Mutation-Specific Guide RNA for Compound Heterozygous Porphyria On-target Scarless Correction by CRISPR/Cas9 in Stem Cells

International audienceCRISPR/Cas9 is a promising technology for gene correction. However, the edition is often biallelic, and uncontrolled small insertions and deletions (indels) concomitant to precise correction are created. Mutation-specific guide RNAs were recently tested to correct dominant inherited diseases, sparing the wild-type allele. We tested an original approach to correct compound heterozygous recessive mutations. We compared editing efficiency and genotoxicity by biallelic guide RNA versus mutant allele-specific guide RNA in iPSCs derived from a congenital erythropoietic porphyria patient carrying compound heterozygous mutations resulting in UROS gene invalidation. We obtained UROS function rescue and metabolic correction with both guides with the potential of use for porphyria clinical intervention. However, unlike the biallelic one, the mutant allele-specific guide was free of on-target collateral damage. We recommend this design to avoid genotoxicity and to obtain on-target scarless gene correction for recessive disease with frequent cases of compound heterozygous mutations

Données médicales

De nos jours, le paradigme du grand nombre (big data) entraîne un renouvellement des représentations médicales du corps (qui se voit fragmenté en autant de données comparables et agençables à merci) et de la relation thérapeutique. L’avènement de la médecine dite personnalisée, de l’intelligence artificielle et de ses dispositifs algorithmiques s’accompagne effectivement de nouveaux imaginaires et rhétoriques soignantes. Ces dernières s’appliquent à déployer un discours prophétique sur les découvertes scientifiques à venir, sur la capacité des sciences à repousser les maladies et même la mort, à renforcer et normaliser les corps ; l’idée d’une médecine toute puissante, en somme. La « vérité » des corps et la résolution de leurs troubles ne se trouveraient que dans ce qui est pensé comme une double objectivation : traitement de données quantitativement nombreuses et réalisation de l’exercice par une machine non douée d’affects. Ce numéro revient sur ces rhétoriques médicales, de saisir ces promesses intellectuelles et techniques dans le temps long, en les articulant aux relations thérapeutiques qu’elles induisent. En investissant d’une part les imaginaires soignants, et d’autre part la place des individus souffrants, nous souhaitons enquêter sur les césures que génère l’émergence des données médicales en grand nombre, comme sur les très fortes permanences, jusqu’aux discours contemporains, de l’idée d’un « progrès » nécessairement obtenu par une mise à distance, de plus en plus importante, de la médiation humaine, pour saisir les corps et leurs pathologies. Nowadays, the paradigm of large numbers (big data) is leading to a renewal of medical representations of the body (which is fragmented into so many comparable data that can be arranged at will) and of the therapeutic relationship. The advent of so-called personalised medicine, of artificial intelligence and its algorithmic devices is indeed accompanied by new imaginary and rhetorical approaches to care. The latter apply themselves to deploying a prophetic discourse on the scientific discoveries to come, on the capacity of science to repel diseases and even death, to reinforce and normalise bodies; the idea of an all-powerful medicine, in short. The 'truth' of bodies and the resolution of their disorders can only be found in what is thought of as a double objectification: the processing of quantitatively numerous data and the carrying out of the exercise by a machine that is not endowed with affects. This issue revisits these medical rhetoric, to grasp these intellectual and technical promises in the long term, by articulating them to the therapeutic relationships they induce. By looking at the imaginary of care-givers on the one hand, and the place of suffering individuals on the other, we wish to investigate the gaps generated by the emergence of large amounts of medical data, as well as the strong persistence, right up to contemporary discourses, of the idea of a "progress" necessarily obtained by distancing human mediation to an increasing extent, in order to grasp the bodies and their pathologies. En la actualidad, el paradigma de los grandes números (big data) está llevando a una renovación de las representaciones médicas del cuerpo (que se ve fragmentado en datos comparables y organizables a voluntad) y de la relación terapéutica. El advenimiento de la llamada medicina personalizada, de la inteligencia artificial y de sus dispositivos algorítmicos va, en efecto, acompañado de nuevos imaginarios y retóricas de los cuidados. Estas últimas se abocan a desplegar un discurso profético sobre los descubrimientos científicos venideros, sobre la capacidad de las ciencias para hacer retroceder a las enfermedades e incluso a la muerte, para reforzar y normalizar los cuerpos; la idea de una medicina todopoderosa, en definitiva. La “verdad” de los cuerpos y la resolución de sus trastornos solo podría encontrarse en lo que se piensa como una doble objetivación: el tratamiento de datos cuantitativamente numerosos y la realización del ejercicio por una máquina desprovista de afectos. Este número revisa esta retórica médica, para captar estas promesas intelectuales y técnicas en una larga duración, articulándolas a las relaciones terapéuticas que inducen. Examinando por un lado los imaginarios del cuidado y, por otro, el lugar de los pacientes, queremos investigar las cesuras generadas por la aparición de los datos médicos en grandes cantidades, así como la fuerte persistencia, hasta los discursos contemporáneos, de la idea de un “progreso” que se obtiene necesariamente poniendo cada vez más a distancia la mediación humana para aprehender los cuerpos y sus patologías

CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations

International audienceCRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nucleaseinduced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on-and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies