De Novo Genome Assembly at Chromosome-Scale of Hermetia illucens (Diptera Stratiomyidae) via PacBio and Omni-C Proximity Ligation Technology.

Hermetia illucens is a species of great interest for numerous industrial applications. A high-quality reference genome is already available for H. illucens. However, the worldwide maintenance of numerous captive populations of H. illucens, each with its own genotypic and phenotypic characteristics, made it of interest to perform a de novo genome assembly on one population of H. illucens to define a chromosome-scale genome assembly. By combining the PacBio and the Omni-C proximity ligation technologies, a new H. illucens chromosome-scale genome of 888.59 Mb, with a scaffold N50 value of 162.19 Mb, was assembled. The final chromosome-scale assembly obtained a BUSCO completeness of 89.1%. By exploiting the Omni-C proximity ligation technology, topologically associated domains and other topological features that play a key role in the regulation of gene expression were identified. Further, 65.62% of genomic sequences were masked as repeated sequences, and 32,516 genes were annotated using the MAKER pipeline. The H. illucens Lsp-2 genes that were annotated were further characterized, and the three-dimensional organization of the encoded proteins was predicted. A new chromosome-scale genome assembly of good quality for H. illucens was assembled, and the genomic annotation phase was initiated. The availability of this new chromosome-scale genome assembly enables the further characterization, both genotypically and phenotypically, of a species of interest for several biotechnological applications.

Strategies for single base gene editing in an immortalized human cell line by CRISPR/Cas9 technology.

The use of CRISPR/Cas9 system has rapidly grown in the last years. Here, the optimization of gene editing of a single-nucleotide polymorphism in a human non-malignant somatic cell line of thyrocytes (Nthy-Ori) was described highlighting strategies for overcoming the problems concerning the delivery and off-targets. We employed both lentivirus and chemical lipids as delivery agents and two strategies for creating the double-strand breaks (DSB). The former induced a DSB by a classical Cas9 nuclease (standard strategy), while the second one employed a modified Cas9 creating a single-strand break (SSB). The knock-in was carried out using a single-stranded donor oligonucleotide or the HR410-PA donor vector (HR). The desired cells could be obtained by combining the double nickase system with the HR vector transfected chemically. This result could be due to the type of DSB, likely processed mainly by non-homologous end joining when blunt (standard strategy) and by HR when overhanging (double nickase). Our results showed that the double nickase is suitable for knocking-in the immortalized Nthy-Ori cell line, while the standard CRISPR/Cas9 system is suitable for gene knock-out creating in/del mutations. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03878-4.