La mutagenèse dirigée est un outil indispensable à toute étude microbiologique, car elle permet d’identifier le rôle de certains locus génétiques identifiés comme acteurs potentiels dans des contextes précis. Cependant, les protocoles de mutagenèse dirigée sont longs et laborieux, et leur mise en œuvre est l’un des points limitants en recherche. L’émergence de CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) comme outil moléculaire a permis d’accélérer et de faciliter ces procédures de mutagenèse par contre-sélection. La limite de ces protocoles se situe dans la régénération de l’espaceur effectuant la contre-sélection. Notre plateforme CRISPR, dénommée Horus, offre une solution à cette limitation. Elle utilise du clonage in vivo afin de raccourcir autant la durée que la charge de travail du protocole, pour aboutir à l'obtention de mutants en une seule étape. Pour se faire nous avons conçu in silico un ARN guide synthétique capable d’agir comme un interrupteur génétique (porte logique ET) et de performer une contre sélection (discriminant les bactéries de types sauvages des mutants) via le système CRISPR-Cas9. / Site-directed mutagenesis is an essential tool for any microbiological study because it makes it possible to identify the role of certain loci identified as potential actors in specific contexts. However, site-directed mutagenesis protocols are long and laborious, and their implementation is one of the limiting points of research. The emergence of CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) as a molecular tool has accelerated the facilitation of these counter-selection mutagenesis protocols. The limitation of these protocols lies in the regeneration of the protospacer mediating the counter selection. Our CRISPR platform, called Horus (HOmologuous Recombination Using SsDNA), offers a solution to this limitation. It uses in vivo cloning to shorten both the duration and the workload of the protocol, allowing to obtain mutants strains in just one step. To do so, we designed in silico a synthetic guide RNA capable of acting as a genetic switch (AND Gate) and performing counter-selection (discriminating WT bacteria from mutants) via the CRISPR-Cas9 system.

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Human cytomegalovirus (HCMV) is a widespread human herpesvirus pathogen and prototypical member of the β-herpesvirus subfamily. Like all herpesviruses, the virus establishes a lifelong latent infection following host exposure, which has the potential to reactivate periodically and contribute to recurrent disease processes. In individuals with weak or compromised immune systems, such reactivation can lead to profound pathology. Understanding how latent infections are maintained is important for uncovering how HCMV causes disease. The study of viral genes that are expressed during latent infection grants insight into how latency is regulated and how it could be therapeutically targeted. To that end, this project has sought to evaluate the functional significance of one such viral gene termed LUNA in the context of latency. In models of experimental latent infection based on primary myeloid cells, levels of viral gene transcription were found to be significantly reduced following infection with LUNA deletion mutant viruses, consistent with corresponding observable changes in post-translational histone modifications over the viral promoters of latency-associated genes. Additionally, using luciferase reporter systems, latency-associated viral gene promoters became activated in response to the expression of wild-type LUNA. Together, these findings argue for a role of LUNA in regulating viral gene expression during latent HCMV infection. One possible mechanism by which LUNA may fulfil its role is by targeting cellular ND10 structures, known intrinsic inhibitors of herpesvirus gene expression, for disruption. In support of this, latently infected cells were found to be devoid of ND10, a phenotype that was recapitulated by the direct expression of wild-type LUNA. Furthermore, mutation studies confirmed the identification of a novel deSUMOylase activity encoded by LUNA that was responsible for mediating ND10 disruption. Use of a catalytically inactive LUNA mutant in transcriptional analyses of latent infection also generated similar results as with the LUNA deletion viruses. Overall, these data support the hypothesis that LUNA serves as an important regulator of viral gene expression during latency, which is likely linked to its ability to target ND10 structures for disruption, thus raising the possibility that inhibition of deSUMOylation may serve as a novel therapeutic strategy to target latent HCMV infection.