Single Adeno-associated Virus Research Articles

All-in-one AAV-mediated Nrl gene inactivation rescues retinal degeneration in Pde6a mice.

Retinitis pigmentosa (RP) is a complex group of inherited retinal diseases characterized by progressive death of photoreceptor cells and eventual blindness. Pde6a, which encodes a cGMP-specific phosphodiesterase, is a crucial pathogenic gene for autosomal recessive RP (RP43); there is no effective therapy for this form of RP. The compact CRISPR/SaCas9 system, which can be packaged into a single adeno-associated virus, holds promise for simplifying effective gene therapy. Here, we demonstrated that all-in-one AAV-SaCas9-mediated Nrl gene inactivation can efficiently prevent retinal degeneration in a RP mouse model with Pde6anmf363/nmf363 mutation. We screened single guide RNAs (sgRNAs) capable of efficiently editing mouse Nrl gene in N2a cells and then achieved effective gene editing by using a single AAV to co-deliver SaCas9 and an optimal Nrl-sg2 into the mouse retina. Excitingly, in vivo inactivation of Nrl improved photoreceptor cell survival and rescued retinal function in treated Pde6a deficient mice. Thus, we showed that a practical, gene-independent method, AAV-SaCas9-mediated Nrl inactivation, holds promise for future therapeutic applications in patients with RP.

Engineered IscB-ωRNA system with expanded target range for base editing.

As the evolutionary ancestor of Cas9 nuclease, IscB proteins serve as compact RNA-guided DNA endonucleases and nickases, making them strong candidates for base editing. Nevertheless, the narrow targeting scope limits the application of IscB systems; thus, it is necessary to find more IscBs that recognize different target-adjacent motifs (TAMs). Here, we identified 10 of 19 uncharacterized IscB proteins from uncultured microbes with activity in mammalian cells. Through protein and ωRNA engineering, we further enhanced the activity of IscB ortholog IscB.m16 and expanded its TAM scope from MRNRAA to NNNGNA, resulting in a variant named IscB.m16*. By fusing the deaminase domains with IscB.m16* nickase, we generated IscB.m16*-derived base editors that exhibited robust base-editing efficiency in mammalian cells and effectively restored Duchenne muscular dystrophy proteins in diseased mice through single adeno-associated virus delivery. Thus, this study establishes a set of compact base-editing tools for basic research and therapeutic applications.

Developing small Cas9 hybrids using molecular modeling

The contraction of CAG/CTG repeats is an attractive approach to correct the mutation that causes at least 15 neuromuscular and neurodegenerative diseases, including Huntington’s disease and Myotonic Dystrophy type 1. Contractions can be achieved in vivo using the Cas9 D10A nickase from Streptococcus pyogenes (SpCas9) using a single guide RNA (sgRNA) against the repeat tract. One hurdle on the path to the clinic is that SpCas9 is too large to be packaged together with its sgRNA into a single adeno-associated virus. Here we aimed to circumvent this problem using the smaller Cas9 orthologue, SlugCas9, and the Cas9 ancestor OgeuIscB. We found them to be ineffective in inducing contractions, despite their advertised PAM sequences being compatible with CAG/CTG repeats. Thus, we further developed smaller Cas9 hybrids, made of the PAM interacting domain of S. pyogenes and the catalytic domains of the smaller Cas9 orthologues. We also designed the cognate sgRNA hybrids using molecular dynamic simulations and binding energy calculations. We found that the four Cas9/sgRNA hybrid pairs tested in human cells failed to edit their target sequences. We conclude that in silico approaches can identify functional changes caused by point mutations but are not sufficient for designing larger scale complexes of Cas9/sgRNA hybrids.

Cell-Specific Single Viral Vector CRISPR/Cas9 Editing and Genetically Encoded Tool Delivery in the Central and Peripheral Nervous Systems.

CRISPR/Cas9 gene editing represents an exciting avenue to study genes of unknown function and can be combined with genetically encoded tools such as fluorescent proteins, channelrhodopsins, DREADDs, and various biosensors to more deeply probe the function of these genes in different cell types. However, current strategies to also manipulate or visualize edited cells are challenging due to the large size of Cas9 proteins and the limited packaging capacity of adeno-associated viruses (AAVs). To overcome these constraints, we developed an alternative gene editing strategy using a single AAV vector and mouse lines that express Cre-dependent Cas9 to achieve efficient cell-type specific editing across the nervous system. Expressing Cre-dependent Cas9 from a genomic locus affords space to package guide RNAs for gene editing together with Cre-dependent, genetically encoded tools to manipulate, map, or monitor neurons using a single virus. We validated this strategy with three common tools in neuroscience: ChRonos, a channelrhodopsin, for studying synaptic transmission using optogenetics, GCaMP8f for recording Ca2+ transients using photometry, and mCherry for tracing axonal projections. We tested these tools in multiple brain regions and cell types, including GABAergic neurons in the nucleus accumbens, glutamatergic neurons projecting from the ventral pallidum to the lateral habenula, dopaminergic neurons in the ventral tegmental area, and proprioceptive neurons in the periphery. This flexible approach could help identify and test the function of novel genes affecting synaptic transmission, circuit activity, or morphology with a single viral injection.

Viral Delivery of Compact CRISPR-Cas12f for Gene Editing Applications.

Treating human genetic conditions invivo requires efficient delivery of the CRISPR gene editing machinery to the affected cells and organs. The gene editing field has seen clinical advances with ex vivo therapies and with invivo delivery to the liver using lipid nanoparticle technology. Adeno-associated virus (AAV) serotypes have been discovered and engineered to deliver genetic material to nearly every organ in the body. However, the large size of most CRISPR-Cas systems limits packaging into the viral genome and reduces drug development flexibility and manufacturing efficiency. Here, we demonstrate efficient CRISPR gene editing using a miniature CRISPR-Cas12f system with expanded genome targeting packaged into AAV particles. We identified efficient guides for four therapeutic gene targets and encoded the guides and the Cas12f nuclease into a single AAV. We then demonstrate editing in multiple cell lines, patient fibroblasts, and primary hepatocytes. We then screened the cells for off-target editing, demonstrating the safety of the therapeutics. These results represent an important step in applying CRISPR editing to diverse genetic sequences and organs in the body.

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-kilobase limit of cargo, SpCas9-mediated base editors can't be packaged into a single AAV vector, which hinders its 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 sgRNA, termed as STUminiBEs. We demonstrated the robust A-to-G conversion (54% on average) by STUminiABE or C-to-T conversion (45% on average) by STUminiCBE. We packaged STUminiCBE 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.

Therapeutic In Vivo Gene Editing Achieved by a Hypercompact CRISPR-Cas12f1 System Delivered with All-in-One Adeno-Associated Virus.

CRISPR-based gene therapies are making remarkable strides toward the clinic. But the large size of most widely used Cas endonucleases including Cas9 and Cas12a restricts their efficient delivery by the adeno-associated virus (AAV) for in vivo gene editing. Being exceptionally small, the recently engineered type V-F CRISPR-Cas12f1 systems can overcome the cargo packaging bottleneck and present as strong candidates for therapeutic applications. In this study, the pairwise editing efficiencies of different engineered Cas12f1/sgRNA scaffold combinations are systemically screened and optimized, and the CasMINI_v3.1/ge4.1 system is identified as being able to significantly boost the gene editing activity. Moreover, packaged into single AAV vectors and delivered via subretinal injection, CasMINI_v3.1/ge4.1 achieves remarkably high in vivo editing efficiencies, over 70% in transduced retinal cells. Further, the efficacy of this Cas12f1 system-based gene therapy to treat retinitis pigmentosa in RhoP23H mice is demonstrated by the therapeutic benefits achieved including rescued visual function and structural preservation. And minimal bystander editing activity is detected. This work advances and expands the therapeutic potential of the miniature Cas12f1 system to support efficient and accurate in vivo gene therapy.

Engineering a transposon-associated TnpB-ωRNA system for efficient gene editing and phenotypic correction of a tyrosinaemia mouse model

Transposon-associated ribonucleoprotein TnpB is known to be the ancestry endonuclease of diverse Cas12 effector proteins from type-V CRISPR system. Given its small size (408 aa), it is of interest to examine whether engineered TnpB could be used for efficient mammalian genome editing. Here, we showed that the gene editing activity of native TnpB from Deinococcus radiodurans (ISDra2 TnpB) in mouse embryos was already higher than previously identified small-sized Cas12f1. Further stepwise engineering of noncoding RNA (ωRNA or reRNA) component of TnpB significantly elevated the nuclease activity of TnpB. Notably, an optimized TnpB-ωRNA system could be efficiently delivered in vivo with single adeno-associated virus (AAV) and corrected the disease phenotype in a tyrosinaemia mouse model. Thus, the engineered miniature TnpB system represents a new addition to the current genome editing toolbox, with the unique feature of the smallest effector size that facilitate efficient AAV delivery for editing of cells and tissues.

Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization

The production of cell-type- and age-specific genetically modified mice is a powerful approach for unraveling unknown gene functions. Here, we present a simple and timesaving method that enables adeno-associated virus (AAV)-mediated cell type- and age-specific recombination in floxed mice. To achieve astrocyte-specific recombination in floxed Ai14 reporter mice, we intravenously injected blood-brain barrier-penetrating AAV-PHP.eB vectors expressing Cre recombinase (Cre) using the astrocyte-specific mouse glial fibrillary acidic protein (mGfaABC1D) promoter. However, we observed non-specific neuron-predominant transduction despite the use of an astrocyte-specific promoter. We speculated that subtle but continuous Cre expression in non-astrocytic cells triggers recombination, and that excess production of Cre in astrocytes inhibits recombination by forming Cre–DNA aggregates. Here, we resolved this paradoxical event by dividing a single AAV into two mGfaABC1D-promoter-driving AAV vectors, one expressing codon-optimized flippase (FlpO) and another expressing flippase recognition target-flanked rapidly degrading Cre (dCre), together with switching the neuron-tropic PHP.eB capsid to astrocyte-tropic AAV-F. Moreover, we found that the FlpO – dCre system with a target cell-tropic capsid can also function in neuron-targeting recombination in floxed mice.

Phage-assisted evolution of compact Cas9 variants targeting a simple NNG PAM.

Compact Cas9 nucleases hold great promise for therapeutic applications. Although several compact Cas9 nucleases have been developed, many genomic loci still could not be edited due to a lack of protospacer adjacent motifs (PAMs). We previously developed a compact SlugCas9 recognizing an NNGG PAM. Here we demonstrate that SlugCas9 displays comparable activity to SpCas9. We developed a simple phage-assisted evolution to engineer SlugCas9 for unique PAM requirements. Interestingly, we generated a SlugCas9 variant (SlugCas9-NNG) that could recognize an NNG PAM, expanding the targeting scope. We further developed a SlugCas9-NNG-based adenine base editor and demonstrated that it could be delivered by a single adeno-associated virus to disrupt PCSK9 splice donor and splice acceptor. These genome editors greatly enhance our ability for in vivo genome editing.

Lysosomal sialidase NEU1, its intracellular properties, deficiency, and use as a therapeutic agent.

Neuraminidase 1 (NEU1) is a lysosomal sialidase that cleaves terminal α-linked sialic acid residues from sialylglycans. NEU1 is biosynthesized in the rough endoplasmic reticulum (RER) lumen as an N-glycosylated protein to associate with its protective protein/cathepsin A (CTSA) and then form a lysosomal multienzyme complex (LMC) also containing β-galactosidase 1 (GLB1). Unlike other mammalian sialidases, including NEU2 to NEU4, NEU1 transport to lysosomes requires association of NEU1 with CTSA, binding of the CTSA carrying terminal mannose 6-phosphate (M6P)-type N-glycan with M6P receptor (M6PR), and intralysosomal NEU1 activation at acidic pH. In contrast, overexpression of the single NEU1 gene in mammalian cells causes intracellular NEU1 protein crystallization in the RER due to self-aggregation when intracellular CTSA is reduced to a relatively low level. Sialidosis (SiD) and galactosialidosis (GS) are autosomal recessive lysosomal storage diseases caused by the gene mutations of NEU1 and CTSA, respectively. These incurable diseases associate with the NEU1 deficiency, excessive accumulation of sialylglycans in neurovisceral organs, and systemic manifestations. We established a novel GS model mouse carrying homozygotic Ctsa IVS6 + 1g/a mutation causing partial exon 6 skipping with simultaneous deficiency of Ctsa and Neu1. Symptoms developed in the GS mice like those in juvenile/adult GS patients, such as myoclonic seizures, suppressed behavior, gargoyle-like face, edema, proctoptosis due to Neu1 deficiency, and sialylglycan accumulation associated with neurovisceral inflammation. We developed a modified NEU1 (modNEU1), which does not form protein crystals but is transported to lysosomes by co-expressed CTSA. In vivo gene therapy for GS and SiD utilizing a single adeno-associated virus (AAV) carrying modNEU1 and CTSA genes under dual promoter control will be created.

Prokaryotic voltage-gated sodium channels are more effective than endogenous Nav1.5 channels in rescuing cardiac action potential conduction: an in silico study.

Methods to augment Na+ current in cardiomyocytes hold potential for the treatment of various cardiac arrhythmias involving conduction slowing. Because the gene coding cardiac Na+ channel (Nav1.5) is too large to fit in a single adeno-associated virus (AAV) vector, new gene therapies are being developed to enhance endogenous Nav1.5 current (by overexpression of chaperon molecules or use of multiple AAV vectors) or to exogenously introduce prokaryotic voltage-gated Na+ channels (BacNav) whose gene size is significantly smaller than that of the Nav1.5. In this study, based on experimental measurements in heterologous expression systems, we developed an improved computational model of the BacNav channel, NavSheP D60A. We then compared in silico how NavSheP D60A expression vs. Nav1.5 augmentation affects the electrophysiology of cardiac tissue. We found that the incorporation of BacNav channels in both adult guinea pig and human cardiomyocyte models increased their excitability and reduced action potential duration. When compared with equivalent augmentation of Nav1.5 current in simulated settings of reduced tissue excitability, the addition of the BacNav current was superior in improving the safety of conduction under conditions of current source-load mismatch, reducing the vulnerability to unidirectional conduction block during premature pacing, preventing the instability and breakup of spiral waves, and normalizing the conduction and ECG in Brugada syndrome tissues with mutated Nav1.5. Overall, our studies show that compared with a potential enhancement of the endogenous Nav1.5 current, expression of the BacNav channels with their slower inactivation kinetics can provide greater anti-arrhythmic benefits in hearts with compromised action potential conduction.NEW & NOTEWORTHY Slow action potential conduction is a common cause of various cardiac arrhythmias; yet, current pharmacotherapies cannot augment cardiac conduction. This in silico study compared the efficacy of recently proposed antiarrhythmic gene therapy approaches that increase peak sodium current in cardiomyocytes. When compared with the augmentation of endogenous sodium current, expression of slower-inactivating bacterial sodium channels was superior in preventing conduction block and arrhythmia induction. These results further the promise of antiarrhythmic gene therapies targeting sodium channels.

Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility.

CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes N4AH and N5HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N3VRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. Combined with deaminases or reverse transcriptases, evoCjCas9 enables robust base and prime editing, with the small size of evoCjCas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems.

A strategy for Cas13 miniaturization based on the structure and AlphaFold

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.

Improved Cardiac Function in Postischemic Rats Using an Optimized Cardiac Reprogramming Cocktail Delivered in a Single Novel Adeno-Associated Virus.

Cardiac reprogramming is a technique to directly convert nonmyocytes into myocardial cells using genes or small molecules. This intervention provides functional benefit to the rodent heart when delivered at the time of myocardial infarction or activated transgenically up to 4 weeks after myocardial infarction. Yet, several hurdles have prevented the advancement of cardiac reprogramming for clinical use. Through a combination of screening and rational design, we identified a cardiac reprogramming cocktail that can be encoded in a single adeno-associated virus. We also created a novel adeno-associated virus capsid that can transduce cardiac fibroblasts more efficiently than available parental serotypes by mutating posttranslationally modified capsid residues. Because a constitutive promoter was needed to drive high expression of these cell fate-altering reprogramming factors, we included binding sites to a cardiomyocyte-restricted microRNA within the 3' untranslated region of the expression cassette that limits expression to nonmyocytes. After optimizing this expression cassette to reprogram human cardiac fibroblasts into induced cardiomyocyte-like cells in vitro, we also tested the ability of this capsid/cassette combination to confer functional benefit in acute mouse myocardial infarction and chronic rat myocardial infarction models. We demonstrated sustained, dose-dependent improvement in cardiac function when treating a rat model 2 weeks after myocardial infarction, showing that cardiac reprogramming, when delivered in a single, clinically relevant adeno-associated virus vector, can support functional improvement in the postremodeled heart. This benefit was not observed with GFP (green fluorescent protein) or a hepatocyte reprogramming cocktail and was achieved even in the presence of immunosuppression, supporting myocyte formation as the underlying mechanism. Collectively, these results advance the application of cardiac reprogramming gene therapy as a viable therapeutic approach to treat chronic heart failure resulting from ischemic injury.

Treatment of autosomal dominant retinitis pigmentosa caused by RHO-P23H mutation with high-fidelity Cas13X in mice

Mutations in Rhodopsin (RHO) gene commonly cause autosomal dominant retinitis pigmentosa (adRP) without effective therapeutic treatment so far. Compared with genomic DNA-targeting CRISPR-Cas9 system, Cas13 edits RNA for therapeutic applications, avoiding the risk of causing permanent changes in the genome. In particular, a compact and high-fidelity Cas13X (hfCas13X) recently has been developed to degrade targeted RNA with minimal collateral effects and could also be packaged in a single adeno-associated virus for efficient invivo delivery. In this study, we engineered single-guide RNA for hfCas13X to specifically knock down human mutant Rhodopsin transcripts RHO-P23H with minimal effect on wild-type transcripts. Moreover, treatment with hfCas13X alleviated the adRP progression in both RHO-P23H overexpression-induced and humanized hRHOP23H/WT mouse models. Our study indicates the potential of hfCas13X in treating adRP caused by RHO mutations and other genetic diseases.

Leverage of nuclease-deficient CasX for preventing pathological angiogenesis

Gene editing with a CRISPR/Cas system is a novel potential strategy for treating human diseases. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) δ suppresses retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Here we show that an innovative system of adeno-associated virus (AAV)-mediated CRISPR/nuclease-deficient (d)CasX fused with the Krueppel-associated box (KRAB) domain is leveraged to block (81.2%± 6.5%) invitro expression of p110δ, the catalytic subunit of PI3Kδ, encoded by Pik3cd. This CRISPR/dCasX-KRAB (4, 269bp) system is small enough to be fit into a single AAV vector. We then document that recombinant AAV serotype (rAAV)1 efficiently transduces vascular endothelial cells from pathologic retinal vessels, which show high expression of p110δ; furthermore, we demonstrate that blockade of retinal p110δ expression by intravitreally injected rAAV1-CRISPR/dCasX-KRAB targeting the Pik3cd promoter prevents (32.1%± 5.3%) retinal p110δ expression as well as pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. These data establish a strong foundation for treating pathological angiogenesis by AAV-mediated CRISPR interference with p110δ expression.

A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9

BackgroundAdenine 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.ResultsWe 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.ConclusionsWe 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.

Discovery of deaminase functions by structure-based protein clustering

The elucidation of protein function and its exploitation in bioengineering have greatly advanced the life sciences. Protein mining efforts generally rely on amino acid sequences rather than protein structures. We describe here the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities. We selected deaminase proteins to analyze and identified many previously unknown properties. We were surprised to find that most proteins in the DddA-like clade were not double-stranded DNA deaminases. We engineered the smallest single-strand-specific cytidine deaminase, enabling efficient cytosine base editor (CBE) to be packaged into a single adeno-associated virus (AAV). Importantly, we profiled a deaminase from this clade that edits robustly in soybean plants, which previously was inaccessible to CBEs. These discovered deaminases, based on AI-assisted structural predictions, greatly expand the utility of base editors for therapeutic and agricultural applications.

Screening an Effective Dual-Adeno-Associated Virus Split-Cytosine Base Editor System for C-to-T Conversion In Vivo.

The cytosine base editor (CBE) has shown promise as a gene editing tool for gene therapy, as it can convert cytidine to thymidine. Adeno-associated virus (AAV) has been widely used for in vivo gene therapy, but its limited 4.7 kb packing capacity presents challenges in delivering CBE by a single AAV. To address this, one feasible solution is to split CBE into two sections for dual-AAV delivery. In this study, we utilized BE3 as an example and constructed 22 potential split-BE3 pairs with the combination of 11 splitting sites and two split-inteins (Npu and Rma). These split-BE3 pairs were initially screened in the green fluorescent protein (GFP) reporter system, with six split-BE3 pairs selected for further evaluation. The subsequent screening of split-BE3 pairs was performed at two endogenous sites in 293T and HeLa cells, revealing that the split-BE3-Rma674, split-BE3-Rma713, and split-BE3-Rma1005 displayed effective C-to-T conversion after transfection. The effectiveness of dual-AAV split-BE3 was further validated in culture cells and adult mouse eyes. Of note, the split-BE3-Rma674 demonstrated the most efficient C-to-T editing after AAV infection, with a maximal editing efficiency of 23.29% ± 10.98% in the mouse retinal pigment epithelium cells in vivo. Overall, our study presents a novel split-BE3 system with effective C-to-T conversion, which could be applied to CBE-based in vivo gene therapy.