This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene knock-ins with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) in bovine and dairy goat fetal fibroblasts.  To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin (MSTN) gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2.  Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in the GFP-PGK-NeoR plasmid background, including a 5´ and 3´ homologous arm flanking the genes humanized Fat-1 (hFat-1) or enhanced green fluorescent protein (eGFP).  Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and the hFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts.  After G418 (Geneticin) selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove.  The gene knock-in events were screened by PCR across the homologous arms.  The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP and hFat-1 gene knock-ins were 13.68 and 0%, respectively.  The efficiencies of CRISPR/Cas9-mediated eGFP and hFat-1 gene knock-ins were 77.02 and 79.01%, respectively.  The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system.  Additionally, the hFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system.  The difference of knock-in efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant (P<0.01).  In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP and hFat-1 gene knock-ins were 32.35 and 26.47%, respectively.  The efficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively.  The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant (P<0.01).  This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs.  The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines. 

Deoxynivalenol (DON) is a type B trichothecenes mycotoxin produced by several Fusarium species, often found in foodstuffs for humans and animals. DON is in great demand for the toxicological researches both in vivo and in vitro. In this work, wheat culture was inoculated with a Fusarium graminearum PH-1 strain for DON production. The solvent system for crude extraction was acetonitrile-water (84:16, v/v). A simple two-step silica gel column chromatography was employed to separate the DON mycotoxin from wheat culture, combined with preparative high performance liquid chromatography (preparative HPLC) to purify the compound. The solvent system for the second silica gel column chromatography was methylene chloride-methanol (17:1, v/v), which provided a good elution effect selected on thin layer chromatography (TLC). The target compound was identified by HPLC, and the chemical structure was confirmed by mass spectrometry (MS) and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. A total of 433 mg of purified DON was obtained from 1 kg of wheat culture, with a purity of 99.01%. The study had provided an easy-operating and cost-effective method to isolate an expensive compound in a simple way.

Bacterial leaf streak (BLS) of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc) is a worldwide destructive disease. Development of resistant varieties is considered to be one of the most effective and eco-friendly ways to control the disease. However, only a few genes/QTLs having resistance to BLS have been identified in rice until now. In the present study, we have identified and primarily mapped a BLS-resistance gene, bls1, from a rice line DP3, derived from the wild rice species Oryza rufipogon Griff. A BC2F2 (9311/DP3//9311) population was constructed to map BLS-resistance gene in the rice line DP3. The segregation of the resistant and susceptible plants in BC2F2 in 1:3 ratio (χ2=0.009, χ2 0.05, 1=3.84, P>0.05), suggested that a recessive gene confers BLS resistance in DP3. In bulked segregant analysis (BSA), two SSR markers RM8116 and RM584 were identified to be polymorphic in resistant and susceptible DNA bulks. For further mapping the resistance gene, six polymorphic markers around the target region were applied to analyze the genotypes of the BC2F2 individuals. As a result, the BLS-resistant gene, designated as bls1, was mapped in a 4.0-cM region flanked by RM587 and RM510 on chromosome 6.