Germline engineering of the chicken genome using CRISPR/Cas9 by in vivo transfection of PGCs.

Development of simple and readily adoptable methods to mediate germline engineering of the chicken genome will have many applications in research, agriculture and industrial biotechnology. We report germline targeting of the endogenous chicken Interferon Alpha and Beta Receptor Subunit 1 (IFNAR1) gene by in vivo transgenic expression of the high-fidelity Cas9 (Cas9-HF1) and guide RNAs (gRNAs) in chickens. First, we developed a Tol2 transposon vector carrying Cas9-HF1, IFNAR1-gRNAs (IF-gRNAs) and green fluorescent protein (GFP) transgenes (pTgRCG) and validated in chicken fibroblast DF1 cells. Next, the pTgRCG plasmid was directly injected into the dorsal aorta of embryonic day (ED) 2.5 chicken embryos targeting the circulating primordial germ cells (PGCs). The resulting chimera roosters generated a fully transgenic generation 1 (G1) hen with constitutive expression of Cas9-HF1 and IF-gRNAs (G1_Tol2-Cas9/IF-gRNA). We detected a spectrum of indels at gRNA-targeted loci in the G1_Tol2-Cas9/IF-gRNA hen and the indels were stably inherited by the G2 progeny. Breeding of the G1_Tol2-Cas9/IF-gRNA hen resulted in up to 10% transgene-free heterozygote IFNAR1 mutants, following null-segregation of the Tol2 insert. The method described here will provide new opportunities for genome editing in chicken and other avian species that lack PGC culture.

Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells.

Exogenous RNA polymerase III (pol III) promoters are commonly used to express short hairpin RNA (shRNA). Previous studies have indicated that expression of shRNAs using standard pol III promoters can cause toxicity in vivo due to saturation of the native miRNA pathway. A potential way of mitigating shRNA-associated toxicity is by utilising native miRNA processing enzymes to attain tolerable shRNA expression levels. Here, we examined parallel processing of exogenous shRNAs by harnessing the natural miRNA processing enzymes and positioning a shRNA adjacent to microRNA107 (miR107), located in the intron 5 of the Pantothenate Kinase 1 (PANK1) gene. We developed a vector encoding the PANK1 intron containing miR107 and examined the expression of a single shRNA or multiple shRNAs. Using qRT-PCR analysis and luciferase assay-based knockdown assay, we confirmed that miR30-structured shRNAs have resulted in the highest expression and subsequent transcript knockdown. Next, we injected Hamburger and Hamilton stage 14-15 chicken embryos with a vector encoding multiple shRNAs and confirmed that the parallel processing was not toxic. Taken together, this data provides a novel strategy to harness the native miRNA processing pathways for shRNA expression. This enables new opportunities for RNAi based applications in animal species such as chickens.

Marker counter-selection via CRISPR/Cas9 co-targeting for efficient generation of genome edited avian cell lines and germ cells.

Efficient isolation of genetically modified cells that are phenotypically indistinguishable from the unmodified cells remains a major technical barrier for the broader utilization of CRISPR/Cas9. Here, we report a novel enrichment approach to select the genome engineered cells by co-targeting a genomically integrated GFP gene along with the endogenous gene of interest (GOI). Using this co-targeting approach, multiple genomic loci were successfully targeted in chicken (DF1) and quail (CEC-32) fibroblast cell lines by transient transfection of Cas9 and guide RNAs (gRNAs). Clonal isolation of co-targeted DF1 cells showed 75% of cell clones had deletion of GFP and biallelic deletion of the GOI. To assess the utility of this approach to generate genome modified animals, we tested it on chicken primordial germ cells (PGCs) expressing GFP by co-targeting with gRNAs against GFP and endogenous ovomucoid (OVM) gene. PGCs enriched for loss of GFP and confirmed for OVM deletion, derived by co-targeting, were injected into Hamburger and Hamilton stage 14-15 chicken embryos, and their ability to migrate to the genital ridge was confirmed. This simple, efficient enrichment approach could easily be applied to the creation of knock-out or edited cell lines or animals.

In Vivo Inhibition of Marek's Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4.

Marek's disease (MD), caused by MD herpesvirus (MDV), is an economically important disease in chickens. The efficacy of the existing vaccines against evolving virulent stains may become limited and necessitates the development of novel antiviral strategies to protect poultry from MDV strains with increased virulence. The CRISPR/Cas9 system has emerged as a powerful genome editing tool providing an opportunity to develop antiviral strategies for the control of MDV infection. Here, we characterized Tol2 transposon constructs encoding Cas9 and guide RNAs (gRNAs) specific to the immediate early infected-cell polypeptide-4 (ICP4) of MDV. We generated transgenic chickens that constitutively express Cas9 and ICP4-gRNAs (gICP4) and challenged them via intraabdominal injection of MDV-1 Woodlands strain passage-19 (p19). Transgenic chickens expressing both gRNA/Cas9 had a significantly reduced replication of MDV in comparison to either transgenic Cas9-only or the wild-type (WT) chickens. We further confirmed that the designed gRNAs exhibited sequence-specific virus interference in transgenic chicken embryo fibroblast (CEF) expressing Cas9/gICP4 when infected with MDV but not with herpesvirus of turkeys (HVT). These results suggest that CRISPR/Cas9 can be used as an antiviral approach to control MDV infection in chickens, allowing HVT to be used as a vector for recombinant vaccines.

Potential benefits of gene editing for the future of poultry farming.

The chicken is an exemplar of efficient intensive animal agriculture and provides two valuable food products, chicken meat and eggs. Only aquaculture is better, by efficiency, but poultry is still top, by mass of animal protein produced as food in the global context. However this efficiency and intensive production comes with a number of challenges. Though the genetics of selective breeding have led to dramatic improvements in yield, efficiency and product quality, traits that relate to disease and welfare outcomes have not been so tractable. These two issues are major impacts to the industry in terms of production and in terms of public perception. Both transgenic technology and genome editing have clear potential for impact in these two important areas. The reproductive biology of birds requires techniques very specific to birds to achieve heritable (germline) edited traits. These are quite involved and, even though they are now well-defined and reliable, there is room for improvement and advances can be expected in the future. Currently the key targets for this technology are modifying chicken genes involved in virus-receptor interactions and cellular response involved in infection. For the egg industry the technology is being applied to the issue of sex-selection for layer hens (and the removal of males), removal of allergens from egg white and the tailoring of eggs system to enhance the yield of influenza vaccine doses. Regulation and trading of the animals generated, and resulting food products, will significantly impact the value and future development of genome editing for poultry.

Overexpressing ovotransferrin and avian ß-defensin-3 improves antimicrobial capacity of chickens and poultry products.

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian ß-defensin-3 (AvßD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvßD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

Innovative approaches to genome editing in avian species.

The tools available for genome engineering have significantly improved over the last 5 years, allowing scientist to make precise edits to the genome. Along with the development of these new genome editing tools has come advancements in technologies used to deliver them. In mammals genome engineering tools are typically delivered into in vitro fertilized single cell embryos which are subsequently cultured and then implanted into a recipient animal. In avian species this is not possible, so other methods have been developed for genome engineering in birds. The most common involves in vitro culturing of primordial germ cells (PGCs), which are cells that migrate through the embryonic circulatory system to the developing gonad and colonize the gonad, eventually differentiating into the gonadocytes which produce either sperm or ova. While in culture the PGCs can be modified to carry novel transgenes or gene edits, the population can be screened and enriched, and then transferred into a recipient embryo. The largest drawback of PGC culture is that culture methods do not transfer well across avian species, thus there are reliable culture methods for only a few species including the chicken. Two newer technologies that appear to be more easily adapted in a wider range of avian species are direct injection and sperm transfection assisted gene editing (STAGE). The direct injection method involves injecting genome engineering tools into the circulatory system of the developing embryo just prior to the developmental time point when the PGCs are migrating to the gonads. The genome engineering tools are complexed with transfection reagents, allowing for in vivo transfection of the PGCs. STAGE utilizes sperm transfection to deliver genome engineering tools directly to the newly fertilized embryo. Preliminary evidence indicates that both methodologies have the potential to be adapted for use in birds species other than the chicken, however further work is needed in this area.

Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).

Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.

Comparing Gene Silencing and Physiochemical Properties in siRNA Bound Cationic Star-Polymer Complexes.

The translation of siRNA into clinical therapies has been significantly delayed by issues surrounding the delivery of naked siRNA to target cells. Here we investigate siRNA delivery by cationic acrylic polymers developed by Reversible Addition-Fragmentation chain Transfer (RAFT) mediated free radical polymerization. We investigated cell uptake and gene silencing of a series of siRNA-star polymer complexes both in the presence and absence of a protein "corona". Using a multidisciplinary approach including quantitative nanoscale mechanical-atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis we have characterized the nanoscale morphology, stiffness, and surface charge of the complexes with and without the protein corona. This is one of the first examples of a comprehensive physiochemical analysis of siRNA-polymer complexes being performed alongside in vitro biological assays, allowing us to describe a set of desirable physical features of cationic polymer complexes that promote gene silencing. Multifaceted studies such as this will improve our understanding of structure-function relationships in nanotherapeutics, facilitating the rational design of polymer-mediated siRNA delivery systems for novel treatment strategies.

Advances in genetic engineering of the avian genome: "Realising the promise".

This review provides an historic perspective of the key steps from those reported at the 1st Transgenic Animal Research Conference in 1997 through to the very latest developments in avian transgenesis. Eighteen years later, on the occasion of the 10th conference in this series, we have seen breakthrough advances in the use of viral vectors and transposons to transform the germline via the direct manipulation of the chicken embryo, through to the establishment of PGC cultures allowing in vitro modification, expansion into populations to analyse the genetic modifications and then injection of these cells into embryos to create germline chimeras. We have now reached an unprecedented time in the history of chicken transgenic research where we have the technology to introduce precise, targeted modifications into the chicken genome, ranging from; new transgenes that provide improved phenotypes such as increased resilience to economically important diseases; the targeted disruption of immunoglobulin genes and replacement with human sequences to generate transgenic chickens that express "humanised" antibodies for biopharming; and the deletion of specific nucleotides to generate targeted gene knockout chickens for functional genomics. The impact of these advances is set to be realised through applications in chickens, and other bird species as models in scientific research, for novel biotechnology and to protect and improve agricultural productivity.

Characterisation of novel microRNAs in the Black flying fox (Pteropus alecto) by deep sequencing.

BACKGROUND: Bats are a major source of new and emerging viral diseases. Despite the fact that bats carry and shed highly pathogenic viruses including Ebola, Nipah and SARS, they rarely display clinical symptoms of infection. Host factors influencing viral replication are poorly understood in bats and are likely to include both pre- and post-transcriptional regulatory mechanisms. MicroRNAs are a major mechanism of post-transcriptional gene regulation, however very little is known about them in bats. RESULTS: This study describes 399 microRNAs identified by deep sequencing of small RNA isolated from tissues of the Black flying fox, Pteropus alecto, a confirmed natural reservoir of the human pathogens Hendra virus and Australian bat lyssavirus. Of the microRNAs identified, more than 100 are unique amongst vertebrates, including a subset containing mutations in critical seed regions. Clusters of rapidly-evolving microRNAs were identified, as well as microRNAs predicted to target genes involved in antiviral immunity, the DNA damage response, apoptosis and autophagy. Closer inspection of the predicted targets for several highly supported novel miRNA candidates suggests putative roles in host-virus interaction. CONCLUSIONS: MicroRNAs are likely to play major roles in regulating virus-host interaction in bats, via dampening of inflammatory responses (limiting the effects of immunopathology), and directly limiting the extent of viral replication, either through restricting the availability of essential factors or by controlling apoptosis. Characterisation of the bat microRNA repertoire is an essential step towards understanding transcriptional regulation during viral infection, and will assist in the identification of mechanisms that enable bats to act as natural virus reservoirs. This in turn will facilitate the development of antiviral strategies for use in humans and other species.

Inhibition of influenza virus in vivo by siRNA delivered using ABA triblock copolymer synthesized by reversible addition-fragmentation chain-transfer polymerization.

AIM: Influenza virus remains a major threat, with outbreaks continuing to occur. Few treatment options are available and drug resistance can emerge rapidly. New drugs that can quickly be adapted to virus mutations are needed. Several highly effective siRNAs targeting influenza that inhibit virus replication are known; however, effective delivery of these siRNAs remains a challenge. The aim of this study was to demonstrate the safety and efficacy of ABA triblock copolymer-delivered siRNA to inhibit influenza virus replication in vivo. MATERIALS & METHODS: We report on the delivery of a siRNA targeting the influenza virus in chicken embryos using an ABA triblock copolymer prepared by reversible addition-fragmentation chain-transfer polymerization, containing a central cationic block and two outer hydrophilic polyethylene glycol blocks. RESULTS: A significant reduction of virus titer was observed with the polymer/anti-influenza siRNA complexes, whereas the control with polymer/control siRNA complexes showed no effect. CONCLUSION: These data suggest that a reversible addition-fragmentation chain transfer-based siRNA delivery platform may be suitable for combating infectious diseases in vivo.

Promotion of Hendra virus replication by microRNA 146a.

Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease.

miRNA_Targets: a database for miRNA target predictions in coding and non-coding regions of mRNAs.

MicroRNAs (miRNAs) are small non-coding RNAs that play a role in post-transcriptional regulation of gene expression in most eukaryotes. They help in fine-tuning gene expression by targeting messenger RNAs (mRNA). The interactions of miRNAs and mRNAs are sequence specific and computational tools have been developed to predict miRNA target sites on mRNAs, but miRNA research has been mainly focused on target sites within 3' untranslated regions (UTRs) of genes. There is a need for an easily accessible repository of genome wide full length mRNA - miRNA target predictions with versatile search capabilities and visualization tools. We have created a web accessible database of miRNA target predictions for human, mouse, cow, chicken, Zebra fish, fruit fly and Caenorhabditis elegans using two different target prediction algorithms, The database has target predictions for miRNA's on 5' UTRs, coding region and 3' UTRs of all mRNAs. This database can be freely accessed at http://mamsap.it.deakin.edu.au/mirna_targets/.

The effect of RAFT-derived cationic block copolymer structure on gene silencing efficiency.

In this work a series of ABA tri-block copolymers was prepared from oligo(ethylene glycol) methyl ether methacrylate (OEGMA(475)) and N,N-dimethylaminoethyl methacrylate (DMAEMA) to investigate the effect of polymer composition on cell viability, siRNA uptake, serum stability and gene silencing. Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization was used as the method of polymer synthesis as this technique allows the preparation of well-defined block copolymers with low polydispersity. Eight block copolymers were prepared by systematically varying the central cationic block (DMAEMA) length from 38 to 192 monomer units and the outer hydrophilic block (OEGMA(475)) from 7 to 69 units. The polymers were characterized using size exclusion chromatography and (1)H NMR. Chinese Hamster Ovary-GFP and Human Embryonic Kidney 293 cells were used to assay cell viability while the efficiency of block copolymers to complex with siRNA was evaluated by agarose gel electrophoresis. The ability of the polymer-siRNA complexes to enter into cells and to silence the targeted reporter gene enhanced green fluorescent protein (EGFP) was measured by using a CHO-GFP silencing assay. The length of the central cationic block appears to be the key structural parameter that has a significant effect on cell viability and gene silencing efficiency with block lengths of 110-120 monomer units being the optimum. The ABA block copolymer architecture is also critical with the outer hydrophilic blocks contributing to serum stability and overall efficiency of the polymer as a delivery system.

Manipulation of estrogen synthesis alters MIR202* expression in embryonic chicken gonads.

Tissue-specific patterns of microRNA (miRNA) expression contribute to organogenesis during embryonic development. Using the embryonic chicken gonads as a model for vertebrate gonadogenesis, we previously reported that miRNAs are expressed in a sexually dimorphic manner during gonadal sex differentiation. Being male biased, we hypothesised that up-regulation of microRNA 202* (MIR202*) is characteristic of testicular differentiation. To address this hypothesis, we used estrogen modulation to induce gonadal sex reversal in embryonic chicken gonads and analyzed changes in MIR202* expression. In ovo injection of estradiol-17beta at Embryonic Day 4.5 (E4.5) caused feminization of male gonads at E9.5 and reduced MIR202* expression to female levels. Female gonads treated at E3.5 with an aromatase inhibitor, which blocks estrogen synthesis, were masculinized by E9.5, and MIR202* expression was increased. Reduced MIR202* expression correlated with reduced expression of the testis-associated genes DMRT1 and SOX9, and up-regulation of ovary-associated genes FOXL2 and CYP19A1 (aromatase). Increased MIR202* expression correlated with down-regulation of FOXL2 and aromatase and up-regulation of DMRT1 and SOX9. These results confirm that up-regulation of MIR202* coincides with testicular differentiation in embryonic chicken gonads.

Sexually dimorphic microRNA expression during chicken embryonic gonadal development.

MicroRNAs are a highly conserved class of small RNAs that function in a sequence-specific manner to posttranscriptionally regulate gene expression. Tissue-specific miRNA expression studies have discovered numerous functions for miRNAs in various aspects of embryogenesis, but a role for miRNAs in gonadal development and sex differentiation has not yet been reported. Using the chicken embryo as a model, microarrays were used to profile the expression of chicken miRNAs prior to, during, and after the time of gonadal sex differentiation (Embryonic Day 5.5 [E5.5], E6.5, and E9.5). Sexually dimorphic miRNAs were identified, and the expression patterns of several were subjected to further validation by in situ hybridization and Northern blot analysis. Expression of one chicken miRNA, MIR202*, was observed to be sexually dimorphic, with upregulation in the developing testis from the onset of sexual differentiation. Additional data from deep sequencing of male and female embryonic gonad RNA samples also indicated upregulation of MIR202* in male gonads. These findings provide the first evidence of sexually dimorphic miRNA expression during vertebrate gonadal sex differentiation and suggest that MIR202* may function in regulating testicular development.

A microRNA catalog of the developing chicken embryo identified by a deep sequencing approach.

MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small regulatory RNAs characterized in chickens we used a deep sequencing approach developed by Solexa (now Illumina Inc.). We sequenced three small RNA libraries prepared from different developmental stages of the chicken embryo (days five, seven, and nine) to produce over 9.5 million short sequence reads. We developed a bioinformatics pipeline to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected almost all of the previously known chicken miRNAs and their respective miRNA* sequences. In addition we discovered 449 new chicken miRNAs including 88 miRNA candidates. Of these, 430 miRNAs appear to be specific to the avian lineage. Another six new miRNAs had evidence of evolutionary conservation in at least one vertebrate species outside of the bird lineage. The remaining 13 putative miRNAs appear to represent chicken orthologs of known vertebrate miRNAs. We discovered 39 additional putative miRNA candidates originating from miRNA generating intronic sequences known as mirtrons.

Genomic comparison of Mycobacterium avium subsp. paratuberculosis sheep and cattle strains by microarray hybridization.

Microarray-based comparisons of three Mycobacterium avium subsp. paratuberculosis isolates, including one sheep strain and two cattle strains, identified three large genomic deletions in the sheep strain, totaling 29,208 bp and involving 24 open reading frames. These deletions may help explain some of the differences in pathogenicity and host specificity observed between the cattle and sheep strains of Mycobacterium avium subsp. paratuberculosis.

Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA.

RNA has been extracted and analysed from in vivo-derived Mycobacterium avium subspecies paratuberculosis recovered from the natural host. The bacteria were selectively extracted from the intestinal tissue of two goats exhibiting clinical signs of Johne's disease. Small intestine was rapidly removed, luminal contents washed away and the mucosa and submucosa harvested. Mycobacteria in this material were released from the macrophages by isotonic lysis and differential centrifugation. RNA was extracted and compared with RNA extracted from bacteria grown in vitro. Real-time polymerase chain reaction was used to analyse the katG gene from the bacterial messenger RNA. The katG mRNA encoding the putative catalase/peroxidase showed differential expression in the in vivo and in vitro-derived samples. We hypothesize that the increase in katG expression for in vivo-derived M. paratuberculosis may represent a response to the oxidative stress encountered within the intra-macrophage environment.