Evaluation of the effect of RNA secondary structure on Cas13d-mediated target RNA cleavage.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13d system was adapted as a powerful tool for targeting viral RNA sequences, making it a promising approach for antiviral strategies. Understanding the influence of template RNA structure on Cas13d binding and cleavage efficiency is crucial for optimizing its therapeutic potential. In this study, we investigated the effect of local RNA secondary structure on Cas13d activity. To do so, we varied the stability of a hairpin structure containing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target sequence, allowing us to determine the threshold RNA stability at which Cas13d activity is affected. Our results demonstrate that Cas13d possesses the ability to effectively bind and cleave highly stable RNA structures. Notably, we only observed a decrease in Cas13d activity in the case of exceptionally stable RNA hairpins with completely base-paired stems, which are rarely encountered in natural RNA molecules. A comparison of Cas13d and RNA interference (RNAi)-mediated cleavage of the same RNA targets demonstrated that RNAi is more sensitive for local target RNA structures than Cas13d. These results underscore the suitability of the CRISPR-Cas13d system for targeting viruses with highly structured RNA genomes.

Cas9 editing of ATXN1 in a spinocerebellar ataxia type 1 mice and human iPSC-derived neurons.

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disease caused by an expansion of the CAG repeat region of the ATXN1 gene. Currently there are no disease-modifying treatments; however, previous work has shown the potential of gene therapy, specifically RNAi, as a potential modality. Cas9 editing offers potential for these patients but has yet to be evaluated in SCA1 models. To test this, we first characterized the number of transgenes harbored in the common B05 mouse model of SCA1. Despite having five copies of the human mutant transgene, a 20% reduction of ATXN1 improved behavior deficits without increases in inflammatory markers. Importantly, the editing approach was confirmed in induced pluripotent stem cell (iPSC) neurons derived from patients with SCA1, promoting the translatability of the approach to patients.

Allele-specific CRISPR-Cas9 editing inactivates a single nucleotide variant associated with collagen VI muscular dystrophy.

The application of allele-specific gene editing tools can expand the therapeutic options for dominant genetic conditions, either via gene correction or via allelic gene inactivation in situations where haploinsufficiency is tolerated. Here, we used allele-targeted CRISPR-Cas9 guide RNAs (gRNAs) to introduce inactivating frameshifting indels at an SNV in the COL6A1 gene (c.868G>A; G290R), a variant that acts as dominant negative and that is associated with a severe form of congenital muscular dystrophy. We expressed SpCas9 along with allele-targeted gRNAs, without providing a repair template, in primary fibroblasts derived from four patients and one control subject. Amplicon deep sequencing for two gRNAs tested showed that single-nucleotide deletions accounted for the majority of indels introduced. While activity of the two gRNAs was greater at the G290R allele, both gRNAs were also active at the wild-type allele. To enhance allele selectivity, we introduced deliberate additional mismatches to one gRNA. One of these optimized gRNAs showed minimal activity at the WT allele, while generating productive edits and improving collagen VI matrix in cultured patient fibroblasts. This study strengthens the potential of gene editing to treat dominant-negative disorders, but also underscores the challenges in achieving allele selectivity with gRNAs.

Synergistic combination of RAD51-SCR7 improves CRISPR-Cas9 genome editing efficiency by preventing R-loop accumulation.

CRISPR-Cas9 has emerged as a powerful tool for genome editing. However, Cas9 genome editing faces challenges, including low efficiency and off-target effects. Here, we report that combined treatment with RAD51, a key factor in homologous recombination, and SCR7, a DNA ligase IV small-molecule inhibitor, enhances CRISPR-Cas9-mediated genome-editing efficiency in human embryonic kidney 293T and human induced pluripotent stem cells, as confirmed by cyro- transmission electron microscopy and functional analyses. First, our findings reveal the crucial role of RAD51 in homologous recombination (HR)-mediated DNA repair process. Elevated levels of exogenous RAD51 promote a post-replication step via single-strand DNA gap repair process, ensuring the completion of DNA replication. Second, using the all-in-one CRISPR-Cas9-RAD51 system, highly expressed RAD51 improved the multiple endogenous gene knockin/knockout efficiency and insertion/deletion (InDel) mutation by activating the HR-based repair pathway in concert with SCR7. Sanger sequencing shows distinct outcomes for RAD51-SCR7 in the ratio of InDel mutations in multiple genome sites. Third, RAD51-SCR7 combination can induce efficient R-loop resolution and DNA repair by enhanced HR process, which leads to DNA replication stalling and thus is advantageous to CRISPR-Cas9-based stable genome editing. Our study suggests promising applications in genome editing by enhancing CRISPR-Cas9 efficiency through RAD51 and SCR7, offering potential advancements in biotechnology and therapeutics.

Suppression of HSV-1 infection and viral reactivation by CRISPR-Cas9 gene editing in 2D and 3D culture models.

Although our understanding of herpes simplex virus type 1 (HSV-1) biology has been considerably enhanced, developing therapeutic strategies to eliminate HSV-1 in latently infected individuals remains a public health concern. Current antiviral drugs used for the treatment of HSV-1 complications are not specific and do not address latent infection. We recently developed a CRISPR-Cas9-based gene editing platform to specifically target the HSV-1 genome. In this study, we further used 2D Vero cell culture and 3D human induced pluripotent stem cell-derived cerebral organoid (CO) models to assess the effectiveness of our editing constructs targeting viral ICP0 or ICP27 genes. We found that targeting the ICP0 or ICP27 genes with AAV2-CRISPR-Cas9 vectors in Vero cells drastically suppressed HSV-1 replication. In addition, we productively infected COs with HSV-1, characterized the viral replication kinetics, and established a viral latency model. Finally, we discovered that ICP0- or ICP27-targeting AAV2-CRISPR-Cas9 vector significantly reduced viral rebound in the COs that were latently infected with HSV-1. In summary, our results suggest that CRISPR-Cas9 gene editing of HSV-1 is an efficient therapeutic approach to eliminate the latent viral reservoir and treat HSV-1-associated complications.

Discovery of novel disease-causing mutation in SSBP1 and its correction using adenine base editor to improve mitochondrial function.

Mutations in nuclear genes regulating mitochondrial DNA (mtDNA) replication are associated with mtDNA depletion syndromes. Using whole-genome sequencing, we identified a heterozygous mutation (c.272G>A:p.Arg91Gln) in single-stranded DNA-binding protein 1 (SSBP1), a crucial protein involved in mtDNA replisome. The proband manifested symptoms including sensorineural deafness, congenital cataract, optic atrophy, macular dystrophy, and myopathy. This mutation impeded multimer formation and DNA-binding affinity, leading to reduced efficiency of mtDNA replication, altered mitochondria dynamics, and compromised mitochondrial function. To correct this mutation, we tested two adenine base editor (ABE) variants on patient-derived fibroblasts. One variant, NG-Cas9-based ABE8e (NG-ABE8e), showed higher editing efficacy (≤30%) and enhanced mitochondrial replication and function, despite off-target editing frequencies; however, risks from bystander editing were limited due to silent mutations and off-target sites in non-translated regions. The other variant, NG-Cas9-based ABE8eWQ (NG-ABE8eWQ), had a safer therapeutic profile with very few off-target effects, but this came at the cost of lower editing efficacy (≤10% editing). Despite this, NG-ABE8eWQ-edited cells still restored replication and improved mtDNA copy number, which in turn recovery of compromised mitochondrial function. Taken together, base editing-based gene therapies may be a promising treatment for mitochondrial diseases, including those associated with SSBP1 mutations.

Evaluation of DNA minicircles for delivery of adenine and cytosine base editors using activatable gene on "GO" reporter imaging systems.

Over 30,000 point mutations are associated with debilitating diseases, including many cancer types, underscoring a critical need for targeted genomic solutions. CRISPR base editors, like adenine base editors (ABEs) and cytosine base editors (CBEs), enable precise modifications by converting adenine to guanine and cytosine to thymine, respectively. Challenges in efficiency and safety concerns regarding viral vectors used in delivery limit the scope of base editing. This study introduces non-viral minicircles, bacterial-backbone-free plasmids, as a delivery vehicle for ABEs and CBEs. The research uses cells engineered with the "Gene On" (GO) reporter gene systems for tracking minicircle-delivered ABEs, CBEs, or Cas9 nickase (control), using green fluorescent protein (GFPGO), bioluminescence reporter firefly luciferase (LUCGO), or a highly sensitive Akaluciferase (AkalucGO) designed in this study. The results show that transfection of minicircles expressing CBE or ABE resulted in significantly higher GFP expression and luminescence signals over controls, with minicircles demonstrating the most substantial editing. This study presents minicircles as a new strategy for base editor delivery and develops an enhanced bioluminescence imaging reporter system for tracking ABE activity. Future studies aim to evaluate the use of minicircles in preclinical cancer models, facilitating potential clinical applications.

Upregulation of utrophin improves the phenotype of Duchenne muscular dystrophy hiPSC-derived CMs.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease. Although it leads to muscle weakness, affected individuals predominantly die from cardiomyopathy, which remains uncurable. Accumulating evidence suggests that an overexpression of utrophin may counteract some of the pathophysiological outcomes of DMD. The aim of this study was to investigate the role of utrophin in dystrophin-deficient human cardiomyocytes (CMs) and to test whether an overexpression of utrophin, implemented via the CRISPR-deadCas9-VP64 system, can improve their phenotype. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) lacking either dystrophin (DMD) or both dystrophin and utrophin (DMD KO/UTRN(+/-)). We carried out proteome analysis, which revealed considerable differences in the proteins related to muscle contraction, cell-cell adhesion, and extracellular matrix organization. Furthermore, we evaluated the role of utrophin in maintaining the physiological properties of DMD hiPSC-CMs using atomic force microscopy, patch-clamp, and Ca2+ oscillation analysis. Our results showed higher values of afterhyperpolarization and altered patterns of cytosolic Ca2+ oscillations in DMD; the latter was further disturbed in DMD KO/UTRN(+/-) hiPSC-CMs. Utrophin upregulation improved both parameters. Our findings demonstrate for the first time that utrophin maintains the physiological functions of DMD hiPSC-CMs, and that its upregulation can compensate for the loss of dystrophin.

Protection of animals against devastating RNA viruses using CRISPR-Cas13s.

The intrinsic nature of CRISPR-Cas in conferring immunity to bacteria and archaea has been repurposed to combat pathogenic agents in mammalian and plant cells. In this regard, CRISPR-Cas13 systems have proved their remarkable potential for single-strand RNA viruses targeting. Here, different types of Cas13 orthologs were applied to knockdown foot-and-mouth disease virus (FMDV), a highly contagious disease of a wide variety of species with genetically diverse strains and is widely geographically distributed. Using programmable CRISPR RNAs capable of targeting conserved regions of the viral genome, all Cas13s from CRISPR system type VI (subtype A/B/D) could comprehensively target and repress different serotypes of FMDV virus. This approach has the potential to destroy all strains of a virus as targets the ultra-conserved regions of genome. We experimentally compared the silencing efficiency of CRISPR and RNAi by designing the most effective short hairpin RNAs according to our developed scoring system and observed comparable results. This study showed successful usage of various Cas13 enzymes for suppression of FMDV, which provides a flexible strategy to battle with other animal infectious RNA viruses, an underdeveloped field in the biotechnology scope.

Functional benefit of CRISPR-Cas9-induced allele deletion for RYR1 dominant mutation.

More than 700 pathogenic or probably pathogenic variations have been identified in the RYR1 gene causing various myopathies collectively known as "RYR1-related myopathies." There is no treatment for these myopathies, and gene therapy stands out as one of the most promising approaches. In the context of a dominant form of central core disease due to a RYR1 mutation, we aimed at showing the functional benefit of inactivating specifically the mutated RYR1 allele by guiding CRISPR-Cas9 cleavages onto frequent single-nucleotide polymorphisms (SNPs) segregating on the same chromosome. Whole-genome sequencing was used to pinpoint SNPs localized on the mutant RYR1 allele and identified specific CRISPR-Cas9 guide RNAs. Lentiviruses encoding these guide RNAs and the SpCas9 nuclease were used to transduce immortalized patient myoblasts, inducing the specific deletion of the mutant RYR1 allele. The efficiency of the deletion was assessed at DNA and RNA levels, and at the functional level after monitoring calcium release induced by the stimulation of the RyR1-channel. This study provides in cellulo proof of concept regarding the benefits of mutant RYR1 allele deletion, in the case of a dominant RYR1 mutation, from both a molecular and functional perspective, and could apply potentially to 20% of all patients with a RYR1 mutation.

Temporal restriction of Cas9 expression improves CRISPR-mediated deletion efficacy and fidelity.

Clinical application of CRISPR-Cas9 technology for large deletions of somatic mutations is inefficient, and methods to improve utility suffer from our inability to rapidly assess mono- vs. biallelic deletions. Here we establish a model system for investigating allelic heterogeneity at the single-cell level and identify indel scarring from non-simultaneous nuclease activity at gRNA cut sites as a major barrier to CRISPR-del efficacy both in vitro and in vivo. We show that non-simultaneous nuclease activity is partially prevented via restriction of CRISPR-Cas9 expression via inducible adeno-associated viruses (AAVs) or lipid nanoparticles (LNPs). Inducible AAV-based expression of CRISPR-del machinery significantly improved mono- and biallelic deletion frequency in vivo, supporting the use of the Xon cassette over traditional constitutively expressing AAV approaches. These data depicting improvements to deletions and insight into allelic heterogeneity after CRISPR-del will inform therapeutic approaches for phenotypes that require either large mono- or biallelic deletions, such as autosomal recessive diseases or where mutant allele-specific gRNAs are not readily available, or in situations where the targeted sequence for excision is located multiple times in a genome.

Base editing rescues acid α-glucosidase function in infantile-onset Pompe disease patient-derived cells.

Infantile-onset Pompe disease (IOPD) results from pathogenic variants in the GAA gene, which encodes acid α-glucosidase. The correction of pathogenic variants through genome editing may be a valuable one-time therapy for PD and improve upon the current standard of care. We performed adenine base editing in human dermal fibroblasts harboring three transition nonsense variants, c.2227C>T (p.Q743∗; IOPD-1), c.2560C>T (p.R854∗; IOPD-2), and c.2608C>T (p.R870∗; IOPD-3). Up to 96% adenine deamination of target variants was observed, with minimal editing across >50 off-target sites. Post-base editing, expressed GAA protein was up to 0.66-fold normal (unaffected fibroblasts), an improvement over affected fibroblasts wherein GAA was undetectable. GAA enzyme activity was between 81.91 ± 13.51 and 129.98 ± 9.33 units/mg protein at 28 days post-transfection, which falls within the normal range (50-200 units/mg protein). LAMP2 protein was significantly decreased in the most robustly edited cell line, IOPD-3, indicating reduced lysosomal burden. Taken together, the findings reported herein demonstrate that base editing results in efficacious adenine deamination, restoration of GAA expression and activity, and reduction in lysosomal burden in the most robustly edited cells. Future work will assess base editing outcomes and the impact on Pompe pathology in two mouse models, Gaa c.2227C>T and Gaa c.2560C>T.

Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47 phox -deficient chronic granulomatous disease.

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox , an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47 phox and p67 phox , ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

Aberrant spliceosome activity via elevated intron retention and upregulation and phosphorylation of SF3B1 in chronic lymphocytic leukemia.

Splicing factor 3b subunit 1 (SF3B1) is the largest subunit and core component of the spliceosome. Inhibition of SF3B1 was associated with an increase in broad intron retention (IR) on most transcripts, suggesting that IR can be used as a marker of spliceosome inhibition in chronic lymphocytic leukemia (CLL) cells. Furthermore, we separately analyzed exonic and intronic mapped reads on annotated RNA-sequencing transcripts obtained from B cells (n = 98 CLL patients) and healthy volunteers (n = 9). We measured intron/exon ratio to use that as a surrogate for alternative RNA splicing (ARS) and found that 66% of CLL-B cell transcripts had significant IR elevation compared with normal B cells (NBCs) and that correlated with mRNA downregulation and low expression levels. Transcripts with the highest IR levels belonged to biological pathways associated with gene expression and RNA splicing. A >2-fold increase of active pSF3B1 was observed in CLL-B cells compared with NBCs. Additionally, when the CLL-B cells were treated with macrolides (pladienolide-B), a significant decrease in pSF3B1, but not total SF3B1 protein, was observed. These findings suggest that IR/ARS is increased in CLL, which is associated with SF3B1 phosphorylation and susceptibility to SF3B1 inhibitors. These data provide additional support to the relevance of ARS in carcinogenesis and evidence of pSF3B1 participation in this process.

Precise correction of a spectrum of ß-thalassemia mutations in coding and non-coding regions by base editors.

ß-thalassemia/HbE results from mutations in the ß-globin locus that impede the production of functional adult hemoglobin. Base editors (BEs) could facilitate the correction of the point mutations with minimal or no indel creation, but its efficiency and bystander editing for the correction of ß-thalassemia mutations in coding and non-coding regions remains unexplored. Here, we screened BE variants in HUDEP-2 cells for their ability to correct a spectrum of ß-thalassemia mutations that were integrated into the genome as fragments of HBB. The identified targets were introduced into their endogenous genomic location using BEs and Cas9/homology-directed repair (HDR) to create cellular models with ß-thalassemia/HbE. These ß-thalassemia/HbE models were then used to assess the efficiency of correction in the native locus and functional ß-globin restoration. Most bystander edits produced near target sites did not interfere with adult hemoglobin expression and are not predicted to be pathogenic. Further, the effectiveness of BE was validated for the correction of the pathogenic HbE variant in severe ß0/ßE-thalassaemia patient cells. Overall, our study establishes a novel platform to screen and select optimal BE tools for therapeutic genome editing by demonstrating the precise, efficient, and scarless correction of pathogenic point mutations spanning multiple regions of HBB including the promoter, intron, and exons.

Engineering miniature CRISPR-Cas Un1Cas12f1 for efficient base editing.

Adeno-associated virus (AAV) is a relatively safe and efficient vector for gene therapy. However, due to its 4.7-kb limit of cargo, SpCas9-mediated base editors cannot be packaged into a single AAV vector, which hinders their clinical application. The development of efficient miniature base editors becomes an urgent need. Un1Cas12f1 is a class II V-F-type CRISPR-Cas protein with only 529 amino acids. Although Un1Cas12f1 has been engineered to be a base editor in mammalian cells, the base-editing efficiency is less than 10%, which limits its therapeutic applications. Here, we developed hypercompact and high-efficiency base editors by engineering Un1Cas12f1, fusing non-specific DNA binding protein Sso7d, and truncating single guide RNA (sgRNA), termed STUminiBEs. We demonstrated robust A-to-G conversion (54% on average) by STUminiABEs or C-to-T conversion (45% on average) by STUminiCBEs. We packaged STUminiCBEs into AAVs and successfully introduced a premature stop codon on the PCSK9 gene in mammalian cells. In sum, STUminiBEs are efficient miniature base editors and could readily be packaged into AAVs for biological research or biomedical applications.

CRISPR-based editing strategies to rectify EYA1 complex genomic rearrangement linked to haploinsufficiency.

Pathogenic structure variations (SVs) are associated with various types of cancer and rare genetic diseases. Recent studies have used Cas9 nuclease with paired guide RNAs (gRNAs) to generate targeted chromosomal rearrangements, focusing on producing fusion proteins that cause cancer, whereas research on precision genome editing for rectifying SVs is limited. In this study, we identified a novel complex genomic rearrangement (CGR), specifically an EYA1 inversion with a deletion, implicated in branchio-oto-renal/branchio-oto syndrome. To address this, two CRISPR-based approaches were tested. First, we used Cas9 nuclease and paired gRNAs tailored to the patient's genome. The dual CRISPR-Cas9 system induced efficient correction of paracentric inversion in patient-derived fibroblast, and effectively restored the expression of EYA1 mRNA and protein, along with its transcriptional activity required to regulate the target gene expression. Additionally, we used CRISPR activation (CRISPRa), which leads to the upregulation of EYA1 mRNA expression in patient-derived fibroblasts. Moreover, CRISPRa significantly improved EYA1 protein expression and transcriptional activity essential for target gene expression. This suggests that CRISPRa-based gene therapies could offer substantial translational potential for approximately 70% of disease-causing EYA1 variants responsible for haploinsufficiency. Our findings demonstrate the potential of CRISPR-guided genome editing for correcting SVs, including those with EYA1 CGR linked to haploinsufficiency.

Context base editing for splice correction of IVSI-110 ß-thalassemia.

ß-Thalassemia is brought about by defective ß-globin (HBB [hemoglobin subunit ß]) formation and, in severe cases, requires regular blood transfusion and iron chelation for survival. Genome editing of hematopoietic stem cells allows correction of underlying mutations as curative therapy. As potentially safer alternatives to double-strand-break-based editors, base editors (BEs) catalyze base transitions for precision editing of DNA target sites, prompting us to reclone and evaluate two recently published adenine BEs (ABEs; SpRY and SpG) with relaxed protospacer adjacent motif requirements for their ability to correct the common HBBIVSI-110(G>A) splice mutation. Nucleofection of ABE components as RNA into patient-derived CD34+ cells achieved up to 90% editing of upstream sequence elements critical for aberrant splicing, allowing full characterization of the on-target base-editing profile of each ABE and the detection of differences in on-target insertions and deletions. In addition, this study identifies opposing effects on splice correction for two neighboring context bases, establishes the frequency distribution of multiple BE editing events in the editing window, and shows high-efficiency functional correction of HBBIVSI-110(G>A) for our ABEs, including at the levels of RNA, protein, and erythroid differentiation.

Expanding RNA editing toolkit using an IDR-based strategy.

RNA base editors should ideally be free of immunogenicity, compact, efficient, and specific, which has not been achieved for C > U editing. Here we first describe a compact C > U editor entirely of human origin, created by fusing the human C > U editing enzyme RESCUE-S to Cas inspired RNA targeting system (CIRTS), a tiny, human-originated programmable RNA-binding domain. This editor, CIRTS-RESCUEv1 (V1), was inefficient. Remarkably, a short histidine-rich domain (HRD), which is derived from the internal disordered region (IDR) in the human CYCT1, a protein capable of liquid-liquid phase separation (LLPS), enhanced V1 editing at on-targets as well as off-targets, the latter effect being minor. The V1-HRD fusion protein formed puncta characteristic of LLPS, and various other IDRs (but not an LLPS-impaired mutant) could replace HRD to effectively induce puncta and potentiate V1, suggesting that the diverse domains acted via a common, LLPS-based mechanism. Importantly, the HRD fusion strategy was applicable to various other types of C > U RNA editors. Our study expands the RNA editing toolbox and showcases a general method for stimulating C > U RNA base editors.

Correction of human nonsense mutation via adenine base editing for Duchenne muscular dystrophy treatment in mouse.

Duchenne muscular dystrophy (DMD) is the most prevalent herediatry disease in men, characterized by dystrophin deficiency, progressive muscle wasting, cardiac insufficiency, and premature mortality, with no effective therapeutic options. Here, we investigated whether adenine base editing can correct pathological nonsense point mutations leading to premature stop codons in the dystrophin gene. We identified 27 causative nonsense mutations in our DMD patient cohort. Treatment with adenine base editor (ABE) could restore dystrophin expression by direct A-to-G editing of pathological nonsense mutations in cardiomyocytes generated from DMD patient-derived induced pluripotent stem cells. We also generated two humanized mouse models of DMD expressing mutation-bearing exons 23 or 30 of human dystrophin gene. Intramuscular administration of ABE, driven by ubiquitous or muscle-specific promoters could correct these nonsense mutations in vivo, albeit with higher efficiency in exon 30, restoring dystrophin expression in skeletal fibers of humanized DMD mice. Moreover, a single systemic delivery of ABE with human single guide RNA (sgRNA) could induce body-wide dystrophin expression and improve muscle function in rotarod tests of humanized DMD mice. These findings demonstrate that ABE with human sgRNAs can confer therapeutic alleviation of DMD in mice, providing a basis for development of adenine base editing therapies in monogenic diseases.