Genome-edited rabbits:Unleashing the potential of a promising experimental animal model across diverse diseases

Animal models are extensively used in all aspects of biomedical research,with substantial contributions to our understanding of diseases,the development of pharmaceuticals,and the exploration of gene functions.The field of genome modification in rabbits has progressed slowly.However,recent advancements,particularly in CRISPR/Cas9-related technologies,have catalyzed the successful development of various genome-edited rabbit models to mimic diverse diseases,including cardiovascular disorders,immunodeficiencies,agingrelated ailments,neurological diseases,and ophthalmic pathologies.These models hold great promise in advancing biomedical research due to their closer physiological and biochemical resemblance to humans compared to mice.This review aims to summarize the novel gene-editing approaches currently available for rabbits and present the applications and prospects of such models in biomedicine,underscoring their impact and future potential in translational medicine.

Genetic improvement of fiber quality in tetraploid cotton:an overview of major QTLs and genes involved in and edited for the quality of cotton fibers

Cotton,an important industrial crop cultivated in more than 70 countries,plays a major role in the livelihood of millions of farmers and industrialists.Cotton is mainly grown for its fiber,an economic component that can be differentiated from its epidermal cells in the outer integument of a developing seed.Fiber length,fiber strength,and fiber fineness are three main attributes that contribute to the quality of cotton fibers.Recent advancements in genomics have identified key genes,which are the most important factors that govern these three traits,can be introduced into cultivars of interest via gene editing,marker-assisted selection,and transgenics,thus the narrow genetic background of cotton can be addressed and its fiber quality traits can be enhanced.Over the past two decades,quantitative trait loci(QTLs)have been mapped for different fiber traits,approximately 1850 QTLs have been mapped for fiber length,fiber strength,and fineness among which a few genes have been edited for quality improvement in cotton.In this background,the current review covers the development and the factors that influence these traits,along with the reported genes,QTLs,and the edited genomes for trait improvement.

Liver Transplantation (in Chinese), revised edition, Edited by Shu-Sen Zheng. Pp843. Beijing: People's Medical Publishing House, 2012. ISBN: 978-7-117-15494-9/R.15495. ￥RMB 158

Liver Transplantation (in Chinese), revised edition, edited by Professor Shu-Sen Zheng and prefaced by Professor Guo-Wei Sang, has just been published. The revised edition is mainly based on the clinical practice of the Liver Transplant Center, the First Affiliated Hospital, Zhejiang University and the experience

EXPLORATION OF THE LATENT RELATION BETWEEN CONTENT AND STYLE THROUGH JOURNALS ON THE TEXTS FROM BOOK~⑧ EDITED BY HUANG YUANSHEN ZHU ZHONGYI

Writing style is the essential issue even at the early stage the beginners who learnto read and write have to confront.From the part-Notes on reading and writing beforethe part of exercises of each lesson in English Book V-VⅢ we can come to see that the ed-itors attempt to mix the content(ideas)with the corresponding techniques.This is

Wheat genome editing expedited by efficient transformation techniques:Progress and perspectives

Genome editing is one of the most promising biotechnologies to improve crop performance.Common wheat is a staple food for mankind. In the past few decades both basic and applied research on common wheat has lagged behind other crop species due to its complex,polyploid genome and difficulties in genetic transformation. Recent breakthroughs in wheat transformation permit a revolution in wheat biotechnology. In this review, we summarize recent progress in wheat genetic transformation and its potential for wheat improvement. We then review recent progress in plant genome editing, which is now readily available in wheat. We also discuss measures to further increase transformation efficiency and potential applications of genome editing in wheat. We propose that, together with a high quality reference genome, the time for efficient genetic engineering and functionality studies in common wheat has arrived.

Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR(TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35 S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.

In silico Prediction and Analysis of Potential Off-Targets and Off-Target Mutation Detection in StERF3-Gene Edited Potato Plants

The imperative aspect of the CRISPR/Cas9 system is a short stretch of 20 nucleotides of gRNA that control the overall specificity.Due to the small size,the chance of its multiple occurrences in the genome increases;however,a few mismatches are tolerated by the Cas9 endonuclease activity.An accurate and careful in silico-based off-target prediction while target selection is preferred to address the issue.These predictions are based on a comprehensive set of selectable parameters.Therefore,we investigated the possible off-target prediction and their screening in StERF3 gene-edited potato plants while developing StERF3-loss-of-function mutants using CRISPR/Cas9 approach.The 201 off-targets for the selected targets of the StERF3 gene were predicted,and 79 werefiltered as potential off-targets.Of these 79,twenty-five off-targets showed scores with defined cut-off values<0.5 and were analyzed in Sterf3-edited potato plants compared to wild-type plants.No off-targeting was found to have occurred in edited plants.

Regulation of gene-edited plants in Europe:from the valley of tears into the shining sun?

Some 20 years ago,the EU introduced complex regulatory rules for the growth of transgenic crops,which resulted in a de facto ban to grow these plants in fields within most European countries.With the rise of novel genome editing technologies,it has become possible to improve crops genetically in a directed way without the need for incorporation of foreign genes.Unfortunately,in 2018,the European Court of Justice ruled that such gene-edited plants are to be regulated like transgenic plants.Since then,European scientists and breeders have challenged this decision and requested a revision of this outdated law.Finally,after 5 years,the European Commission has now published a proposal on how,in the future,to regulate crops produced by new breeding technologies.The proposal tries to find a balance between the different interest groups in Europe.On one side,genetically modified plants,which cannot be discerned from their natural counterparts,will exclusively be used for food and feed and are-besides a registration step-not to be regulated at all.On the other side,plants expressing herbicide resistance are to be excluded from this regulation,a concession to the strong environmental associations and NGOs in Europe.Moreover,edited crops are to be excluded from organic farming to protect the business interests of the strong organic sector in Europe.Nevertheless,if this law passes European parliament and council,unchanged,it will present a big step forward toward establishing a more sustainable European agricultural system.Thus,it might soon be possible to develop and grow crops that are more adapted to global warming and whose cultivation will require lower amounts of pesticides.However,there is still a long way to go until the law is passed.Too often,the storm of arguments raised by the opponents,based on irrational fears of mutations and a naive understanding of nature,has fallen on fruitful ground in Europe.

Long-term,multidomain analyses to identify the breed and allelic effects in MSTN-edited pigs to overcome lameness and sustainably improve nutritional meat production

Beef and mutton production has been aided by breeding to integrate allelic diversity for myostatin(MSTN),but a lack of diversity in the MSTN germplasm has limited similar advances in pig farming.Moreover,insurmountable challenges with congenital lameness and a dearth of data about the impacts of feed conversion,reproduction,and meat quality in MSTN-edited pigs have also currently blocked progress.Here,in a largest-to-date evaluation of multiple MSTN-edited pig populations,we demonstrated a practical alternative edit-site-based solution that overcomes the major production obstacle of hindlimb weakness.We also provide long-term and multidomain datasets for multiple breeds that illustrate how MSTN-editing can sustainably increase the yields of breed-specific lean meat and the levels of desirable lipids without deleteriously affecting feed-conversion rates or litter size.Apart from establishing a new benchmark for the data scale and quality of genome-edited animal production,our study specifically illustrates how gene-editing site selection profoundly impacts the phenotypic outcomes in diverse genetic back-grounds.

Plant Genome Editing Database (PGED): A Call for Submission of Information about Genome-Edited Plant Mutants

Recent advances in genome editing technologies, particularly CRISPR/Cas, enable the alteration of DNA sequences to produce deletions, insertions, and substitutions in genes (Jaganathan et al., 2018), as well as large or entire chromosome deletions in the genomes of plants and animals (Zhou et al., 2014;Adikusuma et al., 2017).

Book Review:Longhorned Woodboring Beetles(Coleoptera:Cerambycidae and Disteniidae):Primary Types of the Smithsonian Institution(edited by S.W.Lingafelter et al.,Smithsonian Institution Scholarly Press,Washington D.C.,USA.2014,390 pages)

This book was written by five authors. I had met two of them, the first and third author. The first author, Steven Wayne Lingafelter, is a research entomologist with the Systematic Entomology Laboratory, USDA, based at the Smithsonian Institution＇s National Museum of Natural History. He has specialized on longhomed woodboring beetles for almost three decades and currently specializes on the Neotropical fauna.

Radiation and Water in the Climate System:Remote Measurements——Edited by Ehrhard Raschke,Springer-Verlag Berlin Heidelberg,Germany,1996 614pp.,ISBN 3-540-61470-2

This book is the proceedings of the NATO Advanced Science Institute“RemoteSensing of Processes Governing Energy and Water Cycles in the Climate System”,held inPln,Germany 1—12 May 1995.In this book,a total of 21 conference papers are includedwith a special part for poster summaries.All the papers are modified or shorten versions

Surrogate production of genome-edited sperm from a different subfamily by spermatogonial stem cell transplantation

The surrogate reproduction technique,such as inter-specific spermatogonial stem cells(SSCs)transplantation(SSCT),provides a powerful tool for production of gametes derived from endangered species or those with desirable traits.However,generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily,by SSCT,has not succeeded.Here,using two small cyprinid fishes from different subfamilies,Chinese rare minnow(gobiocypris rarus,for brief:Gr)and zebrafish(danio rerio),we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure.The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis,resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells.Interestingly,the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior.When pou5f3 and chd knockout Gr SSCs were transplanted,Gr-derived genome-edited sperm was successfully produced in zebrafish.This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT,and surrogate production of genome-edited gametes from another species as well.This method is feasible to be applied to future breeding of commercial fish and livestock.

Experiments that led to the first gene-edited babies: the ethical failings and the urgent need for better governance

The rapid developments of science and technology in China over recent decades, particularly in biomedical research, have brought forward serious challenges regarding ethical governance. Recently, Jian-kui HE, a Chinese scientist, claimed to have "created" the first gene-edited babies, designed to be naturally immune to the human immunodeficiency virus(HIV). The news immediately triggered widespread criticism, denouncement, and debate over the scientific and ethical legitimacy of HE’s genetic experiments. China’s guidelines and regulations have banned germline genome editing on human embryos for clinical use because of scientific and ethical concerns, in accordance with the international consensus. HE’s human experimentation has not only violated these Chinese regulations, but also breached other ethical and regulatory norms. These include questionable scientific value, unreasonable risk-benefit ratio, illegitimate ethics review, invalid informed consent, and regulatory misconduct. This series of ethical failings of HE and his team reveal the institutional failure of the current ethics governance system which largely depends on scientist’s self-regulation. The incident highlights the need for urgent improvement of ethics governance at all levels, the enforcement of technical and ethical guidelines, and the establishment of laws relating to such bioethical issues.

Technological breakthroughs in generating transgene-free and genetically stable CRISPR-edited plants

CRISPR/Cas9 gene-editing technologies have been very effective in editing target genes in all major crop plants and offer unprecedented potentials in crop improvement.A major challenge in using CRISPR gene-editing technology for agricultural applications is that the target gene-edited crop plants need to be transgene free to maintain trait stability and to gain regulatory approval for commercial production.In this article,we present various strategies for generating transgene-free and target geneedited crop plants.The CRISPR transgenes can be removed by genetic segregation if the crop plants are reproduced sexually.Marker-assisted tracking and eliminating transgenes greatly decrease the time and labor needed for identifying the ideal transgene-free plants.Transgenes can be programed to undergo self-elimination when CRISPR genes and suicide genes are sequentially activated,greatly accelerating the isolation of transgene-free and target gene-edited plants.Transgene-free plants can also be generated using approaches that are considered non-transgenic such as ribonucleoprotein transfection,transient expression of transgenes without DNA integration,and nano-biotechnology.Here,we discuss the advantages and disadvantages of the various strategies in generating transgene-free plants and provide guidance for adopting the best strategies in editing a crop plant.

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.

Reflections on the system of evaluation of gene-edited livestock

The rapid development of biotechnology has provided a greater understanding of the biological functions of major candidate genes that have important functions regarding economic traits,and new materials for livestock breeding have been obtained through gene editing(GE)and embryo manipulation with the purpose of improving quality and output and reducing the costs and risk of disease.Public concerns,particularly over safety risks and production performance,must be addressed.Evaluation is the most important component of the regulation of gene-edited livestock and is a crucial guarantee of public safety before the marketing of geneedited animal products.Here,the system of evaluation of gene-edited livestock is discussed in terms of public safety and production performance.The search for safe and ethical applications in the GE of livestock,a case-by-case evaluation strategy,and classification and simplification are used in order to promote a more efficient,objective,comprehensive and operable evaluation system.

Perspectives on the management of synthetic biological and gene edited foods

Synthetic biology,gene editing and transgene technologies that involving gene manipulation are widely used in food research and development.In recent decades,China has formulatedmany policies and regulations to govern the safety of genetically modified agricultural organisms.Based on the comparative analysis of technical connotations on synthetic biology,gene editing and transgenes and related risksregarding the safety of food,environment and the management of genetically modified agricultural organisms,this paper puts forward the following suggestions on 1)management of synthetic biological and gene-edited foods(in this paper,“food”refers to“edible agricultural products and food”),2)promoting the gradual process of industrialization on gene-edited and synthetic biological foods through lessons learned from the practice on genetically modified crops,3)speeding up the industrialization of gene-edited foods and prudently promoting the application of synthetic biological foods,4)urgently establishing and updating policies and regulations related to gene-edited and synthetic biological foods,5)unifying the legislation and supervision of synthetic biological,gene-edited and genetically modified foods 6)establishing ethical principles and bottom lines for genetically manipulated foods.

Genome-edited crops: how to move them from laboratory to market

Recent breakthroughs in CRISPR technology allow specific genome manipulation of almost all crops and have initiated a revolution in precision crop breeding.Rationally-based regulation and widespread public acceptance are needed to propel genome-edited crops from laboratory to market and to translate this innovative technology into agricultural productivity.

Elimination of GGTA1,CMAH,β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

Background:Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic,while immunogenic genes need to be eliminated to improve the immune compatibility between humans and pigs.Current knockout strategies are mainly aimed at the genes causing hyperacute immune rejection(HAR)that occurs in the first few hours while adaptive immune reactions orchestrated by CD4 T cell thereafter also cause graft failure,in which process the MHCⅡmolecule plays critical roles.Methods:Thus,we generate a 4-gene(GGTA1,CMAH,β4GalNT2,and CIITA)knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously.Results:We successfully obtained 4KO piglets with deficiency in all alleles of genes,and at cellular and tissue levels.Additionally,the safety of our animals after gene editing was verified by using whole-genome sequencing and karyotyping.Piglets have survived for more than one year in the barrier,and also survived for more than 3 months in the conventional environment,suggesting that the piglets without MHCⅡcan be raised in the barrier and then gradually mated in the conventional environment.Conclusions:4KO piglets have lower immunogenicity,are safe in genomic level,and are easier to breed than the model with both MHCⅠandⅡdeletion.