[A novel CRISPR/Cas9-hLacI donor adapting system for dsDNA-templated gene editing].

During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.

A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair.

The fast-rising CRISPR-derived gene editing technologies has been widely used in the fields of life science and biomedicine, as well as plant and animal breeding. However, the efficiency of homology-directed repair (HDR), an important strategy for gene knock-in and base editing, remains to be improved. In this study, we came up with the term Donor Adapting System (DAS) to summarize those CRISPR/Cas9 systems modified with adaptor for driving aptamer-fused donor DNA. A set of CRISPR/Cas9-Gal4BD DAS was designed in our study. In this system, Gal4 DNA binding domain (Gal4BD) is used as adaptor to fuse with Cas9 protein, and Gal4 binding sequence (Gal4BS) is used as aptamer to bind to the double-stranded DNA (dsDNA) donor, in order to improve the HDR efficiency. Preliminary results from the HEK293T-HDR.GFP reporter cell line show that the HDR editing efficiency could be improved up to 2-4 times when donor homologous arms under certain length (100-60 bp). Further optimization results showed that the choice of fusion port and fusion linker would affect the expression and activity of Cas9, while the Cas9-Gal4BD fusion with a GGS5 linker was the prior choice. In addition, the HDR efficiency was likely dependent on the aptamer-dsDNA donor design, and single Gal4BD binding sequence (BS) addition to the 5'-end of intent dsDNA template was suggested. Finally, we achieved enhanced HDR editing on the endogenous AAVS1 and EMX1 sites by using the CRISPR/Gal4BD-Cas9 DAS, which we believe can be applied to facilitate animal molecular design breeding in the future.

Alginic acid induces oxidative stress-mediated hormone secretion disorder, apoptosis and autophagy in mouse granulosa cells and ovaries.

Alginic acid (AA) is a kind of polysaccharide extracted from brown seaweeds and has been widely used in food industry. Certain positive effects of AA, such as anti-inflammation and anti-allergy, have been reported. Nevertheless, as a potential chemical contaminant of the environment, its impact on female reproductive system remains to be investigated. The purpose of this study is to explore the impact of AA on ovary and to investigate the further cellular mechanism. Primarily, in vitro cultured mouse ovary granulosa cells (GCs) were treated with AA at a concentration of 10µM for 24 h. The cells and supernatant were collected and subjected to further measures. The results demonstrated that after being treated with 10µM AA for 24 h the levels of estradiol and progesterone in supernatant were down-regulated. And excessive reactive oxygen species (ROS) and declined antioxidant capacity were also determined. Additionally, a large number of apoptotic bodies and autophagic vesicles were found in the experimental cells, and the mitochondria-mediated apoptotic pathway was demonstrated to play a main role in GCs apoptosis. To further investigate the effect of AA on ovary, the female ICR mice were administered with AA (10 mg/ kg bodyweight) intraperitoneally for successive 35 days, and the estrus phase was recorded simultaneously. After exposure, the ovaries and blood samples were collected for further analysis. The results revealed that the estrus period of the mice was shortened and the interestrus period was extended after being treated with AA for 35 days. At the organismal level, the numbers of antral follicles and atresia follicles increased and the levels of pro-apoptosis and autophagy-related proteins were detected upregulated after AA treatment. Taken together, both in vivo and in vitro data suggested that AA has toxicity on female reproduction by disrupting estrogen production and inducing oxidative stress, mitochondria-mediated apoptosis and autophagy. Our results provide new scientific basis and the concern for controlling the increasing use of AA.

Screening of Cucumber Fusarium Wilt Bio-Inhibitor: High Sporulation Trichoderma harzianum Mutant Cultured on Moso Bamboo Medium.

Cucumber fusarium wilt is a soil-borne disease which causes serious production decrease in cucumber cultivation world widely. Extensive using of chemical pesticides has caused serious environmental pollution and economic losses, therefore, it is particularly urgent to develop efficient, safe and pollution-free biopesticide. In this study, a mutant strain of Trichoderma harzianum cultivated in moso bamboo medium was proved to be an efficient bio-inhibitor of the disease. The mutant strain T. harzianum T334, was obtained by three microwave mutagenesis cycles with an irradiation power of 600 W and irradiation time of 40 s. In contrast to the original strain, the inhibition rate on cucumber fusarium wilt of the strain T334 increased from 63 to 78%. In this work, disk milling pretreatment of moso bamboo has shown significant beneficial effects on both biotransformation and sporulation of T334. Its sporulation reached 3.7 × 109 cfu/g in mushroom bags with 90% bamboo stem powder (pretreated by disk milli), 9.5% bamboo leaf powder and 0.5% wheat bran when the ratio of solid to liquid was 4:6, the inoculum amount was 10%, and the culture temperature was 28°C. These results provide an alternative bioinhibitor for the control of cucumber fusarium wilt, and a potential usage of moso bamboo in the production of microbial pesticide.

Effects of Microwave-Assisted Liquid Hot Water Pretreatment on Chemical Composition and Structure of Moso Bamboo.

The effects of microwave assisted liquid hot water (MA-LHW) pretreatment on the chemical composition of Moso bamboo were investigated, and the fiber structure of pretreated residues were studied. The results showed that MA-LHW pretreatment had high selectivity for the degradation of hemicellulose in Moso bamboo, and the extracted hemicellulose could be used to prepare xylooligosaccharide through enzyme depolymerization. The degradation rates of cellulose and lignin after MA-LHW pretreatment were only 14.73% and 7.18%, which were significantly lower than those of LHW pretreatment; 155.0 mg/g xylobiose and 61.0 mg/g xylotrisoe can be obtained after enzymatic hydrolysis, and the yield of xylo-oligosaccharide reached 80.59% of the theoretical conversion rate. MA-LHW pretreatment increased the removal of hemicellulose, lignin, and other non-crystalline parts in bamboo materials, and more cellulose with crystalline structure was retained, which increased the CrI value of Moso bamboo by 14.84%. FTIR spectra showed that the characteristic peak intensity of hemicellulose was significantly reduced after MA-LHW pretreatment, which confirmed the selective degradation of hemicellulose by MA-LAW pretreatment. Moreover, MA-LHW pretreatment also destroyed O-H, C-H, C-O-C, and ß-glucoside bonds in Moso bamboo fiber, caused by the recombination and synthesis of some groups (-CH2 and C=O) of cellulose, hemicellulose, and lignin destroyed under pretreatment conditions.