Fig 1: CRISPR/HDR engineering of hybridoma NLDC145 to obtain sortagable Fab′ fragments against DEC205.(A) Schematic representation of the CRISPR/HDR approach to convert wild-type (WT) hybridomas to Fab′ fragment–producing cell lines. (B) The targeted IgH locus of NLDC-145 with the variable region (VH) and constant regions (CH1, Hinge, CH2, and CH3) annotated is shown. Cas9 is guided by gRNA-H to hinge region and creates a double-stranded break before the first cysteine. The double-stranded break is subsequently repaired by HDR via the donor construct consisting of a sortag and his-tag motif (srt-his), an internal ribosomal entry site (IRES), blasticidin-resistance gene (Bsr), polyA transcription termination signal (pA), and homology arms (5′ HA and 3′ HA). The first 10 amino acids of the hinge before and after successful CRISPR/HDR are indicated. (C) Three days after electroporation, PCR was performed on genomic DNA from WT and CRISPR/HDR-targeted population using primers 1 and 2 (B). Agarose gel shows amplicon of expected size exclusively for the CRISPR/HDR-targeted population, indicating the correct integration of the donor construct within the population. (D) After limiting dilution of CRISPR/HDR-targeted cells, a flow cytometry screen was performed on the supernatant of monoclonal cell suspensions. To this end, DEC205-expressing cells (JAWSII) were incubated with clonal supernatants in combination with secondary antibodies against his-tag (blue) or rIgG2a (red). Exclusive his-tag signal indicates production of Fab′ fragments. (E) Immunoblotting of the supernatant of clones 47 to 52 for his-tag (blue) and rat heavy and light chain (red) confirms flow cytometry results. (F) In competition assay, JAWSII were incubated with a serial dilution (150 to 20 ng/ml) of either purified Fab′DEC205srt (blue), αDEC205 mAb (red), or an isotype control (gray) in combination with fluorescently labeled αDEC205 mAb (1 μg/ml). Decrease in mean fluorescent intensity (MFI) relates to the increase in competition for DEC205 binding with fluorescent-labeled αDEC205 mAb. n = 3, mean ± SEM. (G) Fab′DEC205srt can be C-terminally functionalized with a fluorescently labeled probe [GGGCK(FAM)] by using sortase (3M srt)-mediated ligation. LC, light chain.