A Plug-in system for reprogramming the editing patterns of base editors

Domain: neuroscience
Published: 2025-12-20
Editorial outcome: minor_revision

Abstract
DNA base editors are transformative genome editing tools that enable nucleotide conversions without inducing double-stranded DNA breaks, making them promising for correcting genetic mutations. Current base editors, however, are limited by fixed editing windows and constrained location of deaminases. To address these constraints, we develop a modular system termed Plug-in Base Editor (Plug-in BE), which dynamically programs deaminase positioning via integrating various epitopes and antibody-fused deaminases. This system expands the editing capabilities of base editors by optimizing deaminase’s spatial interaction with DNA, leading to improvements in efficiencies, window restrictions, and safety profiles. We validate Plug-in BE’s versatility and high fidelity in cancer gene therapy and zebrafish embryo editing, demonstrating its potential as a powerful and adaptable tool for basic research and therapeutic applications. This innovation can generate a series of base editors without extensive protein evolution, positioning Plug-in BE as a significant advancement in the field of genome editing.

Introduction
DNA base editors enable the conversion of target nucleotides without introducing double-stranded DNA breaks1, making them highly promising tools for correcting human inherited diseases. Adenine base editors (ABEs) mediate A-to-G conversion2, while cytosine base editors (CBEs) enable C-to-T conversion1. Additionally, glycosylase base editors (CGBEs) implement C-to-G conversion3,4. Through deaminase evolution and the incorporation of certain glycosylases, DNA base editors can achieve other mutation types, such as ACBE converting A-to-C5. Further, replacing SpCas9 with other Cas proteins has led to base editors matching specific PAMs6,7. However, current base editors exhibit a specific editing pattern at a fixed window, limiting their effectiveness in genome editing8,9.

During the optimization of base editors, positioning the deaminase at the N- or C-terminus of nCas9(D10A) has been shown to influence editing efficiency, editing window, byproducts, and Indels1. Further, in inlaid and circularly permuted base editors, these characteristics can be tuned by embedding deaminases into nCas9(D10A) or permuting its domains10–12. However, such recombination alters protein conformation and activity, thereby restricting the number of viable configurations and resulting in a relatively fixed editing pattern and editing window.

In this study, we present a modular system designed to dynamically program the spatial locations of deaminase within DNA base editors. By embedding epitopes at various positions within nCas9(D10A) and fusing deaminase with antibody, we can manipulate the deaminase’s spatial interaction with DNA, thereby altering its editing outcome, including editing efficiency, editing window, byproducts, Indels, and sequence motif preference. These modular components are combined in a plug-in manner, with CRISPR/Cas as a plug-in platform, epitopes as slots, and deaminases as plug-ins to generate a wide array of new base editors with improvements in efficiency, fidelity, and window restriction, which we term Plug-in Base Editor (Plug-in BE). The efficacy of Plug-in BEs has been further validated in both cancer gene therapy and zebrafish embryo editing, highlighting its potential as a powerful and safe tool for basic research and therapeutic applications.

Results
Generation of SunTag plug-in BE via programming deaminase’s spatial location
Current DNA base editors generally restrict the location of deaminases to the N/C-terminal of nCas9(D10A) (Fig. 1A, left). To overcome this limitation, we embedded an epitope at internal positions within nCas9(D10A), thereby enabling the recruitment of antibody-fused deaminases to specific spatial locations (Fig. 1A, right). Here, we developed 62 D10A-GCN4 variants by embedding GCN4 (a 19 amino acids epitope from SunTag system13) primarily within loops between α-helices and β-sheets, thereby minimizing disruption to the function of nCas9(D10A) (Fig. 1B). Based on SpCas9 structure (PDB, 5f9r), the chosen embedded positions are spatially close to the target DNA. Further, structural predictions using AlphaFold314 confirmed that the GCN4 embedment did not significantly alter the conformation of nCas9(D10A) (Fig. 1C), suggesting that D10A-GCN4 variants may retain their functionality.

To establish a mobile deaminase module, we fused AntiGCN4 (a 272 amino acids antibody from SunTag system13) with APOBEC1 and UGI(s)1,15, resulting in constructs named AntiGCN4-BE3 with UGI and AntiGCN4-BE4 with 2xUGI (Fig. 1D). These systems were collectively termed SunTag Plug-in BE3 and SunTag Plug-in BE4. Initially, we selected D10A-231GCN4, D10A-1055GCN4, and D10A-1246GCN4 for editing four gene loci in HEK293T cells. Overall, SunTag Plug-in BE3 improved editing efficiency by 20% to 40% over BE3, BE4, and SunTag Plug-in BE4, making it a promising candidate for further investigation (Fig. 1E).

Subsequently, we systematically assessed the editing efficiencies of all 62 D10A-GCN4 variants within SunTag Plug-in BE3 framework. Notably, 29 variants demonstrated higher efficiency than BE3 (Fig. 1F and Supplementary Fig. 1A). For instance, at the VEGFA site 2, D10A-170/231/1059/1068GCN4 achieved around 90% C-to-T efficiency, outperforming BE3’s 79%. Similar improvements were observed at the FANCF site 1, HEK site 3, and ZAP70.

Interestingly, the GCN4 embedding position within nCas9(D10A) obviously affected the editing window. For example, the editing windows of D10A-170/308/1033GCN4 expanded to the right at the VEGFA site 2 (Fig. 1F). Based on these observations, we classified 62 variants into five groups with distinct editing patterns, according to the editing results at the HEK site 3, ZAP70, and FANCF site 1 (Fig. 1G). The blue group with 10 variants exhibited the highest efficiency and specific rejection to the motifs CC4A, AC9G, GC13T, and GC14A, while the green group with 15 variants rejected CC8T. Remarkably, the variants reduced byproducts (Fig. 1H). For instance, D10A-308GCN4 reduced byproducts from 25% with BE3 to 4% at the FANCF site 1, while D10A-1246GCN4 reduced from 37% to 2% at the VEGFA site 2. These results demonstrate that the strategic spatial programming of deaminase in the modularized Plug-in BE not only enhances editing efficiency but, more importantly, improves editing fidelity.

SunTag Plug-in BE with ABE8eWQ, miniCGBE1, and Target AID
Inspired by the success of SunTag Plug-in BE3, we further incorporated ABE8eWQ16 to develop SunTag Plug-in ABE8eWQ (Fig. 2A). D10A-1/532/945/1068/1072GCN4 exhibited comparable editing efficiencies to ABE8eWQ (Supplementary Fig. 2A). To be noteworthy, the editing window analysis revealed that SunTag Plug-in ABE8eWQ’s editing activity was largely restricted to A6 and A7 (Fig. 2B). Consequently, the contracted window improved single-base editing precision (Fig. 2C). D10A-532GCN4 specifically enhanced the only A8-to-G from 1% with ABE8eWQ to 42% at E21, while D10A-1055GCN4 increased the only A7-to-G from 1% with ABE8eWQ to 90% at OCT4. More importantly, the specific variants further reduced unwanted Indels products (Supplementary Fig. 2C). For example, D10A-532GCN4 reduced Indels from 1.4% of ABE8eWQ to 0.44% at OCT4. Together, these results demonstrate that embedding GCN4 within nCas9(D10A) can refine the activity of ABE8eWQ by narrowing the editing window, enhancing single-base editing precision, and reducing byproducts.

To evaluate whether Plug-in BE could achieve C-to-G conversion, we developed SunTag Plug-in miniCGBE13 and assessed its performance in HeLa cells (Fig. 2D and Supplementary Fig. 3A, C). At FANCF site 4, D10A-1246/1248/1252/1260GCN4 achieved the highest editing efficiency, ranging from 44% to 53%, which was comparable to miniCGBE1’s 46% (Supplementary Fig. 3A). Further analysis revealed that the editing window of SunTag Plug-in miniCGBE1 contracted to C6 (Fig. 2E). In addition, the variants showed reduction in byproducts (Fig. 2F, G). For instance, at PPP1R12C site, whereas miniCGBE1 reached 23% editing efficiency but generated 40% byproducts, D10A-231/1068/1072GCN4 achieved 30% editing efficiency with only 10% byproducts. In summary, SunTag Plug-in miniCGBE1 reduces byproducts, providing a narrow editing window that enhances the editing precision and practical application of CGBEs.

We further integrated Target AID to develop SunTag Plug-in Target AID17. Interestingly, SunTag Plug-in Target AID exhibited a series of wider editing windows and complex editing patterns (Supplementary Fig. 4 and 5).

Taken together, SunTag Plug-in BE is compatible with different deaminases and embedding positions, enabling a wide range of conversions (C-to-T, A-to-G, and C-to-G), while also reprogramming the editing patterns to better achieve diverse editing requirements.

Enhancement of Plug-in BE through integration of MoonTag system
MoonTag system contains a smaller epitope, gp41 (15 amino acids), compared with GCN4 (19 amino acids), and a smaller antibody, Antigp41 (123 amino acids), compared with AntiGCN4 (272 amino acids)18. Given the compact nature of MoonTag system, we hypothesized that it might improve editing performance with Plug-in BE, and therefore investigated the editing outcomes of MoonTag Plug-in BEs (Fig. 3A, E, H).

In MoonTag Plug-in BE3 system, D10A-gp41 variants showed further improvement of editing efficiencies than the D10A-GCN4 counterparts at identical embedding positions (Supplementary Fig. 6A). For instance, at FANCF site 1, D10A-719/768/945/1050/1068/1248gp41 achieved over 70% editing efficiency, while D10A-GCN4 resulted in efficiencies ranging from 50% to 60%. Furthermore, D10A-gp41 variants altered the sequence motif preference and editing window width (Fig. 3B, C). Similar to D10A-GCN4, D10A-gp41 reduced byproducts (Fig. 3D). To evaluate the DNA off-target effects of MoonTag Plug-in BE3, we performed amplicon sequencing at off-target sites of HEK site 3 and VEGFA site 2. Based on the on/off-target ratio, D10A-gp41 variants showed comparable editing specificity to BE3 (Supplementary Fig. 8E–H and Supplementary Note 2).

In MoonTag Plug-in ABE8eWQ system, 24 D10A-gp41 variants exhibited editing efficiency higher than SunTag Plug-in ABE system (Fig. 3F and Supplementary Fig. 2B). Unlike the contracted editing window of SunTag Plug-in ABE8eWQ, MoonTag Plug-in ABE8eWQ exhibited diverse window modes (Fig. 3F). Among the variants, 3 variants (purple group) expanded the editing window to A3 and A8. In addition, 12 variants (green group) had the narrowest editing window with the highest efficiency at A7, while another 9 variants (blue group) contracted their window to A5, A6, and A7. Due to the shifting editing window, MoonTag Plug-in ABE8eWQ enhanced the single-base editing precision (Fig. 3G). Moreover, D10A-gp41 variants further reduced the Indels of ABE8eWQ (Supplementary Fig. 2D). To evaluate off-target effects of MoonTag Plug-in ABE, we conducted amplicon sequencing at off-target sites of OCT4 and HEK site 2, and found that D10A-gp41 variants maintained comparable DNA-level editing specificity to ABE8eWQ (Supplementary Fig. 8A, B). Furthermore, transcriptome analysis confirmed that D10A-gp41 variants had no increase in RNA-level off-target effects compared to ABE8eWQ (Supplementary Fig. 8C, D and Supplementary Note 2).

Next, we expanded MoonTag Plug-in BE to two additional deaminases. MoonTag Plug-in miniCGBE1 showed comparable performance to SunTag Plug-in miniCGBE1 in editing efficiency, editing window, and product purity (Fig. 3H–K and Supplementary Fig. 6E). Surprisingly, MoonTag Plug-in Target AID outperformed SunTag Plug-in Target AID in editing efficiency (Supplementary Figs. 4, 5, and 6C). Cas variants with expanded targeting capacity (e.g., the nearly PAM-less SpRY) can overcome PAM restrictions, thereby broadening the range of base editing. However, even with sgRNA positional adjustments (1–5 bp rightward shifts), SpRY-BE3 failed to efficiently achieve C9-to-T conversion with only 1.4%–16.9% efficiency at HEK site 3 (Supplementary Fig. 7A, B). In contrast, MoonTag Plug-in BE3 (D10A-1246gp41) achieved a higher C9-to-T efficiency of 38.9% without relying on sgRNA redesign (Supplementary Fig. 7B). This finding underscores that expanded PAM compatibility alone does not guarantee precise and efficient base editing, whereas the Plug-in BE system’s programmable deaminase positioning enables targeted edits in regions inaccessible to conventional Cas-fused base editors.

In summary, the compact nature of MoonTag enhances editing efficiencies and improves editing window control in Plug-in BE system, without compromising editing specificity. These findings suggest that MoonTag Plug-in BEs offer a promising strategy for achieving more precise and efficient genome editing.

Expansibility of Plug-in BE with ALFA system for modulating editing patterns
To further validate the expansibility of Plug-in BE as a general strategy for manipulating editing patterns, we developed ALFA Plug-in BE by integrating the ALFA system, which contains a 13 amino acids epitope ALAF and a 132 amino acids antibody Anti-ALFA19,20. Firstly, we constructed ALFA Plug-in CBE and tested D10A-231ALFA and D10A-1246ALFA (Fig. 4A). Notably, the editing windows varied among GCN4, gp41, and ALFA, even when the same embedding positions were used (Fig. 4B). At C15, D10A-231ALFA possessed 36% editing efficiency compared to D10A-231GCN4/gp41’s 5% to 10%. At C3 and C4, D10A-1246ALFA increased editing efficiency by 20% compared to D10A-1246GCN4/gp41. Additionally, D10A-231ALFA and D10A-1246ALFA also reduced byproducts compared to BE3 (Fig. 4C).

Next, we evaluated ALFA Plug-in ABE system at embedding positions 945, 1246, and 1252 (Fig. 4D). As with the previous Plug-in BEs, ALFA’s embedment altered the editing window of ABE8eWQ (Fig. 4D). Notably, D10A-1252ALFA improved editing efficiency by 10% to 40% at A3, A4, and A5, compared to the D10A-GCN4/gp41 counterparts. Similar to GCN4 and gp41, D10A-945/1246ALFA reduced byproducts and improved single-base editing precision, compared with ABE8eWQ (Fig. 4F, G).

The ALFA system generated a distinct editing pattern compared to SunTag and MoonTag when using the same embedding position, demonstrating that the type of Protein-Tagging system can impact editing outcomes. Since ALFA system is an excellent protein purification system19,20, ALFA Plug-in BE could be purified and would possess promising potential for basic and clinical research.

Optimization of plug-in BE via reordering deaminases and antibodies
To examine whether the order of antibody-deaminase fusion influences editing outcomes, we rearrange the components within the Plug-in BE framework. Firstly, we established the four different configurations of antibodies and deaminases within Plug-in ABE (Fig. 5A). As expected, the rearrangement shifted the editing window, as exemplified by ABE8eWQ-1059GCN4, which expanded the window to A9 and improved efficiency from 6% (ABE8eWQ) to 33% (Fig. 5B). Importantly, strategic reordering optimizes editing efficiency (Fig. 5C). For instance, at E21 and HEK site 2, ABE8eWQ-1059GCN4 enhanced efficiency from 5% of 1059GCN4-ABE8eWQ to 36%.

To extend this strategy, we incorporated ABE921, known for its minimal editing window, into Plug-in ABE9 (Fig. 5E). Remarkably, reordering within Plug-in ABE9 resulted in more distinct changes to the editing window of ABE9 (Fig. 5F). For example, ABE9-1059GCN4, ABE9-1059gp41, and 1059gp41-ABE9 exhibited narrower editing windows than ABE9. Likewise, reordering within Plug-in ABE9 led to optimization in editing efficiency and single-base editing precision (Fig. 5G, H).

Consistent with the above results, in Plug-in CGBE1 system3, reordering of eUNG and R33A also improved editing efficiency (Supplementary Fig. 9). Collectively, these results demonstrate that the strategic reordering of deaminases and antibodies can enhance the performance of Plug-in BE systems, allowing for the customization of specific editing patterns.

Given the ability of the Plug-in BE system to generate a diverse range of base editors with distinct editing patterns, we have provided a comprehensive comparative summary of recommended Plug-in BE variants (Supplementary Data 4). This summary outlines the advantages, limitations, and optimal applications for each variant, highlighting key parameters including editing windows, editing efficiency, product purity, and sequence motif preferences.

Systematic characterization of plug-in BE using self-targeting library
To systematically evaluate the editing performance of Plug-in BE, we constructed a Self-Targeting library8 containing 30,230 sgRNA sequences along with the corresponding target sequences (Fig. 6A). In MoonTag Plug-in BE3 system, D10A-532/1055/1246/1252gp41 exhibited wider editing windows, and their efficiencies were approximately 1.5 to 2 times that of BE3 (Fig. 6B). Of note, D10A-1246gp41 possessed a high efficiency at C5, C6, and C14 to C16, while D10A-1055gp41 improved at C1 to C13. Overall, the variants displayed an improvement in the TCN and CCD motifs, and a reduction in the GCN motifs (Fig. 6C). In addition, the variants reduced byproducts C-to-G/A (Fig. 6D, E). Similarly, MoonTag Plug-in Target AID expanded the editing window, reduced byproducts, and altered motif preferences (Supplementary Fig. 10C–H).

Library analysis of MoonTag Plug-in ABE8eWQ system further demonstrated the programmability of Plug-in BE, as different D10A-gp41 variants produced various editing windows (Fig. 6F). D10A-1055gp41 and D10A-231gp41 presented a right-shifted window with comparable editing efficiency, whereas D10A-532gp41 mainly improved the efficiency at A7 to A11. Moreover, the four variants exhibited different motif preferences (Fig. 6G), such as D10A-1055gp41 especially preferring TAC and GAC.

In summary, Self-Targeting library analysis fully demonstrates the detailed relationship between the spatial location of deaminase and editing outcome.

Advantages of Plug-in BE in gene therapy and zebrafish embryo editing
According to ClinVar data, nearly all of pathogenic single nucleotide variants (SNVs) consist of C-to-T, A-to-G, and C-to-G (Supplementary Fig. 11B, top). Importantly, Plug-in BE system expands the editable regions even further, thereby enabling the correction of additional SNVs, with the A-to-G corrections from 23.26% (ABE8eWQ) to 32.87% and the C-to-T correction from 43.53% (BE3/Target AID) to 77.73% (Supplementary Fig. 11B, down).

To demonstrate the therapeutic potential of Plug-in BE, we targeted the E6 and E7 oncogenes of HPV18 in HeLa cells to generate stop codons. The targeted cytosines of chosen sites are located at CCN motif and C11, which are typically edited inefficiently by BE3 and BE48. Remarkably, D10A-1246gp41 achieved an editing efficiency ranging from 10% to 37% at targeted cytosines, producing stop codons and reducing the cell growth rate (Supplementary Fig. 11C–E). Therefore, strategic adjustment of APOBEC1’s spatial position changes its motif preference and editing window, effectively overcoming the limitations of BE3 and BE4 in cancer gene therapy.

To further evaluate the in vivo editing potential of Plug-in BEs, we transcribed mRNAs along with sgRNAs targeting the zebrafish genes TWIST2 and TYR (Fig. 7A). The mRNAs included sequences encoding fluorescent proteins to facilitate the sorting of embryos expressing base editor (Fig. 7B). Firstly, we edited the TWIST2 site using the Plug-in ABE8eWQ system. Larvae edited by ABE8eWQ or D10A-231gp41 displayed phenotypes associated with TWIST2 dysfunction, including curved tails, body trunk malformations, and pericardial edema (Fig. 7C)22,23. Sanger sequencing showed that while ABE8eWQ primarily edited A6-to-G, D10A-231gp41 avoided the CA6G sequence and achieved A8-to-G with a 3.8-fold higher efficiency (Fig. 7C). Furthermore, amplicon sequencing of the top 5 larvae with the highest editing efficiency from Sanger sequencing revealed that D10A-231gp41 increased the only A8-to-G conversion from 10% (ABE8eWQ) to 59% of all mutational reads (Fig. 7D). Furthermore, we used the Plug-in BE3 system to edit the TYR. Sanger sequencing revealed that BE3 achieved approximately 21% C7-to-T, with only 3% C11-to-T, due to its restricted window. In contrast, D10A-1246gp41 achieved an average of 39% C11-to-T, generating a stop codon and resulting in albino larvae (Fig. 7F). To further validate these findings, we performed amplicon sequencing of the top 5 larvae with the highest editing efficiency from Sanger sequencing (Fig. 7G). As expected, sequence type analysis showed that BE3 generated 53.7% of reads with C7-to-G conversion, while almost no C11-to-T reads were detected. In contrast, D10A-1246gp41 produced over 75% of reads with C11-to-T conversion (Fig. 7H).

Together, by overcoming the limitations of traditional base editors, Plug-In BE could achieve both efficient and safe editing in previously un-editable regions in gene therapy and zebrafish embryos with desired phenotypes. The great controllability and high fidelity of Plug-in BE could meet specific editing needs from both basic research and therapeutic applications.

Methods
Ethics statement
The experiments on zebrafish were approved by the Ethics Committee of Zhongnan Hospital of Wuhan University, Wuhan, China. Our experiments on zebrafish complies with the relevant ethical regulations.

Plasmid construction
The following plasmids with Addgene Catalog numbers were used for Plug-in BE plasmid constructions, BE3 (73021), BE4 (100802), ABE8eWQ (161815), ABE9 (194208), Target AID (131300), miniCGBE1 (140253), CGBE1 (140252), pST1374-scFv-APOBEC-UGI-GB1 (113029), pST1374-GCN4-D10A (113022), MoonTag-Nb-GFP (128602), mEGFP-NbALFA (159986). KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KK2602) and 2X MultiF Seamless Assembly Mix (ABclonal, RK21020) were adopted for plasmid construction according to the manufacturer’s protocol. During plasmid construction, pST1374-GCN4-D10A, which carries the resistance gene of blasticidin, served as the plasmid skeleton for the insertion of Plug-in BE elements. The sequences of Plug-in BE variants are listed in Supplementary Data 1.

Cell culture and transfection
HEK293T cells (ATCC, CRL-3216) and HeLa cells (ATCC, CCL-2) were cultured in Dulbecco’s modified Eagle’s medium (Gibco, 11965118) supplemented with 1% penicillin-streptomycin (Gibco, 15140122) and 10% fetal bovine serum (Gibco, A5256701). The cell lines were passaged every 3 days and incubated at 37°C with 5% CO2. The cell lines have been tested to exclude mycoplasma contamination. Cells were cultured in 24-well plates until the confluency was ~70%. Then, protein-expressing plasmids (500ng) were co-transfected into cells with sgRNA plasmids (250ng) using Hieff Trans® Liposomal Transfection Reagent (Yeasen, no. 40802ES08) according to the manufacturer’s protocol. All transfected cells were cultured for four days with blasticidin selection and then harvested for genomic DNA extraction. The sequences of sgRNA are listed in Supplementary Data 2, and the sequences of off-target sites are in Supplementary Data 3.

Detection of cell proliferation rate
The cells after trypsinization were seeded into 96-well plates. Adjust the cell density to 3000 cells per well. In the testing time points (24h, 48h, and 72h), each well was added with 1/10 volume CCK8 (Abbkine, BMU106-CN). The cells were then cultured in a 5% CO2 incubator at 37°C for 2h. Finally, the absorbance value of cells was tested and recorded at 450 nm wavelength.

Genomic DNA extraction and PCR
Genomic DNA of transfected cells and zebrafish larvae was extracted with EasyPure® Genomic DNA Kit (Transgen, TEE101-01) according to the manufacturer’s protocol. The gene loci (200–600bp) were amplified by PCR for Sanger sequencing. Primers are listed in Supplementary Data 2.

Editing efficiency analysis of Sanger sequencing data using EditR
PCR products were sequenced by Sanger sequencing technology (Sangon, Guangzhou). Sanger sequencing data were further quantified to obtain base editing efficiency using the online tool EditR (https://moriaritylab.shinyapps.io/editr_v10/)12.

Amplicon sequencing and data analysis
Genomic DNA was used for two-step PCR with KAPA HiFi HotStart ReadyMix. Primer pairs of first-step PCR with 25 cycles contained a gene-specific region and an outer Illumina-compatible adaptor sequence. Primer pairs of second-step PCR with 11 cycles had the outer-adaptor sequence to append a unique dual index. Then, the amplicon library was sequenced on MGISEQ-T7 platform. Base editing and indels efficiencies were determined by aligning reads to the genomic sequence using CRISPResso2. The percentage of editing product types, i.e., editing efficiency, was calculated as the desired editing reads/total sequencing reads. The percentage of single-base product types, i.e., editing precision, was calculated as the desired DNA editing reads/total mutational reads. Amplicon primers are listed in Supplementary Data 2 and Supplementary Data 3.

mRNA sequencing
HEK293T cells were transfected with protein-expressing plasmids (500ng) and sgRNA plasmid (250ng). At 48h post-transfection, RNA extraction was performed using TRIzol reagent (Invitrogen, 15596018CN). Subsequently, mRNA was reverse transcribed into cDNA using SuperScript III (Invitrogen, 18080051), and underwent high-throughput sequencing on the Illumina Novaseq-150PE platform. RNA-seq data were analyzed using RADAR (v1.0.0) for detection of RNA SNP.

Preparation of self-targeting library
The architecture and sequence of the Self-Targeting library with 30,230 sgRNAs were adopted from a previously reported article8. In brief, each oligonucleotide contains a full-length sgRNA with a corresponding target. The oligonucleotide pool was amplified by PCR with 10 cycles using KAPA HiFi HotStart ReadyMix, and then purified using magnetic beads (Cytiva, 65152105050250). The Lenti-gRNA-Puro plasmid (84752) was digested using BsmBI-v2 (NEB, R0739L). The pool PCR product was introduced into the digested Lenti-gRNA-Puro plasmid using 2X MultiF Seamless Assembly Mix. The recombinant plasmid was transformed into E. coli, and the colonies were scraped for plasmid purification. The lentivirus plasmid library was transfected into HEK293T cells for the construction of the lentivirus library, which was implemented by GURUN Biotechnology (Wuhan, China).

Characteristics of base editor through screening a self-targeting library
The Self-Targeting lentivirus library with an MOI of 1 was transduced into HEK293T cells for the construction of the Self-Targeting cell library. An sgRNA sequence cover at least 1000 cells. After 24h transduction, the cell library was cultured with media containing 2.5 µg/mL puromycin for 10 days. Subsequently, the DNA base editor plasmid was transfected into the cell library. After 5 days of transfection, the genomic DNA of the cells was extracted for PCR amplification and amplicon sequencing.

Preparation of RNAs
mRNA preparation was performed using HiScribe T7 ARCA mRNA Kit (with tailing) (NEB, E2060S) according to the manufacturer’s protocol. The sgRNAs were modified with phosphorothioation and methoxy group, and synthesized by GenScript (Nanjing, China). The sequences of mRNAs are listed in Supplementary Note 1. The sequences of sgRNAs are listed in Supplementary Data 2.

Microinjection in zebrafish embryos
Wild-type AB strain zebrafish was purchased from China Zebrafish Resource Center, Wuhan, China. Zebrafish was maintained at 28°C on a 14:10 hour light:dark cycle. Embryos were obtained from natural spawning of adult fish (3–6 months old, mixed male and female). Embryos at 1-cell stage were microinjected with a 2nL mixture of mRNA (BE3 or ABE8eWQ, 100ng/µL) and sgRNA (200ng/µL), or Plug-in BE mRNAs (D10A-gp41 and Antigp41-deaminase, 100ng/µL) and sgRNA (200ng/µL). A total of 137 injected zebrafish embryos showed detectable fluorescence at 6h post-injection across all experiments. Embryo genomic DNA was extracted at 72h post-fertilization for assessing the base editing efficiency by Sanger sequencing. In each group, the top 5 embryos with the highest editing efficiency from Sanger sequencing were further sequenced by amplicon sequencing. Sex of zebrafish embryos was not considered in the study design because sexual differentiation in zebrafish occurs at approximately 3–4 weeks post-fertilization. The molecular mechanisms of base editing are not expected to differ between sexes at the embryonic stage. The animal experiments on zebrafish were approved by the Ethics Committee of Zhongnan Hospital of Wuhan University.

Statistics & reproducibility
No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The Investigators were not blinded to allocation during experiments and outcome assessment. The cultured cells from a homogeneous and well-mixed population were considered biologically equivalent. For every Plug-in BE combinations (a total of 533 combinations), we tested four genomic loci with two biological replicates (a total of 4264 biological replicates) to assess the base editing efficiency and ensure reproducibility. The Self-Targeting library cells contains 30,230 sgRNAs with >1000 cells per sgRNA, ensuring statistical robustness for identifying the editing pattern of Plug-in BE. Zebrafish embryos from the same clutch were collected sequentially and assigned to injection groups in the order of collection. A total of 137 zebrafish embryos were successfully injected with RNAs, resulting in detectable fluorescence at 6h post-injection across all experiments (ABE8eWQ, n=22; 231gp41-ABE8eWQ, n=35; BE3, n=40; and 1246gp41-BE3, n=40). Statistical analysis was done using GraphPad Prism 8. Experiments were performed with at least two biological replicates. Error bars displayed on graphs represent the mean ± SD of at least three biological replicates. Statistical significance was analyzed using two-sided unpaired student’s t test with 95% confidence interval. The exact p value was represented on the graphs. All attempts at replication were successful.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors present a modular base editing system, termed Plug-in Base Editor (Plug-in BE), which aims to enhance the flexibility, efficiency, and precision of DNA base editors. The manuscript addresses a fundamental limitation of current base editors—fixed deaminase positioning—by leveraging epitope-antibody interactions to program deaminase localization within the Cas9 framework. This strategy expands the editing capabilities of base editors, allowing for more precise genome modifications with improved safety profiles. Overall, the data represents a reasonable effort, as the authors have systematically evaluated 62 different Plug-in BE variants by embedding epitopes at various positions within nCas9(D10A) and employing three epitope-tagging systems (e.g., SunTag, MoonTag, and ALFA) along with multiple types of base editors. However, despite the extensive data set, the presentation of the data requires some restructuring and work. Much of the content consists of a straightforward presentation of data, lacking sufficient analysis and interpretation. To improve the manuscript, I suggest a few points:\nGiven the large number of tools tested, it is unclear how one should select the most appropriate base editor for a specific genomic site. The manuscript would benefit from a comparative summary that clearly defines the strengths, limitations, and optimal use cases of different Plug-in BE variants.\nThe authors introduce new base editing tools that modify the spatial relationship between deaminase and Cas9, a change that could potentially increase off-target effects. However, the manuscript lacks any off-target evaluation at both the DNA and RNA levels. At a minimum, the authors should include a comprehensive off-target analysis for at a couple of representative Plug-in BE systems to ensure that the modifications do not compromise specificity.\nThe authors attempt to demonstrate the in vivo efficacy of their tools using zebrafish experiments; however, the selected target sites may not be optimal. For the Twist2 locus, prior studies, such as “An optimized base editor with efficient C-to-T base editing in zebrafish”, provide a more established reference. The observed phenotypic effects in this manuscript appear to be more indicative of injection-induced toxicity due to excessive dosage rather than specific base-editing outcomes. Can authors show this is not the case? Moreover, the image quality requires further enhancement to improve clarity and interpretability. Regarding the tyr locus, although the authors report an increase in C11 editing efficiency, the concurrent elevation in C7 editing efficiency suggests potential challenges in maintaining site specificity, which warrants further investigation.\nIn Figure 5H, at the OCT4 site, the labels for \"only A7 to G\" and \"only A5 to G\" are switched.\nIn the context of cytosine base editors (CBEs), BE4 typically exhibits higher editing efficiency than BE3. However, in the tools presented by the authors, multiple BE3 variants outperform BE4 in terms of efficiency. Could the authors provide a mechanistic explanation for this observation?", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors developed the Plug-in Base Editor to overcome the limitations of fixed editing windows and the constrained locations of deaminases in base editors. However, current base editors are no longer restricted by these factors, as Cas variants with altered PAMs allow base editing at any desired site, such as SpRY-fused base editors. Additionally, inlaid and circular permutated base editors achieve similar functionality using a single transcript without increasing overall size. Therefore, the impact of the Plug-in Base Editor is minimal unless experimental evidence demonstrates its superiority over these previously reported systems. Furthermore, this work lacks transcriptome and genomic-level off-target analysis data. The resolution of the figures is low, and they contain excessive data not directly related to the issues discussed in the abstract and introduction, making it difficult for readers to grasp the key points of the paper."}, "author_response": "We sincerely thank all reviewers for their thoughtful and constructive feedback that has significantly improved our manuscript. To address reviewers’ concerns, we performed additional experiments and analysis that provided further support for the robustness of the newly-engineered base editors. We have also addressed all other comments by the reviewers by revising the texts and adding relevant discussions. All the changed texts are underlined for easy identification in this manuscript. Below is a point-by-point response to the specific comments of each reviewer.\nCOMMENTS TO AUTHOR:\nReviewer #1: In this manuscript, the authors present a modular base editing system, termed Plug-in Base Editor (Plug-in BE), which aims to enhance the flexibility, efficiency, and precision of DNA base editors. The manuscript addresses a fundamental limitation of current base editors—fixed deaminase positioning—by leveraging epitope-antibody interactions to program deaminase localization within the Cas9 framework. This strategy expands the editing capabilities of base editors, allowing for more precise genome modifications with improved safety profiles. Overall, the data represents a reasonable effort, as the authors have systematically evaluated 62 different Plug-in BE variants by embedding epitopes at various positions within nCas9(D10A) and employing three epitope-tagging systems (e.g., SunTag, MoonTag, and ALFA) along with multiple types of base editors. However, despite the extensive data set, the presentation of the data requires some restructuring and work. Much of the content consists of a straightforward presentation of data, lacking sufficient analysis and interpretation. To improve the manuscript, I suggest a few points:\nComment 1: Given the large number of tools tested, it is unclear how one should select the most appropriate base editor for a specific genomic site. The manuscript would benefit from a comparative summary that clearly defines the strengths, limitations, and optimal use cases of different Plug-in BE variants.\nResponse: Thank you for your constructive feedback on our manuscript. Based on your advice, we have revised the manuscript to include a comprehensive comparative summary of recommended Plug-in BE variants (Supplementary Table 3). This summary table highlights the advantages, limitations, and optimal applications for each variant by outlining the editing windows, editing efficiency, product purity, and sequence motif preferences. In the summary table, we have recommended 1 to 3 variants within each group based on their editing efficiency and specificity. Additionally, we recommended specific editing regions for Plug-in BE variants, which will help users make informed decisions based on target sequence characteristics. The above results were added in our revised manuscript (Page 11, Line 209-213, and Supplementary Table 3).\nSupplementary Table 3. Editing characteristics of recommended Plug-in BE variants in our study.\nBase Editor system Mutation Type Epitope Embedding Position in Editing Window High Editing Efficiency Area Product Preferential Motif Inefficient Motif Recommended Editing\nnCas9(D10A)/Structure Purity Area\nSunTag Plug-in BE3 C-to-T 532, 1033, 1246 C3-C15 40~90% at C6-C11 80~90% TCN CCA, GCT, GCA C3-C15\n231, 1020, 1051 C3-C15 40~90% at C7-C11 80~90% TCN CCA, CCT, GCA C3-C15\nSunTag Plug-in ABE8eWQ A-to-G 532, 945, 1068 A4-A8 50~70% at A5-A7 95~99% TAN AAA, CAA A6 and A7\n231, 1248, 1260 A5-A7 30~50% at A7 95~99% / / A7\nSunTag Plug-in C-to-G 768, 1020, 1246 C5-C7 30-50% at C6 ~55% / / C6\nminiCGBE1 231, 1068, 1072 C5-C8 20~40% at C6-C8 ~70% / TCA C6-C8\nSunTag Plug-in Target C-to-T 213, 231, 1248 C1-C14 40-70% at C3-C11 80-90% GCC, CCA, TCN GCA, ACG C1-C11\nAID 1033 C1-C8 30-50% at C1-C6 80-90% CCA, TCN GCC, C1-C6\n532, 768, 945 C3-C9 40-70% at C3-C7 80-90% GCC, TCN GCA, CCT C3-C9\nMoonTag Plug-in BE3 C-to-T 231, 945, 1033 C3-C11 50-95% at C6-C11 ~80% TCN CCA, ACG, GCN C6-C11\n768, 1055, 1246, C3-C15 50-95% at C6-C11 ~80% TCN CCA, GCN C6-C15\n213, 1252 C6-C11 30-60% at C6-C11 ~80% TCN ACG C6-C11\n308, 868 C6-C15 30-60% at C6-C15 ~80% TCN GCA, GCT, ACG C6-C15\nMoonTag Plug-in A-to-G 532, 1020, 1260 A3-A9 60-80% at A4-A7 90-99% TAN AAA, CAA A4-A7\nABE8eWQ 231, 1055, 1068 A4-A9 40-70% at A6-A8 90-99% TAN AAA, CAA A6-A8\n945, 1246, 1252 A4-A8 40-70% at A5-A7 90-99% TAN AAA, CAA A5-A7\nMoonTag Plug-in C-to-G 1246, 1252, 1260 C5-C7 40-60% at C6 55-60% / / C6\nminiCGBE1 532, 1055 C5-C8 20-40% at C6 and C8 55-70% / TCA C6-C8\nMoonTag Plug-in Target C-to-T 1033, 1246, 1260 C1-C11 40-70% at C1-C8 70-90% / GCC, TCA, ACG C1-C8\nAID 231, 308, 532 C1-C13 40-60% at C2-C13 70-90% / GCC, ACG C2-C13\n213, 945 C1-C9 40-60% at C2-C6 70-90% / GCC C2-C6\n257, 768, 782 C1-C14 40-60% at C4-C9 70-90% / GCA, TCA C4-C9\nALFA Plug-in ABE8eWQ A-to-G 945, 1246, 1252 A4-A7 40-70% at A5-A7 ~95% / / A5-A7\nALFA Plug-in miniCGBE1 C-to-G 1246, 1252 C5-C8 30-40% at C6-C8 50-60% / / C6-C8\nPlug-in ABE9 A-to-G 1059gp41-ABE9, ABE9-1059gp41 A6-A8 ~60% at A7 ~95% / / A7\n532gp41-ABE9, ABE9-532gp41 A4-A8 40-70% at A5-A7 ~95% / / A5-A7\nABE9-945GCN4 A6-A7 ~40% at A6 ~95% / / A6\nABE9-945gp41 A5-A7 40-60% at A5-A7 ~95% / / A6-A7\nComment 2: The authors introduce new base editing tools that modify the spatial relationship between deaminase and Cas9, a change that could potentially increase off-target effects. However, the manuscript lacks any off-target evaluation at both the DNA and RNA levels. At a minimum, the authors should include a comprehensive off-target analysis for at a couple of representative Plug-in BE systems to ensure that the modifications do not compromise specificity.\nResponse: Thank you for the helpful comment. According to your suggestion, we have conducted the off-target analysis for representative Plug-in ABE variants and Plug-in CBE variants.\n1. Plug-in ABE Variants\n1.1 DNA-level Off-target Analysis\nTo compare the off-target effects of MoonTag Plug-in ABE8eWQ (D10A-231gp41 and D10A-1020gp41) with ABE8eWQ, we conducted amplicon sequencing at the OCT4 site, HEK site 2, and their off-target sites 1-3 (Supplementary Figure 8A and B). D10A-231gp41 and D10A-1020gp41 demonstrated low off-target frequencies (<0.5%) at 15 sites in OCT4 and 19 sites in HEK site 2, comparable to ABE8eWQ. Notably, both variants reduced off-target effects at OT6 and OT7 in OCT4 (D10A-231gp41: p = 0.0332, p = 0.0172; D10A-1020gp41: p = 0.0341, p = 0.0173) and OT1 in HEK site 2 (D10A-231gp41: p = 0.0209; D10A-1020gp41: p = 0.0179). Overall, MoonTag Plug-in ABE8eWQ maintained high DNA-level specificity.\n1.2 RNA-level Off-target Analysis\nAdenine deaminase can deaminate adenine on the RNA chain into hypoxanthine, we therefore assessed RNA-level off-target effects of D10A-231gp41 and D10A-1020gp41 using mRNA sequencing (Supplementary Figure 8C and D). At the OCT4 site, D10A-231gp41 exhibited lower off-target effects than ABE8eWQ (p = 0.0272), while D10A-1020gp41 showed no significant difference. At HEK site 2, both D10A-231gp41 and D10A-1020gp41 had off-target effects comparable to ABE8eWQ. These transcriptome analyses confirm that MoonTag Plug-in ABE8eWQ does not increase RNA-level off-target effects.\n2. Plug-in CBE Variants\nTo evaluate the DNA off-target effects of MoonTag Plug-in BE3 (D10A-1246gp41 and D10A-1252gp41), we conducted amplicon sequencing at HEK site 3, VEGFA site 2, and their off-target sites 4-6 (Supplementary Figure 8E and G). We calculated the on/off-target ratio to quantify the editing specificity (Supplementary Figure 8F and H). At VEGFA site 2, D10A-1246gp41 significantly improved the editing specificity at OT1 (p = 0.0436), OT4 (p = 0.0217), OT5 (p = 0.0207), and OT12 (p = 0.0080). D10A-1252gp41 enhanced the editing specificity at OT3 (p = 0.0077), OT4 (p = 0.0139), OT5 (p = 0.0019), OT6 (p = 0.0044), OT10 (p = 0.0252), and OT12 (p = 0.0153). At HEK site 3, both D10A-1246gp41 and D10A-1252gp41 exhibited similar specificity at four off-target sites. Overall, D10A-1246gp41 and D10A-1252gp41 showed comparable editing specificity to BE3.\nThe above off-target analysis has been included in the revised manuscript on Page 8, Line 141-145; Page 9, Line 154-159; and Supplementary Figure 8.\nSupplementary Figure 8. Off-target analysis of MoonTag Plug-in ABE8eWQ and MoonTag Plug-in BE3.\n(A and B) DNA off-target editing efficiency induced by ABE8eWQ, D10A-231gp41 and D10A-1020gp41 with the sgRNAs targeting OCT4 site and HEK site 2. Data are means ± SD (n = 2).\n(C and D) Transcriptome off-target effects of ABE8eWQ, D10A-231gp41, and D10A-1020gp41 with the sgRNAs targeting OCT4 site and HEK site 2.\n(E and F) DNA off-target frequency and on/off-target ratio of BE3, D10A-1246gp41 and D10A-1252gp41 with the sgRNA targeting VEGFA site 2. Data are means ± SD (n = 2).\n(G and H) DNA off-target frequency and on/off-target ratio of BE3, D10A-1246gp41 and D10A-1252gp41 with the sgRNA targeting HEK site 3. Data are means ± SD (n = 2).\n* p < 0.05, ** p < 0.01, *** p < 0.001.\nComment 3: The authors attempt to demonstrate the in vivo efficacy of their tools using zebrafish experiments; however, the selected target sites may not be optimal. For the TWIST2 locus, prior studies, such as “An optimized base editor with efficient C-to-T base editing in zebrafish”, provide a more established reference. The observed phenotypic effects in this manuscript appear to be more indicative of injection-induced toxicity due to excessive dosage rather than specific base-editing outcomes. Can authors show this is not the case? Moreover, the image quality requires further enhancement to improve clarity and interpretability. Regarding the tyr locus, although the authors report an increase in C11 editing efficiency, the concurrent elevation in C7 editing efficiency suggests potential challenges in maintaining site specificity, which warrants further investigation.\nResponse: Thank you for the valuable comment. Based on your suggestion, we have provided high-quality figures in the revised manuscript. Additionally, we have conducted the control experiment to clarify the observed phenotypic effects by injecting the zebrafish embryos with the same dosage of base editor mRNA and non-targeting sgRNA. The control embryos exhibited normal development, confirming that the observed phenotypes in our experiments are not due to excessive dosage toxicity (Figure 7C and F). In our manuscript, the larvae with edited TWIST2 site exhibited a short trunk and curved tail (Figure 7C). These phenotypic effects resemble those of the TWIST2 E78K mutant zebrafish described in your referenced literature 7. In another literature \"Recurrent mutations in the basic domain of TWIST2 cause ablepharon macrostomia and Barber-Say syndromes\" 8, the TWIST2 E75K mutant zebrafish exhibit a dysmorphic body trunk and pericardial edema, which are similar to the phenotype observed in our larvae.\nSince BE3 was unable to edit C11 due to window limitation, we utilized the D10A-1246gp41 in Plug-in BE3 to extend editing window, and successfully achieved a C11-to-T mutation of up to 95.4% (Figure 7F). However, C7-to-T represents a non-specific edit that occurs when expanding the editing window. In contrast, Plug-in ABE8eWQ, Plug-in ABE9, and Plug-in miniCGBE1 offer the ability to more precisely contract the editing window to A7 (Figure 5F) and C6 (Figure 3I), which could help to minimize bystander edits. Further studies are warranted to achieve a more precise editing window in future studies.\nFigure 7. Targeted editing in challenging sequence regions using Plug-in BE\n(A) Schematic of the mRNA structure used for zebrafish embryo injection. An additional P2A-fluorescent protein element is added to the C-terminal of protein to visualize protein expression.\n(B) Visualization of fluorescent embryos for subsequent DNA sequencing.\n(C) Editing results and phenotype of TWIST2 locus in zebrafish. 231gp41-ABE8eWQ represents the combination of D10A-231gp41 and Antigp41-ABE8eWQ. Data are means ± SD. ABE8eWQ, n=22. 231gp41-ABE8eWQ, n=35.\n(D) Editing product analysis of ABE8eWQ and 231gp41-ABE8eWQ at TWIST2 locus by amplicon sequencing. The samples contain five embryos with the highest efficiency calculated using Sanger sequencing data. Data are means ± SD (n = 5).\n(E) Editing efficiency and specificity of ABE8eWQ and 231gp41-ABE8eWQ at TWIST2 locus.\n(F) Editing results and phenotypes of TYR locus in zebrafish. Data are means ± SD (n = 40).\n(G) Editing product analysis of BE3 and 1246gp41-BE3 at TYR locus by amplicon sequencing. The samples contain five embryos with the highest efficiency calculated using Sanger sequencing data. Data are means ± SD (n = 5).\n(H) Editing efficiency and specificity of BE3 and 1246gp41-BE3 at TYR locus.\nComment 4: In Figure 5H, at the OCT4 site, the labels for \"only A7 to G\" and \"only A5 to G\" are switched.\nResponse: Thank you for the helpful comment. We have carefully checked the errors in our manuscript and corrected the labels in Figure 3G (corresponding to the original Figure 5H). Additionally, we have reviewed and made the necessary corrections in other figures.\nComment 5: In the context of cytosine base editors (CBEs), BE4 typically exhibits higher editing efficiency than BE3. However, in the tools presented by the authors, multiple BE3 variants outperform BE4 in terms of efficiency. Could the authors provide a mechanistic explanation for this observation?\nResponse: Thank you for the constructive comment. As you pointed out, the BE4 with 2 UGIs generally exhibits higher editing efficiency than the BE3 with one UGI, primarily due to the additional UGI further inhibiting C-to-G conversion 9. However, in our study, Plug-in BE3 variants outperformed Plug-in BE4 variants in terms of editing efficiency (Figure 1E and Supplementary Figure 1C).\nThe higher editing efficiency of Plug-in BE3 may be attributed to closer spatial positioning of rAPOBEC1 to the DNA substrate, thereby facilitating more efficient deamination of cytosine to uracil. In the Plug-in BE4 system, the two UGIs may spatially restrict rAPOBEC1's proximity to the DNA substrate, and contributes to the lower editing efficiency of Plug-in BE4 compared to Plug-in BE3.\nReviewer #2:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: Thank you for your helpful comments.\nReviewer #3: The authors developed the Plug-in Base Editor to overcome the limitations of fixed editing windows and the constrained locations of deaminases in base editors. However, current base editors are no longer restricted by these factors, as Cas variants with altered PAMs allow base editing at any desired site, such as SpRY-fused base editors. Additionally, inlaid and circular permutated base editors achieve similar functionality using a single transcript without increasing overall size. Therefore, the impact of the Plug-in Base Editor is minimal unless experimental evidence demonstrates its superiority over these previously reported systems. Furthermore, this work lacks transcriptome and genomic-level off-target analysis data. The resolution of the figures is low, and they contain excessive data not directly related to the issues discussed in the abstract and introduction, making it difficult for readers to grasp the key points of the paper.\nComment 1: The authors developed the Plug-in Base Editor to overcome the limitations of fixed editing windows and the constrained locations of deaminases in base editors. However, current base editors are no longer restricted by these factors, as Cas variants with altered PAMs allow base editing at any desired site, such as SpRY-fused base editors.\nResponse: Thank you for the constructive comment. Based on your suggestion, we directly compared SpRY-fused base editors with MoonTag Plug-in BEs. Specifically, we examined the effects of moving the sgRNA by 1–5 bp to the right in SpRY-fused base editors to match the MoonTag Plug-in BE system (Supplementary Figure 7A). At HEK site 3, moving the sgRNA 1–5 bp to the right did not allow SpRY-BE3 to efficiently convert C9-to-T (1.8% - 18.3%). In contrast, D10A-1246gp41 improved the C9-to-T conversion efficiency from 18.3% (sgRNA with moving 2bp) to 43.0% (Supplementary Figure 7B).\nSupplementary Figure 7. Comparison Between SpRY-Fused Base Editor and MoonTag Plug-in BE3\n(A) Schematic of the sgRNAs with movements of 1-5 bp to the right. The moving sgRNAs are used for SpRY-fused base editors.\n(B) Editing windows and efficiencies of SpRY-BE3 and D10A-1246gp41 with the sgRNAs targeting HEK site 3. Data are means ± SD (n = 2).\nComment 2: Additionally, inlaid and circular permutated base editors achieve similar functionality using a single transcript without increasing overall size.\nResponse: Thank you for the insightful comment. The inlaid and circular permutated base editors achieve similar functionality by embedding deaminases into nCas9(D10A) or permuting nCas9(D10A)’s domains 10. However, identifying the optimal embedding and permuting configurations for deaminases is time-consuming and labor-intensive. This is because that embedding deaminases (much larger than GCN4/gp41 epitopes) into nCas9(D10A) alters protein conformation and activity, limiting the number of viable embedding sites. Further, different deaminases have distinct optimal embedding sites due to variations in size and conformation. For example, mini-Sdd9’s inlaid and circular permutated base editors have 11 configurations, but only one performs well 11. Embedding TadA-TadA* into 43 positions of nCas9(D10A) results in only four variants with editing efficiency comparable to the original base editor 12. Additionally, the inlaid and circular permutated base editors possess relatively fixed editing windows, due to the limited configurations 10-12.\nIn contrast, embedding epitopes into nCas9(D10A) in the Plug-in BE system does not significantly alter the protein’s conformation (Figure 1B), therefore offer more flexibility. At VEGFA site 2, 50 out of 62 D10A-gp41 Plug-in CBE variants showed high C-to-T efficiency (Supplementary Figure 1B). Likewise, 24 out of 62 D10A-gp41 Plug-in ABE variants could achieve efficiently A-to-G conversion (Figure 3F). This flexibility of deaminase spatial localization endows Plug-in BE with more compatible editing window and capability of the Plug-in BE system for gene editing applications (Figure 6 and Supplementary Figure 11A).\nComment 3: Furthermore, this work lacks transcriptome and genomic-level off-target analysis data.\nResponse: Thank you for the helpful comment. According to your suggestion, we have conducted the off-target analysis for representative Plug-in ABE variants and Plug-in CBE variants.\n1. Plug-in ABE Variants\n1.1 DNA-level Off-target Analysis\nTo compare the off-target effects of MoonTag Plug-in ABE8eWQ (D10A-231gp41 and D10A-1020gp41) with ABE8eWQ, we conducted amplicon sequencing at the OCT4 site, HEK site 2, and their off-target sites 1-3 (Supplementary Figure 8A and B). D10A-231gp41 and D10A-1020gp41 demonstrated low off-target frequencies (<0.5%) at 15 sites in OCT4 and 19 sites in HEK site 2, comparable to ABE8eWQ. Notably, both variants reduced off-target effects at OT6 and OT7 in OCT4 (D10A-231gp41: p = 0.0332, p = 0.0172; D10A-1020gp41: p = 0.0341, p = 0.0173) and OT1 in HEK site 2 (D10A-231gp41: p = 0.0209; D10A-1020gp41: p = 0.0179). Overall, MoonTag Plug-in ABE8eWQ maintained high DNA-level specificity.\n1.2 RNA-level Off-target Analysis\nAdenine deaminase can deaminate adenine on the RNA chain into hypoxanthine, we therefore assessed RNA-level off-target effects of D10A-231gp41 and D10A-1020gp41 using mRNA sequencing (Supplementary Figure 8C and D). At the OCT4 site, D10A-231gp41 exhibited lower off-target effects than ABE8eWQ (p = 0.0272), while D10A-1020gp41 showed no significant difference. At HEK site 2, both D10A-231gp41 and D10A-1020gp41 had off-target effects comparable to ABE8eWQ. These transcriptome analyses confirm that MoonTag Plug-in ABE8eWQ does not increase RNA-level off-target effects.\n2. Plug-in CBE Variants\nTo evaluate the DNA off-target effects of MoonTag Plug-in BE3 (D10A-1246gp41 and D10A-1252gp41), we conducted amplicon sequencing at HEK site 3, VEGFA site 2, and their off-target sites 4-6 (Supplementary Figure 8E and G). We calculated the on/off-target ratio to quantify the editing specificity (Supplementary Figure 8F and H). At VEGFA site 2, D10A-1246gp41 significantly improved the editing specificity at OT1 (p = 0.0436), OT4 (p = 0.0217), OT5 (p = 0.0207), and OT12 (p = 0.0080). D10A-1252gp41 enhanced the editing specificity at OT3 (p = 0.0077), OT4 (p = 0.0139), OT5 (p = 0.0019), OT6 (p = 0.0044), OT10 (p = 0.0252), and OT12 (p = 0.0153). At HEK site 3, both D10A-1246gp41 and D10A-1252gp41 exhibited similar specificity at four off-target sites. Overall, D10A-1246gp41 and D10A-1252gp41 showed comparable editing specificity to BE3.\nThe above off-target analysis has been included in the revised manuscript on Page 8, Line 141-145; Page 9, Line 154-159; and Supplementary Figure 8.\nSupplementary Figure 8. Off-target analysis of MoonTag Plug-in ABE8eWQ and MoonTag Plug-in BE3.\n(A and B) DNA off-target editing efficiency induced by ABE8eWQ, D10A-231gp41 and D10A-1020gp41 with the sgRNAs targeting OCT4 site and HEK site 2. Data are means ± SD (n = 2).\n(C and D) Transcriptome off-target effects of ABE8eWQ, D10A-231gp41, and D10A-1020gp41 with the sgRNAs targeting OCT4 site and HEK site 2.\n(E and F) DNA off-target frequency and on/off-target ratio of BE3, D10A-1246gp41 and D10A-1252gp41 with the sgRNA targeting VEGFA site 2. Data are means ± SD (n = 2).\n(G and H) DNA off-target frequency and on/off-target ratio of BE3, D10A-1246gp41 and D10A-1252gp41 with the sgRNA targeting HEK site 3. Data are means ± SD (n = 2).\n* p < 0.05, ** p < 0.01, *** p < 0.001.\nComment 4: The resolution of the figures is low, and they contain excessive data not directly related to the issues discussed in the abstract and introduction, making it difficult for readers to grasp the key points of the paper.\nResponse: Thank you for your constructive feedback. Based on your advice, we have provided high-quality figures in the revised manuscript. To ensure the key points of the paper are more accessible to readers, we have restructured and simplified our data presentation as follow.\nRestructure 1: We simplified the heatmaps in Figure 1, Figure 2, and Figure 3.\nRestructure 2: We highlighted the outstanding ability of Plug-in ABE8eWQ in enhancing the single-base editing accuracy in Figures 2C and Figure 3G, and relocated their product analysis data to Supplementary Figure 2C and D.\nRestructure 3: To emphasize the advantage of Plug-in BE for editing zebrafish embryos in Figure 7, we have moved the Clinvar analysis and HeLa cell’s base editing (the original Figure 7A-E) to Supplementary Figure 11.\nRestructure 4: We created a comprehensive comparative summary (Supplementary Table 3) outlining the advantages, limitations, and recommended applications of Plug-in BE variants to help users select the most appropriate variants based on efficiency and specificity.\nSupplementary Table 3. Editing characteristics of recommended Plug-in BE variants in our study.\nBase Editor system Mutation Type Epitope Embedding Position in Editing Window High Editing Efficiency Area Product Preferential Motif Inefficient Motif Recommended Editing\nnCas9(D10A)/Structure Purity Area\nSunTag Plug-in BE3 C-to-T 532, 1033, 1246 C3-C15 40~90% at C6-C11 80~90% TCN CCA, GCT, GCA C3-C15\n231, 1020, 1051 C3-C15 40~90% at C7-C11 80~90% TCN CCA, CCT, GCA C3-C15\nSunTag Plug-in ABE8eWQ A-to-G 532, 945, 1068 A4-A8 50~70% at A5-A7 95~99% TAN AAA, CAA A6 and A7\n231, 1248, 1260 A5-A7 30~50% at A7 95~99% / / A7\nSunTag Plug-in C-to-G 768, 1020, 1246 C5-C7 30-50% at C6 ~55% / / C6\nminiCGBE1 231, 1068, 1072 C5-C8 20~40% at C6-C8 ~70% / TCA C6-C8\nSunTag Plug-in Target C-to-T 213, 231, 1248 C1-C14 40-70% at C3-C11 80-90% GCC, CCA, TCN GCA, ACG C1-C11\nAID 1033 C1-C8 30-50% at C1-C6 80-90% CCA, TCN GCC, C1-C6\n532, 768, 945 C3-C9 40-70% at C3-C7 80-90% GCC, TCN GCA, CCT C3-C9\nMoonTag Plug-in BE3 C-to-T 231, 945, 1033 C3-C11 50-95% at C6-C11 ~80% TCN CCA, ACG, GCN C6-C11\n768, 1055, 1246, C3-C15 50-95% at C6-C11 ~80% TCN CCA, GCN C6-C15\n213, 1252 C6-C11 30-60% at C6-C11 ~80% TCN ACG C6-C11\n308, 868 C6-C15 30-60% at C6-C15 ~80% TCN GCA, GCT, ACG C6-C15\nMoonTag Plug-in A-to-G 532, 1020, 1260 A3-A9 60-80% at A4-A7 90-99% TAN AAA, CAA A4-A7\nABE8eWQ 231, 1055, 1068 A4-A9 40-70% at A6-A8 90-99% TAN AAA, CAA A6-A8\n945, 1246, 1252 A4-A8 40-70% at A5-A7 90-99% TAN AAA, CAA A5-A7\nMoonTag Plug-in C-to-G 1246, 1252, 1260 C5-C7 40-60% at C6 55-60% / / C6\nminiCGBE1 532, 1055 C5-C8 20-40% at C6 and C8 55-70% / TCA C6-C8\nMoonTag Plug-in Target C-to-T 1033, 1246, 1260 C1-C11 40-70% at C1-C8 70-90% / GCC, TCA, ACG C1-C8\nAID 231, 308, 532 C1-C13 40-60% at C2-C13 70-90% / GCC, ACG C2-C13\n213, 945 C1-C9 40-60% at C2-C6 70-90% / GCC C2-C6\n257, 768, 782 C1-C14 40-60% at C4-C9 70-90% / GCA, TCA C4-C9\nALFA Plug-in ABE8eWQ A-to-G 945, 1246, 1252 A4-A7 40-70% at A5-A7 ~95% / / A5-A7\nALFA Plug-in miniCGBE1 C-to-G 1246, 1252 C5-C8 30-40% at C6-C8 50-60% / / C6-C8\nPlug-in ABE9 A-to-G 1059gp41-ABE9, ABE9-1059gp41 A6-A8 ~60% at A7 ~95% / / A7\n532gp41-ABE9, ABE9-532gp41 A4-A8 40-70% at A5-A7 ~95% / / A5-A7\nABE9-945GCN4 A6-A7 ~40% at A6 ~95% / / A6\nABE9-945gp41 A5-A7 40-60% at A5-A7 ~95% / / A6-A7"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have satisfactorily addressed all of my previous concerns. The revised manuscript is clearly written, the data support the conclusions, and the additional analyses and clarifications further strengthen the work.\nMinor comments:\nFigure 7: Replace the scale bar label “200 uM” (micromolar) with “200 μm” (micrometre), and ensure consistent usage throughout the figure and caption (e.g., Scale bar, 200 μm).", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors thoroughly addressed the concerns of reviewer 3.\nOne minor concern: the data collected comparing SpRY to Plug-in BE (Supplementary Figure 7) is excellent, but I don't see it specifically detailed in the text of the manuscript or in either of the supplementary notes. This data should be discussed explicitly in text -- it is worth remarking upon the observation that Cas domains with expanded targeting capacity do not necessarily permit unrestricted and precise base editing, thus highlighting the utility of tools like Plug-in BE."}, "author_response": "Dear Reviewers,\nWe sincerely appreciate the valuable insights and constructive comments provided by reviewers for our manuscript (Title: A Plug-in System for Reprogramming the Editing Patterns of Base Editors). We greatly value these insights, as they have helped enhance the clarity, rigor, and scientific relevance of our work. We sincerely appreciate the time and effort you have devoted to reviewing our manuscript.\nWe have carefully addressed all two comments raised by reviewers and fully incorporated the revisions into the updated manuscript. Below, we provide a detailed point-by-point response to each comment.\nCOMMENTS TO AUTHOR:\nReviewer #1: The authors have satisfactorily addressed all of my previous concerns. The revised manuscript is clearly written, the data support the conclusions, and the additional analyses and clarifications further strengthen the work.\nMinor comments: Figure 7: Replace the scale bar label “200 uM” (micromolar) with “200 μm” (micrometre), and ensure consistent usage throughout the figure and caption (e.g., Scale bar, 200 μm).\nResponse: Thank you very much for pointing out this detail. We have revised Figure 7 as suggested, replacing the scale bar label \"200 uM\" with \"200 μm\". Meanwhile, we have thoroughly checked the figure captions and all figures throughout the manuscript to ensure the consistent use of the scale bar format. The revised Figure 7 has been updated in the revised manuscript for your review.\nReviewer #2: I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: Thank you for your constructive feedback on our manuscript.\nReviewer #4: The authors thoroughly addressed the concerns of reviewer 3.\nOne minor concern: the data collected comparing SpRY to Plug-in BE (Supplementary Figure 7) is excellent, but I don't see it specifically detailed in the text of the manuscript or in either of the supplementary notes. This data should be discussed explicitly in text -- it is worth remarking upon the observation that Cas domains with expanded targeting capacity do not necessarily permit unrestricted and precise base editing, thus highlighting the utility of tools like Plug-in BE.\nResponse: Thank you for your valuable suggestion. We fully agree that the comparative data between SpRY-BE3 and MoonTag Plug-in BE3 (Supplementary Figure 7) merits explicit discussion in the manuscript, as it highlights a key advantage of the Plug-in BE system.\nAs you noted, Cas variants with expanded targeting capacity (e.g., the nearly PAM-less SpRY) overcome PAM restrictions, but do not address the core limitations of fixed editing windows and dependence on highly active sgRNA. Our comparative analysis demonstrates that even with sgRNA positional adjustments (1-5 bp rightward shifts), SpRY-BE3 failed to efficiently achieve C9-to-T conversion with only 1.4%-16.9% efficiency at HEK site 3 (Supplementary Figure 7A and B). In contrast, MoonTag Plug-in BE3 (D10A-1246gp41) achieved a higher C9-to-T efficiency of 38.9% without relying on sgRNA redesign (Supplementary Figure 7B). This finding underscores that expanded PAM compatibility alone does not guarantee precise and efficient base editing, whereas the Plug-in BE system’s programmable deaminase positioning enables targeted edits in regions inaccessible to conventional Cas-fused base editors.\nWe have now integrated this discussion into the revised manuscript (Page 9-10, Lines 165-173) to emphasize the complementary value of Plug-in BE alongside Cas variants with broad targeting scope."}]

Decision letter