Coiled-coil heterodimer-mediated split base editing systems enable flexible and robust nucleotide substitutions

Domain: chemistry
Published: 2026-01-17
Editorial outcome: major_revision

Abstract
Base editors (BEs) enable precise base substitutions, but their size exceeds the packaging capacity of adeno-associated virus (AAV), impeding in vivo applications. Here we design a split BE system that recruits deaminases to Cas9 nickase via coiled-coil heterodimers, resulting in various coiled-coil heterodimers-mediated base editors (CC-BEs), including cytidine base editor (CC-CBE), adenine base editor (CC-ABE), and their derivatives. We reveal that CC-BEs maintain and even improve the editing efficiency of the original unsplit BEs across various cell types and editing scopes, achieving maximum enhancements of 9.6-fold in human immortalized cells and 12.4-fold in primary somatic cells for CC-CBE. Using CC-ABE, we validate in vivo editing efficiency and successfully achieve A-to-G conversion in the Pcsk9 and Dmd genes via dual-AAV vectors in mice. Altogether, we develop a simple and universal strategy to address the challenges posed by the large size of BEs without compromising editing efficiency for base substitutions in vivo.

Introduction
As a precise gene editing tool, the DNA base editor is constructed based on the CRISPR/Cas system in conjunction with deaminases, glycosylases, or their derivatives, enabling base pair substitutions without inducing double-strand DNA breaks or necessitating exogenous donor templates1,2. Currently, a variety of base editors have been developed to enable multiple types of base pair substitutions, such as ABE (A-to-G)3, CBE (C-to-T)4,5, CGBE (C-to-G)6–8, AYBE (A-to-Y)9, DAF-BEs (T-to-G, C-to-G)10, and TSBE (T-to-G, T-to-C)8,11. Base editing technology demonstrates significant potential applications in crop breeding12, generation of animal models relevant to human diseases13–15, genetic screening16, and lineage tracing17,18, particularly offering substantial possibilities for the treatment of numerous genetic diseases associated with pathogenic point mutations19.

Given its relatively low pathogenicity, low immunogenicity, broad tissue tropism, mature production processes, and ability to establish long-term gene expression, adeno-associated virus (AAV) emerges as a potent platform for gene therapy delivery20,21 compared to other delivery tools like lipid nanoparticles (LNPs)22, virus-like particles (VLPs)23, enveloped delivery vehicles (EDVs)24, and selective endogenous encapsidation for cellular delivery (SEND)25. However, the AAV can only package cargo up to about 4.7 kb, inherently smaller than the size of SpCas9-based base editors26,27, impeding their therapeutic application. To bypass this limitation, intein-mediated split base editors with dual-AAV strategy27–31 and miniature base editors32–36 delivered via a single AAV particle have been developed. These strategies have their own limitations in terms of application. Split inteins self-assemble and undergo the ligation of the N- and C-termini of a functional protein through post-translational protein trans-splicing, thereby forming a fully functional protein and excising the intein in the process37. However, the split intein-mediated protein trans-splicing strategy could potentially alter protein function due to the adjacent residues at the extein/intein boundaries on which inteins heavily rely, which are left behind as “footprints”38. Another disadvantage is that selecting the split sites of functional proteins is challenging and may compromise the efficiency of the protein39,40. Miniature nucleases, such as Cas12f, TnpB, and IscB, are utilized to develop miniature base editors through combinations with cytosine and adenine deaminases32–35,41,42. Nevertheless, these miniature gene editing tools exhibit low editing activity and limited editing scope, fundamentally restricting precision targeting43,44. Therefore, there is an unmet need to develop a flexible and efficient base editing system for in vivo single-base substitutions.

Heterodimeric coiled-coil (CC) peptides are typically composed of two alpha helices winding around each other through hydrophobic and electrostatic interactions, enabling highly specific and tight dimerization45,46. Dimerization of the base editor system via CC peptides can offer distinct advantages over the existing split strategies, as this approach does not require selecting splitting sites for the nuclease or impose an additional burden to the AAV vector capacity. Here, we develop a CC-BE system that recruits deaminase to Cas9 nickase via CC dimer-forming modules and demonstrate that CC-BE can serve as a general base substitution platform for conferring high efficiency and flexibility. We generate 5 different versions of CC-BEs, including CC-ABE8e, CC-BE3, CC-ABE9, CC-TadCBE, and CC-AYBE. All 5 versions of CC-BEs exhibit similar or even higher efficiencies of base editing compared to the unsplit BE at most of the loci we tested. Furthermore, CC-BEs enhance or maintain the editing efficacy of unsplit BE across various cell types (HEK293T, MEF, and PFF) and targeting scopes (NGG PAM and NG PAM), robustly demonstrating the versatility of the CC-BE system. Finally, we evaluate the in vivo application of CC-BE using dual-AAV vectors to edit the mouse Pcsk9 gene and correct the Dmd gene, demonstrating high targeted base editing efficiencies of up to 79.0% and 21.9%, respectively. This validates their considerable potential as a simple, flexible, and efficient gene-editing tool for therapeutic applications.

Results
To advance towards a split cytosine base editor, we split CBE into two parts (APOBEC3 and nCas9-UGI) and each part was fused to coiled-coil dimer-forming peptides, P3–P4 pair or N5–N6 pair, so that the N- and C-terminus of CBE were subject to AAV packaging size restriction. We fused P3 or N5 to the C-terminus of APOBEC3 and P4 or N6 to the N-terminus of nCas9 (Cas9 nickase, D10A) (Fig. 1a, b). AlphaFold 3 3D structure predictions47 showed that in the presence of two sections, the pair P3 and P4 or N5 and N6 heterodimerizes, bringing APOBEC3 and nCas9-UGI into proximity and facilitating their assembly into a functional unit (Fig. 1a). As the control, a direct split CBE system without affinity modules and the previously mentioned split-intein CBE system27, in which the inteins splice at Cys574 or Ser714, were constructed (Fig. 1b).

To more accurately assess editing efficiency and eliminate the impact of transfection efficiency, we introduced EGFP into the N-terminal vector of CC-BE, mCherry into the C-terminal vector of CC-BE, and EGFP into the unsplit BE vector. Fluorescence-activated cell sorting was performed to evaluate base editing efficiency after transfection (Supplementary Fig. 1a). Untreated cells were used as a control for setting the gates for cell sorting (Supplementary Fig. 1b). For the sBE-P3P4, sBE-N5N6, and sBE-ctrl groups, mCherry and EGFP double-positive cells were collected; for the unsplit BE group, EGFP-positive cells were collected for subsequent analysis (Supplementary Fig. 1c).

We first compared editing efficiency of CC-CBE with unsplit CBE, direct split CBE, and split-intein CBE across 2 endogenous loci in sorted HEK293T cells (Fig. 1c). The results showed that the precise editing efficiencies of sCBE-P3P4 and sCBE-N5N6 were higher than those of unsplit CBE and direct split CBE (Fig. 1c). And compared to split-intein CBE systems, CC-CBE showed superior or similar efficiencies (Fig. 1c). As we further investigated, the levels of undesirable byproducts of CC-CBE were similar or slightly lower than those of intact CBE (Fig. 1d). Accordingly, these data suggest that the performance of CC-CBE is superior to or comparable with the widely used split CBE strategies. To demonstrate that two CBE components can reassemble into an active form upon coiled-coil heterodimers, the direct split CBE system without affinity modules was used as a control (hereafter sCBE-ctrl) in subsequent studies.

We next examined the universality of CC-CBE at 9 endogenous loci to proceed to a broader scale (Fig. 1e–g). HEK293T cells were transfected with the unsplit CBE, CC-CBE, or directly split CBE system with various sgRNAs to target 9 sites, then sorted to evaluate base editing efficiencies. Consistent with the results presented above, CC-CBE exhibited significantly higher on-target activity at all tested loci compared to the unsplit CBE and sCBE-ctrl (Fig. 1e, f). Among the 9 genomic sites tested, the sCBE-P3P4 demonstrated a mean C-to-T conversion rate of 69.8% (ranging from 41.9% to 90.6%), achieved an average 2.8-fold improvement compared to intact BE3 (ranging from 6.3% to 38.2%, averaging 24.9%) (Fig. 1e). The maximum efficiency improvement for sCBE-P3P4 was 9.6-fold at RNF2 site 2 (from 6.3% to 60.4%) (Fig. 1e). The sCBE-N5N6 exhibited a mean C-to-T conversion rate of 71.4% (ranging from 44.5% to 92.7%), achieved an average 2.9-fold improvement relative to intact BE3 with a maximum improvement of 7.1-fold at RNF2 site 2 (from 6.3% to 44.5%) (Fig. 1e). And the indel frequencies of CC-CBE were similar or lower than those of intact CBE at 4 loci (Fig. 1g). In contrast, the direct split CBE showed unstable performance in terms of both editing efficiencies and indels (Fig. 1e–g).

We next examined the editing windows of CC-CBE, direct split CBE, and intact CBE. The observed editing window for CC-CBE across all loci spanned position 3–13, while direct split CBE displayed unpredictable pattern owing to the absence of affinity modules. (Fig. 1f, h). These results showed that CC-CBE exhibited broader editing window compared to unsplit CBE, thereby extending the utility of CBE beyond the original editing window (position 4–8).

To provide a more comprehensive characterization of CC-CBE, we analyzed its base editing performance in transfected bulk HEK293T cells (Supplementary Fig. 2a–d). The data showed that sCBE-P3P4 exhibited higher precise editing efficiencies (range from 12.65% to 28.11%) than original unsplit CBE (range from 6.08% to 17.37%) at five out of six loci, while sCBE-N5N6 achieved higher efficiencies (range from 9.83% to 33.70%) at all the tested loci (Supplementary Fig. 2a, b). The sCBE-P3P4 induced lower indel frequencies than the unsplit CBE at all six loci, while sCBE-N5N6 showed variable indel frequencies across loci yet remained within a tolerable range (Supplementary Fig. 2c). A broad editing window of CC-CBE was observed in bulk transfected cells (Supplementary Fig. 2d). The data presented here were largely consistent with those obtained from sorted cells.

We noticed that the constructs for CC-BE contain a total of 4×NLS, whereas the unsplit BE contains only 2×NLS. We therefore hypothesized that the differences in editing efficiency between CC-CBE and CBE were due to the varying numbers of NLS. To test this hypothesis, we constructed an unsplit CBE containing 4×NLS for the evaluation of editing efficiency (Supplementary Fig. 3a). Our results indicated that the C-to-T efficiency of 4×NLS-CBE was slightly higher than or comparable to that of 2×NLS-CBE at 3 loci out of 4 edits; however, it still did not exceed that of CC-CBE (Supplementary Fig. 3b). The indel frequencies of 4×NLS-CBE were similar to or higher than those of 2×NLS-CBE (Supplementary Fig. 3c). This suggests that the increased editing efficiency of CC-CBE is not attributable to differences in the number of NLS. We also compared the Cas9 protein expression levels between CC-CBE and intact CBE using Western blot analysis (Supplementary Fig. 4). No obvious differences were observed, suggesting that the differences in editing efficiency may not arise from variations in Cas9 protein expression levels. AI-based prediction of the 3D protein structures of intact CBE and CC-CBE could be used to better elucidate the variances in editing efficiencies (Fig. 1a). Due to the presence of coiled-coil heterodimers, the base editor was reconstituted, bringing UGI and APOBEC3 together compared to intact CBE. We reasoned that the conformational change contributed to a particularly noticeable change in the overall performance of the CC-CBE. Therefore, the results above support the conclusion that CC-CBE demonstrates a highly adaptable strategy for enhancing the efficiency of CBE.

To facilitate the flexibility of adenine base editor, we constructed CC-ABE system by splitting ABE into two fragments using coiled-coil heterodimers. As ABE8e is the most efficient and versatile ABE variant48, we first chose this to test our CC-ABE system. Through the structure predicted by AlphaFold 3, we found that coiled-coil heterodimers allow TadA8e and nCas9 to dimerize at the target site (Fig. 2a). In the design of CC-ABE8e, the C-terminus of TadA8e was fused to P3 or N5 and P4 or N6 was attached to the N-terminus of nCas9 (Fig. 2b). We designated these ABE systems as sABE8e-P3P4 and sABE8e-N5N6, respectively (Fig. 2b). As a control for CC-ABE8e system, we devised the direct split ABE (sABE8e-ctrl) omitted the respective affinity modules (Fig. 2b). To investigate the performance of CC-ABE in sorted HEK293T cells, we compared its editing efficiency with that of intact ABE8e, sABE8e-ctrl, and split-intein ABE8e (Npu C574, Npu S714) at three human endogenous genomic sites (Fig. 2c). We observed that CC-ABE8e achieved at least equivalent level of precise editing efficiencies compared to Npu C574, Npu S714, intact ABE8e and sABE8e-ctrl at two loci. sABE8e-P3P4 exhibited better performance compared to Npu S714 and sABE8e-ctrl, while showing lower efficiency to Npu C574 and intact ABE8e at FANCF site 3 (Fig. 2c). The undesired indel byproducts generated by CC-ABE8e were lower than those of sABE8e-ctrl, intact ABE8e, and split-intein ABE8e at 2 of the 3 target sites (Fig. 2d).

Next, we compared the editing efficiencies of CC-ABE8e with sABE8e-ctrl and intact ABE8e at 13 human genomic loci in sorted HEK293T cells, aiming to explore the performance of CC-ABE8e across a wider range (Fig. 2e–h). We found that compared to sABE8e-ctrl (3.23% to 95.52%, averaging 64.88%), CC-ABE8e (sABE8e-P3P4, 6.61% to 96.75%, averaging 76.07%; and sABE8e-N5N6, 2.64% to 92.48%, averaging 67.23%) had the higher editing efficiency, which displayed similar level to intact ABE8e (9.80% to 95.12%, averaging 76.13%) (Fig. 2e, f). The undesired indel rates of CC-ABE8e were lower than those of sABE8e-ctrl at 7 of the 13 target sites (Fig. 2g). Compared to unsplit ABE8e, CC-ABE8e showed lower undesired indel rates at 4 loci, and comparable rates at 6 loci (Fig. 2g). Subsequently, the average A-to-G conversion rates within the protospacers of all loci were calculated. The results demonstrated that CC-ABE8e (sABE8e-P3P4 and sABE8e-N5N6) induced a higher frequency of A-to-G conversions at positions A9–A14 compared to intact ABE and sABE8e-ctrl, while the efficiency at A5–A8 remained similar to intact ABE (Fig. 2h).

We next evaluated the base editing of CC-ABE in transfected bulk HEK293T cells (Supplementary Fig. 2e–h). CC-ABE showed higher or minimally comparable editing efficiencies relative to sABE-ctrl, and comparable or lower efficiencies compared with the unsplit ABE (Supplementary Fig. 2e). Meanwhile, sABE-P3P4 demonstrated better performance than sABE-N5N6. (Supplementary Fig. 2e, f). In terms of indels, sABE-P3P4 exhibited lower efficiencies than unsplit ABE at 4 out of 6 loci, while sABE-N5N6 achieved lower efficiencies at 5 out of 6 loci (Supplementary Fig. 2g). The relative A-to-G conversion efficiencies within the protospacers were consistent with those observed in sorted cells (Supplementary Fig. 2h).

The A-to-G efficiency of 4×NLS-ABE was slightly higher than or comparable to that of 2×NLS-ABE, while it was slightly lower than or comparable to that of CC-ABE (Supplementary Fig. 3d). The indel frequencies of 4×NLS-ABE were similar to or lower than those of 2×NLS-ABE (Supplementary Fig. 3e). The Cas9 expression levels of CC-ABE and intact ABE were comparable (Supplementary Fig. 4). Given the high efficiencies observed in previous results, we hypothesize that splitting CC-ABE into two separate components (TadA8e-P3/N5 and P4/N6-nCas9) may increase the off-target efficiency, as both components would exhibit greater flexibility within the genome. We then developed an iterative version of CC-ABE (split Cas9-based CC-ABE), in which the nCas9 was split into two parts before Cys574, which may potentially decrease Cas9-dependent off-target activities and restrict the deaminase’s flexibility. We compared the editing efficiencies and undesired indels of split Cas9-based CC-ABE8e with those of sABE8e-ctrl and intact ABE8e at the same human genomic loci in bulk and sorted HEK293T cells (Supplementary Fig. 5a–d). The results were consistent with the data shown in Fig. 2. The split Cas9-based CC-ABE displayed generally similar editing efficiencies to the unsplit ABE system without increasing the indels in either bulk or sorted cells (Supplementary Fig. 5a–d). These findings suggest that the editing efficiency of CC-ABE8e is comparable to intact ABE, characterized by low indel rates and higher peak activity within the editing window.

To evaluate the potential of applying our CC-BE system to other base editing tools, we engineered CC-based TadCBE49, ABE950, and AYBE9. Evolved from TadA8e, TadA-derived cytosine base editors (TadCBEs) exhibit enhanced on-target editing activity, reduced size, and minimized off-target effects49. We split the TadCBE into two parts before Cys574 in the nCas9 domain, then fused each part to coiled-coil heterodimers, resulting in CC-TadCBE (Fig. 3a and Supplementary Fig. 6a). The C-to-T editing efficiency of CC-TadCBE (sTadCBE-P3P4, range 33.40%–80.54%, mean 57.17%; and sTadCBE-N5N6, range 49.51%–82.73%, mean 64.92%) was substantially higher than sTadCBE-ctrl (range 7.67%–20.48%, mean 14.67%) and the unsplit TadCBE (range 9.96%–32.47%, mean 19.75%) (Fig. 3b). Compared to intact TadCBE, the maximum performance improvements achieved were 4.12-fold by sTadCBE-P3P4 at FANCF site 2 and 4.97-fold by sTadCBE-N5N6 at RNF2 site 2 (Fig. 3b and Supplementary Fig. 6d). CC-TadCBE, sTadCBE-ctrl, and intact TadCBE generally showed low residual A-to-G editing across adenosines (Fig. 3c, f). The purity of C-to-T was much higher than that of A-to-G at all target sites (Fig. 3d). CC-TadCBE exhibited a significantly high peak editing efficiency at positions C3–C8 compared to intact TadCBE and sCBE-ctrl (Fig. 3e).

ABE9 precisely edits adenines within a 1–2 nucleotide window and demonstrates the highest efficiency at A550. We fused coiled-coil heterodimers to the C-terminal of TadA9 and N-terminal of nCas9, resulting in CC-ABE9 (Fig. 3g and Supplementary Fig. 6b). The A-to-G editing efficiency of CC-ABE9 was improved when compared with sABE-ctrl at 4 target sites, and was comparable to or slightly lower than intact ABE9 at 3 of the 4 loci (Fig. 3h and Supplementary Fig. 6e). The undesired indel byproducts generated by CC-ABE9 were lower than or similar to those of sABE-ctrl and intact ABE9 at 2 of the 4 loci (Fig. 3i). As shown in Fig. 3J, CC-ABE9 precisely edited the A5 position within the protospacer sequence, which was consistent with the previous study50.

AYBE can achieve A-to-C and A-to-T transversion editing through the fusion of ABE with human N-methylpurine DNA glycosylase protein (MPG)9. We divided the AYBE into two fragments before Cys574 in the nCas9 domain, fused each part to coiled-coil heterodimers, and generated CC-AYBE (Fig. 3k and Supplementary Fig. 6c). The editing efficiency and A-to-Y purity of CC-AYBE were higher than sAYBE-ctrl and comparable to, or lower than intact AYBE at all target sites (Fig. 3l–n, s). The effects of A>C, A>T, and A>G induced by the separate components of CC-AYBE were minimal (Supplementary Fig. 7a–c). The undesired indel byproduct generated by CC-AYBE was generally either lower than or comparable to that of intact AYBE at all loci (Fig. 3o). CC-AYBE exhibited editing efficiency for A-to-Y transversion at A7 and A8 that was similar to or slightly lower than that of intact AYBE (Fig. 3p–r). Taken together, these results demonstrate that coiled-coil heterodimers can be widely adapted to various base editing tools, significantly enhancing flexibility.

To broaden the targeting scope of CC-BE system, we developed NG-CC-BE (NG-CC-BE3, NG-CC-ABE8e) using SpCas9 variant (SpCas9-NG), which had been engineered to recognize relaxed NG PAM51. We tested NG-CC-BE3 (NG-sCBE-P3P4, NG-sCBE-N5N6) and NG-CC-ABE8e (NG-sABE-P3P4, NG-sABE-N5N6) in HEK293T cells at three endogenous genomic sites with NG PAM for each editor. The results showed that NG-CC-BE3 was substantially better than NG-BE3-ctrl, NG-intein-BE3 (Npu C574, Npu S714), and intact NG-BE3 at the tested loci, accordingly obtained slightly higher indel rates at SHANK3 (Fig. 4a–e). Notably, the A-to-G conversion level of NG-sABE-P3P4 was similar to or slightly higher than that of the intact NG-ABE8e and NG-intein-ABE8e (Npu C574, Npu S714), and more efficient than NG-sABE8e-ctrl, while the indel rate of NG-sABE-P3P4 was comparable to or lower than that of intact NG-ABE8e at all tested loci (Fig. 4f–j). We should recognize that a broader editing window may increase the risk of bystander editing, necessitating careful evaluation of the suitability of selected gRNAs.

These data demonstrate that the CC-BE system is broadly compatible with previously characterized SpCas9 variants, thereby broadening the targeting scope and potential applications of the CC-BE through enhanced PAM flexibility.

To further authenticate the editing activity of CC-BE in other mammalian cells, we selected 4 endogenous sites (GHR, HMGA2, IGF1, and PUM2) for CC-CBE and 4 endogenous sites (GHR, IGF1, PUM2, and SMAD2) for CC-ABE in porcine fetal fibroblasts (PFF) cells. These genes were previously reported to affect body size in mice, dogs, and pigs52–54. We electrotransfected PFF cells with site-specific sgRNAs and CC-BE expression plasmids. The electrotransfection efficiency, measured using a reporter system, was approximately 80% (Supplementary Fig. 8a). Genomic DNA of the transfected bulk cells was extracted 72h post-transfection, and targeted deep sequencing was employed to determine the editing efficiency.

According to the results, the CC-CBE demonstrated substantially improved editing efficiencies compared to the unsplit CBE system with peak activity at 4–7 positions (Fig. 4k, l). Strikingly, the increased editing efficiency of CC-CBE was most evident at the IGF1 site with a 12.4-fold change. The sCBE-ctrl exhibited unstable efficiencies in comparation to the unsplit CBE, but still lower than CC-CBE, which was consistent with the data above (Fig. 4k). Compared to unsplit CBE system, the CC-CBE induced lower or similar unwanted genomic alterations at 2 loci (Fig. 4m). Comparable base editing efficiencies were attained by the sABE-P3P4 and unsplit ABE system (Fig. 4n). And the sABE-N5N6 exhibited somewhat lower editing efficiencies (Fig. 4n). In contrast, the direct split ABE system exhibited substantially lower efficiencies compared to CC-ABE and the unsplit ABE system across all 4 edits in PFF cells (Fig. 4n). Both CC-ABE and unsplit ABE system demonstrated high efficiencies with observed peak activity at 4-8 positions (Fig. 4o). The indel frequencies of both CC-ABE and the directly split ABE were lower than those of the unsplit ABE (Fig. 4p). Computing an average editing efficiency at each position, the results showed that editing window was much wider when base editors were divided into two parts by coiled-coil heterodimers (Fig. 4l, o). Thus, CC-BE systems were able to efficiently edit positions that were previously challenging to target with canonical BE systems in PFF cells. In conclusion, our findings highlight that CC-ABE and CC-CBE achieve promising base editing efficiencies in PFF cells, suggesting their capability for mammalian gene editing and potential for broad applications.

Noting the high degree of CC-BE for on-target editing, we next considered the editing fidelities of CC-BE. BEs may bind to the off-target site that are similar to the target sgRNA protospacer, leading to sgRNA-dependent off-targeting. For the assessment of these rates, several off-target sites within 3 nucleotide mismatches have been identified for unsplit base editors, direct split base editors, and CC-BEs via in silico prediction (Supplementary Fig. 9a). The on- and off-target activities of these base editor effectors were measured by deep sequencing. The split and unsplit systems showed similar performance at Chr4 site and EMX1 site 4 (Supplementary Fig. 9b, c). At HIRA site 1, both sCBE-P3P4 and sCBE-N5N6 moderately increased the rates of sgRNA-dependent off-targeting (Supplementary Fig. 9b). And at Chr15 site, the sABE-N5N6 showed slightly lower off-target efficiencies than intact ABE system, while the sABE-P3P4 exhibited somewhat higher off-target efficiencies (Supplementary Fig. 9c). It is worth noting that the sgRNA-dependent off-target generated by CC-BE was not evidently increased compared to the untreated groups in general (Supplementary Fig. 9b). The off-target rate of CC-CBE was below 0.6% for all tested alleles, and the CC-ABE typically exhibited off-target efficiencies below 0.5%, except for sABE-P3P4 at Chr15 site OT-1 (Supplementary Fig. 9b, c).

Besides sgRNA-dependent off-target base editing, the deamination that occurs when the deaminase domain of the base editor binds to DNA without the involvement of sgRNA represents a distinct form of off-target base editing55,56. Thus, we turned our attention towards sgRNA-independent off-target editing. We used the orthogonal R-loop assay48 with dead SaCas9 to generate off-target R-loops and capture the guide-independent DNA editing mediated by CC-BE, direct split BE, and unsplit BE systems (Supplementary Fig. 10a). Amplicon deep sequencing was used to evaluate the on- and off-target activities of these base editors, focusing on the guide-target sites as well as three genomic R-loops generated by dSaCas9. Using this system, off-target sgRNA-independent deamination at the dSaCas9 R-loop site was readily detectable for sCBE-P3P4, but the ratio of on-target to off-target editing for sCBE-P3P4 was higher than intact BE3 at 2 loci out of 3 edits for Sa site 1 (Supplementary Fig. 10b). Despite having a lower on-target to off-target editing ratio than the intact CBE at other Sa sites, sCBE-P3P4 has much higher precision editing efficiencies (Supplementary Fig. 10b and Fig. 1e). For sCBE-N5N6, off-target activity was lower than or comparable to that of intact BE3 at Sa site 1, which consequently led to a higher ratio of on-target to off-target editing (Supplementary Fig. 10b). Notwithstanding that sCBE-N5N6 showed a lower ratio of on-target to off-target editing at Sa site 6, a notable improvement was achieved in base editing efficiency (Supplementary Fig. 10b and Fig. 1e). The sABE-P3P4 exhibited notable off-target activity compared to the other groups, excluding the direct split ABE system, across all three off-target sites, implying that systems with high on-target efficiency also tend to have high off-target effects (Supplementary Fig. 10c and Fig. 2e). We also found that sABE-N5N6 system was less prone to editing the orthogonal R-loops so that they outperformed others for the on-target: off-target editing ratios at all detected sites (Supplementary Fig. 10c). These results demonstrated that sBE-N5N6 exhibited high targeting specificity. Based on all of the results above, we observed that CC-BE systems offered two options for genetic therapies: high specificity of sBE-N5N6 and high efficiency of sBE-P3P4.

To investigate the off-target effects of CC-BE at the genome-wide level, we performed whole-genome sequencing (WGS) in HEK293T cells. A total of 6 groups were incorporated: 3 CBE groups with or without sgRNA of EMX1 site 4 (sCBE-P3P4, sCBE-N5N6, and unsplit CBE), 3 ABE groups with or without sgRNA of Chr4 site (sABE-P3P4, sABE-N5N6, and unsplit ABE). We transfected plasmids into HEK293T cells. Bulk cells were harvested 72h post-transfection, and 30× WGS was performed. First, we confirmed the on-target base editing efficiency by amplicon deep sequencing (Supplementary Fig. 11). Then, genome-wide single-nucleotide variants (SNVs) and small insertions or deletions (indels) were analyzed based on WGS data in CC-BE3 (sCBE-P3P4, sCBE-N5N6), CC-ABE8e (sABE-P3P4, sABE-N5N6), unsplit BEs (BE3, ABE8e), and the non-targeting groups (with base editors but without sgRNAs, NT). The results revealed comparable levels of SNVs (Fig. 5a, b) and indels (Fig. 5c, d) between CC-BE and unsplit BE, as well as with sgRNA-treated and NT groups. Among the CBE groups, there was no significant difference in the average numbers of C>T SNVs in sCBE-P3P4, sCBE-N5N6, CBE, sCBE-P3P4-NT, sCBE-N5N6-NT, and CBE-NT (Fig. 5e). For the ABE groups (sABE-P3P4, sABE-N5N6, ABE, sABE-P3P4-NT, sABE-N5N6-NT, and ABE-NT), the average numbers of A>G SNVs were similar in each group (Fig. 5f). The base substitution types of SNVs (Supplementary Fig. 12a, b) and lengths of indels (Supplementary Fig. 12c, d) showed almost no bias in each group. We mapped the distribution of SNVs to the genome and found that there were almost no differences among the samples (Fig. 5g, h). For the exonic regions, minimal numbers of C>T SNVs in CBE groups and A>G SNVs in ABE groups were detected, and showed concordance among samples (Fig. 5i, j). The genomic distribution of all SNVs, C>T SNVs in CBE groups and A>G SNVs in ABE groups, revealed by circos plots contributed throughout the genome, and there were no regional and sample biases (Fig. 5k, l). In summary, our results suggest the off-target effects of CC-BE are minimal and similar to unsplit BE.

To explore the therapeutic potential of CC-ABE, we selected the mutant mouse fumarylacetoacetate hydrolase (Fah) gene for validation. Defects in this gene cause hereditary tyrosinemia type I (HTI), a lethal genetic metabolic disorder. The Fah gene encodes a key enzyme in the tyrosine metabolism pathway, and deficiency in this gene leads to the accumulation of toxic metabolic intermediates, resulting in hepatocyte apoptosis and severe liver damage57. We evaluated CC-BE in Fah mutant mouse embryonic fibroblasts (MEFs) carrying a homozygous G-to-A point mutation at the end of exon 8, which induces exon skipping and results in the loss of functional Fah enzyme (Supplementary Fig. 13a). To achieve precise base-editing using ABE, we utilized a previously validated Fah sgRNA for targeting58. Within the base editing window of the sgRNA, there are two adenines located at positions 6 and 9, and the adenine at position 9 (A9) is the pathogenic mutation site. The A-to-G base conversion of bystander A6 does not restore splicing. The MEFs were co-electroporated with Fah-targeting sgRNA and the corresponding nucleases. The transfection efficiency is approximately 50% (Supplementary Fig. 8b). Sanger sequencing revealed that sABE-P3P4 mediated evident A-to-G base editing at position A9, resembling unsplit ABE (Supplementary Fig. 13b). Consistent with the aforementioned results, deep sequencing of the transfected bulk cells confirmed that sABE-P3P4 efficiently converted A9-to-G (Supplementary Fig. 13c). The average conversion efficiencies of sABE-P3P4 were comparable to those of unsplit ABE (14.9% vs 16.4%), demonstrating therapeutic potential (Supplementary Fig. 13c).

To validate the in vivo application of CC-ABE, we selected murine proprotein convertase subtilisin/kexin type 9 (Pcsk9) gene, a candidate target for base editing in the treatment of familial hypercholesterolemia, as its loss leads to a reduction in low-density lipoprotein (LDL) cholesterol59. The sgRNA targets the splice donor site of Pcsk9 intron 1, within which its base editing window includes two adenines located at positions 4 (A4) and 6 (A6) (Fig. 6a). The desired editing involves A6 to G conversion, leading to retention and read-through of intron 1, thereby disrupting Pcsk9, while the bystander A4 is a nonfunctional base within the intron (Fig. 6a). First, we evaluated the base editing efficiency of CC-ABE at Pcsk9 locus in bulk MEFs in vitro. We observed a mean of 45.6% targeted A6 editing with sABE-P3P4, while unsplit ABE was 38.7% (Supplementary Fig. 13d). Considering that the A-to-G conversion efficiencies of sABE-P3P4 were higher than those of sABE-N5N6, we packaged sABE-P3P4 into dual-AAV vectors (Supplementary Fig. 13d). The first vector carried TadA8e tethered with coiled-coil peptide P3 at the C-terminus driven by EF-1a short (EFS) promoter and the sgRNA targeting Pcsk9 driven by U6 promoter (Supplementary Fig. 13e). The second vector carried nCas9 (D10A) tethered with coiled-coil peptide P4 at the N-terminus driven by EFS promoter (Supplementary Fig. 13e). The direct split ABE system without tethers was packaged into AAV as the control (Supplementary Fig. 13e). All four vectors were packaged into AAV serotype 8, which exhibits high tropism for hepatocytes. We delivered the dual-AAV8 sABE-P3P4 or sABE-ctrl (5×1011 viral genomes each) to 1-week-old mice via intraperitoneal injection (Supplementary Fig. 13f). Two weeks later, twelve pieces of liver samples were collected from each mouse, and genomic DNA was isolated for evaluating base editing efficiency. Notably, we obtained unambiguous Sanger sequencing traces for A6-to-G conversion events mediated by sABE-P3P4 compared to sABE-ctrl (Supplementary Fig. 13g). Consistently, high-throughput sequencing showed that the mean of targeted A6 A-to-G conversion mediated by sABE-P3P4 was 20.4%, reaching as high as 33.84%, significantly higher than that of sABE-ctrl (Supplementary Fig. 13h, i). The average indel efficiencies of sABE-P3P4 (n=6) and sABE-ctrl (n=5) were 0.09% and 0.03%, respectively (Supplementary Fig. 13j, k).

Next, we systematically compared editing frequencies in vivo among different injection approaches and AAV doses using split Cas9-based CC-ABE (Fig. 6b). Four-week-old mice were injected intraperitoneally or intravenously with different doses of AAV8 (low-dose condition: 2.5×1011 viral genomes each; high-dose condition: 5×1011 viral genomes each) (Fig. 6c). Six weeks after injection, liver tissues and serum were harvested to assess genome-editing events (Fig. 6c). Sanger sequencing demonstrated strong performance for A6-to-G conversion in the liver injected intraperitoneally with sABE-P3P4, but not with sABE-ctrl under high-dose conditions (Fig. 6d). Deep sequencing of amplicons indicated high frequencies of A6-to-G conversion in the sABE-P3P4-treated groups under both high-dose (~70%) and low-dose conditions (~50%) (Fig. 6e, f). The editing efficiency of intraperitoneal injection (n=2 for the high-dose group and n=3 for the low-dose group) and intravenous injection (n=3 for each group) was comparable, and the efficacy exhibited a dose-dependent manner (Fig. 6e, f). Liver treated with sABE-P3P4 showed much higher editing efficiencies compared to those treated with sABE-ctrl (Fig. 6f). We assessed whether bystander edits could be generated by CC-ABE at this site. As shown in Fig. 6f and Supplementary Fig. 14, bystander edits of A4 were observed, while they occurred in the intronic region and would not impact the transcription of the gene. Furthermore, we demonstrated the functionality of base editing. The levels of Pcsk9, total cholesterol, and LDL cholesterol in the sABE-P3P4-treated mice were decreased compared to those in the PBS-treated controls (Fig. 6g, h, i). The levels of liver function markers, alanine aminotransferase (ALT) and aspartate transaminase (AST), in the AAV-injected mice were not elevated, indicating no acute liver dysfunction (Fig. 6j, k).

Taken together, these data strongly demonstrate that CC-BE can be successfully delivered and achieve base editing in vivo with high efficiency.

Duchenne muscular dystrophy (DMD) is a severe monogenic muscle disorder resulting from mutations in the X-linked Dmd gene, which encodes dystrophin, and affects approximately 1 in 3500–5000 newborn males60. The absence of dystrophin results in membrane fragility and muscle degeneration, leading to walking incapability during adolescence and premature death from cardiac and respiratory failure in early adulthood. Becker muscular dystrophy (BMD) is categorized as a relatively mild muscle degenerative disorder resulting from in-frame deletions of the Dmd gene, supporting the synthesis of dystrophin proteins with partial functionality60. To verify the ability of CC-BE to correct pathogenic variants in non-liver tissue disease, for which the lipid nanoparticles (LNPs) would not be an effective alternative therapy, we employed a dual-AAV-based CC-BE strategy to convert DMD to BMD, aiming to restore dystrophin expression in male mice harboring a deletion of exon 51 (ΔEx51) of the Dmd gene (Supplementary Fig. 15), which represents a prevalent disease-causing mutation in humans61. Proper exon splicing requires 3′ splice acceptor sites (SAS) and 5′ splice donor sites (SDS). To correct the dystrophin reading frame in male ΔEx51 mice (Supplementary Fig. 15), we used the sgRNA (mEx50-sgRNA)62 with NG PAM, which induces exon skipping by disrupting the SDS of exon 50 via NG-ABE-mediated gene targeting (Fig. 7a). First, we evaluated the editing efficiency of NG-CC-ABE8e, NG-ABE8e-ctrl, and unsplit NG-ABE8e in mouse N2a neuroblastoma cells. Consistent with the data above, NG-CC-ABE8e exhibited A-to-G base editing efficiency comparable to that of unsplit NG-ABE8e, and obviously higher than that of NG-ABE8e-ctrl at the SDS of exon 50 (Fig. 7b). NG-sABE8e-P3P4, with NG-sABE8e-ctrl as a control, was chosen for in vivo base editing. AAV vectors were constructed, with each vector containing an EFS-driven half of NG-sABE8e-P3P4 or NG-sABE8e-ctrl and a U6-driven mEx50-sgRNA in the reverse orientation (Fig. 7c). All vectors were packaged into AAV9, which displays preferential tropism for skeletal and cardiac muscles63. Dual-AAV split NG-ABE8e system was initially delivered to 5-week-old male ΔEx51 mice via local intramuscular injection (2.5×1011 viral genomes each) or intraperitoneal injection (5×1011 viral genomes each), with two mice per group (Fig. 7d). Muscles were collected for analysis at 7 weeks post-injection (Fig. 7d). For the intraperitoneally injected mice, the tibialis anterior (TA), gastrocnemius (GCM), diaphragm (DIA), cardiac muscle (CM), and abdominal muscle (ABS) were harvested; for those receiving local intramuscular injection, only the TA and GCM were collected. Four pieces of samples were harvested from each muscle of AAV9-injected male ΔEx51 mice, PBS-injected male ΔEx51 mice, and wild-type mice. Genomic PCR amplification products spanning the target site were subjected to amplicon deep sequencing. In intraperitoneally injected mice, the CM (ranging from 7.8% to 15.6%), GCM (ranging from 2.7% to 5.1%), ABS (ranging from 1.8% to 4.8%), TA (ranging from 1.7% to 3.6%), and DIA (ranging from 0.5% to 1.4%, with one notably higher value of 6.0%) exhibited efficient base editing (Fig. 7e). In mice receiving local intramuscular injection, the efficiencies of GCM ranged from 6.1% to 22.2%, and TA ranged from 4.0% to 8.9% (Fig. 7e). Local injection was more effective than intraperitoneal injection in the TA and GCM (Fig. 7e, f). Notably, regardless of whether the injection was intraperitoneal or local intramuscular, the editing efficiencies of NG-sABE8e-P3P4 surpassed those of NG-sABE8e-ctrl (ranging from 0% to 1.3%) in every muscle type tested (Fig. 7e, f). RT-PCR products derived from the TA, GCM, and CM of male ΔEx51 mice injected with the NG-sABE8e-P3P4 system revealed exon 50-skipping events (Fig. 7g, lower band). These exon 50-skipping events were further confirmed by Sanger sequencing of the reverse transcription products, which demonstrated that exon 49 was spliced to exon 52, restoring the reading frame of the Dmd transcript (Fig. 7h).

Based on the evident gene correction efficiency observed after AAV9 injection, we tested whether dystrophin expression levels were rescued in the TA, GCM, and CM extracted from AAV9-injected mice. Using laminin to label muscular tissue, immunostaining revealed that dystrophin expression was restored in mice injected with NG-sABE8e-P3P4, while it was barely detectable in those injected with NG-sABE8e-ctrl in the TA (Fig. 8a), GCM (Fig. 8b), and CM (Fig. 8c). Fully in line with the results above, dystrophin expression levels in the locally injected TA and GCM were higher than in those injected intraperitoneally (Fig. 8a, b). Additionally, the levels of serum creatine kinase (CK) and α-hydroxybutyrate dehydrogenase (α-HBDH), two important markers of muscle injury, were much closer to those of wild-type mice in those injected with NG-sABE8e-P3P4 (Fig. 8d, e). The levels of lactate dehydrogenase (LDH), ALT, and AST in the NG-sABE8e-P3P4-injected mice were not drastically elevated compared to those of wild-type mice, suggesting that gene therapy did not cause liver or other organ damage and may even have a mitigating effect (Fig. 8f, g, h).

Collectively, these data robustly demonstrate that CC-BE can be effectively delivered for precise in vivo editing in non-liver tissues, highlighting the therapeutic potential of the CC-BE system.

Methods
Mice
For targeting the mPcsk9 gene, experiments were conducted using 16 wild-type (C57BL/6J) mice aged 1 week and 26 wild-type (C57BL/6J) mice aged 4 weeks. When targeting the mPcsk9 gene, sex was not considered because this gene is located on the autosome. Given that Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disorder, only male mice were used for targeting the mDMD gene in vivo. For targeting the mDMD gene, 6 male ΔEx51 mice (C57BL/6JGpt-Dmdem2Cd1406/Gpt) aged 5 weeks were used in the experiments. The mouse housing facilities were kept at temperatures between 20–22°C. The mice were housed in an environment with a 12-h light-dark cycle, provided with continuous access to standard rodent feed and drinking water.

Plasmid construction
The CC-ABE and CC-CBE editors used in this manuscript were constructed by pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, CU101-02) using the pCMV-BE3/pCMV-ABE8e plasmid as the backbone. The P3, P4, N5, and N6 sequences (Supplementary Data 1) were integrated into the vector by primer synthesis. To facilitate enrichment of transfected cells in subsequent experiments, we cloned PGK-mCherry into the expression vectors of nCas9 and nCas9-UGI, and PGK-EGFP into the expression vectors of TadA8e, APOBEC and unsplit BEs, respectively.

To construct CC-ABE9, N108Q/L145T mutations were introduced into pCMV-TadA8e vector. To construct Cas9-split CC-ABE, CC-TadCBE, and CC-AYBE, nCas9 was split into two parts before Cys574. The fragments of T1.46 and MPG sequence were derived from Addgene #193284 and #193967 via PCR. For CC-AYBE, TadA8e-nCas9 (amino acid 1–573)-P3/N5 and P4/N6-nCas9 (amino acid 574–713)-MPG were assembled into the CMV backbone. The CC-TadCBE plasmid was constructed by replacing TadA8e and MPG with T1.46 and 2x UGI, respectively.

Plasmids expressing dSaCas9 and U6-SaCas9 guide RNA were derived from PX601, in which D10A and N580A were induced via mutagenesis strategy, and then guide RNA cassette was inserted through BsaI digestion strategy. The SaCas9 guide RNA and SpCas9 guide RNA used in this study were listed in Supplementary Data 2.

Cell line authentication
The HEK293T (CRL-3216) and mouse N2a neuroblastoma (CCL-131) cell lines used in this study were obtained from the American Type Culture Collection (ATCC). These cell lines were authenticated by ATCC. PFFs were isolated from wild-type E35 pig fetuses, and MEFs were isolated from E13.5 wild-type or HT1 mouse embryos. These cells were validated by our laboratory.

Cell culture and transfection
HEK293T and N2a cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; HyClone) supplemented with 10% fetal bovine serum (FBS; Gibco), and the PFFs or MEFs were cultured in DMEM supplemented with 15% FBS (Gibco), 1% nonessential amino acids (Gibco), 2 mM GlutaMAX (Gibco), and 1 mM sodium pyruvate (Gibco) at 37°C and 5% CO2. The medium was changed daily. Cells were passaged and expanded using 0.25% trypsin once the cell confluency reached approximately 90%.

For HEK293T cells, cells were digested into single cells and seeded into a 24-well plate before transfection. PEI transfection was performed when the cell confluency reached 40–60%. Prior to transfection, the medium is replaced with 2% FBS medium. PEI (Sigma, 408727; final concentration: 1 μg/μL) was gently mixed with 2% FBS medium and incubated at room temperature for 5 min. For each well, 3 μg of PEI and 25 μL of 2% FBS medium were mixed, 1 μg of plasmid (250 ng of sgRNA + 750 ng of base editor or 375 ng of sBE at each), and 25 μL of 2% FBS medium were mixed. After that, the PEI mixture was added to the plasmid mixture dropwise, gently stirred with a pipette, and allowed to sit at room temperature for 20 min. The HEK293T cells were then slowly added to the PEI and plasmid mixture for transfection. 12 h after transfection, cells were cultured in medium containing 10% FBS.

For PFFs and MEFs, cells were digested from 10 cm dishes, washed once with PBS, and then divided into 25 aliquots. Each aliquot was mixed with plasmid (1 μg of gRNA and 3 μg of BE or sBE [1.5 µg + 1.5 μg]) in 110 µL PBS, followed by co-electrotransfection at 1350 V, 30 ms, 1 pulse by using the Neon™ transfection system (Life Technology). The medium was replaced 12 h after transfection, and the bulk cells were harvested 72 h post-transfection.

For N2a cells, Lipo transfection was performed when cell confluency reached 40%–60% in 24-well plates. According to the manufacturer’s protocols, 0.8 µL Lipo 8000TM (Beyotime, C0533) and 500 ng plasmid (125 ng of sgRNA and 375 ng of BE or sBE) were mixed in 25 µL medium containing 2% FBS prior to transfection. The mixture was then added into N2a cells following medium replacement. Cells were cultured in medium containing 10% FBS after 12 h and harvested 72 h after transfection.

FACS, cell lysis, and PCR identification
MoFlo Astrios was used to perform fluorescence-activated cell sorting 72 h after transfection. After washing with PBS and digesting with 0.25% trypsin, cells were collected by centrifugation at 200×g for 3 min and then resuspended in PBS. Double-positive cells with mCherry and EGFP were sorted for CC-BEs, EGFP-positive cells were sorted for unsplit BE systems, indicating the successful transfection of CC-BEs or unsplit BE systems. Further analysis was performed on roughly 10,000–50,000 sorted cells. NP40 was used to lyse the sorted cells under the following conditions: 56°C for 60 min and 96°C for 10 min. Following that, 2× Hieff Canace® Plus PCR Master Mix (With Dye) (YEASEN, 10154ES03) was used to perform PCR amplification of the target site.

High-throughput sequencing
Amplification primers were designed near the target locus for library preparation, with specific primers amplifying fragments of about 190 base pairs. PCR amplification was carried out in two rounds. First round, the primers listed in the Supplementary Data 2 were used for the amplification process. Using cell lysate as the template, amplification was carried out for 30 cycles in a 20 μL reaction volume. For the second round, primers with unique Illumina barcodes were used for amplification. Using the first round PCR product as the template, amplification was performed for 15 cycles in a 50 μL reaction volume. Following purification with the HiPure Gel Pure DNA Mini Kit (Magen, D2111-03) and quantification with the Equalbit™ dsDNA HS Assay Kit (Vazyme), the second round PCR products were prepared for deep sequencing on the Illumina HiSeq X platform (Annoroad Gene Technology Corporation). CRISPResso2 (http://crispresso.pinellolab.org/submission) was used to analyze the high-throughput sequencing data67.

Protein extraction and Western blot analysis
For Western blot analysis, HEK293T cells seeded in 24-well plates were transfected with CC-BE (sABE-P3P4/sCBE-P3P4) and unsplit BE system (unsplit-ABE/CBE), then harvested 72 h later. Cell proteins were extracted using radioimmunoprecipitation analysis buffer (RIPA) supplemented with PMSF. The mixtures were lysed on ice for 5 min, and the supernatant was obtained by centrifugation at 15,000×g for 15 min at 4°C. Protein concentration was determined using the bicinchoninic acid (BCA) assay, and 60 µg of total protein was loaded onto an 8% acrylamide gel. Gels were run at 70 V for 150 min, followed by 120 min transfer to a polyvinylidene fluoride (PVDF) membrane at 200 mA on ice. The blot was incubated with rabbit anti-Cas9 antibody (JM11-55, HUABIO, ET1703-85), and beta-actin antibody (AF7018, Affinity) at 4°C overnight, followed by incubation with HRP-conjugated Goat Anti-Rabbit IgG antibody (GB23303, Servicebio) at room temperature for 1 h. The blot was developed using Western Blotting Luminol Reagent (E411-05, Vazyme).

Off-target analysis
Potential off-target sites were predicted in the human genome with COSMID (https://crispr.bme.gatech.edu)68. Three days after transfection with CC-BE and target sgRNAs (EMX1 site 4 and HIRA site 1 for CBE systems; Chr4 site and Chr15 site for ABE systems), cells were harvested for fluorescence-activated cell sorting. The sorted cells were then lysed with NP40. Sequences flanking the predicted off-target sites were amplified using primers designed by COSMID (Supplementary Data 3) for high-throughput sequencing.

R-loop assay
HEK293T cells were transfected at approximately 40%–60% confluency after being seeded on 24-well plates. 375 ng of intact BE systems or CC-BEs (187.5 + 187.5 ng), 250 ng of SpCas9 guide RNA plasmid, and 375 ng of dSaCas9 with SaCas9 guide RNA plasmid were co-transfected by PEI transfection. Twenty-four hours after transfection, the medium was replaced with medium containing 10% FBS. Three days post-transfection, cells were harvested for fluorescence-activated cell sorting. The sorted cells were lysed by NP40, and the PCR amplification was performed using primers flanking the SaCas9 guide-RNA target sites for high-throughput sequencing (Supplementary Data 4).

Whole genome sequencing data analysis
The WGS libraries and sequencing were prepared by Annoroad Gene Technology (Beijing, China). WGS was performed on Illumina NovaSeq X-25B at an average coverage of 30×. Fastp (v0.23.0) was used to filter raw reads (base quality value ≥ 19). The clean reads were mapped to the human reference genome (GRCh38 from UCSC) using BWA-MEM (v0.7.17). Samtools (v1.22) was used to sort BAM files. Picard (v2.9.0) was used to mark duplicative reads. Variants calling was conducted using GATK HaplotypeCaller (v4.1). Variants were annotated by ANNOVAR (v20220521). Bar plots and heatmaps were generated using R package ggplot2 (v4.0.0). Genome-wide mutations were generated using 200 Kb sliding window and plotted with CIRCOS.

AAV production and injection
PackGene Biotech Co., Ltd. (Guangzhou, China) produced and packaged AAV8 and AAV9 viruses. Prior to injection, the mice were anesthetized with an abdominal injection of 5% chloral hydrate. For in vivo base editing of the mPcsk9 gene, we used AAV8 to package the viral vectors. For 1-week-old wild-type mice, dual-AAV8 sABE-P3P4 (5×1011 genome copies of AAV-TadA8e-P3-sgRNA and 5×1011 genome copies of AAV-P4-nCas9) or sABE-ctrl (5×1011 genome copies of AAV-TadA8e-sgRNA and 5×1011 genome copies of AAV-nCas9) were delivered via intraperitoneal injection. Two weeks after AAV injection, livers were harvested and divided into 12 pieces. For 4-week-old wild-type mice, injections were performed via intraperitoneal injection or intravenous injection with sABE-P3P4 (AAV-TadA8e-nCas9-N-P3-sgRNA and AAV-P4-nCas9-C-sgRNA) or sABE-ctrl (AAV-TadA8e-nCas9-N-Pcsk9-sgRNA and AAV-nCas9-C-Pcsk9-sgRNA) at different doses (low-dose condition: 2.5×1011 viral genomes each; high-dose condition: 5×1011 viral genomes each). After 6 weeks, the mice were euthanized, and liver tissues and serum were collected. TIANamp Genomic DNA Kit (TIANGEN) was used to obtain genomic DNA of liver samples.

For in vivo base editing of the mDmd gene, we used AAV9 to package the viral vectors. Dual AAV9 vectors of sABE-P3P4 or sABE-ctrl were delivered to 5-week-old ΔEx51 mice (5×1011 viral genomes of each AAV9 virus for intraperitoneal injection; 2.5×1011 viral genomes of each AAV9 virus for local intramuscular injection). After 7 weeks, the TA, GCM, DIA, CM, and ABS were harvested from the intraperitoneally injected mice; only the TA and GCM were collected from those receiving local intramuscular injection. Each harvested muscle was divided into four parts, designated for subsequent analyses, including RNA extraction, DNA extraction, cryosectioning, and sample backup.

RT-PCR and Sanger sequencing
Muscles samples were ground by homogenizer (60 Hz, 60 s), and total RNA was extracted with RNA rapid extraction solution (Servicebio, G3013). The cDNA was PCR-amplified using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, RRO47A) according to the manufacturer’s protocol.

The cDNA isolated from WT mice, ΔEx51 mice, and CC-BE-treated mice was subjected to PCR amplification using primers (listed in Supplementary Data 5) to obtain amplicons of 767, 534, and 425 bp size for Dmd Exon 48 to Exon 53. The PCR products were analyzed by 2% agarose gel electrophoresis and purified using the HiPure Gel Pure DNA Mini Kit (Magen, D2111-03) for Sanger sequencing.

Serum biochemistry analysis
Blood samples were collected from mice. The collected blood was allowed to stand at room temperature for 30 min before being centrifuged at 3000 rpm for 15 min to separate the serum. The samples were analyzed for various biochemical markers, including CK, ALT, AST, α-HBDH (HB3919), and LDH levels for wild-type mice, PBS-treated ΔEx51 mice, and AAV9-injected ΔEx51 mice. Additionally, Pcsk9, AST, ALT, total cholesterol, and LDL cholesterol levels were assessed in wild-type mice and AAV8-injected mice. Pcsk9 was measured using the Mouse PCSK9 (Proprotein convertase subtilisin/kexin type 9) QuickTest ELISA Kit (QT-EM0153), following the manufacturer’s instructions.

Immunofluorescence staining of muscles
Fresh muscle tissues were rapidly frozen using liquid nitrogen-cooled isopentane and stored at −80°C for subsequent analysis. All muscles were embedded in OCT compound before being cryo-sectioned at 8 µm. The sections were fixed with acetone for 10 min and air-dried for 10 min at 4°C. The sections were then stored at −20°C.

Before immunofluorescence staining, the sections were rewarmed at room temperature for 15 min, followed by washing with PBS three times, 5 min each. After permeabilizing with 0.5% Triton X-100 for 30 min, the sections were blocked with 10% normal goat serum for 1 h at room temperature. After washing with PBS three times, the sections were incubated overnight at 4°C with the antibody mixture (anti-laminin 2 alpha antibody [4H8-2], abcam, ab11576; dystrophin (PT0821R) PT® Rabbit mAb, Immunoway, YM8580). Each antibody was diluted at a ratio of 1:200. The next day, after washing 5 times, the sections were incubated with secondary antibodies (anti-rat IgG (H+L), CST, 4418; anti-rabbit IgG (H+L), CST, 4412) for 1 h at room temperature. Each secondary antibody was diluted at a ratio of 1:2000. DAPI mounting medium (Sigma-Aldrich, F6057-20ML) was added before applying the coverslip. Images were acquired using Zeiss LSM800.

Statistics and reproducibility
In this study, data analysis was conducted using GraphPad Prism 10.3.1. Error bars indicated the mean ± standard deviation of at least three independent biological replicates. No statistical method was used to predetermine sample size. The sample size is based on the variability observed in independent experiments and is consistent with the standards in related studies. No data were excluded from the analyses. Investigators were not blinded to allocation during experiments or outcome assessment. For assessing significant differences in precise editing efficiency across groups, the two-tailed Student’s t test was employed (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and ns indicating not significant). Exact P values are provided in Source data file.

Ethics statement
C57BL/6J and ΔEx51 mice (C57BL/6JGpt-Dmdem2Cd1406/Gpt, GemPharmatech) were used in this study. All animal experiments were consistent with the guideline of the Animal Welfare and Research Ethics Committee at Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences (IACUC: 2025073).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors report a split system utilizing non-covalent recruitment of deaminases to Cas9 nickase via coiled-coil heterodimers. They demonstrate that this system can improve or at least maintain the editing efficiency of ABEs and CBEs with different PAM preferences in vitro. Additionally, they assess its in vivo application using dual AAV vectors to edit the Pcsk9 gene in mice, achieving 33.84% on-target editing. While developing a flexible and universal split base editing system is valuable, several limitations reduce the manuscript's impact.\n1. In Vitro Editing Efficiency\nFigures 1–6 present data from plasmid transfections comparing the control editor and different split versions. In some cases, CC-BE significantly increased on-target editing efficiency. The authors speculate that coiled-coil heterodimers enhance reconstitution, bringing UGI and APOBEC3 into closer proximity compared to the intact CBE, which may contribute to improved performance. However, the study does not assess whether differences in editing efficiency arise from variations in protein expression. Did the authors verify editor expression under different conditions? A Western blot analysis would be informative in determining whether the observed differences are due to expression levels rather than an intrinsic advantage of the split system.\n2. In Vivo Study Design\nThe in vivo experiments involve delivering dual AAV8 vectors (sABE-P3P4, sABE-N5N6, sABE-ctrl or ABE) via intraperitoneal (IP) injection at 1E12 vg total to 1-week-old mice, with liver harvest at 2 weeks post-injection. This design has several concerns:\no Efficiency and Timing: IP injection generally leads to lower transduction efficiency and a longer time to peak AAV expression compared to intravenous (IV) injection.\no High AAV Dose: A 1E12 vg dose in 1-week-old pups is extremely high and lacks translational relevance, particularly for AAV8, which primarily targets the liver.\no Duration of Expression: Harvesting the liver at 2 weeks post-injection may not be sufficient for full evaluation, especially given the slower kinetics of IP delivery.\n3. Prolonged AAV-Mediated Expression and Off-Target Risks\nA major concern with AAV-mediated delivery is the prolonged expression of the editor, which can lead to cumulative on-target editing but also increases off-target risks, a critical issue for in vivo gene editing. In contrast, Lipid Nanoparticles (LNPs) provide transient expression, minimizing long-term off-target effects. While LNPs currently achieve high efficiency primarily in the liver, AAVs have the advantage of transducing a broader range of tissues. Therefore, to demonstrate the translational impact of the CC split system, the authors should show its ability to correct a pathogenic variant in a non-liver tissue, where LNPs would not be an effective alternative.", "reviewer_2": "Mu et al present a novel approach for splitting base editors, which already made a powerful impact in the treatment of human disease, as several variants are already enrolled in clinical trials. Using coiled-coils (CC) as a non-covalent tethering option, the authors show the applicability of using CCs to improve base-editing, demonstrated at several genomic loci and in several cell lines. The functionality is additionally confirmed in vivo by editing the Pcsk9 gene. Although the authors provide a substantial amount of editing data, confirmed by NGS, the paper lacks several important experiments.\nMajor concerns:\n1. The comparison of different designs may be slightly misleading. The constructs for CC-BE (for CBEs and ABEs) have altogether 4 NLS, whereas the fused ABE or fused CBE (that CC-BE are benchmarked to) only have 2 NLS. The authors did not even mention that in the text. In order to fully evaluate the potential improvement of the genome editing the authors should conduct experiments with constructs (for both CBE and ABE) that have an equal number of NLS at several genomic loci, since the slight improvement in genome editing might be due to the different nuclear translocation of CC-BEs.\n2. The authors also failed to mention in the text that the % of editing is presented on the sorted population of cells. Only when looking at the Method section, the reader may comprehend that sorting was carried out. It would be important to know what is the actual base editing efficiency in the bulk cell population. This goes for all experiments the whole paper (also for MEF, PEF cells etc., and results presented in Fig.7C,E). The authors should provide the representative results of BE on bulk cell population and should correct the figure legends and text. The gating strategy must be included in the paper.\n3. It was surprising that sCBE and sABE led to such a high editing efficiency. This could in principle pose a substantial threat, due to the off-target effects so the authors should conduct a genome-wide determination of the deamination status as shown e.g. in Xie et al.2023 to demonstrate that there are no or very little nondesired mutations.\n4. The authors did check for the potential off-target activity on the computationally predicted off-target sites, but we know that BE can also lead to chromosomal rearrangements, translocations, so the authors should execute CIRCLE-seq, CHANGE-seq to test the in vitro predicted off-target sites and conduct CAST-seq to check for potential major chromosomal aberrations.\n5. The in vivo part on editing the Pcsk9 gene may also be misleading; the authors should compare the efficiency of CC-BE to the original fused BE, used as the state-of-the-art and not to split-BE as they did. Also, the functionality of the in vivo editing should be shown, e.g. PCSK9 levels via ELISA, cholesterol values, liver enzymes etc. As this is also an important part of the paper, authors should examine the bystander edits. Bystander edits can be seen already on the chromatograms (e.g. A12 in the Pcsk9).\n6. The discussion section of the paper could be improved as the authors just summarize their work. In the Discussion section, the authors should discuss the pros/cons of using CC-BE against conventional fused BE, dual AAV vs. single AAV etc. The authors should also mention smaller Cas9 versions, hypercompact Cas proteins (e.g. NanoCas, Cas14f etc) and how these can alter the need to go via dual AAV approach.\nMinor concerns, which need to be addressed:\n- Line 63: include the word “relatively” before the low pathogenicity\n- We could see BE improvement for CC-BE but only in the case of CBEs. Why is this the case? The authors should address this. Additionally, wider BE window is not desirable as this can lead to substantial bystander edits. Please elaborate on this.\n- Line 169: word “intern” (intein?)\n- Line 239/240: “observed improvement on all tested loci” - only one locus was actually edited, this should be corrected.\n- Line 250: the loci tested for CBE and ABE are switched\n- The authors should present the electroporation efficiency for the MEF and PEF cells\n- Line 275: By using Spry Cas or PAM-less Cas editors, researchers are not limited to edit only the genomic targets with PAM sequences, but again, the wider base-editing window is not desirable. The authors should comment on this issue.\n- The part of the paper with experiments, using orthogonal R-loop assay, should be additionally explained (specifically text referencing the Fig.6E-H). Why is the Cas9-independent editing for the ABE system so high (Fig.6G)-explain please? Also, the scheme for the R-loop assay could be presented better.\n- Line 311/316 Fig.1F; should be 6F\n- Line 406: this statement should be rephrased: genome editing improvements is not robust\n- The authors should explain why the AAVs were injected via ip injection and not via a standard i.v. injection? Also, why was the in vivo research carried out on 1-week old animals, as the majority of the papers (where they use the same model) use adult or 5-6 weeks old animals (e.g. Rothgangl et al. 2021)?\n- The Method section for the cell electroporation, off-target analysis, R-loop assay and in vivo part should be substantially expanded, so that the researchers can repeat the methods if needed\n- Figure 1, 2: where are the data on the missing sites (e. g. site 4, 15, 16 in Fig.2?)\n- The authors should discuss why they achieved poor editing with original editors on some loci as these sites have been quite highly edited based on several reports ?\n- Fig.2H: please select different colors of the lines as it is very difficult to point out which editor is in which color\n- The statistic is missing throughout the whole paper\n- In Fig.3E: which site is that?\n- Fig3K-N: the separate parts should be included in the experiments as negative controls\n- There are discrepancies between results. The authors state that the indels are formed less in the CC-BE as compared to the original BE (see 5C, 4E)\n- The authors should discuss why the improvement is seen only for CBE systems in some cases but not in ABEs.\n- Include the references in the Discussion (line 376-408)"}, "author_response": "We appreciate the positive and encouraging comments from the reviewer. Our specific responses are presented below.\n1. In Vitro Editing Efficiency\nFigures 1–6 present data from plasmid transfections comparing the control editor and different split versions. In some cases, CC-BE significantly increased on-target editing efficiency. The authors speculate that coiled-coil heterodimers enhance reconstitution, bringing UGI and APOBEC3 into closer proximity compared to the intact CBE, which may contribute to improved performance. However, the study does not assess whether differences in editing efficiency arise from variations in protein expression. Did the authors verify editor expression under different conditions? A Western blot analysis would be informative in determining whether the observed differences are due to expression levels rather than an intrinsic advantage of the split system.\nResponse: We couldn't agree more with this good suggestion. To verify whether differences in editing efficiency arise from variations in protein expression, we performed Western blot analysis to compare the protein expression levels between the CC-BE and unsplit BE. Consistently, the expression levels of CC-BE and the control base editor were comparable (Supplementary Fig. 4).\n2. In Vivo Study DesignThe in vivo experiments involve delivering dual AAV8 vectors (sABE-P3P4, sABE-N5N6, sABE-ctrl or ABE) via intraperitoneal (IP) injection at 1E12 vg total to 1-week-old mice, with liver harvest at 2 weeks post-injection. This design has several concerns:\no Efficiency and Timing: IP injection generally leads to lower transduction efficiency and a longer time to peak AAV expression compared to intravenous (IV) injection.\no High AAV Dose: A 1E12 vg dose in 1-week-old pups is extremely high and lacks translational relevance, particularly for AAV8, which primarily targets the liver.\no Duration of Expression: Harvesting the liver at 2 weeks post-injection may not be sufficient for full evaluation, especially given the slower kinetics of IP delivery.\nResponse: Thanks very much for the informative and helpful comments. We conducted additional in vivo experiments to fully address above concerns. For the efficiency, timing, and AAV dose, four-week-old mice were injected intraperitoneally or intravenously with different doses of AAV8 (low-dose condition: 2.5 × 1011 viral genomes each; high-dose condition: 5 × 1011 viral genomes each). We harvested liver tissues and total plasma to assess genome-editing events six weeks after injection. Sanger sequencing and deep sequencing of amplicons both indicated high frequencies of A-to-G conversion in the sABE-P3P4-treated groups under both high-dose (~70%) and low-dose conditions (~50%) (Fig. 6e, f). The editing efficiency of intraperitoneal injection and intravenous injection is comparable, and the efficacy exhibits a dose-dependent manner (Fig. 6e, f). Livers treated with sABE-P3P4 showed significantly superior activity compared to those treated with sABE-ctrl.\n3. Prolonged AAV-Mediated Expression and Off-Target Risks\nA major concern with AAV-mediated delivery is the prolonged expression of the editor, which can lead to cumulative on-target editing but also increases off-target risks, a critical issue for in vivo gene editing. In contrast, Lipid Nanoparticles (LNPs) provide transient expression, minimizing long-term off-target effects. While LNPs currently achieve high efficiency primarily in the liver, AAVs have the advantage of transducing a broader range of tissues. Therefore, to demonstrate the translational impact of the CC split system, the authors should show its ability to correct a pathogenic variant in a non-liver tissue, where LNPs would not be an effective alternative.\nResponse: We appreciate the valuable suggestion to correct a pathogenic variant in a non-liver tissue, as it would powerfully demonstrate the translational impact of our CC-BE split system. Thus, we employed a dual-AAV-based CC-BE strategy to convert DMD to BMD, aiming to restore dystrophin expression in mice harboring a deletion of exon 51 of the Dmd gene (Supplementary Fig. 16), which represents a prevalent disease-causing mutation in humans61. The Dmd gene mutation will result in membrane fragility and muscle degeneration, leading to walking incapability during adolescence and premature death from cardiac and respiratory failure in early adulthood. Our data suggest that single-swap genomic editing at the Dmd SDS using the dual-AAV-based CC-BE system is efficient in inducing exon 50 skipping, thereby restoring the open reading frame of the dystrophin transcript and rescuing truncted dystrophin expression.\nIn detail, we added following results in the revised manuscript:\n”Delivery of NG-CC-ABE via dual-AAV9 restores dystrophin expression in ∆Ex51 mice\nDuchenne muscular dystrophy (DMD) is a severe X-linked genetic disorder caused by mutations in the Dmd gene, which encodes dystrophin60. The absence of dystrophin results in membrane fragility and muscle degeneration, leading to walking incapability during adolescence and premature death from cardiac and respiratory failure in early adulthood. Becker muscular dystrophy (BMD) is a relatively mild form of muscle degeneration caused by in-frame deletions of the Dmd gene, which allow the production of partially functional dystrophin proteins60. To verify the ability of CC-BE to correct pathogenic variants in non-liver tissue disease, for which the lipid nanoparticles (LNPs) would not be an effective alternative therapy, we employed a dual-AAV-based CC-BE strategy to convert DMD to BMD, aiming to restore dystrophin expression in mice harboring a deletion of exon 51 (∆Ex51) of the Dmd gene (Supplementary Fig. 15), which represents a prevalent disease-causing mutation in humans61.\nProper exon splicing requires 3' splice acceptor sites (SAS) and 5' splice donor sites (SDS) of exons. To correct the dystrophin reading frame in ∆Ex51 mice (Supplementary Fig. 15), we used the sgRNA (mEx50-sgRNA)62 with NG PAM, which induces exon skipping by disrupting the SDS of exon 50 via NG-ABE for gene targeting (Fig. 7a). Firstly, we evaluated the editing efficiency of NG-CC-ABE8e, NG-ABE8e-ctrl, and unsplit NG-ABE8e in mouse N2a neuroblastoma cells. Consistent with the data above, NG-CC-ABE8e exhibited A-to-G base editing efficiency comparable to that of unsplit NG-ABE8e, and obviously higher than that of NG-ABE8e-ctrl at the SDS of exon 50 (Fig. 7b). NG-sABE8e-P3P4, with NG-sABE8e-ctrl as a control, was chosen for in vivo base editing.\nAAV vectors were constructed, with each vector containing an EFS-driven half of NG-sABE8e-P3P4 or NG-sABE8e-ctrl and a U6-driven mEx50-sgRNA in the reverse orientation (Fig. 7c). All vectors were packaged into AAV9, which displays preferential tropism for skeletal and cardiac muscles63. Dual-AAV split NG-ABE8e system was initially delivered to 5-week-old ∆Ex51 mice via local intramuscular injection (2.5×1011 viral genomes each) or intraperitoneal injection (5×1011 viral genomes each), with two mice per group (Fig. 7d). Muscles were collected for analysis at 7 weeks post-injection (Fig. 7d). For the intraperitoneally injected mice, the tibialis anterior (TA), gastrocnemius (GCM), diaphragm (Dia), cardiac muscle (CM), and abdominal muscle (Abs) were harvested; for those receiving local intramuscular injection, only the TA and GCM were collected. Four pieces of samples were harvested from each muscle of AAV9-injected ∆Ex51 mice, PBS-injected ∆Ex51 mice, and wild-type mice. Genomic PCR amplification products spanning the target site were subjected to amplicon deep sequencing. In intraperitoneally injected mice, the CM (ranging from 7.8% to 15.6%), GCM (ranging from 2.7% to 5.1%), Abs (ranging from 1.8% to 4.8%), TA (ranging from 1.7% to 3.6%), and Dia (ranging from 0.5% to 1.4%, with one notably higher value of 6.0%) exhibited efficient base editing (Fig. 7e). In mice receiving local intramuscular injection, the efficiencies of GCM ranged from 6.1% to 22.2%, and TA ranged from 4.0% to 8.9% (Fig. 7e). Local injection was more effective than intraperitoneal injection in the TA and GCM (Fig. 7e, f). Notably, regardless of whether the injection was intraperitoneal or local intramuscular, the editing efficiencies of NG-sABE8e-P3P4 significantly surpassed those of NG-sABE8e-ctrl (ranging from 0% to 1.3%) in every muscle type tested (Fig. 7e, f). RT-PCR products derived from the TA, GCM, and CM of ∆Ex51 mice injected with the NG-sABE8e-P3P4 system revealed exon 50-skipping events (Fig. 7g, lower band). These exon 50-skipping events were further confirmed by Sanger sequencing of the reverse transcription products, which demonstrated that exon 49 was spliced to exon 52, restoring the reading frame of the Dmd transcript (Fig. 7h).\nBased on the evident gene correction efficiency observed after AAV9 injection, we tested whether dystrophin expression levels were rescued in the TA, GCM, and CM extracted from AAV9-injected mice. Using laminin to label muscular tissue, immunostaining revealed that dystrophin expression was restored in mice injected with NG-sABE8e-P3P4, while it was barely detectable in those injected with NG-sABE8e-ctrl in the TA (Fig. 8a), GCM (Fig. 8b), and CM (Fig. 8c). Fully in line with the results above, dystrophin expression levels in the locally injected TA and GCM were higher than in those injected intraperitoneally (Fig. 8a, b). Additionally, the levels of serum creatine kinase (CK) and α-hydroxybutyrate dehydrogenase (α-HBDH), two important markers of muscle injury, were much closer to wild-type in mice injected with NG-sABE8e-P3P4 (Fig. 8d, e). The levels of lactate dehydrogenase (LDH), ALT, and AST in the NG-sABE8e-P3P4-injected mice were not significantly elevated compared to wild-type mice, suggesting that gene therapy did not cause liver or other organ damage and may even have a mitigating effect (Fig. 8f, g, h).\nCollectively, these data robustly demonstrate that CC-BE can be effectively delivered for precise in vivo editing in non-liver tissues, highlighting the therapeutic potential of the CC-BE system.”\nReviewer #2 (Remarks to the Author):\nMu et al present a novel approach for splitting base editors, which already made a powerful impact in the treatment of human disease, as several variants are already enrolled in clinical trials. Using coiled-coils (CC) as a non-covalent tethering option, the authors show the applicability of using CCs to improve base-editing, demonstrated at several genomic loci and in several cell lines. The functionality is additionally confirmed in vivo by editing the Pcsk9 gene. Although the authors provide a substantial amount of editing data, confirmed by NGS, the paper lacks several important experiments.\nWe appreciate the reviewer's positive assessment of our work.\nMajor concerns:\n1. The comparison of different designs may be slightly misleading. The constructs for CC-BE (for CBEs and ABEs) have altogether 4 NLS, whereas the fused ABE or fused CBE (that CC-BE are benchmarked to) only have 2 NLS. The authors did not even mention that in the text. In order to fully evaluate the potential improvement of the genome editing the authors should conduct experiments with constructs (for both CBE and ABE) that have an equal number of NLS at several genomic loci, since the slight improvement in genome editing might be due to the different nuclear translocation of CC-BEs.\nResponse: Thanks for your valuable comments and suggestions. Theoretically, the more NLS signal sequences carried, the more base editors transported into the cell nucleus, which may consequently enhance the efficiency of genome editing. Therefore, we increased the number of NLS included in the intact ABE and CBE to four, consistent with the number of NLS in the CC-BE. However, our results indicated that the C-to-T efficiency of 4×NLS-CBE was slightly higher than or comparable to that of 2×NLS-CBE at 3 loci out of 4 edits; however, it still did not exceed CC-CBE (Supplementary Fig. 3b). The indel frequencies of 4×NLS-CBE were similar or higher than those of 2×NLS-CBE (Supplementary Fig. 3c). This suggests that the increased editing efficiency of CC-CBE is not due to differences in the number of NLS. The A-to-G efficiency of 4×NLS-ABE was slightly higher than or comparable to that of 2×NLS-ABE, while it was slightly lower than or comparable to that of CC-ABE (Supplementary Fig. 3d).\n2. The authors also failed to mention in the text that the % of editing is presented on the sorted population of cells. Only when looking at the Method section, the reader may comprehend that sorting was carried out. It would be important to know what is the actual base editing efficiency in the bulk cell population. This goes for all experiments the whole paper (also for MEF, PEF cells etc., and results presented in Fig.7C,E). The authors should provide the representative results of BE on bulk cell population and should correct the figure legends and text. The gating strategy must be included in the paper.\nResponse: Thank you for pointing out the importance of clearly distinguishing between the overall base editing efficiency in the bulk cell population, the gating strategy for sorting, and the efficiency measured in the sorted cell population. We provided the representative results of BE on bulk cell population in Supplementary Fig. 2. We also incorporated the gating strategy into the supplementary Fig. 1, and corrected the figure legends and text as suggested.\n3. It was surprising that sCBE and sABE led to such a high editing efficiency. This could in principle pose a substantial threat, due to the off-target effects so the authors should conduct a genome-wide determination of the deamination status as shown e.g. in Xie et al.2023 to demonstrate that there are no or very little nondesired mutations.\nResponse: We thank the reviewer for pointing out that the high editing efficiency of sCBE and sABE could, in principle, raise justifiable concerns regarding potential off-target effects. Whole-genome sequencing was carried out and the corresponding results have been incorporated into the revised manuscript as follows:\n“Off‐target effects of CC‐Base editor\nTo investigate the off-target effects of CC-Base editor at the genomic level, we performed whole-genome sequencing (WGS) in HEK293T cells. A total of 6 groups were incorporated: 3 CBE groups with or without sgRNA of EMX1 site4 (sCBE-P3P4, sCBE-N5N6, unsplit CBE), 3 ABE groups with or without sgRNA of Chr4 site (sABE-P3P4, sABE-N5N6, unsplit ABE). We transfected plasmids into HEK293T cells. Bulk cells were harvested 72h after transfection, and 30× WGS data was collected. First, we confirmed the on-target base editing efficiency by amplicon deep sequencing (Supplementary Fig. 11). Then, genome-wide single-nucleotide variants (SNVs) and small insertions or deletions (indels) were analyzed based on WGS data in CC-BE3 (sCBE-P3P4, sCBE-N5N6), CC-ABE8e (sABE-P3P4, sABE-N5N6), unsplit BEs (BE3, ABE8e), and the non-targeting group (with base editors but without sgRNAs, NT). The results revealed comparable levels of SNVs (Fig. 5a, b) and indels (Fig. 5c, d) between CC-BEs and unsplit BEs, as well as with sgRNA-treated and NT groups. In CBE groups, there was no significant difference in the average numbers of the C>T SNVs in sCBE-P3P4, sCBE-N5N6, CBE, sCBE-P3P4-NT, sCBE-N5N6-NT and CBE-NT (Fig. 5e). For ABE groups (sABE-P3P4, sABE-N5N6, ABE, sABE-P3P4-NT, sABE-N5N6-NT and ABE-NT), the average numbers of the A>G SNVs were similar in each group (Fig. 5f). The base substitution types of SNVs (Supplementary Fig. 12a, b) and lengths of indels (Supplementary Fig. 12c, d) showed almost no bias in each group. We mapped the distribution of SNVs to the genome and found that there was almost no difference among samples (Fig. 5g, h). For the exonic regions, minimal numbers of C>T SNVs in CBE groups and A>G SNVs in ABE groups were detected, and showed concordance among samples (Fig. 5i, j). The genomic distribution of all SNVs, C>T SNVs in CBE groups and A>G SNVs in ABE groups, revealed by circos plots covered the entire genome, and there were no regional and sample biases (Fig. 5k, i). In summary, our results suggest the off‐target effects of CC‐Base editor were minimal and similar to unsplit BEs.”\n4. The authors did check for the potential off-target activity on the computationally predicted off-target sites, but we know that BE can also lead to chromosomal rearrangements, translocations, so the authors should execute CIRCLE-seq, CHANGE-seq to test the in vitro predicted off-target sites and conduct CAST-seq to check for potential major chromosomal aberrations.\nResponse: Thanks for the kind remind that base editor can also lead to chromosomal rearrangements and translocations. Therefore, we conducted CIRCLE-seq to validate the off-target sites predicted in vitro (this choice was made as CIRCLE-seq and CHANGE-seq share similar technical principles, the CIRCLE-seq experiments for this study were performed by DIATRE). As shown in the figure below, for both CC-ABE and CC-CBE, we did not detect more off-target effects compared to the control group.\nWe also deeply recognize the necessity of investigating potential major chromosomal aberrations (as suggested for CAST-seq). Regrettably, due to technical limitations in our laboratory, we were unable to successfully perform CAST-seq in this study. However, we would like to provide additional context here: CC-BEs utilize nickases, which have an extremely low probability of inducing double-strand breaks (DSBs) in the genome. Since chromosomal rearrangements are primarily driven by DSB-mediated repair errors, the use of nickases may inherently reduce the risk of such aberrations.\n5. The in vivo part on editing the Pcsk9 gene may also be misleading; the authors should compare the efficiency of CC-BE to the original fused BE, used as the state-of-the-art and not to split-BE as they did. Also, the functionality of the in vivo editing should be shown, e.g. PCSK9 levels via ELISA, cholesterol values, liver enzymes etc. As this is also an important part of the paper, authors should examine the bystander edits. Bystander edits can be seen already on the chromatograms (e.g. A12 in the Pcsk9).\nResponse: Thanks for the valuable suggestions about using the original fused BE as a control for in vivo Pcsk9 gene editing experiments and conducting more detailed data collection and analysis. Although original fused BE is the superior option, we selected split-BE as an alternative considering the AAV can only package cargo up to about 4.7 kb, inherently smaller than the size of original base editors. We further assessed the functionality of the in vivo Pcsk9 editing. As shown in Fig. 6g, h, I, the levels of Pcsk9, total cholesterol, and LDL cholesterol in the sABE-P3P4-treated mice were decreased compared to those in the PBS-treated controls. The levels of liver function markers, alanine aminotransferase (ALT) and aspartate transaminase (AST), in the AAV-injected mice were not significantly elevated, indicating no acute liver dysfunction (Fig. 6j, k). As for the examination of bystander edits, the bystander edits of A can be observed, however, they do not affect transcript production, as they occur in the intronic region and do not impact the splicing process (Fig. 6f and Supplementary Fig. 15).\n6. The discussion section of the paper could be improved as the authors just summarize their work. In the Discussion section, the authors should discuss the pros/cons of using CC-BE against conventional fused BE, dual AAV vs. single AAV etc. The authors should also mention smaller Cas9 versions, hypercompact Cas proteins (e.g. NanoCas, Cas14f etc) and how these can alter the need to go via dual AAV approach.\nResponse: We are very grateful for this suggestion that can improve the discussion section of our paper. We made the following paragraph to the discussion section:\n“Compared to conventional BEs, CC-BE has evident advantages, including flexible design, improved or comparable base editing efficiency, and the ability to overcome the AAV packaging issue caused by the large size of BEs. The in vivo delivery of CC-BE relies on dual-AAV system, whereas delivering miniature base editor based on hypercompact Cas proteins such as Cas12f, TnpB, and IscB requires single-AAV. However, these miniature gene editing tools demonstrate lower editing activity compared to conventional BEs and have a limited editing scope, fundamentally restricting their capacity for precise targeting. Although dual AAV delivery methods have been explored to address size limitations, these strategies introduce additional manufacturing and safety challenges due to the necessity of co-delivering multiple vectors. In the future, compact nucleases with potent editing capabilities, such as NanoCas variants64, may enable single-AAV delivery of effective base editors in vivo, thus combining the advantages of high efficiency and streamlined delivery.”\nMinor concerns, which need to be addressed:\n- Line 63: include the word “relatively” before the low pathogenicity\nResponse: Thanks a lot for your patient and careful reading of our manuscript. We added the word “relatively” before the low pathogenicity.\n- We could see BE improvement for CC-BE but only in the case of CBEs. Why is this the case? The authors should address this. Additionally, wider BE window is not desirable as this can lead to substantial bystander edits. Please elaborate on this.\nResponse: As we described at the discussion section, “Through 3D structure predictions using AlphaFold 3, we found that in the presence of coiled-coil dimerization peptides, the spatial positions of UGI and APOBEC3 are closer compared to the intact CBE. Therefore, we reasonably infer that the conformational change significantly alters the overall performance of CC-CBE, leading to an improvement in base editing efficiency. However, an obvious improvement is observed in CBE systems, but not in ABEs. This may be due to several reasons. First, the conformational changes induced by CC peptides may enhance the activity of both UGI and APOBEC3, but have no effect on adenine deaminase alone. Second, differences in the reaction kinetics and substrate-binding affinity of cytosine and adenine deaminases may lead to varying gene editing outcomes.”\nA higher editing efficiency also results in a broader editing window. Notably, this expanded range can even enable editing of hard-to-reach positions outside the canonical editing window of traditional CBEs. This can also be beneficial for saturated mutation screening. On the other hand, for gene therapy applications, this must be balanced against the need to select gRNAs that efficiently target the aimed base pair to minimize bystander editing.\n- Line 169: word “intern” (intein?)\nResponse: Sorry for this spelling mistake, we revised the word “intern” using the “intein”.\n- Line 239/240: “observed improvement on all tested loci” - only one locus was actually edited, this should be corrected.\nResponse: We corrected the description as suggested.\n- Line 250: the loci tested for CBE and ABE are switched\nResponse: Sorry for this mistake, we switched the loci tested for CBE and ABE.\n- The authors should present the electroporation efficiency for the MEF and PEF cells\nResponse: Thanks for the great suggestion. We have incorporated flow cytometry results to present the electroporation efficiency for the PEF and MEF cells (Supplementary Fig. 8).\n- Line 275: By using Spry Cas or PAM-less Cas editors, researchers are not limited to edit only the genomic targets with PAM sequences, but again, the wider base-editing window is not desirable. The authors should comment on this issue.\nResponse: Thanks for your suggestion. We added the followed sentence “We should recognize that a broader editing window may increase the risk of bystander editing, necessitating careful evaluation of the suitability of selected gRNAs.”\n- The part of the paper with experiments, using orthogonal R-loop assay, should be additionally explained (specifically text referencing the Fig.6E-H). Why is the Cas9-independent editing for the ABE system so high (Fig.6G)-explain please? Also, the scheme for the R-loop assay could be presented better.\nResponse: We substantially expanded the experiment Method section for the R-loop assay. The inherent properties of the deaminase itself including single strand DNA binding affinity, enzymatic kinetics and size, will significantly influence off-target editing efficiency examined by R-loop. We added the scheme for the R-loop assay as suggested in the revised paper.\n- Line 311/316 Fig.1F; should be 6F\nResponse: Sorry for this mistake, we have corrected the serial number.\n- Line 406: this statement should be rephrased: genome editing improvements is not robust\nResponse: We have removed this inaccurate description.\n- The authors should explain why the AAVs were injected via ip injection and not via a standard i.v. injection? Also, why was the in vivo research carried out on 1-week old animals, as the majority of the papers (where they use the same model) use adult or 5-6 weeks old animals (e.g. Rothgangl et al. 2021)?\nResponse: We thank the reviewer for raising the concerns regarding the AAV injection method and the age of the animals used. To address these points, we have performed additional experiments utilizing four-week-old mice and administering AAV8 via both intraperitoneal and intravenous routes at different doses.\nWe added following results in the revised manuscript：\n“Next, we systematically compared editing frequencies in vivo among different injection approaches and AAV doses using split Cas9-based CC-ABE (Fig. 6b). Four-week-old mice were injected intraperitoneally or intravenously with different doses of AAV8 (low-dose condition: 2.5 × 1011 viral genomes each; high-dose condition: 5 × 1011 viral genomes each) (Fig. 6c). Six weeks after injection, liver tissues and serum were harvested to assess genome-editing events (Fig. 6c). Sanger sequencing demonstrated strong performance for A6-to-G conversion in the liver injected intraperitoneally with sABE-P3P4, but not with sABE-ctrl under high-dose conditions (Fig. 6d). Deep sequencing of amplicons indicated high frequencies of A6-to-G conversion in the sABE-P3P4-treated groups under both high-dose (~70%) and low-dose conditions (~50%) (Fig. 6e, f). The editing efficiency of intraperitoneal injection (n = 2 for the high-dose group and n = 3 for the low-dose group) and intravenous injection (n = 3 for each group) is comparable, and the efficacy exhibits a dose-dependent manner (Fig. 6e, f). Livers treated with sABE-P3P4 showed significantly higher editing efficiencies compared to those treated with sABE-ctrl (Fig. 6f). We assessed whether bystander edits could be generated by CC-ABE at this site. As shown in Fig. 6f and Supplementary Fig. 14, bystander edits of A4 were observed, while they occurred in the intronic region and would not impact the transcription of the gene. Furthermore, we demonstrated the functionality of base editing. The levels of Pcsk9, total cholesterol, and LDL cholesterol in the sABE-P3P4-treated mice were decreased compared to those in the PBS-treated controls (Fig. 6g, h, i). The levels of liver function markers, alanine aminotransferase (ALT) and aspartate transaminase (AST), in the AAV-injected mice were not significantly elevated, indicating no acute liver dysfunction (Fig. 6j, k).”\n- The Method section for the cell electroporation, off-target analysis, R-loop assay and in vivo part should be substantially expanded, so that the researchers can repeat the methods if needed\nResponse: Thanks very much for the suggestion on improving the quality of our paper writing. We substantially expanded the Method section for the cell electroporation, off-target analysis, R-loop assay, and in vivo part in the revised manuscript.\n- Figure 1, 2: where are the data on the missing sites (e. g. site 4, 15, 16 in Fig.2?)\nResponse: During the experiment, we encountered issues such as low alignment rates in some amplicon sequencing results. These problems may have been caused by mutations in the regions of the genome where primers bind, leading to the exclusion of some data. To enhance readability, we have updated the nomenclature for the loci.\n- The authors should discuss why they achieved poor editing with original editors on some loci as these sites have been quite highly edited based on several reports ?\nResponse: Differences including cell culturing conditions and transfection methods may lead to inconsistent editing efficiencies compared to previously reported results. However, all control experiments across different groups targeting the same loci were conducted under identical experimental conditions to ensure the reliability of our data.\n- Fig.2H: please select different colors of the lines as it is very difficult to point out which editor is in which color\nResponse: We have adjusted the colors and shades of the line graph to enhance its readability.\n- The statistic is missing throughout the whole paper\nResponse: Thanks for your kind reminder. We have added the statistical analysis throughout the whole paper.\n- In Fig.3E: which site is that?\nResponse: This figure represents our integrated analysis of C base positions and their editing efficiencies across all 4 test loci for TadCBE.\n- Fig3K-N: the separate parts should be included in the experiments as negative controls\nResponse: The separate parts of Figures 3K-3N were included in the experiments to serve as negative controls (see Supplementary Fig. 7).\n- There are discrepancies between results. The authors state that the indels are formed less in the CC-BE as compared to the original BE (see 5C, 4E)\nResponse: We appreciate this comment. Regarding indels, the performance of CC-BE varies across different cell types, nuclease types, and target loci.\n- The authors should discuss why the improvement is seen only for CBE systems in some cases but not in ABEs.\nResponse: AlphaFold 3 3D structure predictions revealed that in the presence of two sections, the pair P3 and P4 or N5 and N6 heterodimerizes, bringing deaminase and Cas9 nickase into proximity and facilitating their assembly into a functional complex. However, an obvious improvement is observed in CBE systems, but not in ABEs. This may be due to several reasons. First, the conformational changes induced by CC peptides may enhance the activity of both UGI and APOBEC3, but have no effect on adenine deaminase alone. Second, differences in the reaction kinetics and substrate-binding affinity of cytosine and adenine deaminases may lead to varying gene editing outcomes.\n- Include the references in the Discussion (line 376-408)\nResponse: Thank you for pointing this out. The references have been added to the Discussion section as suggested."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All of my questions have been fully addressed by the authors, and I have no additional concerns.", "reviewer_2": "Authors did an impressive work in the revision of the paper and responded to all raised issues. I believe it is now better than before and in my opinion deserved to be published without any further delay."}, "author_response": "Many thanks for taking the effort to review our manuscript, and your uplifting comments hold great significance.\nThank you for the invaluable comments. Your positive feedback are deeply appreciated."}]

Decision letter