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.

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.