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.

A booming field of large animal model research

Animal models are integral to the study of fundamental biological processes and the etiology of human diseases.Small animal models,especially those involving mice,have yielded abundant and significant insights,greatly enhancing our understanding of biological phenomena and disease mechanisms.

In vivo evaluation of guide-free Cas9-induced safety risks in a pig model

The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy.However,there are concerns about the safety of this system,specifically related to the use of guide-free Cas9.Previous studies have shown that guidefree Cas9 can induce genomic instability in vitro.However,the in vivo safety risks associated with guide-free Cas9 have not been evaluated,which is necessary for the development of gene therapy in clinical settings.In this study,we used doxycycline-inducible Cas9-expressing pigs to evaluate the safety risks of guide-free Cas9 in vivo.Our findings demonstrated that expression of guide-free Cas9 could induce genomic damages and transcriptome changes in vivo.The severity of the genomic damages and transcriptome changes were correlate with the expression levels of Cas9 protein.Moreover,prolonged expression of Cas9 in pigs led to abnormal phenotypes,including a significant decrease in body weight,which may be attributable to genomic damage-induced nutritional absorption and metabolic dysfunction.Furthermore,we observed an increase in whole-genome and tumor driver gene mutations in pigs with long-term Cas9 expression,raising the risk of tumor occurrence.Our in vivo evaluation of guide-free Cas9 in pigs highlights the necessity of considering and monitoring the detrimental effects of Cas9 alone as genome editing via the CRISPR/Cas9 system is implemented in clinical gene therapy.This research emphasizes the importance of further study and implementation of safety measures to ensure the successful and safe application of the CRiSPR/Cas9 system in clinical practice.

2008： year of the rat for stem cell research

2008 breakthrough of the year went to reprogramming as announced by magazine Science recently , highlighting a stem cell revolution in the scientific world underway commencing at 2006. For the field of stem cell and developmental biology, 2008 ended with a truly exciting achievement for the Rat, i.e., the generation of germline competent embryonic stem cells from rat blastocysts （Figure 1） .

Organoids as preclinical models of human disease:progress and applications

In the field of biomedical research,organoids represent a remarkable advancement that has the potential to revolutionize our approach to studying human diseases even before clinical trials.Organoids are essentially miniature 3D models of specific organs or tissues,enabling scientists to investigate the causes of diseases,test new drugs,and explore personalized medicine within a controlled laboratory setting.Over the past decade,organoid technology has made substantial progress,allowing researchers to create highly detailed environments that closely mimic the human body.These organoids can be generated from various sources,including pluripotent stem cells,specialized tissue cells,and tumor tissue cells.This versatility enables scientists to replicate a wide range of diseases affecting different organ systems,effectively creating disease replicas in a laboratory dish.This exciting capability has provided us with unprecedented insights into the progression of diseases and how we can develop improved treatments.In this paper,we will provide an overview of the progress made in utilizing organoids as preclinical models,aiding our understanding and providing a more effective approach to addressing various human diseases.

Generation of gene-target dogs using CRISPR/Cas9 system

Dear Editor,Dogs(Canis familiaris)serve as human companions and are raised to herd livestock,aid hunters,guard homes,perform police and rescue work,and guide the blind.Dogs exhibit close similarities to humans in terms of metabolic,physiological,and anatomical characteristics,and thus are ideal genetic and clinical models to study human diseases(Tsai et al.,2007).Gene target technology is a powerful tool to create new strains of animals with favorable traits.However,thus far,gene-target dogs have not been developed due to their unique species-specific reproductive characteristics,which limits the applications of dogs especially in the field of biomedical research.Recently,clustered regularly interspaced short palindromic repeats(CRISPRs)/CRISPR-associated(Cas)9 system was applied to edit specific genes with a high efficiency(Cong et al.,2013;Mali et al.,2013).Here we attempt to explore the feasibility of producing gene knockout(KO)dogs by using this technology.Beagle dog,the most widely used breed in biomedical research,was used as our animal model.Myostatin(MSTN)was chosen as the first gene of interest.

Lactate and protein lactylation:the ugly duckling of energy as the sculpture artist of proteins

Metabolites play important roles in numerous cell biology processes,such as cell proliferation,differentiation,stress response,and cell death[1].Recently,lactate and lactate-derived lysine residue lactylation(Kla)have emerged as newly discovered epigenetic modifications that play critical roles in various physiological and pathological processes.In the history of lactate research,we can categorize the studies into three mile stones(Fig.S1 online).

Multiple gene modifications of pigs for overcoming obstacles of xenotransplantation

Xenotransplantation,involving animal organ transplantation into humans to address the human organ shortage,has been studied since the 17th century.Early attempts to obtain organs from animals such as goats,dogs,and non-human primates proved unsuccessful.In the 1990s,scientists agreed that pigs were the most suitable donor animals for xenotransplantation.However,immune rejection between pig and human has hindered the application.To overcome these challenges,researchers developed genetically modified pigs that deactivate xenoreactive antigen genes and express human protective genes.These advances extended xenograft survival from days to years in non-human primates,resulting in the first human heart xenotransplant trial.Using genetically engineered pigs for the organ shortage is promising.This review provides an overview of potential incompatibilities of immunogenicity and functional proteins related to xenotransplantation between humans and pigs.Furthermore,it elucidates possible approaches for multiplex gene modification to breed better-humanized pigs for clinical xenotransplantation.

Integration of Computational Analysis and Spatial Transcriptomics in Single-cell Studies

Recent advances of single-cell transcriptomics technologies and allied computational methodologies have revolutionized molecular cell biology.Meanwhile,pioneering explorations in spatial transcriptomics have opened up avenues to address fundamental biological questions in health and diseases.Here,we review the technical attributes of single-cell RNA sequencing and spatial transcriptomics,and the core concepts of computational data analysis.We further highlight the challenges in the application of data integration methodologies and the interpretation of the biological context of the findings.

Generation of functional posterior spinal motor neurons from hPSCs-derived human spinal cord neural progenitor cells

Spinal motor neurons deficiency results in a series of devastating disorders such as amyotrophic lateral sclerosis(ALS),spinal muscular atrophy(SMA)and spinal cord injury(SCI).These disorders are currently incurable,while human pluripotent stem cells(hPSCs)-derived spinal motor neurons are promising but suffered from inappropriate regional identity and functional immaturity for the study and treatment of posterior spinal cord related injuries.In this study,we have established human spinal cord neural progenitor cells(hSCNPCs)via hPSCs differentiated neuromesodermal progenitors(NMPs)and demonstrated the hSCNPCs can be continuously expanded up to 40 passages.hSCNPCs can be rapidly differentiated into posterior spinal motor neurons with high efficiency.The functional maturity has been examined in detail.Moreover,a co-culture scheme which is compatible for both neural and muscular differentiation is developed to mimic the neuromuscular junction(NMJ)formation in vitro.Together,these studies highlight the potential avenues for generating clinically relevant spinal motor neurons and modeling neuromuscular diseases through our defined hSCNPCs.

Generation of pigs with humanized type Ⅱ collagen by precise human COL2A1 gene knock-in

Osteoarthritis(OA)is the most common chronic disease,characterized by progressive cartilage breakdown,subchondral bone sclerosis,and aberrant bone outgrowth(Yucesoy et al.,2015;Hussain et al.,2016).OA is one of the leading causes of cartilage damage.Patients with severe cartilage damage require transplantation of articular cartilage to improve their quality of life.Type Ⅱ collagen is a major component of articular cartilage and intervertebral discs and plays an important role in the structure and strength of connective tissues that support muscles and joints(Byers,1994).

Variant PRC1 subunit RYBP/YAF2 forms condensate with RING1B and promotes H2AK119ub deposition

Dear Editor,Polycomb group(Pc G)proteins represent important roles in repressing gene expression throughout development.The Polycomb repressive complexes(PRCs)have been subdivided into two central protein complexes,PRC1 and PRC2.PRC1 catalyzes H2AK119ub and PRC2catalyzes H3K27me1/2/3(Fursova et al.,2019).PRC1 is further categorized as CBX-containing canonical PRC1(c PRC1)and RYBP/YAF2-containing variant PRC1(v PRC1)(Blackledge and Klose,2021).

乳酸化修饰参与调控DNA损伤修复和癌细胞化疗敏感性

The phenomenon in which cells prefer glycolysis to oxidative phosphorylation with increased lactate production is known as the Warburg effect and is found prevalent in cancer cells and pluripotent stem cells [1]. In addition to being an intermediate metabolite of glycolysis, lactate has been reported as a signal involved in multiple important biological processes, such as innate immunity [2], the cell cycle [3], hippocampal neurogenesis [4].

Biomolecular condensates and disease pathogenesis

Biomolecular condensates or membraneless organelles(MLOs)formed by liquid-liquid phase separation(LLPS)divide intracellular spaces into discrete compartments for specific functions.Dysregulation of LLPS or aberrant phase transition that disturbs the formation or material states of MLOs is closely correlated with neurodegeneration,tumorigenesis,and many other pathological processes.Herein,we summarize the recent progress in development of methods to monitor phase separation and we discuss the biogenesis and function of MLOs formed through phase separation.We then present emerging proof-of-concept examples regarding the disruption of phase separation homeostasis in a diverse array of clinical conditions including neurodegenerative disorders,hearing loss,cancers,and immunological diseases.Finally,we describe the emerging discovery of chemical modulators of phase separation.

Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells

Background:Tooth is vital not only for a good smile,but also good health.Yet,we lose tooth regularly due to accidents or diseases.An ideal solution to this problem is to regenerate tooth with patients’own cells.Here we describe the generation of tooth-like structures from integration-free human urine induced pluripotent stem cells(ifhU-iPSCs).Results:We first differentiated ifhU-iPSCs to epithelial sheets,which were then recombined with E14.5 mouse dental mesenchymes.Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30%for 8 different iPSC lines,comparable to H1 hESC.We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures,possessing physical properties such as elastic modulus and hardness found in the regular human tooth.Conclusion:Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.

Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process.Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline.Previous pig models with tetracycline regulatory elements were generated through random integration.This process often resulted in uncertain expression and unpredictable phenotypes,thus hindering their applications.Here,by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus,respectively,a double knock-in reporter pig model was generated.We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo.Two att P sites were arranged to flank the td Tomato to switch reporter gene.Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos.To display the flexible application of this system,we generated a pig strain with Dox-inducing h KRASexpression through phiC31 integrase-mediated cassette exchange.After eight months of Dox administration,squamous cell carcinoma developed in the nose,mouth,and scrotum,which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis.Overall,the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

Single cell atlas of developing mouse dental germs reveals populations of CD24^(+)and Plac8^(+)odontogenic cells

The spatiotemporal relationships in high-resolution during odontogenesis remain poorly understood.We report a cell lineage and atlas of developing mouse teeth.We performed a large-scale(92,688 cells)single cell RNA sequencing,tracing the cell trajectories during odontogenesis from embryonic days 10.5 to 16.5.Combined with an assay for transposase-accessible chromatin with high-throughput sequencing,our results suggest that mesenchymal cells show the specific transcriptome profiles to distinguish the tooth types.Subsequently,we identified key gene regulatory networks in teeth and bone formation and uncovered spatiotemporal patterns of odontogenic mesenchymal cells.CD24^(+)and Plac8^(+)cells from the mesenchyme at the bell stage were distributed in the upper half and preodontoblast layer of the dental papilla,respectively,which could individually induce nonodontogenic epithelia to form tooth-like structures.Specifically,the Plac8^(+)tissue we discovered is the smallest piece with the most homogenous cells that could induce tooth regeneration to date.Our work reveals previously unknown heterogeneity and spatiotemporal patterns of tooth germs that may lead to tooth regeneration for regenerative dentistry.

A tunable, rapid, and precise drug control of protein expression by combining transcriptional and post-translational regulation systems

Rapid,precise,and tunable regulation of protein abundance would be significantly useful in a variety of biotechnologies and biomedical applications.Here,we describe a system that allows tunable and rapid drug control of gene expression for either gene activation or inactivation in mammalian cells.We construct the system by coupling Tet-on 3 G and small molecule-assisted shutoff systems,which can respectively induce transcriptional activation and protein degradation in the presence of corresponding small molecules.This dual-input drug inducer regulation system facilitates a bidirectional control of gene expression.The gene of interest can be precisely controlled by dual small molecules in a broad dynamic range of expression from overexpression to complete silence,allowing gene function study in a comprehensive expression profile.Our results reveal that the bidirectional control system enables sensitive dosage-and time-dependent regulation for either turn-on or shutoff of gene expression.We also apply this system for inducible genome editing and gene activation mediated by clustered regularly interspaced short palindromic repeats.The system provides an integrated platform for studying multiple biological processes by manipulating gene expression in a more flexible way.

Epigenome-Metabolome-Epigenome signaling cascade in cell biological processes

Cell fate determination as a fundamental question in cell biology has been extensively studied at different regulatory levels for many years.However,the mechanisms of multilevel regulation of cell fate determination remain unclear.Recently,we have proposed an Epigenome-Metabolome-Epigenome(E-M-E)signaling cascade model to describe the cross-over cooperation during mouse somatic cell reprogramming.In this review,we summarize the broad roles of E-M-E signaling cascade in different cell biological processes,including cell differentiation and dedifferentiation,cell specialization,cell proliferation,and cell pathologic processes.Precise E-M-E signaling cascades are critical in these cell biological processes,and it is of worth to explore each step of E-M-E signaling cascade.E-M-E signaling cascade model sheds light on and may open a window to explore the mechanisms of multilevel regulation of cell biological processes.

Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation

Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs)into mouse blastocysts with deficiency of Flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS)of Flk-1^EGFP/ECFP rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.