligand affinity assays at different concentrations were analysed using Graphpad Prism v10.5.
Base editing and prime editing of cell lines
Human K562 cells were cultured in IMDM supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (10,000 U ml−1), 2% l-glutamine (200 mM). For base-editing and prime-editing experiments, K562 cells were collected and resuspended in electroporation solution59 supplemented with 500 ng base editor plasmid and 150-360 pmol of sgRNA (IDT) in a 20 μl electroporation volume. Cells were electroporated using Lonza 4D-Nucleofector system (FF-120 pulse) and cultured for 72 h before evaluation by flow cytometry or gDNA collection.
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