RESUMEN
Precise genome editing requires the resolution of nuclease-induced DNA double strand breaks (DSBs) via the homology-directed repair (HDR) pathway. In mammals, this is typically outcompeted by non-homologous end-joining (NHEJ) that can generate potentially genotoxic insertion/deletion mutations at DSB sites. Because of higher efficacy, clinical genome editing has been restricted to imperfect but efficient NHEJ-based approaches. Hence, strategies that promote DSB resolution via HDR are essential to facilitate clinical transition of HDR-based editing strategies and increase safety. Here we describe a novel platform that consists of a Cas9 fused to DNA repair factors to synergistically inhibit NHEJ and favor HDR for precise repairing of Cas-induced DSBs. Compared to canonical CRISPR/Cas9, the increase in error-free editing ranges from 1.5-fold to 7-fold in multiple cell lines and in primary human cells. This novel CRISPR/Cas9 platform accepts clinically relevant repair templates, such as oligodeoxynucleotides (ODNs) and adeno-associated virus (AAV)-based vectors, and has a lower propensity to induce chromosomal translocations as compared to benchmark CRISPR/Cas9. The observed reduced mutational burden, resulting from diminished indel formation at on- and off-target sites, provides a remarkable gain in safety and advocates this novel CRISPR system as an attractive tool for therapeutic applications depending on precision genome editing.
Asunto(s)
Proteína 9 Asociada a CRISPR , Edición Génica , Humanos , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Reparación del ADN por RecombinaciónRESUMEN
DNA is the source of genetic information, and preserving its integrity is essential in order to sustain life. The genome is continuously threatened by different types of DNA lesions, such as abasic sites, mismatches, interstrand crosslinks, or single-stranded and double-stranded breaks. As a consequence, cells have evolved specialized DNA damage response (DDR) mechanisms to sustain genome integrity. By orchestrating multilayer signaling cascades specific for the type of lesion that occurred, the DDR ensures that genetic information is preserved overtime. In the last decades, DNA repair mechanisms have been thoroughly investigated to untangle these complex networks of pathways and processes. As a result, key factors have been identified that control and coordinate DDR circuits in time and space. In the first part of this review, we describe the critical processes encompassing DNA damage sensing and resolution. In the second part, we illustrate the consequences of partial or complete failure of the DNA repair machinery. Lastly, we will report examples in which this knowledge has been instrumental to develop novel therapies based on genome editing technologies, such as CRISPR-Cas.
Asunto(s)
Daño del ADN/fisiología , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Daño del ADN/genética , Reparación del ADN/genética , Reparación del ADN/fisiología , Edición Génica , HumanosRESUMEN
The formation and function of highly specialized cells and tissues in a multicellular organism from a single genome are enabled through differential spatiotemporal access to the information contained in the genomic DNA. The epigenome plays an essential role in how DNA information can be accessed, and in the last decade the link between epigenetic aberrations and pathologies has become increasingly clear. Methods to precisely modify the epigenome are hence attracting interest as potential novel therapeutics. We recently described a platform, designer epigenome modifier (DEM), capable of precisely editing the epigenome of a cell to control the expression of selected genes. Here, we provide a detailed protocol to streamline the process of identifying DEMs that efficiently and selectively bind to their intended target site and inactivate expression of the target gene. Further, we describe the procedure to simultaneously regulate the expression of up to three genes in a multiplexed fashion. The protocol is divided into four stages that guide the user through the generation of the multicolor reporter cell line and its use for selecting functional DEMs. The duration of the whole procedure described varies from ~6 weeks when using a single reporter up to 13 weeks for fine-tuning the multiplex epigenome editing abilities of selected DEMs using three reporters. Given the great interest in epigenome editing in various fields of biomedical research, this protocol will help scientists to explore these novel technologies for their research.