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.

MiR-106a targets ATG7 to inhibit autophagy and angiogenesis after myocardial infarction

Background:Myocardial infarction(MI)is an acute condition in which the heart mus-cle dies due to the lack of blood supply.Previous research has suggested that au-tophagy and angiogenesis play vital roles in the prevention of heart failure after MI,and miR-106a is considered to be an important regulatory factor in MI.But the specific mechanism remains unknown.In this study,using cultured venous endothelial cells and a rat model of MI,we aimed to identify the potential target genes of miR-106a and discover the mechanisms of inhibiting autophagy and angiogenesis.Methods:We first explored the biological functions of miR-106a on autophagy and angiogenesis on endothelial cells.Then we identified ATG7,which was the down-stream target gene of miR-106a.The expression of miR-106a and ATG7 was investi-gated in the rat model of MI.Results:We found that miR-106a inhibits the proliferation,cell cycle,autophagy and angiogenesis,but promoted the apoptosis of vein endothelial cells.Moreover,ATG7 was identified as the target of miR-106a,and ATG7 rescued the inhibition of autophagy and angiogenesis by miR-106a.The expression of miR-106a in the rat model of MI was decreased but the expression of ATG7 was increased in the infarction areas.Conclusion:Our results indicate that miR-106a may inhibit autophagy and angiogenesis by targeting ATG7.This mechanism may be a potential therapeutic treatment for MI.

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.

Base editors:development and applications in biomedicine

Base editor(BE)is a gene-editing tool developed by combining the CRISPR/Cas system with an individual deaminase,enabling precise single-base substitution in DNA or RNA without generating a DNA double-strand break(DSB)or requiring donor DNA templates in living cells.Base editors offer more precise and secure genome-editing effects than other conventional artificial nuclease systems,such as CRISPR/Cas9,as the DSB induced by Cas9 will cause severe damage to the genome.Thus,base editors have important applications in the field of biomedicine,including gene function investigation,directed protein evolution,genetic lineage tracing,disease modeling,and gene therapy.Since the development of the two main base editors,cytosine base editors(CBEs)and adenine base editors(ABEs),scientists have developed more than 100 optimized base editors with improved editing efficiency,precision,specificity,targeting scope,and capacity to be delivered in vivo,greatly enhancing their application potential in biomedicine.Here,we review the recent development of base editors,summarize their applications in the biomedical field,and discuss future perspectives and challenges for therapeutic applications.

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.

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases

Dear Editor, Recently, zinc finger nuclease, transcrip- tion activator-like effector nuclease, and RNA-guided Cas9 endonuciease （Cas9） have emerged as powerful means for genome editing （Conklin, 2013; Gaj eta[., 2013）. These nucleases are efficient in gen- erating double-strand breaks in the genome that can be repaired by error-prone nonho- mologous end joining leading to a functional knockout （KO） of the targeted gene or used to integrate a DNA sequence at a specific locus through homologous recombination.

Genetically Modified Pig Models for Human Diseases

Genetically modified animal models are important for understanding the pathogenesis of human disease and developing therapeutic strategies. Although genetically modified mice have been widely used to model human diseases, some of these mouse models do not replicate important disease symptoms or pathology. Pigs are more similar to humans than mice in anatomy, physiology, and genome. Thus, pigs are considered to be better animal models to mimic some human diseases. This review describes genetically modified pigs that have been used to model various diseases including neurological, cardiovascular, and diabetic disorders. We also discuss the development in gene modification technology that can facilitate the generation of transgenic pig models for human diseases,

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.

Genetically humanized pigs exclusively expressing human insulin are generated through custom endonuclease-mediated seamless engineering

Dear Editor,Type1diabetes(T1D)isa lifelong(chronic)disease and a major health problem throughout the world.This disease can be treatedby either insulin injection or islet transplantation.Islet transplantation is considered as a better treatment for T1D patients,because islets can produce and release insulin at the appropriate time,resulting in tight blood glucose control.

Efficient and high-fidelity base editor with expanded PAM compatibility for cytidine dinucleotide

Cytidine base editor(CBE),which is composed of a cytidine deaminase fused to Cas9 nickase,has been widely used to induce C-to-T conversions in a wide range of organisms.However,the targeting scope of current CBEs is largely restricted to protospacer adjacent motif(PAM)sequences containing G,T,or A bases.In this study,we developed a new base editor termed“nNme2-CBE”with excellent PAM compatibility for cytidine dinucleotide,significantly expanding the genome-targeting scope of CBEs.Using nNme2-CBE,targeted editing efficiencies of 29.0%-55.0%and 17.3%—52.5%were generated in human cells and rabbit embryos,respectively.In contrast to conventional nSp-CBE,the nNme2-CBE is a natural high-fidelity base editing platform with minimal DNA off-targeting detected in vivo.Significantly increased efficiency in GC context and precision were determined by combining nNme2Cas9 with rationally engineered cytidine deaminases.In addition,the Founder rabbits with accurate single-base substitutions at Fgf5 gene loci were successfully generated by using the nNme2-CBE system.These novel nNme2-CBEs with expanded PAM compatibility and high fidelity will expand the base editing toolset for efficient gene modification and therapeutic applications.

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.

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.

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.

Production of transgenic pigs over-expressing theantiviral gene Mx1

The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interestingcandidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cellnuclear transfer (SCNT) to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs expressapproximately 15–25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was alsomarkedly enhanced. We challenged fibroblast cells isolated from the ear skin of transgenic and control pigs withinfluenza A virus and classical swine fever virus (CFSV). Indirect immunofluorescence assay (IFA) revealed a profounddecrease of influenza A proliferation in Mx1 transgenic cells. Growth kinetics showed an approximately 10-foldreduction of viral copies in the transgenic cells compared to non-transgenic controls. Additionally, we found thatthe Mx1 transgenic cells were more resistant to CSFV infection in comparison to non-transgenic cells. These resultsdemonstrate that the Mx1 transgene can protect against viral infection in cells of transgenic pigs and indicate thatthe Mx1 transgene can be harnessed to develop disease-resistant pigs.

Generation of multi-gene knockout rabbits using the Cas9/gRNA system

The prokaryotic clustered regularly interspaced short palindromic repeat(CRISPR)-associated system(Cas)is a simple,robust and efficient technique for gene targeting in model organisms such as zebrafish,mice and rats.In this report,we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA(gRNA)into the cytoplasm of pronuclear-stage embryos.We achieved biallelic gene knockout(KO)rabbits by injection of 1 gene(IL2rg)or 2 gene(IL2rg and RAG1)Cas9 mRNA and gRNA with an efficiency of 100%.We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100%for 3 genes(IL2rg,RAG1 and RAG2)and 33.3%for 5 genes(IL2rg,RAG1,RAG2,TIKI1 and ALB).Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.

Efficient multinucleotide deletions using deaminase-Cas9 fusions in human cells

CRISPR/Cas9 system is a robust genome editing platform in biotechnology and medicine.However,it generally produces small insertions/deletions(indels,typically 1-3 bp)but rarely induces larger deletions in specific target sites.Here,we report a cytidine deaminase-Cas9 fusion-induced deletion system(C-DEL)and an adenine deaminase-Cas9 fusion-induced deletion system(A-DEL)by combining Cas9 with rat APOBEC1(r A1)and Tad A 8e,respectively.Both C-DEL and A-DEL improve the efficiency of deletions compared with the conventional Cas9 system in human cells.In addition,the C-DEL system generates a considerable fraction of predictable multinucleotide deletions from 5’-deaminated C bases to the Cas9-cleavage site and increases the proportion of larger deletions at the target loci.Taken together,the CDEL and A-DEL systems provide a practical strategy for producing efficient multinucleotide deletions,expanding the CRISPR/Cas9 toolsets for gene modifications in human cells.

Generation of knockout rabbits using transcription activator-like effector nucleases

Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platformscontributing to redefine the boundaries of modern biological research. They are composed of a non-specificcleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications byinducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases havebeen employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively.This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies,biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbitsusing transcription activator-like effector nucleases, and a perspective of the field.

Reprogramming mature terminally differentiated adipocytes to induced pluripotent stem cells

Mature adipocytes are terminally differentiated somatic cells. Here, we report the successful generation of induced pluripotent stem （iPS） cells from mouse mature adipocytes by forced expression of six transcription factors （Oct4, Sox2, c-Myc, Klf4, Rarγ, and Lrh1） with a piggyBac transposon-based strategy. The resulting iPS cells were pluripotent as evidenced by the fact that they stained positive for alkaline phosphatase, expressed high levels of key pluripotency markers including Oct4, Nanog, and SSEA1, and remained pluripotent on a 2i media. In vitro differen- tiation of the iPS cells showed that the cell derivatives of all three germ layers could be readily obtained through forma- tion of embryoid bodies. Most importantly, these adipocyte- derived iPS cells were capable of producing chimera with high frequencies when reintroduced into early-stage em- bryos and transmitted through the germ line. This study demonstrates that the new six-factor reprogramming tech- nology facilitates the reset of the terminally differentiated adipocytes to the ground state of pluripotency, enabling us to fully explore the potential of mature adipocytes as a viable cell source for regenerative medicine.

Generation of ApoE deficient dogs via combination of embryo injection of CRISPR/Cas9 with somatic cell nuclear transfer

Atherosclerotic cardiovascular disease is the leading cause of death in the world which is resulted from complex interactions among multiple genetic and environmental factors （WHO）. Athero- sclerosis is a chronic inflammatory disease characterized by accumulation of lipids in the arterial wall （Gofman and Lindgren, 1950）. Tremendous clinical and experimental efforts have been made to reveal the pathogenesis of the disease. Nevertheless, the mechanism of atherosclerosis is still unclear. A suitable animal model to study metabolic disorders and subsequent atherosclerosis is a necessity. The traditional method by feeding high fat diet to establish animal models of atherosclerosis disease is time- consuming and laborious, and in many circumstances, the pheno- types are not consistent among the individual models.