Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0153-5) contains supplementary material, which is available to authorized users.

SummaryThe complete genome of spring viraemia of carp virus (SVCV) strain A-1 isolated from cultured common carp (Cyprinus carpio) in China was sequenced and characterized. Reverse transcriptionpolymerase chain reaction (RT-PCR) derived clones were constructed and the DNA was sequenced. It showed that the entire genome of SVCV A-1 consists of 11,100 nucleotide base pairs, the predicted size of the viral RNA of rhabdoviruses. However, the additional insertions in bp 4633-4676 and bp 4684-4724 of SVCV A-1 were different from the other two published SVCV complete genomes. Five open reading frames (ORFs) of SVCV A-1 were identified and further confirmed by RT-PCR and DNA sequencing of their respective RT-PCR products. The 5 structural proteins encoded by the viral RNA were ordered 3 0 -N-P-M-G-L-5 0 . This is the first report of a complete genome sequence of SVCV isolated from cultured carp in China. Phylogenetic analysis indicates that SVCV A-1 is closely related to the members of the genus Vesiculovirus, family Rhabdoviridae.

Hepatic injury is a prevalent ailment worldwide. The hepatoprotective effects of corn peptides (CPs) were evaluated against carbon tetrachloride (CCl4)‐induced liver injury in mice. Low molecular weight CP (Mw < 5 kD) were obtained by alcalase hydrolysis combined with ultrafiltration technology. Results showed that the hepatoprotective effects of CP (200 mg/kg body weight [bw]) were manifested by a significant decrease of the aminotransferase (aspartate transaminase, alanine transaminase) activities in serum and malondialdehyde level in liver, and by a significant increase of superoxide dismutase activity and glutathione level in liver (P < 0.01), compared with the model group. The biochemical findings were supplemented by histopathological examination of liver sections. The hepatoprotective effect was close to that of positive control group – Silymarin (50 mg/kg bw). In conclusion, this study demonstrated that the pretreatment of CP at the dose of 200 mg/kg bw showed a significant protective effect to alleviate CCl4‐induced hepatocellular injury, and the hepatoprotective effect was due to the CP's antioxidant capability and high Fischer's ratio. PRACTICAL APPLICATIONS There is an ever‐increasing need of an agent that could protect liver from various damages. Therapies developed along the principles of Western medicine are often limited in efficacy, carry the risk of adverse effects and are often too costly, especially for the developing world. Corn peptides (CPs) were obtained from corn gluten meal, which was the by‐product of corn industry. It will broaden the application and increase the value of corn gluten meal. CPs, especially those with low molecular weight, have been reported with many bioactive functions, which were isolated with potent free radical scavenging capacities and high Fischer's ratio. In general CP could become a safe and potent hepatoprotective agent and could be manufactured into various forms, such as powder, tinctures, tablet, capsule and syrup. The future applications of CP would be mostly as a medicine, supplement or functional foods.

Porcine circovirus type 2 (PCV2), a single-stranded DNA virus, is associated with postweaning multisystemic wasting syndrome (PMWS). ORF2 protein (capsid) of PCV2 was recently demonstrated to be a major immunogenable to induce protection in pigs with a prime-boost protocol. In this study, the ORF2 gene of PCV2 was expressed in insect cells. The product self-assembled into particles that were structurally and antigenically indistinguishable from regular PCV2 capsids. To evaluated the immunogenicity of these virus-like particles, PCV2-free piglets were vaccinated with the crude lysate from recombinant baculovirus (Ac.ORF2)-infected insect cells, at doses of 0.1 ml (10(6) cells), 0.5 mL (5 x 10(6) cells) or 1.0 ml (10(7) cells). The immune response was monitored by an indirect enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and lymphocyte proliferation assay. The ELISA results indicated that primary immune response was elicited with 0.5 ml or 1.0 ml of crude lysate from Ac.ORF2. After boost immunization, relatively higher levels of PCV2 antibody were elicited in 0.5-ml or 1.0-ml vaccinated groups, compared to the 0.1-ml group. In addition, higher PCV2 specific lymphocyte proliferation response was developed in piglets vaccinated with 0.5 ml or 1.0 ml of crude lysate, especially in those vaccinated with with 1.0 ml of crude lysate. Thus, the expressed ORF2 protein has significant potential as a subunit vaccine against PCV2 infection.

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Certain bioactive peptides are reported to be able to alleviate hepatic fibrosis. Our previous work has confirmed the hepatoprotective effect of corn peptides (CPs) that are prepared from a high protein by-product, corn gluten meal, on acute liver injury in an animal model. However, the antifibrotic activity of CPs remained to be elucidated. In this study, the hepatoprotective effect of CPs on thioacetamide (TAA)-induced liver fibrosis was tested. Results showed that CPs (100 mg/kg body weight) significantly decreased the levels of alanine transaminase/aspartate transaminase, laminin, type IV collagen, and type III collagen in serum and increased the serum albumin levels and total antioxidant capacity. Additionally, with CP treatment (100 mg/kg body weight), a significant decrease was observed in the levels of malondialdehyde, nitric oxide, hydroxyproline, transforming growth factor β1, and lactate dehydrogenase activity as well as the liver index, while the activity of superoxidedismutase was significantly increased in livers. The histological and morphological analysis showed that the hepatocyte structure in CP-treated rats was superior to that of TAA-injured rats, and inflammation and fibrosis were also ameliorated. Therefore, CPs can be used as an option for prevention and adjuvant therapy of liver fibrosis.

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Efficient generation of plants carrying mutations in multiple genes remains a challenge. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations, but assembly of these systems requires a robust, high-capacity toolkit. Here, we describe MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), an extensively updated toolkit for assembly of two or more CRISPR/Cas systems. We developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. We validated the utility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, and by creating transgenic Arabidopsis thaliana that constitutively or inducibly overexpress multiple genes. We then demonstrated that the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors facilitated the assembly of two orthogonal CRISPR/Cas systems including SpCas9 and SaCas9, and thus facilitated the creation of transgenic lines harboring these systems. We anticipate that MISSA 2.0 will enable substantial advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems, as well as in plant synthetic biology.