Figure 1.

Enhanced Cas9-driven HDR by designing a self-linearized donor plasmid. (a) Schematic shows the PnP workflow to reprogram mRuby hybridomas to express a selected antibody following incorporation of a recombinant synthetic antibody in the V_H locus.²⁷ (b) Optimized workflow in which a version of the PnP-mRuby cells constitutively expressing Cas9²⁶ is used together with an HDR donor plasmid harboring a recognition site for the same Cas9 gRNA that is used to cleave mRuby (protospacer adjacent motif is indicated in red). Following entry of the plasmid and the gRNA complex into the nucleus, Cas9, which is also targeted to the nucleus due to its nuclear localization signal, is recruited to both induce a DSB in the genomic mRuby coding sequence and linearize the plasmid, rendering it more prone to integration by HDR. (c) In vitro testing of plasmid cleavage by recombinant Cas9. Three versions of the self-linearizing plasmid were generated, bearing the cleavage site upstream of the 5ʹ homology arm (pPnP-lin5ʹ), downstream of the 3ʹ homology arm (pPnP-lin3ʹ) and at both sites (pPnP-lin5ʹ/3ʹ). As expected, in the first two cases Cas9 cleavage produced a linearized construct, while the double-cut plasmid underwent both single cleavage at either site or simultaneous cleavage at both, resulting in a shorter construct. (d) Exemplary flow cytometry dot plots show evaluation of HDR integration. The improved plasmids were compared to the unmodified plasmid (pPnP) and to the PCR-linearized donor (PCR). HDR efficiency is evaluated in terms of surface antibody expression 3 d after transfection. (e) Fold improvement of HDR rates of all the linearized donor formats compared to the mean of unmodified plasmid. HDR efficiency is quantified as described in (d). The plot is representative of n = 3 replicates and the error bars indicate standard deviation. Flow cytometry dot plots of all replicates are shown in Figure S1.