Viruses are excellent tools for gene transfer applications. Understanding the molecular biology of viruses will further contribute to the development of optimized gene delivery tools.
Recent years have witnessed tremendous advancements in the development of technologies for genome editing in particular with the exploitation of programmable nuclease, such as CRISPR/Cas.
Our laboratory is interested in developing molecular tools for precise and efficient gene therapy strategies by exploiting viral and non-viral delivery systems as well as programmable nucleases.
DEVELOPMENT OF MOLECULAR TOOLS FOR PRECISE AND EFFICIENT GENOME EDITING (Funded by Horizon 20202 EU-FP: UPGRADE and Progetto Sofia):
This goal will be achieved through a series of research activities:
Development of evolutionary diversified inactive CRISPR/Cas into active tools for editing of mammalian cells. We will adapt evolutionary diversified CRISPR-Cas systems for their use in eukaryotic cells through a random mutagenesis process and in vivo selection. Several CRISPR–Cas systems have been reported, yet they are inactive in eukaryotic cells. These new variants offer the possibility to expand the genome editing tool box in terms of PAM requirements and molecular size.
Development of novel high-fidelity Cas9 or Cas9 orthologs variants.
The yeast-based screening platform developed in our lab (Nature Biotech, 36 265-271 2018) will be used to select new variants with improved specificity and preserved on-target activity by starting from smaller orthologues
IMPROVING GENE SUBSTITUTION BY HOMOLOGOUS DIRECTED REPAIR (Funded by Horizon 20202 EU-FP: UPGRADE):
Identification of cellular factors that increase efficiency of targeted genome editing.
We are performing cellular screening to identify factors that in combination with CRISPR/Cas9 and specific DNA donor templates, may improve the efficiency of homology directed repair. New reporter cell lines are used and high-content screening technologies employed.
Directed evolution based on CRISPR/Cas9 technology
Directed evolution platform is employed to evolve programmable nucleases. The platform is also applied to reverse functional impairment of cellular factors mutated in genetic diseases. This strategy should be relevant for those genetic defects that cannot be repaired with genome editing strategies (such as deletions or incompatible with PAM requisites).
APPLICATION OF GENOME EDITING TOOLS FOR THE DEVELOPMENT OF GENE THERAPY STRATEGIES (Funded by Fondazione Ricerca Fibrosi Cistica, FFC and by progetto Sofia):
The genome editing tools are used in the lab for the development of novel molecular strategies for the treatment of genetic diseases. We are mainly focused in repairing genetic defects of the CFTR gene in Cystic Fibrosis both at the level of the introns well as in exons of the mutated gene. CRISPR/Cas9 directed evolution strategies are also applied for genetic defects that cannot be fixed by “standard” genome editing approaches.
The lab is working in finding genomic repair strategies for the treatment of genetic defects in Cornelia de Lange syndrome (CdLS) with a specific focus on the rare G5483A substitution in the NIPBL gene (progetto Sofia).
- Anna Cereseto, PI
- Antonio Casini, post doctoral fellow
- Claudia Montagna, post doctoral fellow
- Gianluca Petris, post doctoral fellow
- Giulia Maule, PhD student
- Michele Demozzi, PhD student
- Zeger Debyser, KU Leuven, Belgium
- Annarita Miccio, Institute of genetic diseases Imagine, INSERM, Francia
- Thomas Gillingwater, College of Medicine & Veterinary Medicine, University of Edinburgh
- Alessandra Recchia, Università di Modena
Montagna C, Petris G, Casini A, Maule G, Franceschini GM, Zanella I, Conti L, Arnoldi F, Burrone OR, Zentilin L, Zacchigna S, Giacca M, Cereseto A. VSV-G Enveloped vesicles for traceless delivery of CRISPR-Cas9. Mol. Therapy NA 2018; 12: 453-462.
Casini A, Olivieri M, Petris G, Montagna C, Reginato R, Maule G, Lorenzin F, Prandi D, Romanel A, Demichelis F, Inga A, Cereseto A. In vivo screening of highly specific SpCas9 variants. Nat Biotechnol 2018; 36:265-271.
Romanel A, Garritano S, Stringa B, Blattner M, Dalfovo D, Chakravarty D, Soong D, Cotter KA, Petris G, Dhingra P, Gasperini P, Cereseto A, Elemento O, Sboner A, Khurana E, Inga A, Rubin MA, Demichelis F. Inherited determinants of early recurrent somatic mutations in prostate cancer. Nat Commun 2017;8:48.
Petris G, Casini A, Montagna C, Lorenzin F, Prandi D, Romanel A, Zasso J, Conti L, Demichelis F, Cereseto A. Hit and go CAS9 delivered through a lentiviral based self-limiting circuit. Nat Commun 2017;8:15334.
Cereseto A, Giacca M. Imaging HIV-1 nuclear pre-integration complexes. Methods Mol Biol Clifton NJ 2014;1087:47–54.
Di Primio C, Quercioli V, Allouch A, Gijsbers R, Christ F, Debyser Z, Arosio D, Cereseto A. Single-cell imaging of HIV-1 provirus (SCIP). Proc Natl Acad Sci U S A 2013;110:5636–5641.
Allouch A, Di Primio C, Alpi E, Lusic M, Arosio D, Giacca M, Cereseto A. The TRIM family protein KAP1 inhibits HIV-1 integration. Cell Host Microbe 2011;9:484–495.
Manganaro L, Lusic M, Gutierrez MI, Cereseto A, Del Sal G, Giacca M. Concerted action of cellular JNK and Pin1 restricts HIV-1 genome integration to activated CD4+ T lymphocytes. Nat Med 2010;16:329–333.
Christ F, Thys W, De Rijck J, Gijsbers R, Albanese A, Arosio D, Emiliani S, Rain J-C, Benarous R, Cereseto A, Debyser Z. Transportin-SR2 imports HIV into the nucleus. Curr Biol CB 2008;18:1192–1202.
Albanese A, Arosio D, Terreni M, Cereseto A. HIV-1 pre-integration complexes selectively target decondensed chromatin in the nuclear periphery. PloS One 2008;3:e2413.