• RNAi
• CRISPR/Cas9
• splicing variants
• BRAF V600E
• melanoma

BRAF (V-Raf Murine Sarcoma Viral Oncogene Homolog B) is a gene on chromosome 7 (7q34) encoding for a protein belonging to the RAF proteins family and characterized by Serine/Threonine kinase activity.
BRAF protein is involved in the MAPK cascade (Mitogen-Activated Protein Kinase), that is responsible for the regulation of those processes that support cell survival, mainly cell proliferation, differentiation, and programmed cell death.
Oncogenic activating mutations in BRAF lead to constitutive activation of the MAPK pathway, resulting in uncontrolled cell proliferation and apoptosis inhibition.
BRAF V600E is an activating missense mutation in codon 600 of exon 15 of BRAF gene that is responsible for the Valine 600 to Glutamic Acid substitution. This mutation has been observed in several cancers such as adenocarcinoma, thyroid gland papillary carcinoma, lung adenocarcinoma, but mainly in melanoma, since approximately 50% of all melanomas contains BRAF V600E.

In 2015 and later in 2017, Marranci et al., from the lab where I have been working, experimentally demonstrated the existence of a BRAF isoform, only predicted before that time, named “X1”, that is both transcribed and translated in the cell.
The canonical (named “reference”, “ref”) and the X1 isoforms of BRAF are splicing variants and they are co-expressed inside the cell. They differ at the transcript level for their sequence and length, mainly because of their 3’-UTR: the reference’s 3’-UTR is about 100 nucleotides long, while the X1’s is indeed as long as 7000 nucleotides. In addition, the two isoforms also differ at the coding sequence level: the ref’s ORF consists in 18 exons, while the X1’s contains a shorter exon 18 (subjected to splicing), followed by an additional exon (exon 19). This leads also to a difference at the protein level, because of the different aminoacidic sequence resulting at the C-term of the BRAF-ref and BRAF-X1 proteins.
The fact that these two isoforms are co-expressed inside the cell, but also show differences in terms of transcript and protein length and sequence, led to think that they may have distinct functions in the cell.

The aim of my thesis project is to create a human melanoma cell line that stably expresses only one of the two isoforms of BRAF V600E, the ref or the X1, to better understand their functions.

The strategy I adopted is based on the use of the CRISPR/Cas9 system in the AAVS1 genomic locus (docking site) to achieve stable expression of one of the two BRAF isoform, together with RNAi (RNA interference) against the endogenous BRAF to prevent the co-expression of the two isoforms inside the cell.
Specifically, I cloned the coding sequences of either BRAF V600E ref or X1 inside the Gen2 expression vector that contains the Tet-ON system for Doxy-inducible expression of the gene of interest. Thanks to the homology arms for the AAVS1 locus, when melanoma cells are transfected with this vector and are provided with the Cas9 protein and a guide RNA that targets the AAVS1 locus itself, a double-strand break (DSB) occurs, followed by homology-directed repair (HDR). The result is the construct integration inside the cell genome (AAVS1 locus).
I also designed an shRNA (short hairpin RNA) targeting BRAF in order to prevent the co-expression of ref and X1 endogenous BRAF isoforms.
Moreover, to avoid the shRNA silencing action on the exogenous BRAF, the coding sequences of BRAF V600E ref and X1, before the cloning in the Gen2 vector, underwent site-directed mutagenesis to modify the nucleotide sequence of the region that is recognized by the shRNA, without altering the aminoacidic sequence of the encoded proteins (synonymous mutation).
I cloned the shBRAF inside a lentiviral vector containing, again, the Tet-ON system allowing Doxy-inducible shRNA expression once delivered inside melanoma cells through infection.

The final result is a human melanoma cell line that, after Doxy treatment, undergoes endogenous BRAF knockdown and, at the same time, starts expressing the BRAF V600E isoform of interest, ref or X1.

This human melanoma cell line will be crucial to study the role of the two isoforms of BRAF V600E and their involvement in different cellular and metabolic pathways to understand if BRAF V600E ref and X1 have distinct functions and if they regulate different processes inside the cell.