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The genome of resting CD4+ T cells can be edited without their activation
Maria Moshareva, PCR.news
Scientists from Germany for the first time successfully carried out CRISPR-Cas9 editing of resting CD4+ T cells without their activation. To do this, they introduced a ribonucleoprotein complex of guide RNA and recombinant Cas9 protein with a nuclear localization signal into the cell. Scientists have demonstrated that their methodology can be used in research on the treatment of HIV infection.
Unlike activated, resting CD4+ T cells have a fairly high resistance to HIV. The analysis of the causes of resistance is difficult due to the lack of effective protocols for editing the genomes of such cells. To study resting CD4+ T cells, an alternative protocol is sometimes used — they are edited during activation and then the cells are returned to a state phenotypically similar to the resting state. However, there remains a risk that such cells are not sufficiently similar to resting cells to use them as a model. In a new paper, scientists from Germany have developed a method that allows you to effectively edit resting CD4+ T cells.
Resting CD4+ T cells do not divide, and it is very important to provide them with optimal cultivation conditions after extraction from the donor's blood. The authors found that culturing cells in the presence of IL-7 and IL-15 interleukins allows them to be maintained in a viable state for six weeks. Analysis of the expression levels of activation markers showed that the cells do not switch to the activated state.
Usually, transfection of resting CD4+ T cells is ineffective, since the penetration of the introduced DNA into the nucleus occurs during cell division. Scientists have applied nucleofection, a method that allows reagents to penetrate directly into the nucleus. During nucleofection, not DNA, but ribonucleoprotein complexes from guide RNA and recombinant Cas9 protein with a nuclear localization signal were delivered to the cells to obtain knockouts. If it is necessary to introduce foreign genetic material into the genome, the system is supplemented with a fragment of double-stranded DNA for homologous recombination.
The knockout efficiency of CD46 and PSGL-1 genes was 85% and 93%, respectively, and two weeks after nucleofection, these proteins were almost not detected in cells. In addition, it was possible to produce multiple knockout of five genes that play an important role in the development of HIV infection (CD4, CXCR4, PSGL-1, TRIM5a and CPSF6), as well as the CD46 gene. In this case, the proteins of all six genes were also not detected in the cells after two weeks. The viability of cells with a single knockout almost did not decrease, and in the case of multiple knockout decreased by only 20%.
The protein does not disappear from the cell immediately, this is due to the degradation time of its mRNA and the protein itself, which is different for each gene. For example, the CXCR4 receptor is not detected in cells within a week after editing, and the soluble protein SAMHD1 ceases to be detected only after six weeks. In addition, the authors introduced fluorescent reporter genes into the genome with an efficiency of 1 to 20%, followed by sorting.
The CXCR4 knockout made the cells resistant to HIV-1 infection, and the SAMHD1 knockout led to a decrease in resistance to HIV-1, but not to the hybrid virus, which includes the Vpx protein. This correlates with the known role of these proteins. MX2 knockout had virtually no effect on the infection of HIV-1 cells, despite the important role of this protein in the infection of other cell types. And the effect of CPSF6 loss, on the contrary, turned out to be more significant than in other cell types.
The authors emphasize that genome modification may be of interest not only for studying the interaction of HIV and resting CD4+ T cells, but also for studying the biology of immune cells.
Article by Albanese et al. Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses is published in the journal Nature Methods.
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