[48]
Hou et al. networks in T ZJ 43 cells and more importantly has the potential to revolutionise T cell-based therapies. and genes, respectively, which can be subdivided into a variable and a constant region signified by appending the gene RAC3 name with either and with the populations of CTLA-4? and PD-1? T cells reaching up to ~90%. In both cases T cells were restimulated for 48h prior to flow cytometric analysis. Given the high editing efficiencies achieved using this method, it has become a benchmark for editing of stimulated murine T cells [16]. Similarly to resting human T cells, resting murine T cells have a poor proliferative capacity and die in the absence of stimulation. To date, two strategies for in vitro knockout in unstimulated murine T cells have been developed, both of which are based on RNP complex electroporation. The first strategy involves stimulation of the resting T cells two hours after electroporation and this approach can lead to editing efficiencies of up to ~60% [16]. Despite the affordable editing performance, the short time period between RNP delivery and stimulation means that the protein encoded by the targeted gene is usually unlikely to have been degraded by the time the edited T cells are activated. Therefore, this technique is not suitable to study the role of the disrupted gene in T cell activation and differentiation. The other strategy takes advantage of the finding that the immediate stimulation can be effectively replaced by incubation of the T cells with IL-7, which is known to promote T cell survival, before and after RNP nucleofection. This method can result in ~70% reduction in expression of a target protein in resting T cells and the incubation with IL-7 leads to no apparent activation of the T cells [16]. Murine cells ZJ 43 provide a platform for editing T cells in vivo and Beil-Wagner and co-workers were first to carry out such a knockout in unstimulated murine T cells [32]. In their study, oocytes were microinjected with DNA constructs encoding promoter, respectively. Perhaps unsurprisingly, the knockout population constituted only ~1% of T cells isolated from the transgenic mice. The limited success of this approach is likely to have ZJ 43 been caused by the inability of the knockout population of T ZJ 43 cell precursors to undergo thymic selection, and it is possible that targeting a gene that is not involved in immune cell interactions would have resulted in a better efficiency. A more viable strategy to perform in vivo knockouts is usually to directly edit lymphoid cells isolated from constitutive Cas9-expressing mice, which circumvents the need to deliver Cas9 into the cells. To this end, Cas9-expressing mice were developed by Platt et al. by insertion of a Cas9-P2A-EGFP expression cassette into the Rosa26 locus, which has been leveraged by LaFleur et al. to devise a chimera-based Cas9-sgRNA delivery system termed chimera immune editing (CHIME) [33,34]. This technique does not involve direct editing of Cas9-expressing T cells per se, but instead T cell progenitors are edited prior to differentiation into T cells. Briefly, hematopoietic stem cells (HSC) are isolated from the bone marrow of a Cas9 transgenic mouse, lentivirally transduced with a relevant sgRNA and transplanted into irradiated mice. Following the reconstitution of the immune system, T cells are isolated from the chimeric animals and analysed. When CHIME was used to ablate Pdcd1, ~80% of T cells gained indels in the target locus and up to ~90% reduction in expression of Pdcd1 was observed following T cell activation. Mouse strains expressing Cas9-P2A-EGFP have been broadly adopted over the past few years and now are commonly used to isolate Cas9 expressing cells for editing of single and multiple genes or even for performing genome-wide screens. Furthermore, novel constitutive Cas9-expressing mouse ZJ 43 strains were developed with improved expression of Cas9 [35]. More recently, Nssing et al. have developed a method which permits modification of T cells isolated from mice that do not express Cas9. This technique involves RNP delivery into the resting T cells followed by transfer into a congenic mouse strain. In their report, P14 T cells bearing a TCR specific for a lymphocytic choriomeningitis virus (LCMV) antigen were electroporated with or and loci in TCR-transgenic T cells is usually highly desirable. To this end, dsDNA HDRT and Cas9 RNP electroporation has been effectively used to knockin a polycistronic construct encoding both transgenic TCR chains into one of the TCR loci and.