Genotoxic effects of base and prime editing in human hematopoietic stem cells

Abstract

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.

Identification of a human hematopoietic stem cell subset that retains memory of inflammatory stress

“…Future functional studies are required to complement the computational signatures we found here for which we need immunophenotypic markers for prospective isolation of HSC-iM. Furthermore, we do not know if HSC-I and HSC-iM originate from the same pool of HSC, nor do we know whether they are static or dynamic states, which may be addressed with lineage tracing ( 82 ). In summary, we have established an inflammation-recovery xenotransplantation model and an inflammatory memory framework to mechanistically interrogate the role of HSC in infection, aging and disease.…”

HLA-Based Banking of Human Induced Pluripotent Stem Cells in Saudi Arabia

“…Additionally, gene editing technologies like, CRISPR/Cas9 has been shown to introduce unintended genomic aberrations and may render the cells not useful for therapy (Fu et al, 2013; Cradick et al, 2013). Even base and prime editing that does not involve double strand breaks have recently been shown to induce significant genotoxicity in human cells (Fiumara et al, 2023).…”

Nucleotide metabolism constrains prime editing in hematopoietic stem and progenitor cells

“…2b ). Strikingly, PE efficiency rose from undetectable levels with the original B2M epegRNA 15 to an average of 25% and 61% with one and two C•C mismatches, respectively ( Fig. 2b ).…”

“…4a ). While Vpx mRNA co-delivery and dN supplementation improved editing at ATP1A1 , modulation of nucleotide metabolism had minimal impact on PE in quiescent HSPCs with the HBB epegRNA and an additional epegRNA designed to knockout B2M 15 ( Supplementary Fig. 4b , c ).…”