RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

Mapping Transgene Insertion Sites Reveals the α-Cre Transgene Expression in Both Developing Retina and Olfactory Neurons

The Tg(Pax6-cre,GFP)2Pgr (α-Cre) mouse (MGI:3052661) is a commonly used Cre line thought to be retinal-specific. Using targeted locus amplification (TLA), we mapped the insertion site of the transgene, and defined primers useful to deduce zygosity. Further analyses revealed four tandem copies of the transgene. The insertion site mapped to clusters of vomeronasal and olfactory receptor genes. Using R26R and Ai14 Cre reporter mice, we confirmed retinal Cre activity, but also detected expression in olfactory neurons, implicating the influence of neighbouring regulatory elements. RT-PCR and the buried food pellet (BFP) test did not detect any effects of the transgene on flanking genes in the nasal mucosa and retina. Together, these data precisely map α-Cre, show that it does not affect surrounding loci, but reveal previously unanticipated transgene expression in olfactory neurons. The α-Cre mouse can be a valuable tool in both retinal and olfactory research.

Influence of transgenesis on genome variability in cucumber lines with a thaumatin II gene

The development of new plant varieties by genetic modification aims at improving their features or introducing new qualities. However, concerns about the unintended effects of transgenes and negative environmental impact of genetically modified plants are an obstacle for the use of these plants in crops. To analyze the impact of transgenesis on plant genomes, we analyze three cucumber transgenic lines with an introduced thaumatin II gene. After genomes sequencing, we analyzed the transgene insertion site and performed variant prediction. As a result, we obtained similar number of variants for all analyzed lines (average of 4307 polymorphisms), with high abundance in one region of chromosome 4. According to SnpEff analysis, the presence of genomic variants generally does not influence the genome functionality, as less than 2% of polymorphisms have high impact. Moreover, analysis indicates that these changes were more likely induced by in vitro culture than by the transgenesis itself. The insertion site analysis shows that the region of transgene integration could cause changes in gene expression, by gene disruption or loss of promoter region continuity.

Rapid and Detailed Characterization of Transgene Insertion Sites in Genetically Modified Plants via Nanopore Sequencing

Molecular characterization of genetically modified plants can provide crucial information for the development of detection and identification methods, to comply with traceability, and labeling requirements prior to commercialization. Detailed description of the genetic modification was previously a challenging step in the safety assessment, since it required the use of laborious and time-consuming techniques. In this study an accurate, simple, and fast method was developed for molecular characterization of genetically modified (GM) plants, following a user-friendly workflow for researchers with limited bioinformatic capabilities. Three GM events from a diverse array of crop species—perennial ryegrass, white clover, and canola—were used to test the approach that exploits long-read sequencing by the MinION device, from Oxford Nanopore Technologies. The method delivered a higher degree of resolution of the transgenic events within the host genome than has previously been possible with the standard Illumina short-range sequencing strategies. The flanking sequences, copy number, and presence of backbone sequences, and overall transgene insertion structure were determined for each of the plant genomes, with the additional identification of moderate-sized secondary insertions that would have previously been missed. The proposed workflow takes only about 1 week from DNA extraction to analyzed result, and the method will complement the existing approaches for molecular characterization of GM plants, since it makes the process faster, simpler, and more cost-effective.

Sexually Dimorphic Growth Stimulation in a Strain of Growth Hormone Transgenic Coho Salmon (Oncorhynchus kisutch)

Growth hormone (GH) transgenic fish often exhibit remarkable transformations in growth rate and other phenotypes relative to wild-type. The 5750A transgenic coho salmon strain exhibits strong sexually dimorphic growth, with females possessing growth stimulation at a level typical of that seen for both sexes in other strains harbouring the same gene construct (e.g. M77), while males display a modest level of growth stimulation. GH mRNA levels were significantly higher in females than in males of the 5750A strain but equivalent in the M77 strain, indicating sex and transgene insertion locus altered transgene expression. We found that acute estradiol treatments did not influence GH expression in either strain (5750A and M77) or the transgene promoter (metallothionein-B), suggesting that estradiol level was not a significant factor influencing transgene activity. The feminization of XX and XY fish of the 5750A and M77 strains generated all-female groups and resulted in equalized growth of the two genetic sexes, suggesting that the presence of the Y chromosome was not directly capable of influencing the GH transgene–mediated growth in a physiological female conditions. These data suggest that the difference in growth rate seen between the sexes in the 5750A strain arises from non-estradiol-mediated sex influences on gene regulation at the transgene locus. This study shows how genetic factors and transgene insertion sites can influence transgene expression with significant consequent effects on phenotype.

Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells

Baculovirus expression vectors are successfully used for the commercial production of complex (glyco)proteins in eukaryotic cells. The genome engineering of single-copy baculovirus infectious clones (bacmids) in E. coli has been valuable in the study of baculovirus biology, but bacmids are not yet widely applied as expression vectors. An important limitation of first-generation bacmids for large-scale protein production is the rapid loss of gene of interest (GOI) expression. The instability is caused by the mini-F replicon in the bacmid backbone, which is non-essential for baculovirus replication in insect cells, and carries the adjacent GOI in between attTn7 transposition sites. In this paper, we test the hypothesis that relocation of the attTn7 transgene insertion site away from the mini-F replicon prevents deletion of the GOI, thereby resulting in higher and prolonged recombinant protein expression levels. We applied lambda red genome engineering combined with SacB counterselection to generate a series of bacmids with relocated attTn7 sites and tested their performance by comparing the relative expression levels of different GOIs. We conclude that GOI expression from the odv-e56 (pif-5) locus results in higher overall expression levels and is more stable over serial passages compared to the original bacmid. Finally, we evaluated this improved next-generation bacmid during a bioreactor scale-up of Sf9 insect cells in suspension to produce enveloped chikungunya virus-like particles as a model vaccine.

Engineered expression of the invertebrate-specific scorpion toxin AaHIT reduces adult longevity and female fecundity in the diamondback moth Plutella xylostella

BACKGROUNDPrevious Genetic Pest Management (GPM) systems in diamondback moth (DBM) have relied on expressing lethal proteins (‘effectors’) that are ‘cell-autonomous’ i.e. do not leave the cell they are expressed in. To increase the flexibility of future GPM systems in DBM, we aimed to assess the use of a non cell-autonomous, invertebrate-specific, neurotoxic effector – the scorpion toxin AaHIT. This AaHIT effector was designed to be secreted by expressing cells, potentially leading to effects on distant cells, specifically neuromuscular junctions.RESULTSExpression of AaHIT caused a ‘shaking/quivering’ phenotype which could be repressed by provision of an antidote (tetracycline); a phenotype consistent with the AaHIT mode-of-action. This effect was more pronounced when AaHIT expression was driven by the Hr5/ie1 promoter (82.44% of males, 65.14% of females) rather than Op/ie2 (57.35% of males, 48.39% of females). Contrary to expectations, the shaking phenotype and observed fitness costs were limited to adults where they caused severe reductions in mean longevity (–81%) and median female fecundity (–93%). qPCR of AaHIT expression patterns and analysis of piggyBac-mediated transgene insertion sites suggest that restriction of observed effects to the adult stages may be due to influence of local genomic environment on the tetO-AaHIT transgene.CONCLUSIONWe have demonstrated the feasibility of using non cell-autonomous effectors within a GPM context for the first time in the Lepidoptera, one of the most economically damaging orders of insects. These findings provide a framework for extending this system to other pest Lepidoptera and to other secreted effectors.

Effect of Transgene Location, Transcriptional Control Elements and Transgene Features in Armed Oncolytic Adenoviruses

Clinical results with oncolytic adenoviruses (OAds) used as antitumor monotherapies show limited efficacy. To increase OAd potency, transgenes have been inserted into their genome, a strategy known as “arming OAds”. Here, we review different parameters that affect the outcome of armed OAds. Recombinant adenovirus used in gene therapy and vaccination have been the basis for the design of armed OAds. Hence, early region 1 (E1) and early region 3 (E3) have been the most commonly used transgene insertion sites, along with partially or complete E3 deletions. Besides transgene location and orientation, transcriptional control elements, transgene function, either virocentric or immunocentric, and even the codons encoding it, greatly impact on transgene levels and virus fitness.