• de novo genome assembly
• genome editing
• Hermetia illucens

Due to rapid growth of world population, global food demand is expected to increase by nearly 100% from 2005 to 2050, thus posing future fundamental challenges related to finding new sources of nutrients for human consumption and animal feed production. In the context of sources for new nutrients, edible insects gained relevant attention: numerous studies have demonstrated that they can complement traditional sources of fat and proteins commonly used in feed formulation, without negatively impacting on growth performance, animal quality and health. Hermetia illucensm (Diptera: Stratiomyidae), commonly known as black soldier fly (BSF), is among the edible insects that have the greatest potential for use in animal feed and it’s already used as a supplement of poultry and swine feed, but also in the aquaculture of several fish species.
However, despite its important nutritional characteristics, the introduction of BSF in animal feed is still limited by a number of elements, including its high lipid content and unbalanced fatty acid profile.
For this reason, BSF could be a possible future candidate for site-specific genome editing applications, such as the introduction of the human Apolipoprotein A-I, with particular regards to the Milano variant (ApoA-IMilano, AIM) gene coding sequence, involved in cholesterol and lipid metabolism, and associated with anti-inflammatory properties. Indeed, the site-specific expression of this gene should lead to further improved nutritional characteristics, thus favouring its wider application in animal nutrition. In literature are shown several studies of animal diseases related to the lipid content of diets normally administered in intensive farming and aquaculture, so the introduction of AIM genetically modified BSF could improve animal welfare, with important advantages both from an ethical point of view and in terms of the final food quality.
The aim of this thesis was the identification and the characterization of 5 genes of BSF genome, as potential candidates for the successive genome editing approach as mentioned above, through site-specific insertion of multicistronic constructs by means of the CRISPR/Cas strategy, in order to ensure stage- and time-specific expression of the human AIM gene in BSF.
At first, the identification of the potential candidate genes for genome editing was performed on databases such as FlyBase, KEGG Pathways, NCBI, UniProt via an in silico approach. The selection process was based on the following criteria: high levels of expression in those larval stages relevant for the use of BSF as feed supplement; high levels of expression in Drosophila melanogaster, in those tissue and in those larval stages relevant for the use of BSF as feed supplement; protein processing and protein subcellular localisation similar to that of the AIM protein in humans. A list of 8 interesting genes were then compiled and the successive part of the project focused on the further characterization of these genes. The subsequent characterization has been performed by means of RNA extraction from BSF pupae, cDNA reverse transcription, amplification of coding sequence (CDS) of a candidate gene by end-point PCR, inserts cloning into pGEM T-easy vectors (Promega) and their subsequent sequencing.
In order to identify and characterize possible candidate genes, it is necessary to consider that BSF is not a model organism and that structural and functional knowledge about this genome is limited. For this reason, the studies described above were carried out alongside a de novo genome assembly, commissioned to the company Dovetail Genomics (USA). A first, still preliminary, output of the sequencing analysis has been released by the Dovetail Genomics with the following features: 1.210.120 number of reads, 16,3 Gb of base pairs and a coverage of 14x. The actual step of assembly is the genome assembled by means of hifiasm (an assembler for HiFi reads which delivers haplotype-resolved assembly). The de novo genome assembly has then been followed by the analysis of the sequences received from Dovetail Genomics (USA), using various bioinformatic tools such as AUGUSTUS, BUSCO and other sequence alignment tools, with the final goal to define a chromosome-scale genome assembly and to start the annotation analysis of the genome itself. This allowed to quantify the assembled genome completeness, to perform ab initio gene prediction (which returned 54.204 predicted genes), to associate part of the predicted genes with a possible function.
Overall, the definition of a high resolution assembly for BSF, combined with the acquisition of information on possible target genes for genome editing strategies, could be preliminary step for a future genetic modification approach, in order to increase the potential use of this species in the field of feed formulation for various terrestrial and aquatic animal species.