When using 1 µg linear dsDNA instead, knock-in rates were lower (13.9–20%), but cell viability and recovery were improved and became comparable to the control condition, where transfection with Cas9-RNP was performed without dsDNA ( Fig. However, this amount of dsDNA impaired cell viability and resulted in low T cell recovery ( Fig. We found that 4 µg of linear dsDNA maximized the knock-in rate of 28.8–32.1% across four independent T cell donors ( Fig. Further, we titrated the amount of linear and plasmid DNA side-by-side and determined knock-in efficiency, cell viability, and cell recovery by flow cytometry 3 d after electroporation. All studies were performed using the R691A HiFi-Cas9 variant to minimize CRISPR/Cas9 off-target events ( Vakulskas et al., 2018). The CD8 + T cells were cultured for 48 h before being nucleofected (4D nucleofection system Lonza) with Cas9-RNPs containing a chemically-synthesized single guide RNA (sgRNA) targeting exon 1 of the TRAC locus together with the respective donor DNA template. Here, we first cultured isolated human CD8 + T cells in PRIME-XV media supplemented with the cytokines IL-7 and IL-15 and stimulated them with TransAct, a bead-free colloidal polymeric nanomatrix conjugated to humanized CD3 and CD28 agonists. Similar to our previous approach to CRISPR/Cas9-mediated gene knock-out in T cells ( Seki and Rutz, 2018), we optimized the process individually for CD8 + and CD4 + T cells, rather than working with a mixed cell population. We generated this construct as linear dsDNA, as a pUC57 plasmid, or as a nanoplasmid ( Fig. We designed a donor template with 500-bp homology arms targeting exon 1 of the TRAC locus and encoding the α chain of the NY-ESO-1–specific 1G4 TCR ( Li et al., 2005) fused with the fluorescent protein mNeonGreen (mNG Shaner et al., 2013). Since plasmid DNA is toxic to T cells ( Mandal et al., 2014 Su et al., 2016), the use of these small backbone vectors could help to reduce the amount of DNA needed for transfection. Commercially available nanoplasmids consist of a <0.5-kb backbone ( Luke et al., 2009 Williams et al., 2006). In addition to conventional plasmid backbones, which are ∼2.5 kb in size (i.e., pUC57), several smaller circularized DNA backbones including minicircles, midges, and nanoplasmids have been described for cell engineering applications ( Hardee et al., 2017). To circumvent the labor-intensive steps involved in the generation and purification of PCR-based linear dsDNA, and to facilitate T cell engineering with sequence-verified templates, we investigated the use of plasmid DNA. However, production and purification of AAV not only represents a significant clinical manufacturing challenge ( Loo and van derWright, 2016 Halbert et al., 2018 Davidsson et al., 2020), it also limits more widespread use of this approach in the research community.īuilding on previous work, including a protocol for CRISPR/Cas9-mediated gene knock-out in human and murine T cells ( Seki and Rutz, 2018 Oh et al., 2019) and a report describing the use of linear dsDNA as repair template ( Roth et al., 2018), we set out to develop a robust, efficient, and scalable protocol for nonviral CRISPR/Cas9-mediated gene knock-in in primary human T cells. Several studies have subsequently reported high editing efficiencies using AAV-based repair templates ( Choi et al., 2019 Vakulskas et al., 2018 Dai et al., 2019). This approach was used, for instance, to insert a CAR construct into the T cell receptor α constant ( TRAC) region locus, which placed the CAR under the control of the endogenous TCR promotor, thus improving its performance ( Eyquem et al., 2017). Viral vectors, in particular adeno-associated viruses (AAV), have been used to deliver donor DNA templates for HDR-mediated target gene knock-in in T cells ( Sather et al., 2015 Wang et al., 2016 Eyquem et al., 2017 Choi et al., 2019). Homology-directed repair (HDR) of double-strand DNA breaks introduced by targeted gene editing methods, such as transcription activator–like effector nucleases, zinc finger nucleases, or CRISPR/Cas9, can be used to make precise changes to a genomic sequence, including the insertion of long stretches of DNA at a defined genomic location ( Li et al., 2020 Singh et al., 2017).
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