Enhancing fatty acid and omega-3 production in Schizochytrium sp. using developed safe-harboring expression system.

BACKGROUND: Schizochytrium, a group of eukaryotic marine protists, is an oleaginous strain, making it a highly promising candidate for the production of lipid-derived products such as biofuels and omega-3 fatty acids. However, the insufficient advancement of genetic engineering tools has hindered further advancements. Therefore, the development and application of genetic engineering tools for lipid enhancement are crucial for industrial production. RESULTS: Transgene expression in Schizochytrium often encounters challenges such as instability due to positional effects. To overcome this, we developed a safe-harbor transgene expression system. Initially, the sfGFP gene was integrated randomly, and high-expressing transformants were identified using fluorescence-activated cell sorting. Notably, HRsite 2, located approximately 3.2 kb upstream of cytochrome c, demonstrated enhanced sfGFP expression and homologous recombination efficiency. We then introduced the 3-ketoacyl-ACP reductase (KR) gene at HRsite 2, resulting in improved lipid and docosahexaenoic acid (DHA) production. Transformants with KR at HRsite 2 exhibited stable growth, increased glucose utilization, and a higher lipid content compared to those with randomly integrated transgenes. Notably, these transformants showed a 25% increase in DHA content compared to the wild-type strain. CONCLUSION: This study successfully established a robust homologous recombination system in Schizochytrium sp. by identifying a reliable safe harbor site for gene integration. The targeted expression of the KR gene at this site not only enhanced DHA production but also maintained growth and glucose consumption rates, validating the efficacy of the safe-harbor approach. This advancement in synthetic biology and metabolic engineering paves the way for more efficient biotechnological applications in Schizochytrium sp.

Safe-Harbor-Targeted CRISPR/Cas9 System and Cmhyd1 Overexpression Enhances Disease Resistance in Cordyceps militaris.

Fungal disease of mushroomCordyceps militaris (CM) caused byCalcarisporium cordycipiticola (CC) is destructive to fruiting body cultivation, resulting in significant economic loss and potential food safety risks. CRISPR/Cas9 genome editing has proven to be a powerful tool for crop improvement but seldom succeeded in mushrooms. Here, the first genomic safe-harbor site, CmSH1 locus, was identified in the CM genome. A safe-harbor-targeted CRISPR/Cas9 system based on an autonomously replicating plasmid was designed to facilitate alien gene integration at the CmSH1 locus. Cmhyd1, one of the hydrophobin genes, was confirmed as a defensive factor against CC infection, and Cmhyd1 overexpression by this system showed enhancement of disease resistance with negligible effect on the agronomic traits of CM. No off-target events and residues of plasmid sequence were tested by PCR and genome resequencing. This study provided the first safe harbor site for genetic manipulations, a safe harbor-targeted CRISPR/Cas9 system, and the first disease-resistant gene-editing breeding system in mushrooms.

Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain.

Our group has developed several approaches for stable, non-viral integration of inducible transgenic elements into the genome of mammalian cells. Specifically, a piggyBac tetracycline-inducible genetic element of interest (pB-tet-GOI) plasmid system allows for stable piggyBac transposition-mediated integration into cells, identification of cells that have been transfected using a fluorescent nuclear reporter, and robust transgene activation or suppression upon the addition of doxycycline (dox) to the cell culture or the diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. More recently, we have developed a transgenic system as an alternative to piggyBac called mosaic analysis by dual recombinase-mediated cassette exchange (MADR), as well as additional in vitro transfection techniques and in vivo dox chow applications. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning of respective genetic element of interest (GOI) into response plasmid Basic Protocol 2: In vitro nucleofection of iPSC-derived human/mouse neural progenitor cells and subsequent derivation of stable inducible cell lines Alternate Protocol: In vitro electroporation of iPSC-derived human/mouse neural progenitor cells Support Protocol: Recovery stage after in vitro transfection Basic Protocol 3: Adding doxycycline to cells to induce/reverse GOI Basic Protocol 4: Assessing gene expression in vitro by non-invasive bioluminescence imaging of luciferase activity.

Efficient Nuclease-Directed Integration of Lentivirus Vectors into the Human Ribosomal DNA Locus.

Lentivirus vectors (LVs) are efficient tools for gene transfer, but the non-specific nature of transgene integration by the viral integration machinery carries an inherent risk for genotoxicity. We modified the integration machinery of LVs and harnessed the cellular DNA double-strand break repair machinery to integrate transgenes into ribosomal DNA, a promising genomic safe-harbor site for transgenes. LVs carrying modified I-PpoI-derived homing endonuclease proteins were characterized in detail, and we found that at least 21% of all integration sites localized to ribosomal DNA when LV transduction was coupled to target DNA cleavage. In addition to the primary sequence recognized by the endonuclease, integration was also enriched in chromatin domains topologically associated with nucleoli, which contain the targeted ribosome RNA genes. Targeting of this highly repetitive region for integration was not associated with detectable DNA deletions or negative impacts on cell health in transduced primary human T cells. The modified LVs characterized here have an overall lower risk for insertional mutagenesis than regular LVs and can thus improve the safety of gene and cellular therapy.

New Human Chromosomal Sites with "Safe Harbor" Potential for Targeted Transgene Insertion.

This study identified 35 new sites for targeted transgene insertion that have the potential to serve as new human genomic "safe harbor" sites (SHS). SHS potential for these 35 sites, located on 16 chromosomes, including both arms of the human X chromosome, and for the existing human SHS AAVS1, hROSA26, and CCR5 was assessed using eight different desirable, widely accepted criteria for SHS verifiable with human genomic data. Three representative newly identified sites on human chromosomes 2 and 4 were then experimentally validated by in vitro and in vivo cleavage-sensitivity tests, and analyzed for population-level and cell line-specific sequence variants that might confound site targeting. The highly ranked site on chromosome 4 (SHS231) was further characterized by targeted homology-dependent and -independent transgene insertion and expression in different human cell lines. The structure and fidelity of transgene insertions at this site were confirmed, together with analyses that demonstrated stable expression and function of transgene-encoded proteins, including fluorescent protein markers, selectable marker cassettes, and Cas9 protein variants. SHS-integrated transgene-encoded Cas9 proteins were shown to be capable of introducing a large (17 kb) gRNA-specified deletion in the PAX3/FOXO1 fusion oncogene in human rhabdomyosarcoma cells and as a Cas9-VPR fusion protein to upregulate expression of the muscle-specific transcription factor MYF5 in human rhabdomyosarcoma cells. An engineering "toolkit" was developed to enable easy use of the most extensively characterized of these new human sites, SHS231, located on the proximal long arm of chromosome 4. The target sites identified here have the potential to serve as additional human SHS to enable basic and clinical gene editing and genome-engineering applications.