Gene Editing and Molecular Biology (GEMBio)

The gene editing and molecular biology facility (GEMBio) offers support to the University's scientific research, teaching and third mission activities in the fields of molecular biology and gene expression regulation.

The GEMBio facility (directed by Prof. Marco Trerotola, Cancer Pathology Unit, Molecular Medicine Laboratory) can support the researchers in the field of molecular biology, from standard gene subcloning to the most recent technologies for targeted regulation of gene expression (including RNA interference and CRISPR/Cas9 gene editing).

The services of the GEMBio facility apply to various scientific fields, in particular  biomedical sciences, and are provided to public and private bodies, through scientific collaborations and project consultancy, or through fee-for-service modalities.

Some of the main services offered by the GEMBio facility are listed below.

1. CRISPR/Cas9 editing for functional inactivation of target genes

The service includes:

(1) design and synthesis of 3 pairs of sgRNA oligonucleotides for each target gene to be inactivated;

(2) annealing of sgRNA oligonucleotide pairs;

(3) insertion of sgRNA oligonucleotides into specific lentiviral plasmids;

(4) sequencing of recombinant plasmids;

(5) transfection of packaging cells (HEK-293T) for generation of recombinant virions;

(6) collection of supernatant from HEK-293T containing the recombinant virions;

(7) transduction of target cells (provided by the user);

(8) selection of resistant cells and expansion of single-cell clones (3 x 96-well plates for each sgRNA);

(9) extraction of genomic DNA from at least 10 cell clones for each sgRNA;

(10) sequencing of the edited region;

(11) bioinformatic analysis of sequenced DNAs;

(12) expansion of positive cell clones (at least 3 for each sgRNA).

2. Fluorescent chimeric proteins

The service includes the generation of fluorescent chimeras of target genes fused to spectral variants of the green fluorescent protein (GFP) or the red fluorescent protein (RFP). The user is required to provide the gene of interest and the corresponding sequence in electronic format. The GEMBio facility will perform PCR amplification and subcloning into the required destination vectors.

3. Site-specific mutagenesis

The user is required to provide the gene of interest within a plasmid vector, for subsequent modification (point mutations, triplet insertions/deletions) using Q5 site directed mutagenesis technology. The GEMBio facility will provide the user with the vector containing the mutated gene of interest, after verifying that mutagenesis has occurred by Sanger sequencing.

Base-editing/Prime editing technology based on CRISPR will also be available soon, for the introduction of site-specific mutations into the genome of target cells.

4. RNA interference

Stable inhibition of the expression of target genes, based on the use of shRNA. The user is asked to provide the sequence of the gene of interest, and the GEMBio facility will design 3 pairs of shRNA oligonucleotides for each gene to be silenced. These oligonucleotides will then be inserted into specific plasmid vectors. The GEMBio facility will sequence the recombinant plasmids, and will provide the user with 3 products corresponding to the 3 pairs of oligonucleotides designed for the specific silencing experiment.

5. “Ready-to-use” plasmids

The GEMBio facility has a collection of expression vectors for proteins (native, mutated, tagged, fluorescent, etc.) that can be expressed in bacteria or higher organisms (a complete and updated list will be periodically published on the CAST website), and can be used for study of molecular signaling, for the marking of organelles and specific regions of the cellular environment, for heterologous productions and for advanced biochemical analyses (protein-protein interactions). Distribution of such plasmids is subject to Material Transfer Agreement limitations that will be implemented with end users.