A homing rescue gene drive with multiplexed gRNAs reaches high frequency in cage populations but generates functional resistance.

CRISPR homing gene drives have considerable potential for managing populations of medically and agriculturally significant insects. They operate by Cas9 cleavage followed by homology-directed repair, copying the drive allele to the wild-type chromosome and thus increasing in frequency and spreading throughout a population. However, resistance alleles formed by end-joining repair pose a significant obstacle. To address this, we create a homing drive targeting the essential hairy gene in Drosophila melanogaster. Nonfunctional resistance alleles are recessive lethal, while drive carriers have a recoded "rescue" version of hairy. The drive inheritance rate is moderate, and multigenerational cage studies show drive spread to 96%-97% of the population. However, the drive does not reach 100% due to the formation of functional resistance alleles, despite using four gRNAs. These alleles have a large deletion but likely utilize an alternate start codon. Thus, revised designs targeting more essential regions of a gene may be necessary to avoid such functional resistance. Replacement of the rescue element's native 3' UTR with a homolog from another species increases drive inheritance by 13%-24%. This was possibly because of reduced homology between the rescue element and surrounding genomic DNA, which could also be an important design consideration for rescue gene drives.

Gene drive and genetic sex conversion in the global agricultural pest Ceratitis capitata.

Homing-based gene drives are recently proposed interventions promising the area-wide, species-specific genetic control of harmful insect populations. Here we characterise a first set of gene drives in a tephritid agricultural pest species, the Mediterranean fruit fly Ceratitis capitata (medfly). Our results show that the medfly is highly amenable to homing-based gene drive strategies. By targeting the medfly transformer gene, we also demonstrate how CRISPR-Cas9 gene drive can be coupled to sex conversion, whereby genetic females are transformed into fertile and harmless XX males. Given this unique malleability of sex determination, we modelled gene drive interventions that couple sex conversion and female sterility and found that such approaches could be effective and tolerant of resistant allele selection in the target population. Our results open the door for developing gene drive strains for the population suppression of the medfly and related tephritid pests by co-targeting female reproduction and shifting the reproductive sex ratio towards males. They demonstrate the untapped potential for gene drives to tackle agricultural pests in an environmentally friendly and economical way.

A lead-free Cs2ZnCl4 perovskite nanocrystals fluorescent probe for highly selective detection of norfloxacin.

The abuse of antibiotics would seriously affect human health and has become of worldwide critical concern, thus it is urgent to develop an environmentally friendly and nontoxic fluorescent probe for antibiotics sensing. In this work, a lead-free Cs2ZnCl4 perovskite nanocrystals (PNCs) probe was fabricated for sensing norfloxacin (NOR) employing a modified ligand-assisted reprecipitation method. The prepared Cs2ZnCl4 PNCs probe had strong blue emission around 440 nm, and the characteristics of PNCs were systematically characterized by X-ray photoelectric spectroscopy (XPS), Fourier transforms infrared spectroscopy (FTIR), transmission electron microscope (TEM) and powder X-ray diffraction (XRD). The results revealed that the fluorescence intensity of the Cs2ZnCl4 PNCs was significantly enhanced after the introduction of norfloxacin. The Cs2ZnCl4 PNCs can be used as a fluorescent probe to selectively and sensitively detect norfloxacin in the concentration range from 0.2 to 50.0 µM, with a correlation coefficient (R2) of 0.9954 and the limit of detection (LOD, 3σ) of 0.1499 µM. The preparation and application of a lead-free perovskite fluorescent probe for norfloxacin would promote the application of perovskite fluorescent probes in biochemical assays.

Optimization of Initial Cation Concentrations for L-Lactic Acid Production from Fructose by Lactobacillus pentosus Cells.

In this study, Box-Behnken design was applied to optimize the initial concentrations of 4 cations for L-lactic acid production from fructose by homologous batch fermentation of Lactobacillus pentosus cells. The optimum initial cation concentrations were obtained as 6.542 mM Mg2+, 3.765 mM Mn2+, 2.397 mM Cu2+, and 3.912 mM Fe2+, respectively. The highest L-lactic acid yield and productivity were obtained as 0.935 ± 0.005 g/g fructose and 1.363 ± 0.021 g/(L × h), respectively, with a maximum biomass concentration of 7.97 ± 0.17 g/L. The effectiveness of the optimization by Box-Behnken design was confirmed based on the small errors between predicted results and experimental results shown as 0.3%, - 0.2%, and - 1.2%, respectively. The quadratic models with high accuracy and reliability can be applied to mathematically forecasted the fermentation performance. After the optimization, the lactic acid yield and productivity were significantly improved by 3.7% and 21.0%, respectively.

L-Lactic acid production from fructose by chitosan film-coated sodium alginate-polyvinyl alcohol immobilized Lactobacillus pentosus cells and its kinetic analysis.

Under the optimal conditions of immobilization and fermentation, the highest LA yield of 0.966 ± 0.006 g/g fructose and production rate of 2.426 ± 0.018 g/(L × h) with an error of -0.5% and -0.2% to the predicted results were obtained from batch fermentation by the CS film-coated SA-PVA immobilized L. pentosus cells. The LA yield and production rate of these immobilized cells were 2.7% and 10.1% higher than that of normal SA-PVA immobilized cells respectively, and they were 5.7% and 48.4% higher than that of free cells, respectively. The effect of temperature on different types of immobilized cells and free cells was significantly different, but the effect of pH on different types of cells was not much different. The kinetic models could effectively describe the different fermentation performances of three types of cells. The immobilized cells have excellent reusability to conduct 9 runs of repeated batch fermentation.