RESUMEN
Base editors (BEs) introduce base substitutions without double-strand DNA cleavage. Besides precise substitutions, BEs generate low-frequency 'stochastic' byproducts through unclear mechanisms. Here, we performed in-depth outcome profiling and genetic dissection, revealing that C-to-G BEs (CGBEs) generate substantial amounts of intermediate double-strand breaks (DSBs), which are at the centre of several byproducts. Imperfect DSB end-joining leads to small deletions via end-resection, templated insertions or aberrant transversions during end fill-in. Chromosomal translocations were detected between the editing target and off-targets of Cas9/deaminase origin. Genetic screenings of DNA repair factors disclosed a central role of abasic site processing in DSB formation. Shielding of abasic sites by the suicide enzyme HMCES reduced CGBE-initiated DSBs, providing an effective way to minimize DSB-triggered events without affecting substitutions. This work demonstrates that CGBEs can initiate deleterious intermediate DSBs and therefore require careful consideration for therapeutic applications, and that HMCES-aided CGBEs hold promise as safer tools.
Asunto(s)
Ácidos Alcanesulfónicos , Roturas del ADN de Doble Cadena , Translocación Genética , Humanos , Reparación del ADN por Unión de Extremidades , Reparación del ADN/genética , Sistemas CRISPR-CasRESUMEN
An essential step for SARS-CoV-2 infection is the attachment to the host cell receptor by its Spike receptor-binding domain (RBD). Most of the existing RBD-targeting neutralizing antibodies block the receptor-binding motif (RBM), a mutable region with the potential to generate neutralization escape mutants. Here, we isolated and structurally characterized a non-RBM-targeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a KD of 5.6 nM, neutralizes SARS-CoV-2 including the current Variants of Concern such as B.1.1.7, B.1.351, P.1, and B.1.617.2 (Delta), displays modest cross-reactivity against SARS-CoV, and reduces viral replication in hamsters. The epitope coincides with a predicted "ideal" vulnerability site with high functional and structural constraints. Mutation of the residues of the conserved epitope variably affects FD20-binding but confers little or no resistance to neutralization. Finally, in vitro mode-of-action characterization and negative-stain electron microscopy suggest a neutralization mechanism by which FD20 destructs the Spike. Our results reveal a conserved vulnerability site in the SARS-CoV-2 Spike for the development of potential antiviral drugs.
Asunto(s)
COVID-19 , SARS-CoV-2 , Anticuerpos Antivirales , Humanos , Glicoproteína de la Espiga del CoronavirusRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide severe coronavirus disease 2019 (COVID-19) pandemic since December 2019. There is a great demand for effective therapies for the prevention and treatment of COVID-19. Developing therapeutic neutralizing antibodies (NAbs), which could block viral infection, is such a promising approach, as NAbs have been successfully applied to the treatment of other viral infections. The recent advances of antibody technology have greatly accelerated the discovery of SARS-CoV-2 NAbs, and many of which are now actively tested in clinical trials. Here, we review the approaches applied for SARS-CoV-2 NAb development, and discuss the emerging technologies underlining the antibody discovery. We further summarize the common features of these antibodies including the shared neutralizing epitopes and sequence features.
Asunto(s)
Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , COVID-19/inmunología , COVID-19/terapia , SARS-CoV-2/inmunología , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/aislamiento & purificación , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/aislamiento & purificación , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Diversidad de Anticuerpos , COVID-19/virología , Descubrimiento de Drogas , Epítopos/química , Epítopos/inmunología , Humanos , Ratones , Modelos Moleculares , Pandemias , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V(D)J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturbation screening we here identify the Snf2-family helicase-like ERCC6L2 as one such factor. We show that ERCC6L2 promotes double-strand break end-joining and facilitates optimal CSR in mice. At the cellular levels, ERCC6L2 rapidly engages in DNA repair through its C-terminal domains. Mechanistically, ERCC6L2 interacts with other end-joining factors and plays a functionally redundant role with the XLF end-joining factor in V(D)J recombination. Strikingly, ERCC6L2 controls orientation-specific joining of broken ends during CSR, which relies on its helicase activity. Thus, ERCC6L2 facilitates programmed recombination through directional repair of distant breaks.
Asunto(s)
ADN Helicasas/metabolismo , ADN/metabolismo , Mamíferos/genética , Recombinación V(D)J/genética , Animales , Sistemas CRISPR-Cas/genética , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Redes Reguladoras de Genes , Células HEK293 , Humanos , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/metabolismo , Ratones Noqueados , Mutación/genética , Unión ProteicaRESUMEN
Base editors (BEs) are emerging tools used for precision correction or diversifying mutation. It provides a potential way to recreate somatic hypermutations (SHM) for generating high-affinity antibody, which is usually screened from antigen-challenged animal models or synthetic combinatorial libraries. By comparing somatic mutations in the same genomic context, we screened engineered deaminases and CRISPR-deaminase coupling approaches and updated diversifying base editors (DBEs) to generate SHM. The deaminase used in DBEs retains its intrinsic nucleotide preference and mutates cytidines at its preferred motifs. DBE with AID targets the same hotspots as physiological AID does in vivo, while DBE with other deaminases generates distinct mutation profiles from the same DNA substrate. Downstream DNA repair pathways further diversified the sequence, while Cas9-nickase restricted mutation spreading. Finally, application of DBE in an antibody display system achieved antibody affinity maturation ex vivo. Our findings provide insight of DBE working mechanism and an alternative antibody engineering approach.