Characterization and reverse genetic establishment of cattle derived Akabane virus in China

Background Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. Results An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. Conclusion We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

phiC31 integrase for recombination mediated single copy insertion and genome manipulation in C. elegans

C. elegans benefits from a large set of tools for genome manipulation. Yet, the precise single-copy insertion of very large DNA constructs (>10 kb) and the generation of inversions are still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that serves as a landing pad for integration of transgenes by recombination mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations were obtained for insert sizes up to ∼33.4 kb. Taking advantage of this integration method we establish a dual color fluorescent operon reporter system able to study post-transcriptional regulation of mRNA. Last, we show that large chromosomal segments can be inverted using phiC31 integrase. Thus, the phiC31 integrase system should be a useful addition to the C. elegans toolkit.

Genetically engineered birds; pre-CRISPR and CRISPR era

Generating biopharmaceuticals in genetically engineered bioreactors continues to reign supreme. Hence, genetically engineered birds have attracted considerable attention from the biopharmaceutical industry. Fairly recent genome engineering methods have made genome manipulation an easy and affordable task. In this review, we first provide a broad overview of the approaches and main impediments ahead of generating efficient and reliable genetically engineered birds, and various factors that affect the fate of a transgene. This section provides an essential background for the rest of the review, in which we discuss and compare different genome manipulation methods in the pre-CRISPR and CRISPR era in the field of avian genome engineering.

Applications of piggyBac Transposons for Genome Manipulation in Stem Cells

Transposons are mobile genetic elements in the genome. The piggyBac (PB) transposon system is increasingly being used for stem cell research due to its high transposition efficiency and seamless excision capacity. Over the past few decades, forward genetic screens based on PB transposons have been successfully established to identify genes associated with drug resistance and stem cell-related characteristics. Moreover, PB transposon is regarded as a promising gene therapy vector and has been used in some clinically relevant stem cells. Here, we review the recent progress on the basic biology of PB, highlight its applications in current stem cell research, and discuss its advantages and challenges.

Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing. Their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a program to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in the insertion of gene fragments to essential genes (median 52.1%), the complete deletion of an open reading frame (median 78.9%), and the introduction of single amino acid substitutions (median 79.2%). The backbone vectors with the program would provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.

Genetic tools for the development of recombinant lactic acid bacteria

Lactic acid bacteria (LAB) are a phylogenetically diverse group with the ability to convert soluble carbohydrates into lactic acid. Many LAB have a long history of safe use in fermented foods and are recognized as food-grade microorganisms. LAB are also natural inhabitants of the human intestinal tract and have beneficial effects on health. Considering these properties, LAB have potential applications as biotherapeutic vehicles to delivery cytokines, antigens and other medicinal molecules. In this review, we summarize the development of, and advances in, genome manipulation techniques for engineering LAB and the expected future development of such genetic tools. These methods are crucial for us to maximize the value of LAB. We also discuss applications of the genome-editing tools in enhancing probiotic characteristics and therapeutic functionalities of LAB.

Methods for the Manipulation of Herpesvirus Genome and the Application to Marek’s Disease Virus Research

Herpesviruses are a group of double-strand DNA viruses that infect a wide range of hosts, including humans and animals. In the past decades, numerous methods have been developed to manipulate herpesviruses genomes, from the introduction of random mutations to specific genome editing. The development of genome manipulation methods has largely advanced the study of viral genes function, contributing not only to the understanding of herpesvirus biology and pathogenesis, but also the generation of novel vaccines and therapies to control and treat diseases. In this review, we summarize the major methods of herpesvirus genome manipulation with emphasis in their application to Marek’s disease virus research.

Characterization and Reverse Genetic Establishment of Cattle Derived Akabane Virus in China

Background: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed in the tropical and temperate zones of Asia and Africa and is considered as a great threat in herbivores.Results: An AKAV defined as TJ2016 was firstly isolated from the serum of cattle in China in 2016. Sequence analysis of the S and M segments suggested that the isolated TJ2016 was closely related to the strains JaGAr39 and JaLAB39, which belonged to genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ-2016 S, M, and L segment was separately constructed into the TVT7R plasmid under the control of T7 promoter which named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. Then, the three constructed plasmids were transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to rescue AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to have similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells.Conclusion: We successfully isolated a AKAV strain TJ2016 from the serum of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool which can be used for further AKAV pathogenesis and even vaccine studies.