Stefano Beretta

Base and prime editors (BEs and PEs) may provide more precise genetic engineering than nuclease-based approaches because they bypass the dependence on DNA double-strand breaks. However, little is known about their cellular responses and genotoxicity. Here, we compared state-of-the-art BEs and PEs and Cas9 in human hematopoietic stem and progenitor cells with respect to editing efficiency, cytotoxicity, transcriptomic changes and on-target and genome-wide genotoxicity. BEs and PEs induced detrimental transcriptional responses that reduced editing efficiency and hematopoietic repopulation in xenotransplants and also generated DNA double-strand breaks and genotoxic byproducts, including deletions and translocations, at a lower frequency than Cas9. These effects were strongest for cytidine BEs due to suboptimal inhibition of base excision repair and were mitigated by tailoring delivery timing and editor expression through optimized mRNA design. However, BEs altered the mutational landscape of hematopoietic stem and progenitor cells across the genome by increasing the load and relative proportions of nucleotide variants. These findings raise concerns about the genotoxicity of BEs and PEs and warrant further investigation in view of their clinical application.

HIV-1 insertions targeting BACH2 or MLK2 are enriched and persist for decades in hematopoietic cells from patients under combination antiretroviral therapy. However, it is unclear how these insertions provide such selective advantage to infected cell clones. Here, we show that in 30/87 (34%) patients under combination antiretroviral therapy, BACH2, and STAT5B are activated by insertions triggering the formation of mRNAs that contain viral sequences fused by splicing to their first protein-coding exon. These chimeric mRNAs, predicted to express full-length proteins, are enriched in T regulatory and T central memory cells, but not in other T lymphocyte subsets or monocytes. Overexpression of BACH2 or STAT5B in primary T regulatory cells increases their proliferation and survival without compromising their function. Hence, we provide evidence that HIV-1-mediated insertional activation of BACH2 and STAT5B favor the persistence of a viral reservoir in T regulatory cells in patients under combination antiretroviral therapy.HIV insertions in hematopoietic cells are enriched in BACH2 or MLK2 genes, but the selective advantages conferred are unknown. Here, the authors show that BACH2 and additionally STAT5B are activated by viral insertions, generating chimeric mRNAs specifically enriched in T regulatory cells favoring their persistence.