CRISPR/Cas9 gene editing in a chicken model: current approaches and applications

Improvements in genome editing technology in birds using primordial germ cells (PGCs) have made the development of innovative era genome-edited avian models possible, including specific chicken...     

植物基因组编辑检测方法

以CRISPR/Cas9技术为代表的基因组编辑在生物领域的革命性应用使得生命科学研究迈入新篇章。该技术以其灵活性、易用性且扩展性强等优势,大大加快了基因工程研究,也加速了植物分子育种的步伐。但是,遗传转化过程中产生大量潜在的基因编辑植株,使得早期高通量快速筛选和检测目标编辑植株面临很大挑战。本文综述了近年来植物基因组编辑检测的各种方法,比较了其优缺点和适用范围;同时,还对近几年植物基因组编辑检测方法的发展趋势进行了深入分析和展望,以期对基因组编辑技术在植物中的应用提供参考。     

Germline genome editing versus preimplantation genetic diagnosis: Is there a case in favour of germline interventions?

CRISPR is widely considered to be a disruptive technology. However, when it comes to the most controversial topic, germline genome editing (GGE), there is no consensus on whether this technology has any substantial advantages over existing procedures such as embryo selection after in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD). Answering this question, however, is crucial for evaluating whether the pursuit of further research and development on GGE is justified. This paper explores the question from both a clinical and a moral viewpoint, namely whether GGE has any advantages over existing technologies of selective reproduction and whether GGE could complement or even replace them. In a first step, I review an argument of extended applicability. The paper confirms that there are some scenarios in which only germline intervention allows couples to have (biologically related) healthy offspring, because selection will not avoid disease. In a second step, I examine possible moral arguments in favour of genetic modification, namely that GGE could save some embryos and that GGE would provide certain benefits for a future person that PGD does not. Both arguments for GGE have limitations. With regard to the extended applicability of GGE, however, a weak case in favour of GGE should still be made.     

Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos

This study reports for the first time the production of chicken germline chimeras by transfer of embryonic germ (EG) cells into recipient embryos of different strain. EG cells were established by the subculture of gonadal tissue cells retrieved from stage 28 White Leghorn (WL) embryos with I/I gene. During primary culture (P(0)), gonadal primordial germ cells (gPGCs) in the stromal cells began to form colonies after 7 days in culture with significant (P < 0.0001) increase in cell population. Colonized gPGCs were then subcultured with chicken embryonic fibroblast monolayer for EG cell preparation. Prepared EG cells or gPGCs at P(0) were transferred to stage 17 Korean Ogol chicken (KOC) embryos with i/i gene. The recipient chickens were raised for 6 months to sexual maturity, then a testcross analysis by artificial insemination was conducted for evaluating germline chimerism. As results, transfer of EG cells and gPGCs yielded total 17 germline chimeras; 2 out of 15 (13.3%) and 15 of 176 sexually matured chickens (8.5%), respectively. The efficiency of germline transmission in the chimeras was 1.5-14.6% in EG cells, while 1.3-27.6% in gPGCs. In conclusion, chicken germline chimeras could be produced by the transfer of EG cells, as well as gPGCs, which might enormously contribute to establishing various innovative technologies in the field of avian transgenic research for bioreactor production.     

i-GONAD: A method for generating genome-edited animals without ex vivo handling of embryos

The recent development of genome editing technologies has enabled the creation of genome-edited animals, with alterations at the desired target locus. The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for this purpose because it is simpler and more efficient than other genome editing technologies. The conventional methods for creation of genome-edited animals involve ex vivo handling of embryos (zygotes) for microinjection or in vitro electroporation. However, this process is laborious and time-consuming, and relatively large numbers of animals are used. Furthermore, these methods require specialized skills for handling embryos. In 2015, we reported a novel method for the creation of genome-edited animals without ex vivo handling of embryos. The technology known as Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) involved intraoviductal instillation of genome editing components into a pregnant female and subsequent in vivo electroporation of an entire oviduct. The genome editing components present in the oviductal lumen are transferred to preimplantation embryos in situ for introducing insertion or deletion (indel) mutations at the desired loci. This technology was further improved by optimizing several parameters to develop improved GONAD (i-GONAD) for the efficient generation of mutant or knock-in animals. In this review, we discuss the historical background, potential applications, advantages, and future challenges of GONAD/i-GONAD technology.     

Applications of avian transgenesis

The ability to introduce foreign DNA into the genome of an organism has proven to be one of the most powerful tools in modern biology. Methods for the manipulation of the animal genome have been developed at an impressive pace for 3 decades, but only in the past 5 years have useful tools for avian transgenesis emerged. The most efficient technique involves the use of replication-deficient lentiviral vectors to deliver foreign DNA into the avian germline. Although lentiviral-mediated transgenesis presents some constraints, progress in this area has garnered interest in both industry and academia for its potential applications in biological research, biotechnology, and agriculture. In this review we evaluate methods for the production of transgenic birds, focusing on the advantages and limitations of lentiviral-mediated transgenesis. We also provide an overview of future applications of this technology. The most exciting of these include disease-resistant transgenic poultry, genetically modified hens that produce therapeutic proteins in their eggs, and transgenic songbirds that serve as a model to study communication disorders. Finally, we discuss technological advances that will be necessary to make avian transgenesis a more versatile tool.     

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.     

Male germline control of transposable elements

Repetitive sequences, especially transposon-derived interspersed repetitive elements, account for a large fraction of the genome in most eukaryotes. Despite the repetitive nature, these transposable elements display quantitative and qualitative differences even among species of the same lineage. Although transposable elements contribute greatly as a driving force to the biological diversity during evolution, they can induce embryonic lethality and genetic disorders as a result of insertional mutagenesis and genomic rearrangement. Temporary relaxation of the epigenetic control of retrotransposons during early germline development opens a risky window that can allow retrotransposons to escape from host constraints and to propagate abundantly in the host genome. Because germline mutations caused by retrotransposon activation are heritable and thus can be deleterious to the offspring, an adaptive strategy has evolved in host cells, especially in the germline. In this review, we will attempt to summarize general defense mechanisms deployed by the eukaryotic genome, with an emphasis on pathways utilized by the male germline to confer retrotransposon silencing.     

Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans.     

动物转基因新技术研究进展

动物转基因技术是21世纪发展最为迅速的生物高新技术之一, 它是指通过基因工程技术将外源基因整合到受体动物基因组中, 从而使其得以表达和遗传的生物技术。动物转基因的关键限制因素是转基因效率和基因表达的精确调控。目前有多种转基因技术, 每一种技术各有其优缺点, 仍然需要进一步研究。随着研究的深入, 转基因技术必将在探讨基因功能、动物遗传改良、生物反应器、动物疾病模型、器官移植等领域有广阔的应用前景。文章综述了近年发展的提高转基因效率的生殖干细胞法、提高转基因精确性的基因打靶法、RNA干扰(RNAi)介导的基因沉默技术和诱导多能干细胞(iPS)转基因技术。新的转基因技术为转基因动物的研究提供了更好的平台, 可以加快促进人类医药卫生、畜牧生产等领域的发展。     

Applications and potential of genome editing in crop improvement

Genome-editing tools provide advanced biotechnological techniques that enable the precise and efficient targeted modification of an organism’s genome. Genome-editing systems have been utilized in a wide variety of plant species to characterize gene functions and improve agricultural traits. We describe the current applications of genome editing in plants, focusing on its potential for crop improvement in terms of adaptation, resilience, and end-use. In addition, we review novel breakthroughs that are extending the potential of genome-edited crops and the possibilities of their commercialization. Future prospects for integrating this revolutionary technology with conventional and new-age crop breeding strategies are also discussed.     

A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals:A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis

The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species.Moreover,these novel tools have become easier to use and have resulted in a great increase of applications.Whilst gene knockout(KO) or knockin(KI) animal models are relatively easy to achieve,there is a bottleneck in the detection and analysis of these mutations.Although several methods exist to detect these targeted mutations,we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process.The HMA-CE method uses a simple PCR amplification of genomic DNA(gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes(HD) signature for each mutation.This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.     

Improving the efficiency for generation of genome-edited zebrafish by labeling primordial germ cells

Although CRISPR/Cas, a new versatile genome-editing tool, has been widely used in a variety of species including zebrafish, an important vertebrate model animal for biomedical research, the low efficiency of germline transmission of induced mutations and particularly knockin alleles made subsequent screening for heritable offspring tedious, time-consuming, expensive and at times impossible. In this study, we reported a method for improving the efficiency of germline transmission screening for generation of genome-edited zebrafish mutants. Co-microinjecting yfp-nanos3 mRNA with Cas9 mRNA, sgRNA and single strand DNA donor to label the distribution of microinjected nucleotides in PGCs (primordial germ cells), we demonstrated that founders carrying labeled PGCs produced much higher numbers of knockin and knockout progeny. In comparison with the common practice of selecting founders by genotyping fin clips, our new strategy of selecting founders with tentatively fluorescent-labeled PGCs significantly increase the ease and speed of generating heritable knocking and knockout animals with CRISPR/Cas9.     

Blurring the germline: Genome editing and transgenerational epigenetic inheritance

Sperm, eggs and embryos are made up of more than genes, and there are indications that changes to non-genetic structures in these elements of the germline can also be inherited. It is, therefore, a mistake to treat phrases like ‘germline inheritance’ and ‘genetic inheritance’ as simple synonyms, and bioethical discussion should expand its focus beyond alterations to the genome when considering the ethics of germline modification. Moreover, additional research on non-genetic inheritance draws attention to a variety of means whereby differences can be inherited in offspring generations that do not rely on differences in germline structures. Research on these diverse forms of inheritance challenges the notion that there is some special form of ethical concern that falls on germline interventions in general, and on interventions to the nuclear genome within the germline in particular.     

小RNAs在生殖细胞发育过程中的作用

很多动物可以产生具调节作用的小RNAs,根据产生方式和作用机制可以将它们分为三类:微小RNAs(miRNAs)、与Piwi相互作用的RNAs(piRNAs)和内源小干扰RNAs(endo-siRNAs),这些小RNAs可以在生物生殖细胞发育过程中发挥重要作用。其中miRNAs的主要作用是调节蛋白质基因的表达;piRNAs主要的作用是沉默转座因子,但piRNAs主要存在于生殖细胞中;endo-siRNAs则可能具有上述两种主要作用。该文论述了这三种小RNAs在生物生殖细胞发育过程中的作用,同时也讨论了它们在治疗生物不育及其在生物节育方面的应用前景。     

Mitochondrial DNA Purifying Selection in Mammals and Invertebrates

Numerous mitochondrial quality control mechanisms exist within cells, but none have been shown to effectively assess and control the quality of mitochondrial DNA (mtDNA). One reason such mechanisms have yet to be elucidated is that they do not appear to be particularly active in most somatic cells, where many studies are conducted. The female germline, the cell lineage that gives rise to eggs, appears to be an exception. In the germline, strong purifying selection pathways act to eliminate deleterious mtDNA. These pathways have apparently evolved to prevent pathogenic mtDNA mutations from accumulating over successive generations and causing a decline of species via Muller's ratchet. Despite their fundamental biological importance, the mechanisms underlying purifying selection remain poorly understood, with no genes involved in this process yet identified. In this review, we discuss recent studies exploring mechanisms of germline mtDNA purifying selection in both mammalian and invertebrate systems. We also discuss the challenges to future major advances. Understanding the molecular basis of purifying selection is not only a fundamental outstanding question in biology, but may also pave the way to controlling selection in somatic tissues, potentially leading to treatments for people suffering from mitochondrial diseases.     

Targeted genome engineering via zinc finger nucleases

With the development of next-generation sequencing technology, ever-expanding databases of genetic information from various organisms are available to researchers. However, our ability to study the biological meaning of genetic information and to apply our genetic knowledge to produce genetically modified crops and animals is limited, largely due to the lack of molecular tools to manipulate genomes. Recently, targeted cleavage of the genome using engineered DNA scissors called zinc finger nucleases (ZFNs) has successfully supported the precise manipulation of genetic information in various cells, animals, and plants. In this review, we will discuss the development and applications of ZFN technology for genome engineering and highlight recent reports on its use in plants.     

Rapid and efficient production of genome-edited animals by electroporation into oocytes injected with frozen or freeze-dried sperm

Sperm preservation is a useful technique for maintaining valuable animal strains. Rat sperm could be frozen or freeze-dried in a simple Tris-EDTA solution (TE buffer), and oocytes that were fertilized with these sperm by intracytoplasmic sperm injection (ICSI) developed into offspring. Genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system enables the rapid production of genetically modified rats. The recent innovative method, named the TAKE method, could easily produce genome edited rats by electroporation of endonucleases into embryos. Although various rat strains have been applied for genome editing, genome editing using strains that were preserved as sperm took longer because it required collecting embryos after maturation of animals regenerated from sperm. To reduce the production period, we directly electroporated Cas9 protein and gRNA into oocytes that were injected with frozen or freeze-dried sperm in TE buffer. No effect of electroporation until 30 V to ICSI-embryos derived from frozen or freeze-dried sperm were shown in the development of offspring. Furthermore, the rate of genome editing in offspring was high (56% for frozen and 50% for freeze-dried sperm). These results concluded that the combination of ICSI and the TAKE method was useful for the rapid production of genome-edited animals from sperm that have been preserved as genetic resources.