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Abstract 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 guide-free 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.

Abstract Background Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging. Results Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control. Conclusions Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.

Abstract Background Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency. Results Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol. Conclusions Our innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.

Abstract Conditional gene regulation systems can control gene expression in predefined tissues or organs at a desired time. Site‐specific recombinase systems and chemically induced gene expression systems are the two most widely used approaches for creating genetically modified (GM) animals with conditional regulation of gene expression. Generation of GM pigs with controllable elements, usually involving multiple gene editing, used to be a major challenge due to a lack of germ line‐competent pluripotent stem cells. With the emergence of artificial endonuclease‐mediated gene editors, a variety of GM pigs with recombinase‐specific recognition elements or chemically induced elements for conditional regulation of gene expression have been generated by the combination of site‐directed knock‐in of somatic cells and somatic cell nuclear transfer technology, allowing conditional deletion of endogenous genes or overexpression of exogenous genes in pigs. These inducible tool pig models will greatly facilitate the production of GM pigs and broaden the applications of transgenic pigs in biomedicine and agriculture fields. In this paper, we review the progress in the construction and application of pigs with controllable elements using gene editing techniques.

Abstract Background Nme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency. Results Our results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797Smu and Nme2ABE8e-797−C, respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice. Conclusions These novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications.

Abstract The small size of the Cas nuclease fused with various effector domains enables a broad range of function. Although there are several ways of reducing the size of the Cas nuclease complex, no efficient or generalizable method has been demonstrated to achieve protein miniaturization. In this study, we establish an Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturization and generate five compact variants of Cas13 with full RNA binding and cleavage activity comparable the wild-type enzymes based on a combination of IDC strategy and AlphaFold2. In addition, we construct an RNA base editor, mini-Vx, and a single AAV (adeno-associated virus) carrying a mini-RfxCas13d and crRNA expression cassette, which individually shows efficient conversion rate and RNA-knockdown activity. In summary, these findings highlight a feasible strategy for generating downsized CRISPR/Cas13 systems based on structure predicted by AlphaFold2, enabling targeted degradation of RNAs and RNA editing for basic research and therapeutic applications.

Prime editor (PE) is a versatile genome editing tool that does not need extra DNA donors or inducing double-strand breaks. However, in vivo implementation of PE remains a challenge because of its oversized composition. In this study, we screened out the smallest truncated Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) with the F155Y mutation to keep gene editing efficiency. We discovered the most efficient gene editing variants of MMLV RT with the smallest size. After optimization of the pegRNAs and incorporation with nick sgRNAs, the mini-PE delivered up to 10% precise editing at target sites in human and mouse cells. It also edited the mouse Hsf1 gene in the mouse retina precisely after delivery with adeno-associated viruses (AAVs), although the editing efficiency was lower than 1%. We will focus on improving the editing efficiency of mini-PE and exploiting its therapeutic potential against human genetic diseases.

Cas9 protein without sgRNAs can induce genomic damage at the cellular level in vitro. However, whether the detrimental effects occur in embryos after Cas9 treatment remains unknown. Here, using pig embryos as subjects, we observed that Cas9 protein transcribed from injected Cas9 mRNA can persist until at least the blastocyst stage. Cas9 protein alone can induce genome damage in preimplantation embryos, represented by the increased number of phosphorylated histone H2AX foci on the chromatin fiber, which led to apoptosis and decreased cell number of blastocysts. In addition, single-blastocyst RNA sequencing confirmed that Cas9 protein without sgRNAs can cause changes in the blastocyst transcriptome, depressing embryo development signal pathways, such as cell cycle, metabolism, and cellular communication-related signal pathways, while activating apoptosis and necroptosis signal pathways, which together resulted in impaired preimplantation embryonic development. These results indicated that attention should be given to the detrimental effects caused by the Cas9 protein when using CRISPR-Cas9 for germline genome editing, especially for the targeted correction of human pathological mutations using germline gene therapy.

African swine fever (ASF) is an acute and highly contagious lethal infectious disease in swine that severely threatens the global pig industry. At present, a safe and efficacious vaccine is urgently required to prevent and control the disease. In this study, we evaluated the safety and immunogenicity of replication-incompetent type-2 adenoviruses carrying African swine fever virus (ASFV) antigens, namely CP204L (p30), E183L (p54), EP402R (CD2v), B646L (p72), and B602L (p72 chaperone). A vaccine cocktail delivered by simultaneous intramuscular (IM) and intranasal (IN) administration robustly elicited both systemic and mucosal immune responses against AFSV in mice and swine and provided highly effective protection against the circulating ASFV strain in farmed pigs. This multi-antigen cocktail vaccine was well tolerated in the vaccinated animals. No significant interference among antigens was observed. The combined IM and IN vaccination using this adenovirus-vectored antigen cocktail vaccine warrants further evaluation for providing safe and effective protection against ASFV infection and transmission.

Abstract Background Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size. Results We discovered that ABE8e can tolerate large single deletions in the REC2 (Δ174-296) and HNH (Δ786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes. Conclusions We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications.

The RAN Guanine Nucleotide Release Factor (RANGRF) gene encodes the protein MOG1, which binds to Nav1.5 and facilitates its transport to the cell membrane. Nav1.5 mutations have been linked to various cardiac arrhythmias and cardiomyopathy. To investigate the role of RANGRF in this process, we utilized the CRISPR/Cas9 gene editing system to generate a homozygous RANGRF knockout hiPSC line. The availability of the cell line will prove to be an invaluable asset in the study of disease mechanisms and the testing of gene therapies for cardiomyopathy.

Abstract Background CRISPR-based toolkits have dramatically increased the ease of genome and epigenome editing. SpCas9 is the most widely used nuclease. However, the difficulty of delivering SpCas9 and inability to modulate its expression in vivo hinder its widespread adoption in large animals. Results Here, to circumvent these obstacles, a doxycycline-inducible SpCas9-expressing (DIC) pig model was generated by precise knock-in of the binary tetracycline-inducible expression elements into the Rosa26 and Hipp11 loci, respectively. With this pig model, in vivo and/or in vitro genome and epigenome editing could be easily realized. On the basis of the DIC system, a convenient Cas9-based conditional knockout strategy was devised through controlling the expression of rtTA component by tissue-specific promoter, which allows the one-step generation of germline-inherited pigs enabling in vivo spatiotemporal control of gene function under simple chemical induction. To validate the feasibility of in vivo gene mutation with DIC pigs, primary and metastatic pancreatic ductal adenocarcinoma was developed by delivering a single AAV6 vector containing TP53-sgRNA, LKB1-sgRNA, and mutant human KRAS gene into the adult pancreases. Conclusions Together, these results suggest that DIC pig resources will provide a powerful tool for conditional in vivo genome and epigenome modification for fundamental and applied research.

Interscapular brown adipose tissue (iBAT) of both rabbits and humans exhibits a similar whitening phenomenon under physiological conditions. However, a detailed characterization of iBAT whitening in them is still lacking. Here, we chose rabbits as a model to gain a better understanding of the molecular signature changes during the whitening process of iBAT by transcriptomic analysis of rabbit iBAT at day 1, day 14, 1 month and 4 months after birth. We applied non-invasive MRI imaging to monitor the whitening process and correlated these changes with analysis of morphological, histological and molecular features. Principal component analysis (PCA) of differentially expressed genes delineated three major phases for the whitening process as Brown, Transition and Whitened BAT phases. RNA-sequencing data revealed that whitening of iBAT was an orchestrated process where multiple types of cells and tissues participated in a variety of physiological processes including neovascularization, formation of new nervous networks and immune regulation. Several key metabolic and signalling pathways contributed to whitening of iBAT, and immune cells and immune regulation appeared to play an overarching role.

A Neisseria cinerea Cas9 variant is characterised in PAM preference, editing efficiency (for both nuclease and base editing) and anti-CRISPR protein blocking, with a PAM of (N)4GYAT reported alongside a mouse zygote assay.

Cas12a-mediated cytosine (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded target scopes are developed, by combining highly active deaminases with Cas12a variants, enCas12a, RR and RVR, recognizing non-TTTV PAMs.

Introduction: The ABO blood group system has important clinical significance in the safety of blood transfusion and organ transplantation. Numerous ABO variations, especially variations in the splice sites, have been identified to be associated with some ABO subtypes.Methods: Here, we performed the c.767T>C substitution of the ABO gene in human induced pluripotent stem cells (hiPSCs) by the adenosine base editor (ABE) system and described its characteristics at the genome level in detail.Results: The hiPS cell line with c.767T>C substitution maintained a normal karyotype (46, XX), expressed pluripotency markers, and showed the capability to spontaneously differentiate into all three germ layers in vivo. The genome-wide analysis demonstrated that the c.767T>C substitution in the ABO gene did not cause any detected negative effect in hiPSCs at the genome level. The splicing transcript analysis revealed that splicing variants were observed in the hiPSCs with ABO c.767T>C substitutions.Conclusion: All these results indicated that some splicing variants occurred in hiPSCs with c.767 T>C substitution of ABO gene, which probably had a significant effect on the formation of the rare ABO*Ael05/B101 subtype.

Schizophrenia is a group of common psychosis of unknown etiology and GRIN2A gene has been a risk gene for schizophrenia. In order to understand the relationship between the GRIN2A and schizophrenia, we generated a GRIN2A-KO human embryonic stem cell line by CRISPR/Cas9 system, which could provide a valuable resource for investing pathogenic mechanisms underlying schizophrenia and facilitating the development of targeted medicine.

Primary microcephaly (PMCPH) is a rare autosomal recessive neurodevelopmental disorder with a global prevalence of PMCPH ranging from 0.0013% to 0.15%. Recently, a homozygous missense mutation in YIPF5 (p.W218R) was identified as a causative mutation of severe microcephaly. In this study, we constructed a rabbit PMCPH model harboring YIPF5 (p.W218R) mutation using SpRY-ABEmax mediated base substitution, which precisely recapitulated the typical symptoms of human PMCPH. Compared with wild-type controls, the mutant rabbits exhibited stunted growth, reduced head circumference, altered motor ability, and decreased survival rates. Further investigation based on model rabbit elucidated that altered YIPF5 function in cortical neurons could lead to endoplasmic reticulum stress and neurodevelopmental disorders, interference of the generation of apical progenitors (APs), the first generation of progenitors in the developing cortex. Furthermore, these YIPF5-mutant rabbits support a correlation between unfolded protein responses (UPR) induced by endoplasmic reticulum stress (ERS), and the development of PMCPH, thus providing a new perspective on the role of YIPF5 in human brain development and a theoretical basis for the differential diagnosis and clinical treatment of PMCPH. To our knowledge, this is the first gene-edited rabbit model of PMCPH. The model better mimics the clinical features of human microcephaly than the traditional mouse models. Hence, it provides great potential for understanding the pathogenesis and developing novel diagnostic and therapeutic approaches for PMCPH.

Abstract Background Previous studies have identified the carbohydrate epitope Galα1–3Galβ1–4GlcNAc-R (termed the α-galactosyl epitope), known as the α-Gal antigen as the primary xenoantigen recognized by the human immune system. The α-Gal antigen is regulated by galactosyltransferase (GGTA1), and α-Gal antigen-deficient mice have been widely used in xenoimmunological studies, as well as for the immunogenic risk evaluation of animal-derived medical devices. The objective of this study was to develop α-Gal antigen-deficient rabbits by GGTA1 gene editing with the CRISPR/Cas9 system. Results The mutation efficiency of GGTA1 gene-editing in rabbits was as high as 92.3% in F0 pups. Phenotype analysis showed that the α-Gal antigen expression in the major organs of F0 rabbits was decreased by more than 99.96% compared with that in wild-type (WT) rabbits, and the specific anti-Gal IgG and IgM antibody levels in F1 rabbits increased with increasing age, peaking at approximately 5 or 6 months. Further study showed that GGTA1 gene expression in F2-edited rabbits was dramatically reduced compared to that in WT rabbits. Conclusions α-Gal antigen-deficient rabbits were successfully generated by GGTA1 gene editing via the CRISPR/Cas9 system in this study. The feasibility of using these α-Gal antigen-deficient rabbits for the in situ implantation and residual immunogenic risk evaluation of animal tissue-derived medical devices was also preliminarily confirmed.

Adenine base editors (ABEs), composed of an evolved adenine deaminase fused to the Cas9 nickase, enable efficient and precise A-to-G conversion in various organisms. However, the base editing of some challenging loci with the ABE7.10 system in rabbits was inefficient in our previous study. Here, we show that ABE8.17 and SpRY-ABE8.17 can efficiently induce base editing in mouse and rabbit embryos. In addition, this strategy can be used to precisely mimic clinical point mutations in rabbits. Furthermore, by eliminating the linker in ABE8.17, we created ABE8.17-NL, which achieved efficient base editing within a narrowed window (2–4 nts) in human HEK293FT cells. Collectively, these findings show that ABE8.17 systems can efficiently induce efficient A-to-G base editing at desired sites and that the ABE7.10 system is inefficient, thus providing an efficient way to generate ideal disease models in rabbits.

Pluripotent stem cells (PSCs) are promising in regenerative medicine. A major challenge of PSC therapy is the risk of teratoma formation because of the contamination of undifferentiated stem cells. Constitutive promoters or endogenous SOX2 promoters have been used to drive inducible caspase-9 (iCasp9) gene expression but cannot specifically eradicate undifferentiated PSCs. Here, we inserted iCasp9 gene into the endogenous OCT4 locus of human and mouse PSCs without affecting their pluripotency. A chemical inducer of dimerization (CID), AP1903, induced iCasp9 activation, which led to the apoptosis of specific undifferentiated PSCs in vitro and in vivo. Differentiated cell lineages survived because of the silence of the endogenous OCT4 gene. Human and mouse PSCs were controllable when CID was administrated within 2 weeks after PSC injection in immunodeficient mice. However, an interval longer than 2 weeks caused teratoma formation and mouse death because a mass of somatic cells already differentiated from the PSCs. In conclusion, we have developed a specific and efficient PSC suicide system that will be of value in the clinical applications of PSC-based therapy.

Abstract Background Adoptive cell therapy (ACT) is a particularly promising area of cancer immunotherapy, engineered T and NK cells that express chimeric antigen receptors (CAR) are being explored for treating hematopoietic malignancies but exhibit limited clinical benefits for solid tumour patients, successful cellular immunotherapy of solid tumors demands new strategies. Methods Inactivation of BCL11B were performed by CRISPR/Cas9 in human T cells. Immunophenotypic and transcriptional profiles of sgBCL11B T cells were characterized by cytometer and transcriptomics, respectively. sgBCL11B T cells are further engineered with chimeric antigen receptor. Anti-tumor activity of ITNK or CAR-ITNK cells were evaluated in preclinical and clinical studies. Results We report that inactivation of BCL11B in human CD8+ and CD4+ T cells induced their reprogramming into induced T-to-natural killer cells (ITNKs). ITNKs contained a diverse TCR repertoire; downregulated T cell-associated genes such as TCF7 and LEF1; and expressed high levels of NK cell lineage-associated genes. ITNKs and chimeric antigen receptor (CAR)-transduced ITNKs selectively lysed a variety of cancer cells in culture and suppressed the growth of solid tumors in xenograft models. In a preliminary clinical study, autologous administration of ITNKs in patients with advanced solid tumors was well tolerated, and tumor stabilization was seen in six out nine patients, with one partial remission. Conclusions The novel ITNKs thus may be a promising novel cell source for cancer immunotherapy. Trial registration ClinicalTrials.gov, NCT03882840 . Registered 20 March 2019-Retrospectively registered.

Anti-deaminases can inhibit APOBEC3, a component of cytosine base editors. Here Zhanjun Li and colleagues repurposed anti-deaminase proteins derived from viruses to inhibit base editors for use in efficient regulation of base editors’ activity in gene modification and therapeutic applications.

Animal models are important for understanding the pathogenesis of human diseases and for developing and testing new drugs. Pigs have been widely used in the research on the cardiovascular, skin barrier, gastrointestinal, and central nervous systems as well as organ transplantation. Recently, pigs also become an attractive large animal model for the study of neurodegenerative diseases because their brains are very similar to human brains in terms of mass, gully pattern, vascularization, and the proportions of the gray and white matters. Although adeno-associated virus type 9 (AAV9) has been widely used to deliver transgenes in the brain, its utilization in large animal models remains to be fully characterized. Here, we report that intravenous injection of AAV9-GFP can lead to widespread expression of transgene in various organs in the pig. Importantly, GFP was highly expressed in various brain regions, especially the striatum, cortex, cerebellum, hippocampus, without detectable inflammatory responses. These results suggest that intravenous AAV9 administration can be used to establish large animal models of neurodegenerative diseases caused by gene mutations and to treat these animal models as well.

Abdominal aortic aneurysm (AAA) is pathologically characterized by intimal atherosclerosis, disruption and attenuation of the elastic media, and adventitial inflammatory infiltrates. Although all these pathological events are possibly involved in the pathogenesis of AAA, the functional roles contributed by adventitial inflammatory macrophages have not been fully documented. Recent studies have revealed that increased expression of matrix metalloproteinase-12 (MMP-12) derived from macrophages may be particularly important in the pathogenesis of both atherosclerosis and AAA. In the current study, we developed a carrageenan-induced abdominal aortic adventitial inflammatory model in hypercholesterolemic rabbits and evaluated the effect of adventitial macrophage accumulation on the aortic remodeling with special reference to the influence of increased expression of MMP-12. To accomplish this, we compared the carrageenan-induced aortic lesions of transgenic (Tg) rabbits that expressed high levels of MMP-12 in the macrophage lineage to those of non-Tg rabbits. We found that the aortic medial and adventitial lesions of Tg rabbits were greater in degree than those of non-Tg rabbits, with the increased infiltration of macrophages and prominent destruction of elastic lamellae accompanied by the frequent appearance of dilated lesions, while the intimal lesions were slightly increased. Enhanced aortic lesions in Tg rabbits were focally associated with increased dilation of the aortic lumens. RT-PCR and Western blotting revealed high levels of MMP-12 in the lesions of Tg rabbits that were accompanied by elevated levels of MMP-2 and -3, which was caused by increased number of macrophages. Our results suggest that adventitial inflammation constitutes a major stimulus to aortic remodeling and increased expression of MMP-12 secreted from adventitial macrophages plays an important role in the pathogenesis of vascular diseases such as AAA.

Recently, a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize a minimal NG protospacer adjacent motif (PAM) was reported to expand the targeting scope in genome editing. However, increased genome-wide off-target mutations with this variant compared with SpCas9 were reported in previous studies. In addition, lower base editing frequencies and higher unintended off-target mutations were also found in Hoxc13-ablated rabbits generated by NG-BE4max in our study. Here, a high-fidelity base editor, NG-HiFi, in comparison to NG-BE4max, showed retention of on-target activity while exhibiting significantly decreased off-target activity in Hoxc13-ablated rabbits. Collectively, the improved specificity and reduced off-target effect of SpCas9-NG assisted in cytidine base editing with the NG-HiFi system, providing a promising tool to precisely model human diseases in rabbits.

Introduction: Since xenografts offer a wide range of incomparable advantages, they can be a better option than allografts but only if the possibility of immunological rejection can be eliminated. In this study, we investigated the ability of α1,3-galactosyltransferase (α1,3-GT) gene knockout (GTKO) pig cancellous bone to promote the repair of a femoral condyle bone defect and its influence on heterologous immune rejection.Materials and methods: Cylindrical bone defects created in a rhesus monkey model were transplanted with GTKO bone, WT bone or left empty. For immunological evaluation, T lymphocyte subsets CD4+ and CD8+ in peripheral blood were assayed by flow cytometry, and the IL-2 and IFN-γ contents of peripheral blood serum were analyzed by ELISA at 2, 5, 7, 10, and 14 days post-surgery. Micro-CT scans and histological assessment were conducted at 4 and 8 weeks after implantation.Results: Compared with WT-pig bone, the heterologous immunogenicity of GTKO-pig bone was reduced. The defect filled with fresh GTKO-pig bone was tightly integrated with the graft. Histological analysis showed that GTKO-pig cancellous bone showed better osseointegration and an appropriate rate of resorption. Osteoblast phenotype progression in the GTKO group was not affected, which revealed that GTKO-pig bone could not only fill and maintain the bone defect, but also promote new bone formation.Conclusion: GTKO-pig cancellous bone decreased the ratio of CD4+ to CD8+ T cells and cytokines (IFN-γ and IL-2) to inhibit xenotransplant rejection. Moreover, GTKO group increased more bone formation by micro-CT analysis and osteoblastic markers (Runx2, OSX and OCN). Together, GTKO-pig cancellous bone showed better bone repair than WT-pig cancellous bone.

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

Gene-modified miniature pigs serve as alternative tissue and organ donors for xenotransplantation to alleviate the shortage of human allogenic organs. However, the high copy number of porcine endogenous retrovirus (PERV) genomes integrates with the porcine genome, which has a potential risk of cross-species transmission and hinders the clinical practice of xenotransplantation. Recently, CRISPR/Cas9 has been used to inactivate PERVs. However, Cas9 also triggers severe DNA damage at multiple integrated PERV sites in the porcine genome, which induces senescence and apoptosis of porcine cells. In this study, the cytosine base editor (CBE), an efficient and safe editor that does not cause DNA double strand breaks (DSBs), was used for PERV editing to reduce cytotoxic effects. Seven sgRNAs were set to target gag and pol loci of PERVs to induce premature stop codons. We found that approximately 10% of cell clones were completely inactivated for PERVs in pig ST cells, and the plasmid that was used for editing the PERVs did not integrate into host genome and influence the karyotype of the modified cells. Our studies offer a powerful and safe strategy for further generating PERV-knockout pigs using base editors.

Previous work suggested that histone demethylase JMJD3 is detrimental to somatic cell reprogramming. Here, the authors show that while JMJD3 has a context-independent detrimental effect on early stages of reprogramming, during late stages it activates epithelial and pluripotency genes together with Klf4.

CRISPR-Cas9-mediated gene knockout and base-editing-associated induction of STOP codons (iSTOP) have been widely used to exterminate the function of a coding gene, while they have been reported to exhibit side effects. In this study, we propose a novel and practical alternative method referred to as CRISPR Start-Loss (CRISPR-SL), which eliminates gene expression by utilizing both adenine base editors (ABEs) and cytidine base editors (CBEs) to disrupt the initiation codon (ATG). CRISPR-SL has been verified to be a feasible strategy on the cellular and embryonic levels (mean editing efficiencies up to 30.67% and 73.50%, respectively) and in two rabbit models mimicking Otc deficiency (Otc gene) and long hair economic traits (Fgf5 gene).

CRISPR-Cas9 and base editors (BEs) systems are poised to become the gene-editing tool of choice in clinical contexts; however, large-fragment deletion was found in Cas9-mediated mutation cells and mice. In this study, by analyzing 16 gene-edited rabbit lines (including 112 rabbits) generated using SpCas9, BEs, xCas9, and xCas9-BEs with long-range PCR genotyping and long-read sequencing by the PacBio platform, we show the extension of thousands of base fragment deletions in single-guide RNA/Cas9 and xCas9 system mutation rabbits, but no deletions were found in BE-induced mutation rabbits. Thus, we first validated that no large-fragment deletion was induced by the BEs system, suggesting that BE systems can be beneficial tools for the further development of highly accurate and secure gene therapy for the clinical treatment of human genetic disorders.

Abstract Background Many favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site. Results In this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system. Conclusion The newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.

Abstract Background Cytidine base editors (CBEs), composed of a cytidine deaminase fused to Cas9 nickase (nCas9), enable efficient C-to-T conversion in various organisms. However, current base editors can induce unwanted bystander C-to-T conversions when multiple Cs are present in the ~ 5-nucleotide activity window of cytidine deaminase, which negatively affects their precision. Here, we develop a new base editor which significantly reduces unwanted bystander activities. Results We used an engineered human APOBEC3G (eA3G) C-terminal catalytic domain with preferential cytidine-deaminase activity in motifs with a hierarchy CCC>CCC>CC (where the preferentially deaminated C is underlined), to develop an eA3G-BE with distinctive CC context-specificity and reduced generation of bystander mutations. Targeted editing efficiencies of 18.3–58.0% and 54.5–92.2% with excellent CC context-specificity were generated in human cells and rabbit embryos, respectively. In addition, a base editor that can further recognize relaxed NG PAMs is achieved by combining hA3G with an engineered SpCas9-NG variant. The A3G-BEs were used to induce accurate single-base substitutions which led to nonsense mutation with an efficiency of 83–100% and few bystander mutations in Founder (F0) rabbits at Tyr loci. Conclusions These novel base editors with improved precision and CC context-specificity will expand the toolset for precise gene modification in organisms.

Base editors can efficiently produce single nucleotide alterations without requiring a double-strand break. Here the authors show base editing at multiple sites simultaneously in pigs.

A key challenge is to find/re-purpose approved drugs that could be used in humans to induce autophagy-associated clearance of neurodegenerative proteins. Here, authors demonstrate that felodipine, an anti-hypertensive drug, can induce autophagy and clear a variety of aggregated neurodegenerative disease-associated proteins in mouse brains at plasma concentrations similar to those that would be seen in humans taking the drug.

Conventional human pluripotent stem cells (hPSCs) fail to contribute to interspecies chimaeras when injected into mouse blastocysts. Here the authors show that forced expression of BMI1 overcomes apoptosis of hPSCs in blastocysts of mouse, rabbit and pig allowing them to contribute to chimaeras.

Background: Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by reduced melanin that are caused by mutations in the gene encoding tyrosinase (TYR), which is the rate-limiting enzyme in the production of the pigment melanin. Many studies or meta-analyses have suggested an association between the TYR T373K SNP and OCA1, but there is limited biochemical and genetic evidence to support this association. Methods: We overexpressed TYR-WT and TYR-T373K mutants on HK293T cells and tested the changes of melanin production and tyrosinase activity. Then we generated TYR-K373T knock-in (KI) rabbits by microinjection of ssDNA and synthesized RNAs targeting C1118A using CRISPR/Cas9-HDR to observe the formation of melanin. Findings: We demonstrated that the T373K mutation in TYR can reduce tyrosinase activity, leading to an absence of melanin synthesis at the cell-level. The gene-edited TYR-K373T rabbits exhibited rescued melanin production in hair follicles and irises, as inferred from the evident decrease in pigmentation in TYR-T373K rabbits, thus providing functional validation of the albinism-associated T373K SNP at the animal level. Interpretation: Our study provides the first animal-level functional validation of the albinism-associated TYR K373T SNP in rabbits, and these results will facilitate gene therapy of OCA1 in pre-clinical settings in the future. Fund: The National Key Research and Development Program of China Stem Cell and Translational Research, the Strategic Priority Research Program of the Chinese Academy of Sciences, the Guangdong Province Science and Technology Plan Project, and the Program for JLU Science and Technology Innovative Research Team. Keywords: Tyrosinase, OCA, T373K, Point mutation, Rabbit, CRISPR/Cas9

Abstract In previous studies, CRISPR/Cas9 was shown to induce unexpected exon skipping; however, the mechanism by which this phenomenon is triggered is controversial. By analyzing 22 gene-edited rabbit lines generated using CRISPR/Cas9, we provide evidence of exon skipping at high frequency in premature termination codon-mutated rabbits but not in the rabbits with a premature termination codon mutation in exon 1 rabbits with non-frameshift or missense mutations. Our results suggest that CRISPR-mediated exon skipping depends on premature termination codon mutation-induced nonsense-associated altered splicing.

Transcription activator-like effectors (TALEs) have been effectively used for targeted genome editing, transcriptional regulation, epigenetic modification, and locus-specific DNA imaging. However, with the advent of the clustered regularly interspaced short palindromic repeat/Cas9 system, an easy-to-use tool with the same function as TALEs, TALEs have recently been abandoned because of their complexity, time consumption, and difficult handling in common labs. Here, we described a degenerated codon-based TALE assembly system for simple, rapid, and efficient TALE assembly. TALE trimers with nonrepetitive DNA sequences were amplified by PCR and sequentially assembled via Gibson assembly. Our method is cost-effective, requires only commonly used basic molecular biology reagents, and takes only 2 h from target sequence analysis to completion. This simple, rapid, and lab-friendly TALE assembly method will restore the value of TALEs in DNA targeting.

Base editors can make targeted changes without inducing a double-stranded break. Here, the authors apply the BE3 and ABE7.10 systems to rabbit to create highly efficient targeted base substitutions and various mutation types, and show reduced frequency of undesired by-products with the updated BE4-Gam system.

Summary: Pig cloning by somatic cell nuclear transfer (SCNT) remains extremely inefficient, and many cloned embryos undergo abnormal development. Here, by profiling transcriptome expression, we observed dysregulated chromosome-wide gene expression in every chromosome and identified a considerable number of genes that are aberrantly expressed in the abnormal cloned embryos. In particular, XIST, a long non-coding RNA gene, showed high ectopic expression in abnormal embryos. We also proved that nullification of the XIST gene in donor cells can normalize aberrant gene expression in cloned embryos and enhance long-term development capacity of the embryos. Furthermore, the increased quality of XIST-deficient embryos was associated with the global H3K9me3 reduction. Injection of H3K9me demethylase Kdm4A into NT embryos could improve the development of pre-implantation stage embryos. However, Kdm4A addition also induced XIST derepression in the active X chromosome and thus was not able to enhance the in vivo long-term developmental capacity of porcine NT embryos. : In this article, Liangxue Lai and colleagues show that nullification of the XIST gene in donor cells can normalize aberrant gene expression in cloned embryos and result in high cloning efficiency in pigs. Keywords: pig somatic cell nuclear transfer, XIST, H3K9me3, H3K9me3 demethylases, H3K9me3 methyltransferases

miR-122 is the most abundant miRNA in the human liver, accounting for 52% of the entire hepatic miRNome. Previous studies have demonstrated that miR-122 plays key roles in hepatocyte growth, metabolism, and homeostasis. Here, we created three miR-122 knockout human embryonic stem cell line lines, WAe001-A-7, WAe001-A-8, and WAe001-A-9, using the CRISPR/Cas9 technique. These mutated cell lines retained their pluripotency, in vitro differentiation potential, normal morphology, and karyotype.

The MEN1 gene is cytogenetically located at 11q13.1 and encodes the nuclear protein menin, which is involved in cell proliferation, apoptosis, differentiation, and metabolism. Here, we generated two MEN1 knockout human embryonic stem cell lines, WAe001-A-4 and WAe001-A-5, by targeting exon-2 and exon-9 of MEN1 using the CRISPR/Cas9 technique. These cell lines maintained their pluripotency, in vitro differentiation potential, normal morphology, and karyotype. These human MEN1-mutated cell lines not only enlarge the pool of lab resources but also provide ideal models to dissect the detailed physio-pathological roles of the menin protein.

Abstract Immunodeficient mice engrafted with either normal or cancerous human cells are widely used in basic and translational research. In particular, NOD/SCID/IL2rg−/− mice can support the growth of various types of human cancer cells. However, the hairs of these mice interfere with the observation and imaging of engrafted tissues. Therefore, novel hairless strains exhibiting comparable immunodeficiency would be beneficial. Recently, the CRISPR/Cas9 system has been used for efficient multiplexed genome editing. In the present study, we generated a novel strain of nude NOD/SCID/IL2rg−/− (NSIN) mice by knocking out Foxn1 from NOD/SCID/IL2rg−/− (NSI) mice using the CRISPR/Cas9 system. The NSIN mice were deficient in B, T, and NK cells and not only showed impaired T cell reconstitution and thymus regeneration after allogeneic bone marrow nucleated cell transplantation but also exhibited improved capacity to graft both leukemic and solid tumor cells compared with NSI, NOG, and NDG mice. Moreover, the NSIN mice facilitated the monitoring and in vivo imaging of both leukemia and solid tumors. Therefore, our NSIN mice provide a new platform for xenograft mouse models in basic and translational research.

The gene asialoglycoprotein receptor 1 (ASGR1) encodes a subunit of the asialoglycoprotein receptor. Here we report the generation of a human embryonic stem cell line WAe001-A-6 harbouring homozygous ASGR1 mutations using CRISPR/Cas9. The mutation involves a 37 bp deletion, resulting in a frame shift. The homozygous knockout WA01 cell line maintains a normal karyotype, typical stem cell morphology, pluripotency and differentiation potential in vitro.