RESUMEN
The development of CRISPR-derived genome editing technologies has enabled the precise manipulation of DNA sequences within the human genome. In this review, we discuss the initial development and cellular mechanism of action of CRISPR nucleases and DNA base editors. We then describe factors that must be taken into consideration when developing these tools into therapeutic agents, including the potential for unintended and off-target edits when using these genome editing tools, and methods to characterize these types of edits. We finish by considering specific challenges associated with bringing a CRISPR-based therapy to the clinic, including manufacturing, regulatory oversight, and considerations for clinical trials that involve genome editing agents.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Terapia Genética , Animales , Proteína 9 Asociada a CRISPR , Ensayos Clínicos como Asunto , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica/métodos , Técnicas de Transferencia de Gen , Ingeniería Genética , Terapia Genética/métodos , Terapia Genética/tendencias , Humanos , Modelos Animales , ARN Guía de Kinetoplastida , Reparación del ADN por Recombinación , Investigación Biomédica Traslacional/métodos , Investigación Biomédica Traslacional/tendenciasRESUMEN
Base editors (BEs) are genome editing agents that install point mutations with high efficiency and specificity. Due to their reliance on uracil and inosine DNA damage intermediates (rather than double-strand DNA breaks, or DSBs), it has been hypothesized that BEs rely on more ubiquitous DNA repair pathways than DSB-reliant genome editing methods, which require processes that are only active during certain phases of the cell cycle. We report here the first systematic study of the cell cycle-dependence of base editing using cell synchronization experiments. We find that nickase-derived BEs (which introduce DNA backbone nicks opposite the uracil or inosine base) function independently of the cell cycle, while non-nicking BEs are highly dependent on S-phase (DNA synthesis phase). We found that synchronization in G1 (growth phase) during the process of cytosine base editing causes significant increases in Câ¢G to Aâ¢T "byproduct" introduction rates, which can be leveraged to discover new strategies for precise Câ¢G to Aâ¢T base editing. We observe that endogenous expression levels of DNA damage repair pathways are sufficient to process base editing intermediates into desired editing outcomes, and the process of base editing does not significantly perturb transcription levels. Overall, our study provides mechanistic data demonstrating the robustness of nickase-derived BEs for performing genome editing across the cell cycle.