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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.

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.

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.

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.

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.

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.

The limb girdle muscular dystrophy type 2L (LGMD2L) is caused by mutations of the ANO5 gene in humans which encodes a 913 amino-acid integral membrane protein. Although cardiomyopathy has been reported in patients with an ANO5 mutation, the ANO5 mutant mice did not recapitulate this phenotype in previous studies. This study demonstrated that the ANO5−/− rabbits recapitulated the typical signs of cardiomyopathy with decreased ejection fraction (EF) and fraction shortening (FS) with increased interstitial fibrosis. This ANO5−/− rabbit model would promote basic research to comprehend the pathogenesis and mechanism of ANO5-related cardiomyopathy.

Various clinical differences have been observed between patients with the FBN1 gene mutation and those with the classical Marfan phenotype. Although FBN1 knockout (KO) or dominant-negative mutant mice are widely used as an animal model for Marfan syndrome (MFS), these mice cannot recapitulate the genotype/phenotype relationship of Marfanoid-progeroid-lipodystrophy (MPL) syndrome, which is caused by a mutation in the C-terminus of fibrillin-1, the penultimate exon of the FBN1 gene. Here, we describe the generation of a rabbit MPL model with C-terminal truncation of fibrillin-1 using a CRISPR/Cas9 system. FBN1 heterozygous (FBN1 Het) rabbits faithfully recapitulated the phenotypes of MFS, including muscle wasting and impaired connective tissue, ocular syndrome and aortic dilation. Moreover, skin symptoms, lipodystrophy, growth retardation and dysglycemia were also seen in these FBN1 Het rabbits, and have not been reported in other animal models. In conclusion, this novel rabbit model mimics the histopathological changes and functional defects of MPL syndrome, and could become a valuable model for studies of pathogenesis and drug screening for MPL syndrome.

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. [ABSTRACT FROM AUTHOR]

Premature aging syndromes are rare genetic disorders mimicking clinical and molecular features of aging. Products of the LMNA gene, primarily lamin A and C, are major components of the nuclear lamina. A recently identified group of premature aging syndromes was related to mutations of the LMNA gene. Although LMNA disorders have been identified in premature aging syndromes, affect specifically the skeletal muscles, cardiac muscles, and lipodystrophy, understanding the pathogenic mechanisms still need to be elucidated. Here, to establish a rabbit knockout (KO) model of premature aging syndromes, we performed precise LMNA targeting in rabbits via co-injection of Cas9/sgRNA mRNA into zygotes. The LMNA-KO rabbits exhibited reduced locomotion activity with abnormal stiff walking posture and a shortened stature, all of them died within 22 days. In addition, cardiomyopathy, muscular dystrophy, bone and joint abnormalities, as well as lipodystrophy were observed in LMNA-KO rabbits. In conclusion, the novel rabbit LMNA-KO model, displayed typical features of histopathological defects that are observed in premature aging syndromes, and may be utilized as a valuable resource for understanding the pathophysiological mechanisms of premature aging syndromes and elucidating mysteries of the normal process of aging in humans. [ABSTRACT FROM AUTHOR]

Cytidine base editors, which are composed of a cytidine deaminase fused to clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) nickase, enable the efficient conversion of the C·G base pair to T·A in various organisms. However, the currently used rat apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1(rA1)-based BE3 is often inefficient in target Cs that are immediately downstream of a G (GC context). Here, we observed that, with an 11-nt editing window, an optimized activation-induced cytidine deaminase (AID)-Cas9 fusion can efficiently convert C to T in a variety of sequence contexts in rabbits. Strikingly, the enhanced AID-Cas9 fusion (eAID-BE4max) has significant effectiveness of inducing Tyr p.R299H mutation in GC contexts (from 16.67 to 83.33%) in comparison with BE3 in founder rabbits. Furthermore, the engineered AID-Cas9 variants were produced with reduced bystander activity [eAID (N51G)-BE4max] and increased genome-targeting scope (eAID-NG-BE4max). Overall, this work provides a series of improved tools that were generated using optimized AID-Cas9 fusions and associated engineered variants that can be used for efficient and versatile C-to-T base editing, especially in GC contexts. [ABSTRACT FROM AUTHOR]

Paired-homeodomain transcription factor 4 (PAX4) gene encodes a transcription factor which plays an important role in the generation, differentiation, development, and survival of insulin-producing b-cells during mammalian pancreas development. PAX4 is a key diabetes mellitus (DM) susceptibility gene, which is associated with many different types of DM, including T1DM, T2DM, maturity onset diabetes of the young 9 (MODY9) and ketosis prone diabetes. In this study, a novel PAX4 gene knockout (KO) model was generated through co-injection of clustered regularly interspaced short palindromic repeats (CRISPR)- associated protein 9 (Cas9) mRNA/sgRNA into rabbit zygotes. Typical phenotypes of growth retardation, persistent hyperglycemia, decreased number of insulin-producing b cells and increased number of glucagon- producing a cells were observed in the homozygous PAX4 KO rabbits. Furthermore, DM associated phenotypes including diabetic nephropathy, hepatopathy, myopathy and cardiomyopathy were also observed in the homozygous PAX4 KO rabbits but not in the wild type (WT) controls and the heterozygous PAX4 KO rabbits. In summary, this is the first PAX4 gene KO rabbit model generated by CRISPR/Cas9 system. This novel rabbit model may provide a new platform for function study of PAX4 gene in rabbit and gene therapy of human DM in clinical trails [ABSTRACT FROM AUTHOR]