RESUMEN
CRISPR-Cas9 provides a tool to treat autosomal dominant disease by non-homologous end joining (NHEJ) gene disruption of the mutant allele. In order to discriminate between wild-type and mutant alleles, Streptococcus pyogenes Cas9 (SpCas9) must be able to detect a single nucleotide change. Allele-specific editing can be achieved by using either a guide-specific approach, in which the missense mutation is found within the guide sequence, or a protospacer-adjacent motif (PAM)-specific approach, in which the missense mutation generates a novel PAM. While both approaches have been shown to offer allele specificity in certain contexts, in cases where numerous missense mutations are associated with a particular disease, such as TGFBI (transforming growth factor ß-induced) corneal dystrophies, it is neither possible nor realistic to target each mutation individually. In this study, we demonstrate allele-specific CRISPR gene editing independent of the disease-causing mutation that is capable of achieving complete allele discrimination, and we propose it as a targeting approach for autosomal dominant disease. Our approach utilizes natural variants in the target region that contain a PAM on one allele that lies in cis with the causative mutation, removing the constraints of a mutation-dependent approach. Our innovative patient-specific guide design approach takes into account the patient's individual genetic make-up, allowing on- and off-target activity to be assessed in a personalized manner.
Asunto(s)
Alelos , Sistemas CRISPR-Cas , Edición Génica , Genes Dominantes , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/terapia , Terapia Genética , Mutación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Línea Celular , Genómica/métodos , Haplotipos , Humanos , Polimorfismo de Nucleótido Simple , Medicina de Precisión , ARN Guía de Kinetoplastida , Factor de Crecimiento Transformador beta1/genéticaAsunto(s)
Amiloidosis Familiar/genética , Distrofias Hereditarias de la Córnea/genética , Proteínas de la Matriz Extracelular/genética , Mutación Puntual , Factor de Crecimiento Transformador beta/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Amiloidosis Familiar/diagnóstico , Amiloidosis Familiar/etnología , Amiloidosis Familiar/cirugía , Canadá/epidemiología , Niño , Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/etnología , Distrofias Hereditarias de la Córnea/cirugía , Análisis Mutacional de ADN , Femenino , Estudios de Seguimiento , Humanos , Italia/etnología , Queratomileusis por Láser In Situ , Masculino , Persona de Mediana Edad , Linaje , Reacción en Cadena de la Polimerasa , Secuenciación del Exoma , Adulto JovenRESUMEN
CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at "off-target" sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated.