Anti-CRISPR protein AcrIIA5 can enhance the activity and security of prime editing

Domain: neuroscience
Published: 2025-12-19
Editorial outcome: accept

Abstract
Prime editing (PE) enables the precise installation of intended base substitutions, small deletions or small insertions into the genome of living cells. While the use of Cas9 nickase can avoid DNA double-strand breaks (DSB), undesired insertions and deletions (indels) often accompany the corrected its, particularly when PE activity increased. Here we show that the anti-CRISPR (Acr) protein AcrIIA5 can significantly enhance PE activity by up to 8.2-fold while markedly reducing byproduct indels. Further investigation reveals that AcrIIA5 can promote PE across various approaches (PE2, PE3, PE4, PE5, and PE6), edit types (substitutions, insertions and deletions), and endogenous loci. Mechanistically, AcrIIA5 appears to inhibit the re-nicking activity of PE complex rather than enhancing the core editing machinery itself, suggesting a distinct mode of interaction with Cas9. Overall, we demonstrate that a known “inhibitor” Acr protein can unexpectedly acting as an “enhancer” of CRISPR/Cas-based genome editing, providing an effective strategy to optimize PE specificity and activity.

Introduction
Gene editing tools enable targeted changes in the genome of living cells, advancing the life sciences and therapeutic applications1. Programmable nucleases, such as ZFNs2, TALENs3, or CRISPR/Cas nucleases4–6, induced targeted DSBs, which can either promote non-homologous end joining (NHEJ)-mediated random insertions and deletions (indels), or facilitate homology-directed repair (HDR)-mediated precise corrections in the presence of donor DNA templates. However, DSBs have been proven to carry significant risks, including large DNA rearrangements7,8, deletions8,9, or translocations10, which pose non-negligible safety concerns. Instead, base editors, composed of a deaminase and a Cas protein, can install base transitions like C-to-T11 or A-to-G12, or base transversions like G-to-T13 or T-to-G14,15 without introducing DSBs. However, despite the advantages above, base editors have suffered from insufficient product purity11,13–16, undesired bystander editing11,12 and off-targeting17,18, severely undermining the accuracy and security.

Advantageously, PE system can precisely install base substitutions, DNA insertions, or DNA deletions in genomes without the need for DSBs or donor DNA templates19. The commonly used PE2 system minimally consists of a modified PE2 protein (Cas9-H840A nickase fused with an engineered Moloney murine leukemia virus reverse transcriptase, M-MLV RT) and a pegRNA that contains both a spacer sequence and a 3’ extension encoding the desired edit. Guided by pegRNA, the PE2 protein binds the target DNA and nicks the PAM-containing strand, forming an exposed 3’ end that pairs to the primer bindings site (PBS) in the pegRNA extension. Then, M-MLV RT synthesizes the corresponding sequences guided by RT template (RTT) in pegRNA extension, into the nicked non-target strand (NTS), and the edits are ultimately incorporated into the genome.

To improving PE2 editing efficiency, another sgRNA was introduced in PE3 system to nick the non-edited strand19, while another MLH1dn was fused with PE2 protein in PE4 system to inhibit DNA mismatch repair (MMR) which lead to non-mutated edit20. PE6 systems, which utilized engineered Cas9 proteins or different types of RT, have also demonstrated improved efficiency in certain edits21. Despite avoiding DSBs, PE systems still resulted in noticeable undesired indels which were significantly increased in PE3 or PE5. Besides, PE4 performs well in MMR-active cell strains like K562, but showed marginal improvements in MMR-inactive cell strains like HEK293T. Additionally, MLH1dn fused in PE4 was relatively large (752 amino acids), complicating its incorporation into adeno-associated viruses (AAV), which have a limited encapsulation capacity.

Phage-derived anti-CRISPR (Acr) proteins are a class of small proteins (usually <150 amino acids) that strongly inhibit various Cas proteins through diverse mechanisms22. Among them, AcrIIA5 can effectively block the DNA cleavage activity of Streptococcus pyogenes Cas9 (SpCas9)23–26 and its derived base editors27.

In this study, we identify that AcrIIA5 can enhance the activity of PE, and consequently develop the AcrIIA5-assisted PE (aPE) system, showing significantly improved editing efficiency and decreased indels simultaneously. We further confirm that aPE performs well across different architectures (PE2, PE3, PE4, PE5, and PE6) and endogenous loci, enhancing substitutions, deletions, and insertions by up to 8.2-fold, 3.2-fold, and 5.2-fold, respectively. Meanwhile, we also observe that aPE can produce variable editing outcomes at different positions within the same target site. Further analyses reveal that edits causing PAM-silencing tended to decrease aPE editing efficiency, whereas edits at PAM-proximal or PAM-distal positions generally benefit from aPE. This position-dependent effect is likely due to AcrIIA5 preventing re-targeting and re-nicking by the SpCas9-H840A of the PE complex. Notably, this regulatory mechanism appears to be unique to AcrIIA5 among tested Acr proteins, highlighting its potential as a versatile modulator for PE systems.

Results
AcrIIA5 can enhance PE activity
The anti-CRISPR protein AcrIIA5 can inhibit the gene editing ability of Type II Cas9 by suppressing its cleavage activity25,26. To explore the potential of AcrIIA5 in regulating the CRISPR/Cas9 and its derived systems, we co-delivered it with SpCas9, base editors, and the PE2 system into human HEK293T cells in equimolar (Fig. 1a–c). To eliminate transfection-related effects caused by additional AcrIIA5-expressing plasmid load, we also delivered an equal amount of an empty vector pcDNA3.1(-) without AcrIIA5 as control.

After SpCas9 induces DSBs in the target DNA, cells repair the breaks via NHEJ pathway, resulting in the formation of indels4–6. We observed that SpCas9 caused indels of 71.6% at HEK3 locus, while the indels decreased to 20.6% with AcrIIA5 co-transfected (Fig. 1a), indicating that AcrIIA5 significantly inhibited the SpCas9 activity.

The cytosine base editor (CBE), composed of SpCas9-D10A and deaminases, can convert specific cytosines (C) in the target DNA to thymine (T). We observed that CBEs with cytosine deaminases such as hA3A28,29, hA3Bctd30, hA3Gctd31, and mAPOBEC111 as catalytic cores induce efficient C-to-T conversions at positions C3, C4, C5, and C9 in HEK3 locus (Fig. 1b and Supplementary Fig. 1). However, the addition of AcrIIA5 significantly decreased these C-to-T conversions, indicating that AcrIIA5 effectively inhibited the activity of these CBEs.

The PE2 system enables the introduction of the +1 T-to-A conversion at HEK3 target guided by a specific pegRNA. Surprisingly, when AcrIIA5 was co-delivered with the PE2 into HEK293T cells, the intended T-to-A editing efficiency increased from 6.7% to 9.8%, suggesting that AcrIIA5 may enhance the activity of the PE system (Fig. 1c).

Previous reports have proven that varying doses of AcrIIA5 can inhibit SpCas927 and base editors26 to different extents. Therefore, we hypothesized that the dosage of AcrIIA5 might similarly affect the performance of PE system. Then, we co-delivered AcrIIA5 and the PE2 system into HEK293T cells at molar ratios of 0.1/0.2/0.3/0.4/0.5/0.6/0.8/1.0/1.5/2.0/2.5/3.0 to introduce +1 T-to-A at HEK3 locus (Fig. 1d). The results showed that the PE2 alone caused conversion of 6.7%, and co-delivery with varying doses of the empty vector resulted in similar efficiencies.

In stark contrast, different doses of AcrIIA5 had a significant impact on PE2 activity, with the maximum +1 T-to-A conversion reached 24.9% at 0.1 AcrIIA5:PE2 molar ratio. As the molar ratio increased from 0.1 to 3.0, the PE2 efficiency gradually decreased from 24.9% to 8.8%. Additionally, PE2 alone led to 1.4% indels, which remained stable when co-delivered with different doses of the empty vector (Fig. 1e). Theoretically, the indels should decrease with increasing AcrIIA5 dosage. However, even at low molar ratios of 0.1 and 0.2, the indels significantly dropped to 0.5-fold and 0.14-fold of the original rate, respectively.

These results confirmed that AcrIIA5 can enhance the performance of the PE2 by improving activity and decreasing indels. Hence, we named this system as aPE. Considering that with the increasing dosage of AcrIIA5, the increase of PE2 efficiency gradually declined, thus we selected 0.1 molar ratio of AcrIIA5:PE in subsequent tests except other explanations, aiming to achieve the optimal enhancement of PE activity.

Investigation of aPE-introduced base conversions
Previous results have confirmed that aPE2 showed higher editing efficiency in +1 T-to-A edits at HEK3 target. To determine whether this enhancement can extend to other types of base conversions, we unbiasedly tested all possible base mutations across +1 to +6 positions (Fig. 1f). To our surprise, aPE2 exhibited two distinct windows with markedly different outcomes. In the +1 to +4 region, the efficiencies of all aPE2-introduced base conversions were remarkably enhanced compared to PE2, with an average increase of 3.4-fold and a maximum increase of up to 5.3-fold (+4 T-to-C). In contrast, all aPE2-introduced conversions in +5 and +6 location dramatically decreased to average 0.42-fold of PE2. Notably, aPE2-induced indels (average 0.49%) were significantly reduced almost in all base conversions across +1 to +6 compared to PE2 (average 1.28%) (Fig. 1g).

The above results suggested that the improvement of PE2 activity by AcrIIA5 maybe influenced more by the position of the edited base than by the type of base conversion. To verify this assumption, we evaluated each base conversion, including both transitions and transversions, at position +1 to +6 in three additional targets. Similar to HEK3 locus, aPE2-introduced conversions efficiencies in +1 to +4 at HEK2 locus increased by an average of 1.9-fold, while conversions at positions +5 and +6 were reduced (Supplementary Fig. 2a). Somewhat differently, aPE2-introduced base conversions efficiencies were only improved in +1 to +3 at HEK5 (average 3.2-fold) (Supplementary Fig. 2b) and HEK6 (average 2.2-fold) (Supplementary Fig. 2c), while those conversions in +4 to +6 regions were inhibited. Meanwhile, almost all indels alongside the edits across the three additional targets obviously decreased (Supplementary Fig. 2d–f).

After analyzing all the edits introduced by PE2 and aPE2 across the four tested targets (Fig. 1h), we observed that the aPE2 activity for base conversions at positions +1 to +4 increased to 2.7-fold, 3.0-fold, 2.5-fold, and 1.9-fold of that observed by PE2, respectively. While the base conversion efficiencies by aPE2 were reduced to 0.42-fold and 0.48-fold of those observed by PE2 at positions +5 and +6. Interestingly, PE2 showed higher editing efficiency at positions +5 and +6 and lower editing efficiency at positions +1 to +4. Moreover, the indels comparison of HEK2, HEK3, and HEK5 (HEK6 was excluded as high background) targets revealed that the average aPE2-induced indels across positions +1 to +6 were reduced to 0.29-fold, 0.26-fold, 0.34-fold, 0.30-fold, 0.54-fold, and 0.45-fold, respectively, of that caused by PE2 (Fig. 1i). These results confirmed that the enhancement of PE2 activity assisted by AcrIIA5 was independent of the base conversion types and was solely related to the position of the edited base within the targets. Additionally, almost all indels across positions +1 to +6 caused by aPE2 were obviously inhibited compared to PE2, proving the high security and accuracy of aPE2.

Compatibility of aPE with various constructs
As the most commonly used version, PE2 showed limitations in activity. PE3 employed additional sgRNA to nick the non-edited strand, greatly enhancing the PE2 activity19. PE4 showing high activity was engineered through the fusion of PE2 and MLH1dn protein which inhibited the MMR, and PE5 was constructed with the addition of a sgRNA nicking the non-edited strand20. However, PE3 and PE5 usually induced significantly increased indels, reducing the purity of editing outcomes. Besides, although PE4 performed well in MMR-active cell lines like K562, but showed only slight improvement in MMR-inactive cell lines like HEK293T. Considering the outstanding performance of aPE2 in enhancing the editing efficiency and accuracy, we hypothesized AcrIIA5 may also improve the PE3, PE4, and PE5 architectures. Subsequently, we tested these PE and aPE systems at three individual edits in three different targets.

The results indicated that AcrIIA5 can advance the editing efficiency of all tested PE architectures at the HEK2, +3 C-to-A edits (Fig. 2a). Specifically, additional sgRNAs causing –36 nick and –40 nick did not enhance the activity of PE2 or PE4, while only the introduction of +21 nick increased the performance of PE2 and PE4, raising their efficiencies from 23.8% (PE3+21 nick) and 28.0% (PE5+21 nick), to 31.2% (aPE3+21 nick) and 29.2% (aPE5+21 nick), respectively. Besides, AcrIIA5 significantly boosted the efficiencies of PE2 (from 16.2% to 37.2%) and PE4 (from 17.0% to 35.6%), suggesting that AcrIIA5’s assistance may be more effective for PE2 and PE4 compared to PE3 and PE5. Additionally, all aPE constructs exhibited significantly reduced indels compared to PE constructs (Supplementary Fig. 3a). Similar results can be observed at HEK5, +2 T-to-C edits (Fig. 2b and Supplementary Fig. 3b) and HEK6, +2 G-to-A edits (Fig. 2c and Supplementary Fig. 3c).

Comprehensive comparison clearly revealed distinction between PE and aPE systems (Fig. 2d). In the intra-comparison within the PE system, the editing efficiency of PE2 (7.5%), PE4 (8.2%), PE3 (8.6%), and PE5 (10.7%) showed a progressive increase, suggesting that in the MMR-deficient HEK293T cell line, nicking the non-editing strand (PE3) can enhance PE2 activity more effectively than fused MLH1dn (PE4), and these two enhancements were additive (PE5) (Fig. 2d). However, in the intra-comparison within the aPE system, the editing efficiency of aPE2 (19.2%), aPE4 (17.7%), aPE3 (15.6%), and aPE5 (11.6%) showed a progressive decrease.

In the inter-comparison between PE and aPE, aPE exhibited significantly higher editing activity (Fig. 2d). For instance, the average editing efficiencies of aPE2, aPE4, aPE3, and aPE5 were 2.6-fold, 2.2-fold, 1.8-fold, and 1.1-fold higher, respectively, compared to their corresponding PE systems. Notably, PE3 and PE4 only increased PE2 activity to 1.31-fold and 1.05-fold, respectively, which were significantly lower than the 2.6-fold increase achieved by aPE2, indicating that AcrIIA5 was more effective than MLH1dn (PE4) and the additional nicking sgRNA (PE3) (Fig. 2d). Moreover, aPE2 reached 1.8-fold of the activity of PE5 which was considered as the most effective architecture, confirming the strong potency of AcrIIA5.

In the comprehensive statistics of indels, PE2, PE4, PE3, and PE5 showed average indels of 5.05%, 3.04%, 8.09%, and 6.53%, respectively (Fig. 2e), which proved the high efficacy of MLH1dn in reducing by-products and confirmed that the additional nicking sgRNA in PE3 or PE5 would significantly increase the indels. Notably, all aPE constructs exhibited superb decrease of indels compared to according PE constructs (Fig. 2e). For example, aPE2, aPE3, aPE4, and aPE5 showed average indels of 1.11%, 0.77%, 1.47%, and 0.81%, respectively. Notably, MLH1dn reduced indels by 1.66-fold (PE4) and 1.24-fold (PE5) for PE2 and PE3, respectively, while AcrIIA5 reduced indels by 4.5-fold (aPE2) and 5.5-fold (aPE3) for PE2 and PE3, respectively, suggesting that AcrIIA5 was more effective than MLH1dn in reducing by-product indels. Besides, MLH1dn had a length of 752 amino acids (2256 bp), while AcrIIA5 was only 140 amino acids (420 bp). Therefore, AcrIIA5 may be more suitable for integration into AAV vectors with a 4.7 kb packaging limit.

Furthermore, we speculated on the reason for the progressively decreased editing activity observed from aPE2 to aPE5. We guessed that this phenomenon may stem from structural and expression-related constraints inherent to these more complex architectures. In PE4 and PE5, the MMR inhibitor MLH1dn is co-expressed via a P2A self-cleaving peptide. However, the cleavage efficiency of the P2A sequence has been reported to be incomplete in HEK293T cells32. This incomplete cleavage may lead to a subset of SpCas9-RT fusion proteins retaining a covalently linked MLH1dn domain. Given that MLH1dn is a relatively large protein, this residual fusion could introduce steric hindrance that interferes with AcrIIA5’s interaction with SpCas9, thereby diminishing its enhancing effect in PE4 and PE5.

To investigate whether the genetic linkage between the PE2 expression cassette and MLH1dn via a P2A sequence affects the regulatory impact of AcrIIA5, we constructed an additional system in which MLH1dn is expressed from a separate plasmid. For clarity, we refer to the original PE4 system as PE2+iMLH1dn (integrated MLH1dn) and the system with independent MLH1dn expression as PE2+sMLH1dn (separate MLH1dn). We then evaluated editing efficiencies across the PE2–PE5 architectures under these modified conditions (Supplementary Fig. 3d, e). Consistent with our earlier findings, aPE2 exhibited higher editing efficiency than aPE2+iMLH1dn, and similarly, aPE3 outperformed aPE3+iMLH1dn (Supplementary Fig. 3e). Strikingly, both aPE2+sMLH1dn and aPE3+sMLH1dn showed even greater editing efficiencies than their respective aPE2 and aPE3 counterparts. This unexpected result further supports our hypothesis that incomplete P2A cleavage in PE4 can lead to residual MLH1dn interfering with AcrIIA5–SpCas9 interactions, thereby reducing its regulatory effect. Moreover, the improved performance of aPE systems when MLH1dn is delivered in trans highlights the modularity and extensibility of the aPE platform.

Besides, a recent study reported that the La protein can significantly enhance the editing efficiency of PE system through binding to the poly(U) at the 3’ end of pegRNA33. Consistent with this, we observed that PE7 increased the HEK3, +1 T-to-A conversion to a level comparable to that of aPE2, although it produced higher indel frequencies than aPE2 (Supplementary Fig. 3f). Interestingly, however, PE7 exhibited markedly reduced activity when combined with AcrIIA5, suggesting that the presence of the La protein may counteract the effect of AcrIIA5. Given that the only difference between PE2 and PE7 is the addition of the La protein, which stabilizes the pegRNA by binding its 3’ poly(U) tail, we hypothesized that this component is a key factor influencing the response of aPE7 to AcrIIA5.

According to previous reports26, AcrIIA5 can bind to sgRNA in SpCas9 complex, suggesting it may also interact with pegRNA. We speculated that AcrIIA5 and La protein might interfere with each other on the pegRNA scaffold. Besides, like iMLH1dn tested above, the La protein alone may also prevent the AcrIIA5-SpCas9 interaction through steric hindrance. To investigate these possibilities, we engineered a system in which the poly(U) tail was removed by inserting a self-cleaving HDV ribozyme between the pegRNA and the poly(U) sequence (Supplementary Fig. 3g). This ribozyme cleaves cotranscriptionally, producing pegRNA transcripts lacking the poly(U) extension. We then compared the editing efficiencies of PE2, PE7, aPE2, and aPE7 systems with or without the poly(U) tail.

We first observed that aPE2 with HDV-cleaved pegRNA exhibited higher editing efficiency than the corresponding PE2 control, confirming that AcrIIA5 functions independently of the poly(U) sequence and does not act through direct binding to it (Supplementary Fig. 3h). Next, we found that PE7 with HDV-cleaved pegRNA restored editing activity to levels comparable to PE2, consistent with La protein requiring poly(U) to exert its function. Notably, aPE7 with HDV-cleaved pegRNA also regained activity similar to aPE2, proving that the size of La protein alone can not prevent the AcrIIA5-SpCas9 interaction through steric hindrance. Together, these results support a refined model in which the poly(U) tail enables La protein binding to the 3’ end of pegRNA, and this bound La protein indirectly interferes with AcrIIA5 function. This spatial interference likely impairs AcrIIA5’s ability to fine-tune the SpCas9–pegRNA complex, ultimately leading to the decreased editing efficiency observed in the aPE7 system.

Overall, we have proven AcrIIA5 was compatible with a wide range of PE architectures. Especially, aPE2 not only demonstrated higher activity than PE3 but also maintained lower indels compared to PE4, reflecting both high editing activity and accuracy.

In addition to introducing extra non-edited strand nicking sgRNAs and MLH1dn proteins that inhibited MMR, modifying the pegRNA, SpCas9-H840A, and RT in the PE systems can also affect editing outcomes. Recent study confirmed that the epegRNA with a 3’ exonuclease protection motif can improve the PE performance34. Furthermore, through extensive amino acid mutations screening and phage-assisted directed evolution, series of novel PE systems were developed, including PE6a (SpCas9-H840A+evoEc48 RT), PE6c (SpCas9-H840A+evoTf1 RT), PE6d (with additional T128N+V223Y+D200C mutations in the M-MLV RT and deletion of the RNase H domain), and PE6e (with additional K918A+K775R mutations in SpCas9-H840A)21.

In this study, by introducing a variety of potentially effective amino acid mutations into the M-MLV RT35 of PE2, we successfully identified mutations such as L139P, E302R, and V223A that can enhance editing efficiency (Supplementary Fig. 4a). We hypothesized that combinations of these mutations could further enhance PE2 activity. Consequently, we generated PE2 variants with additional mutations in M-MLV RT, including PE2-PR (additional L139P+E302R), PE2-PA (additional L139P+V223A), PE2-AR (additional V223A+E302R), and PE2-PAR (additional L139P+V223A+E302R). Subsequently, we tested these PE variants with changes in SpCas9 or RT and with the epegRNA with 3’ evopreQ1 motif. The results showed all tested PE variant showed higher activity and lower indels with the assistance of AcrIIA5 in +1 T-to-A edit at HEK3 target (Supplementary Fig. 4b). In detail, aPE variants showed 1.05-fold to 2.40-fold of the PE variants activity. Among them, PE2-PA exhibited the highest initial editing efficiency of 17.76%, which was further increased to 20.33% with the assistance of AcrIIA5, and aPE2 showed highest activity in aPE variants. Similarly to previous results, indels caused by aPE variants substantially decreased to 0.16-fold to 0.40-fold compared to PE variants.

To further assess the generalizability of the aPE strategy, we compared aPE2 with several recently developed PE variants. We tested PE2, PE_Y1836, PEmax**37, PE2_NC38, and PE2_SB39 at three representative loci with or without AcrIIA5 and found that aPE2 consistently outperformed all of these systems (Supplementary Fig. 5a, b). Moreover, each of the tested variants exhibited further improvements in editing efficiency and reductions in indels formation when combined with AcrIIA5 (Supplementary Fig. 5c, d). This enhancement was also evident in epiPE240 (Supplementary Fig. 5e), whose activity and precision were comprehensively elevated by AcrIIA5 (Supplementary Fig. 5f, g). In addition, we examined the compatibility of aPE2 with engineered pegRNAs, including apegRNA41 and mpegRNA42 (Supplementary Fig. 6a, b). While both designs improved the performance of wtpegRNA-PE2, their combination with aPE2 resulted in even greater activity together with markedly lower indels formation (Supplementary Fig. 6c, d). Notably, the indels formation of mpegRNA-PE2 was reduced from 1.81% to 0.29%, and was further decreased to 0.16% by mpegRNA-aPE2, underscoring the critical role of AcrIIA5 in enhancing both activity and specificity across diverse PE architectures.

Additionally, our study found that the enhancement of base conversions by aPE systems might be position-dependent. However, the SpCas9-H840A in the PE system can only recognize NGG PAM, which limited the positions where aPE system can improve. To address this issue, we replaced SpCas9-H840A in aPE2ΔRNaseH with the PAM-less SpRY-H840A, and tested it at several targets with both conventional and non-conventional PAMs (Fig. 2f). The results showed that in the +1 T-to-A edits at targets with the conventional GGG PAM, the PAM-less PE2ΔRNaseH achieved an editing efficiency of 10.33%, which increased to 14.52% with AcrIIA5. At targets with non-conventional PAMs such as TGC, GAT, and AAA, the PAM-less PE2ΔRNaseH achieved editing efficiencies of 2.33%, 5.02%, and 2.96% for +1 A-to-T, +1 C-to-G, and +1 A-to-T edits, respectively. With the assistance of AcrIIA5, these efficiencies were increased to 5.68%, 10.03%, and 6.96%, respectively. Simultaneously, the indels were significantly reduced in all tested edits. Consequently, the compatibility of AcrIIA5 with the PAM-less PE enhanced the applicability of this strategy.

In conclusion, AcrIIA5 was compatible with most known PE architectures, including various engineered pegRNA, RTs, and SpCas9 variants, as well as the addition of extra MLH1dn protein and nicking sgRNA.

Assessment of aPE-introduced deletions and insertions
PE cannot only introduce various types of base conversions but also delete small DNA fragments. To validate whether AcrIIA5 can enhance the performance of PE-mediated deletions, we then examined four targets for testing increasing deletion lengths ranging from 1 to 6 nt. At the HEK3 target (Fig. 3a, b), PE2 showed efficiencies of 17.7%, 20.4%, and 9.1% for 1 nt, 2 nt, and 3 nt deletions, while aPE2 showed that of 38.3%, 42.3%, and 12.5%, respectively. However, aPE2 exhibited lower activity for 4 nt, 5 nt, and 6 nt deletions compared to PE2, suggesting that aPE2-mediated deletions might also be position-dependent. Meanwhile, most unintended indels by aPE2 considerably decreased (Fig. 3c). Similar results were observed at HEK2 (Supplementary Fig. 7a, b), HEK5 (Supplementary Fig. 7c, d), and HEK6 (Supplementary Fig. 7e, f) targets, even though there were some distinction about the enhancement potency.

The comprehensive analysis revealed that the average aPE2-introduced deletion efficiencies of 1 nt, 2 nt, and 3 nt were 1.73-fold, 1.68-fold, and 1.14-fold of PE2, respectively (Fig. 3d). While PE2 exhibited higher activity than aPE2 in the deletions of 4 nt, 5 nt, and 6 nt. This position-dependent augmentation was highly similar to that observed in base conversion edits, although the latter were achieved by aPE2 with greater efficiency than PE2 at the +1 to +4 position within the targets. Consistently, aPE2 only showed 0.31-fold, 0.39-fold, 0.57-fold, 0.30-fold, 0.36-fold, and 0.42-fold of indels formation compared to PE2 accompanying the deletions of 1 nt to 6 nt (Fig. 3e), confirming that AcrIIA5 was also highly effective in reducing unintended by-products during deletions.

Previous results validated that the closer aPE-mediated edits was to the +1 position within the target, the more likely it was to be augmented by AcrIIA5 (Figs. 1g, 3d). We then attempted to insert DNA fragments of varying lengths at the +1 position within four different targets. Excellently, most aPE2-mediated insertions at the +1 position showed obvious improvement compared to PE2 (Fig. 3f, g and Supplementary Fig. 7g–i), and all indels were also reduced simultaneously (Fig. 3h and Supplementary Fig. 7j–l). The comprehensive analysis showed that the average aPE2-introduced insertion efficiencies of 1 nt to 6 nt were 1.92-fold, 1.37-fold, 1.36-fold, 1.30-fold, 1.24-fold, and 1.14-fold, respectively, compared to PE2 (Fig. 3i), indicating the strong potency of AcrIIA5, although its effectiveness decreased as the length of the inserted fragments increased. Persistently, all insertions by aPE2 decreased to an average of 0.43-fold compared to PE2 (Fig. 3j).

Examination of pathogenic mutations introduced by aPE
PE enables the precise introduction of base conversions, insertions, and deletions, making it highly promising for correcting genetic errors, albeit with limitation of low activity and increased indels when applying PE3 construct. Our previous results showcased that the aPE system possessed strong activity and exceptional security. Therefore, we aimed to evaluate aPE system in manipulating 12 pathogenic mutations21 including both base conversions and deletions.

The results showed that aPE2 exhibited 1.14-to 8.21-fold higher efficiency compared to PE2 in installing a half pathogenic mutation, represented by DMD, c.7098+1 G-to-T (Fig. 4a). However, the efficiency of the other half of the mutations was not significantly improved by aPE2. In addition, we observed that the indels in 10/12 edits were reduced by aPE2 (Supplementary Fig. 8a). The holistic statistics showed that aPE2 improved editing efficiency and reduced indels in 50% of the pathogenic mutations (6/12), while 25% of the mutations (3/12) had unchanged efficiency but also reduced indels (Fig. 4b), which meant that aPE2 improved the performance of 75% of the tested pathogenic mutations.

Duchenne muscular dystrophy (DMD) is the most common pediatric neuromuscular disorder, caused by mutations in the largest gene in the human genome, the dystrophin gene43. To further explore the therapeutic potential of aPE system, we attempted to evaluate the efficiency of various aPE variants in correcting the DMD, c.5287C>T mutation, which converted the 1763rd. amino acid in the dystrophin protein into a stop codon (Fig. 4c). The results exhibited that PE6c had the highest initial editing efficiency of 1.51% and increased to 2.75% with a 1.82-fold enhancement by aPE6c. PE2ΔRNaseH showed an efficiency of 0.69% and increased to 4.49% with the assistance of AcrIIA5, the highest among tested aPE variants, with a 6.56-fold improvement. Meanwhile, all tested aPE variants showed decreased indels (Supplementary Fig. 8b).

Besides, we also examined the installation of NF1, c.1059del mutation related to neurofibromatosis44, by PE or aPE system. The results showed PE6c possessed higher editing efficiency (12.5%) than PE2 (7.4%), and was further enhanced to 17.45% by aPE6c (Fig. 4d). Similar to DMD, c.5287C>T mutation, the aPE2ΔRNaseH showed the highest editing efficiency of 18.4%. Among them, most edits also exhibited reduced indels by aPE variants, albeit not as pronounced as observed in base conversion type (Supplementary Fig. 8c).

Overall, the aPE systems exhibited excellent performance in installing pathogenic mutations, especially aPE2ΔRNaseH. Additionally, previous results confirmed that aPE functioned effectively in a position-dependent manner rather than in a editing type-dependent manner (Fig. 1h and Fig. 3d), thus the efficiency of installing pathogenic mutations also highlighted the potential in treating according genetic errors.

Assessment of the aPE generalizability in different cell lines
To explore whether the improved performance of the aPE system extends to other cellular contexts, we tested it in additional human cell lines, specifically HeLa and U2OS, by transfecting them with either PE2 or aPE2 systems. Targeted amplicon sequencing at HEK2, +2 G-to-A (Fig. 4e) and HEK3, +1 T-to-A (Fig. 4f) demonstrated that aPE2 consistently achieved higher intended editing efficiencies while maintaining comparable or lower indel frequencies compared to PE2 in both cell lines. These results indicated that the aPE system retains its enhanced performance across different cellular backgrounds, highlighting its broad applicability and potential for diverse biomedical applications.

AAV delivery evaluation of aPE
AAVs are clinically verified and FDA approved for in vivo gene editing applications45. Recent reports shown that PE2 can be divided into two parts and then reconstituted into intact functional PE2 through intein-based trans splicing46. Encouraged by this strategy, we splitted PE2ΔRNaseH and fused it with Npu-intein, then packaged them into AAV_PE_1 and AAV_PE_2 vectors (Supplementary Fig. 9a). Meanwhile, we also constructed AAV_AcrIIA5 containing AcrIIA5 under CMV promoter. By co-transducting the AAV_PE_1 and AAV_PE_2 into HEK293T cells, we observed the +1 T-to-A editing efficiency of 2.97% at HEK3 target (Supplementary Fig. 9b). Upon adding a high concentration of AAV_AcrIIA5 containing AcrIIA5, the editing efficiency was suppressed to a minimal level. As the concentration of AcrIIA5 gradually decreased, the efficiency increased to 3.00% at 5−5 molar ratio. However, we did not observe any enhancement of PE2 by AcrIIA5. We hypothesized that the split PE2 protein required enough time to reassemble into a functional enzyme; thus, early expression of AcrIIA5 might hinder this process. Therefore, we attempted to add AcrIIA5 12h after the delivery of AAV_PE_1 and AAV_PE_2. As the concentration of AcrIIA5 decreased, the previously suppressed editing efficiency gradually increased, reaching a peak of 3.73% at 5−5 molar ratio, which was 1.33-fold higher than the PE2 efficiency of 2.80% (Supplementary Fig. 9b). Although delaying AcrIIA5 addition partially restored editing efficiency in the split PE2 system, the overall enhancement remained modest. This results suggested that the reassembly process might still limit the full benefit of AcrIIA5 when using AAV-based delivery. To address this constraint and better leverage the full potential of AcrIIA5, we next explored an alternate delivery strategy using lipid nanoparticles (LNPs).

LNP delivery evaluation of aPE
Unlike AAVs, LNPs allow direct cytoplasmic delivery of full-length mRNA, eliminating the need for protein splitting and subsequent intein-mediated reconstitution. This method enables immediate translation of the complete PE complex, which could minimize premature interactions with AcrIIA5 and maximize editing performance. We first in vitro transcribed full-length PE2ΔRNaseH and AcrIIA5 mRNAs and encapsulated them with pegRNA in LNPs for co-delivery into HEK293T cells (Fig. 4g). Our results demonstrated that LNP-mediated delivery of PE2ΔRNaseH mRNA alone resulted in an intended editing efficiency of 7.5% at the HEK3, +1 T-to-A edit. When co-delivered with AcrIIA5 mRNA at molar ratios of 0.5 and 0.1 relative to PE2ΔRNaseH, the editing efficiency of the aPE2 system increased to 11.1% (1.48-fold of PE2ΔRNaseH) and 13.5% (1.80-fold of PE2ΔRNaseH), respectively, representing a notable enhancement compared to PE2ΔRNaseH alone. Moreover, the frequency of undesired indels was consistently lower in the aPE2ΔRNaseH groups than in the PE2ΔRNaseH-only group. These results suggest that LNP-based delivery of the full-length aPE system is both feasible and effective, enhancing its therapeutic potential.

Hypercholesterolemia is a major risk factor for cardiovascular disease, and gain-of-function mutations or elevated expression of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) have been firmly established as key drivers of impaired LDL clearance through enhanced degradation of LDL receptors (LDLRs)47. Therapeutic strategies that disrupt PCSK9 function have shown substantial clinical benefit, underscoring this gene as a highly relevant target for evaluating genome editing tools47. To assess the therapeutic potential of the aPE system, we examined its performance at the PCSK9 locus in hepatocyte HepG2 cells. We selected the intron 1 splice donor site GT for introducing a +1 G-to-A mutation to disrupt the PCSK9. The results showed that LNP-delivered PE (mRNA-PE2ΔRNaseH) achieved a +1 G-to-A substitution with an efficiency of 8.9%, whereas LNP-aPE (co-delivery of AcrIIA5 and PE2ΔRNaseH mRNAs at a 0.1:1 ratio) markedly improved the editing efficiency to 24.7%, while maintaining low indels (Fig. 4i). This precise disruption of the GT splice donor site resulted in a functional knockdown of PCSK9. ELISA analysis showed that secreted PCSK9 protein levels caused by LNP-aPE were reduced by 32.6% relative to the control group and by 21.3% compared with LNP-PE treatment (Fig. 4j). Functionally, this translated into enhanced LDL clearance, as measured by flow cytometry with fluorescently labeled Dil-LDL. Compared with controls, LNP-PE increased LDL uptake by 8.5%, while LNP-aPE further boosted Dil-LDL internalization by 18.0%, proving that LNP-aPE editing of PCSK9 could effectively restore the capacity for LDL metabolism in HepG2 cells (Fig. 4k and Supplementary Fig. 10). Together, these results demonstrated that the LNP-aPE system can precisely and efficiently disrupt PCSK9 splicing in hepatocyte-derived cells, leading to a tangible reduction of PCSK9 activity and improved LDL uptake. Considering the critical role of PCSK9 in hypercholesterolemia, these results highlight the translational promise of aPE for therapeutic editing at clinically relevant loci.

PBS is not the determinant of aPE activity
While the above results confirmed that the aPE system can enhance editing efficiency across multiple delivery strategies, we also observed that its effect varied at certain target sites. To better understand the factors underlying this variability, we further investigated the sequence and thermodynamic properties associated with aPE activity. We first analyzed the maximum fold increase of aPE (Supplementary Fig. 11a, b). The results indicated that when a target showed Tm value between 50 to 55 °C, the PE efficiency was more likely to be enhanced by AcrIIA5. Conversely, when target characterized with Tm value around 65 °C, the PE efficiency cannot be enhanced by AcrIIA5 and may even be inhibited.

In fact, since the PBS sequence in pegRNA is partially complementary to the spacer, the modulation of PE system activity by AcrIIA5 might also be related to PBS (Supplementary Fig. 11c). To test this hypothesis, we adjusted the PBS length in pegRNAs responsible for HEK3 +1 T-to-A (Supplementary Fig. 11d), HEK2 +4 G-to-A (Supplementary Fig. 11e), and HEK2 +5 G-to-A (Supplementary Fig. 11f) to alter their Tm values. The results showed that varying the PBS length led to different changes in PE editing efficiencies for these edits. However, the effect of AcrIIA5 in enhancing or inhibiting these edits remained consistent, though the degree of the effect varied slightly. Notably, indels were significantly reduced across all edits (Supplementary Fig. 11g–i). These findings confirmed that altering the PBS length did not obviously affect AcrIIA5’s ability to regulate PE activity, thereby alleviating constraints in pegRNA design when using aPE.

RTT is not the determinant of aPE activity
As previously mentioned, the regulation of PE-mediated base conversions by AcrIIA5 was position-dependent, aligning with the statistical findings that the edited base closer to +1 position was more likely to be improved by AcrIIA5 (Supplementary Fig. 11j). In fact, we have defined a fixed 14 nt RTT length when designing pegRNA. Thus, the base edited at a specific position also indicated a corresponding RHA (Right homology arm) (Supplementary Fig. 11a), meaning that PE activity with longer RHAs were more possible to be enhanced by AcrIIA5 (Supplementary Fig. 11j). To examine this hypothesis, we altered the RHA lengths for several PE-mediated edits, expecting that shortening the RHA might suppress edits originally enhanced by AcrIIA5, while extending the RHA might boost edits originally inhibited by AcrIIA5. However, the results showed that neither extending nor shortening the RHA obviously altered AcrIIA5’s regulatory effect on the respective edits, whether they were base conversions (Supplementary Fig. 11k, i and Supplementary Fig. 12a–f) or DNA deletions (Supplementary Fig. 12g, h). We also observed that the indels decrease caused by aPE was not significantly affected by varying RHA lengths (Supplementary Fig. 12). These findings indicated that RHA length in pegRNA was not a determining factor in AcrIIA5’s regulation on PE activity, suggesting that RTT length did not need to be a concern when designing pegRNAs for aPE systems.

Mechanistic insights into the position-dependent effect of aPE
Since we have confirmed that the pegRNA composition is not the determinant of aPE activity, we put our focus on the position itself. Specifically, the key issue lies in explaining the observed phenomenon that PE2 exhibits higher editing efficiency at the +1 to +3/+4 positions, but significantly lower efficiency at the +5 or +6 positions. Considering that protein abundance might directly influence PE outcomes, we first examined the expression levels of PE2 protein in the presence or absence of AcrIIA5 using Western blot analysis at the HEK2 locus across the +1 to +6 positions (Supplementary Fig. 13). The results showed that aPE2 exhibited expression levels comparable to those of PE2 across +1 to +6 positions, indicating that AcrIIA5 does not interfere with PE2 protein expression. Therefore, the changes in editing efficiency observed between PE and aPE systems are not attributable to differences in protein abundance.

Next, considering that the aPE system exhibited a sharp decrease in editing activity at the +5 and +6 positions, which correspond to the PAM sequence, we guessed that edits within or outside the PAM region might differentially influence aPE activity. To test this hypothesis, we further evaluated the impact of AcrIIA5 co-expression on PE2 at PAM downstream positions, specifically from +7 to +14, at the HEK2 locus (Fig. 5a, b). Interestingly, the editing efficiencies of aPE2 were consistently higher than that of PE2 across all positions in this region. Besides, we also examined aPE2-mediated insertions and deletions at positions downstream of the PAM sequence. The results showed that aPE2 consistently achieved higher activity than PE2 for these edits as well (Supplementary Fig. 14).

Subsequently, we examined the combined effects of PAM-proximal and PAM-silencing edits (Fig. 5c, d). When only PAM-proximal positions (+1 to +4) were edited, aPE2 showed higher efficiency than PE2. In contrast, at the PAM-silencing position (+5), the editing efficiency of aPE2 was lower than that of PE2. Notably, when PAM-proximal edits were introduced together with a PAM-silencing mutation, the editing efficiency of aPE2 was significantly reduced compared to PE2. These findings suggest that PAM-silencing edits may be a key factor influencing the editing outcome of the aPE system. To probe this hypothesis in more detail, we utilized SpG, an engineered variant of SpCas9 that recognizes a relaxed N G N PAM, to construct an SpG-based PE2 system with expanded targetable regions. We then evaluated the editing outcomes of both SpCas9-PE2 (mainly N G G PAM and slightly N G A PAM) and nSpG-PE2 at the +5 and +6 positions (Fig. 5e, f).

Consistent with our previous findings, the editing efficiency of SpCas9-aPE2 at both +5 and +6 positions was lower than that of SpCas9-PE2. For the nSpG-based system, aPE2 exhibited lower editing efficiency than PE2 at the +5 position, but higher efficiency at the +6 position. This contrasting outcome observed in the nSpG-based system is consistent with its recognition of the N G N PAM. These observations suggest that PAM-silencing edits are critical modulators of aPE activity and raise the possibility that their position relative to the PAM influences re-targeting dynamics and editing efficiency.

We then attempted to establish a model to explain the mechanism of the aPE system. Previous studies have demonstrated that PAM-silencing edits can enhance PE activity and reduce undesired indel byproducts, presumably by preventing re-recognition and re-nicking of the edited strand19. Such re-nicking can lead to reversion of the edit or promote indel formation, especially in the context of PE3 systems. Inspired by these findings, we speculated that the activity of aPE might also be influenced by a similar prevention of re-nicking mechanism. Specifically, for PAM-proximal or distal edits, AcrIIA5 may suppress SpCas9-H840A nicking activity just enough to slow re-targeting of the edited strand, without completely abolishing the initial nicking required for initiating the PE process (Fig. 5g). Moreover, once the intended mutations are installed, the edited targets may exhibit reduced binding affinity for the original SpCas9-H840A-pegRNA complex due to sequence mismatches within the spacer region, making them more resistant to re-targeting and potentially further delaying re-nicking in the presence of AcrIIA5. In contrast, for PAM-silencing edits, the inhibition provided by the PAM mutation itself may already be sufficient to prevent re-nicking. Thus, the additional suppression from AcrIIA5 could overly weaken the initial nicking activity, thereby reducing the overall editing efficiency.

Based on this model, we hypothesized that the performance of aPE is closely tied to the intrinsic DNA cleavage activity of SpCas9 at each tested target site. At loci with high cleavage activity, initial nicking is more likely to occur even in the presence of AcrIIA5, allowing the PE process to proceed efficiently. In such cases, AcrIIA5 may primarily function to suppress excessive re-nicking, thereby enhancing editing precision and efficiency. Conversely, at sites with low SpCas9 activity, AcrIIA5 may overly inhibit nicking, ultimately reducing editing efficiency.

To examine this hypothesis, we next sought to determine whether a correlation exists between SpCas9 cleavage activity and aPE performance. Given that in vitro SpCas9 cleavage assays may not accurately reflect chromatin accessibility and other cellular factors influencing cleavage efficiency in vivo, we turned to a cell-based predictive approach. We utilized DeepSpCas9, a deep learning model which predicts SpCas9 cleavage activity across thousands of genomic loci in HEK293T cells48. By inputting the sequences of our tested loci into this model, we obtained predicted cleavage scores (Fig. 5g), where higher scores indicate stronger SpCas9 activity in cellular contexts. By correlating the DeepSpCas9 scores with the observed fold change in editing efficiency between aPE and PE systems (aPE:PE ratio) (Fig. 5h), we found a strong association that higher predicted SpCas9 activity scores generally linked to greater enhancement by AcrIIA5. Specifically, at target sites with DeepSpCas9 scores above 50, aPE consistently outperformed PE. In contrast, at sites with scores below 50, aPE often showed reduced efficiency compared to PE.

To further validate this correlation, we selected an additional set of four loci with predicted DeepSpCas9 scores >50 and four with scores <50 (Fig. 5i). The results showed that all four high-score loci exhibited enhanced editing efficiency with aPE compared to PE, whereas all four low-score loci showed reduced aPE activity. These findings support the conclusion that the performance of aPE is indeed influenced by initial SpCas9 cleavage activity at the target locus, and that DeepSpCas9 scores can be prospectively used to guide target selection, thereby improving the predictability and applicability of aPE in practical genome editing scenarios.

Molecular basis of AcrIIA5-mediated enhancement of PE activity
We have preliminarily demonstrated the relationship between SpCas9 cleavage activity and aPE performance, but the underlying mechanism remains uncertain. To further explore the molecular basis of this observation and validate our proposed model, we focused on dissecting the individual contributions of SpCas9 and AcrIIA5 to aPE function. As is well known, the generation of indels in the PE system is directly linked to the cleavage activity of SpCas9-H840A. For example, further reducing the cleavage activity of SpCas9-H840A on the non-edited strand significantly decreases indels byproduct49, whereas introducing additional cleavage on the non-edited strand, as in the PE3 architecture, markedly increases indels. Consistent with this, we observed that AcrIIA5 significantly suppressed PE-associated indels across various edits, confirming its inhibitory effect on SpCas9-H840A cleavage. To clarify this, we systematically tested conditions altering the nickase and RT functions. We replaced the SpCas9-H840A with SpCas9-H840A+D10A (dCas9) and Cas9, or discarded the RT in PE2 protein. At HEK5 +2 C-to-T edit (Fig. 6a), the presence of AcrIIA5 suppressed the indels, confirming AcrIIA5’s inhibitory potency on SpCas9. Furthermore, when SpCas9-H840A was replaced with dCas9, no obvious +2 C-to-T conversion efficiency was observed, regardless of the presence of AcrIIA5, indicating that the enhancement of PE efficiency by AcrIIA5 occurred after the cleavage of the edited strand. We also observed that SpCas9 efficiency was significantly suppressed by AcrIIA5 (from 10.95% to 5.89%), and accordingly, the PE efficiency based on SpCas9 also slightly declined (from 4.09% to 3.31%). However, only when SpCas9-H840A was combined with RT did AcrIIA5 enhanced editing efficiency (from 1.22% to 6.37%) while simultaneously reducing indels (from 0.65% to 0.41%). Similar results that aPE worked only in the presence of SpCas9-H840A and RT were observed in another event at the HEK5 +6 G-to-T edit (Fig. 6b). These findings confirmed that AcrIIA5 enhances PE activity only when the edited strand is nicked and reverse transcription can proceed, demonstrating that its enhancement effect coexists with its suppression of SpCas9 cleavage.

Given that the enhancing effect of AcrIIA5 on PE efficiency depends on the precise balance between SpCas9 nicking and inhibition, we next sought to investigate whether AcrIIA5 preferentially targets a specific nuclease domain of SpCas9 to exert its regulatory function. Understanding this interaction could help explain how AcrIIA5 selectively reduces undesired indels while still permitting sufficient nicking to enable reverse transcription. To this end, we designed a series of biochemical experiments using SpCas9 variants with distinct catalytic activities (Supplementary Fig. 15). Specifically, we purified wild-type SpCas9, SpCas9-D10A (HNH-active, RuvC-inactive), SpCas9-H840A (RuvC-active, HNH-inactive), and AcrIIA5, and reconstituted SpCas9-sgRNA ribonucleoprotein (RNP) complexes with each SpCas9 variant. These RNPs were incubated with a supercoiled pUC57 plasmid substrate in the presence of increasing concentrations of AcrIIA5 (at molar ratios of 0, 0.1, 1.0, and 10 relative to SpCas9). In the absence of AcrIIA5, wild-type SpCas9 mainly converted the supercoiled (SC) plasmid into the linear (LIN) form. Similarly, SpCas9-D10A nicked the targeted strand, producing predominantly open circular (OC) DNA, while SpCas9-H840A, which nicks the opposite strand, also generated OC products, albeit with slightly different efficiency.

Upon titration of AcrIIA5, we observed a progressive reduction in cleavage/nicking activity for wild-type SpCas9 and SpCas9-H840A. At a 1:1 molar ratio of AcrIIA5 to SpCas9, both enzymes exhibited a considerable suppression of activity, with most DNA remaining in the SC form. This indicates effective inhibition of both double-strand cleavage and RuvC-mediated nicking. In contrast, SpCas9-D10A retained substantial nicking activity even at AcrIIA5 molar ratios of 0.1 and 1.0, and only at the highest ratio (10:1) did we observe notable reduction in OC product formation. These results suggest that the RuvC domain is more susceptible to AcrIIA5 inhibition than the HNH domain, supporting our hypothesis that AcrIIA5 fine-tunes PE activity by selectively dampening redundant re-nicking events while preserving the initial nicking required to initiate prime editing.

Moreover, the phenomenon that SpCas9-H840A exhibited progressively reduced but not fully abolished nicking activity inhibited by AcrIIA5 (Supplementary Fig. 15c), as evidenced by the persistent presence of nicked OC plasmid, indicates that AcrIIA5 cannot completely inhibit SpCas9-H840A. This finding is precisely consistent with our proposed aPE mechanistic model (Fig. 5g), in which such “soft” inhibition is sufficient to prevent excessive re-nicking of the newly edited NTS while still permitting residual nicking of unedited DNA strands to initiate prime editing.

Furthermore, to determine whether this mechanism is unique to AcrIIA5, we tested other anti-CRISPR proteins with distinct inhibition modes. For example, AcrIIA1 reduced SpCas9 activity by degradation50; AcrIIA251 and AcrIIA452 both blocked binding of SpCas9 and target DNA binding by occupying the PAM-interaction region. We applied these Acrs to regulate PE performance and tested them at the HEK5 +2 and +6 conversion edits. Additionally, we tested AcrIIA5-D1126, which shared 87% homology with AcrIIA5.

The results showed that AcrIIA1 and AcrIIA4 consistently and strongly inhibited PE efficiency at both the +2 and +6 positions, while AcrIIA2 exhibited a weaker inhibitory effect (Fig. 6c, d). Surprisingly, AcrIIA5-D1126 suppressed the +2 C-to-T conversion, whereas AcrIIA5 enhanced this conversion by 1.41-fold. However, AcrIIA5 reduced the +6 G-to-T conversion efficiency from 8.6% to 0.4%, and AcrIIA5-D1126 also lowered this efficiency to 0.8%. Comparatively, AcrIIA5-D1126 decreased the efficiency at the +2 position by 26.4% and at the +6 position by 91.3%, indicating that the activity of AcrIIA5-D1126 was also position-dependent like AcrIIA5. Sequence alignment revealed that the most significant distinction between AcrIIA5 and AcrIIA5-D1126 lied in the N-terminal intrinsically disordered region (IDR) and the α1 region (Supplementary Fig. 16), which have been shown to influence the inhibition of AcrIIA5 to SpCas926, suggesting that they may play a critical role in regulating PE activity, especially considering the starkly different PE editing outcomes by AcrIIA5 and AcrIIA5-D1126. These findings strongly support that AcrIIA5 promotes PE efficiency through its canonical SpCas9 inhibition pathway, which appears to be distinct from other Acr proteins tested.

To pinpoint the specific regions responsible for this effect, we next performed mutational analyses of both AcrIIA5 and SpCas9. We performed a split-function mutation analysis of SpCas9 and AcrIIA5 to investigate whether the observed enhancement of PE2 activity is driven by their interaction. Although no structural complex of SpCas9 and AcrIIA5 has been reported to date, previous studies have identified two key regions through which AcrIIA5 inhibits SpCas926. They include the positively charged N-terminal IDR and α1 helix, and the loop between α3 and α4 along with the surrounding region.

We first conducted stepwise deletions of the N-terminal IDR (Fig. 6e). While short deletions did not significantly impact the function of AcrIIA5, the AcrIIA5Δ7 mutant exhibited a significantly reduced enhancement of PE2 activity compared to wild-type AcrIIA5, indicating that the N-terminal IDR plays a critical role in modulating PE2 activity. Next, we replaced the positively charged residues with alanine inside IDR. The AcrIIA5 and AcrIIA5 mutants showed a modest reduction in their ability to enhance PE2 activity, whereas the AcrIIA5 mutant almost completely lost its enhancing effect and even impaired PE2 editing efficiency (Fig. 6e). These findings are consistent with previous in vitro SpCas9 cleavage assays26, which showed that RKR mutations or extended deletions within the IDR abolish the inhibitory function of AcrIIA5, indicating that effective SpCas9 inhibition is essential for its enhancement of PE activity. Furthermore, as reported24, mutations within or near the α3–α4 loop region also abolish AcrIIA5’s inhibitory function. We also introduced alanine substitutions in this region (Fig. 6f), and the resulting mutants showed a marked decrease in aPE2 activity.

Interestingly, we observed a concurrent decline in both editing efficiency and indel suppression when the activity of aPE2 was reduced, suggesting that mutations weakening AcrIIA5’s inhibitory function compromise its dual role in enhancing editing and minimizing byproducts of PE system (Supplementary Fig. 17). In summary, our data confirm that mutations previously shown to disrupt AcrIIA5’s inhibition of SpCas9 in both biochemical and cellular settings also markedly reduce its ability to enhance PE activity. This parallel loss of function indicates that the enhancement of PE activity by AcrIIA5 fundamentally relies on its inhibitory interaction with SpCas9. Thus, although AcrIIA5 appears to act as an enhancer in the context of PE, its effect ultimately stems from its canonical role as a SpCas9 inhibitor.

Additionally, to identify residues critical for the PE2-enhancing function of AcrIIA5, we performed single-point reversion mutations on AcrIIA5-D1126, which differs from wild-type AcrIIA5 by 18 amino acids (Fig. 6g). We aimed to determine whether specific mutations could restore its ability to enhance PE2 activity. The results revealed that K28Q, K29E, and K31D were key mutations that significantly improved the activity of AcrIIA5-D1126 toward wild-type AcrIIA5 levels. When mapping all three functional regions onto the 3D structure of AcrIIA5, we found that they are spatially clustered on the same surface. This observation suggested that these residues may contribute to the interaction with SpCas9 and its subsequent inhibition (Fig. 6h).

To further investigate the key regions in SpCas9 that mediate its interaction with AcrIIA5, we sought to identify specific residues involved in this response. Both our results (Fig. 6e, f) and previous studies26 have demonstrated that positively charged residues in the IDR region of AcrIIA5 are critical for its inhibitory activity. Moreover, our in vitro cleavage assays indicated that the RuvC domain of SpCas9 is likely the primary target of AcrIIA5. Based on this, we hypothesized that AcrIIA5 may interact with negatively charged regions within the RuvC domain through electrostatic forces. To test this, we generated alanine substitutions at a series of candidate acidic residues in the RuvC domain, specifically aspartic acid (D) and glutamic acid (E), and evaluated PE2 editing efficiency with or without AcrIIA5 (Supplementary Fig. 18). Unexpectedly, none of these mutations obviously affected the ability of AcrIIA5 to modulate PE2 activity, suggesting that the tested residues are unlikely to be directly involved in the interaction. This finding indicates that the binding interface between AcrIIA5 and SpCas9 is more complex than initially anticipated, and future studies using high-resolution structural analysis or systematic mutagenesis will be needed to clarify the precise mechanism of AcrIIA5-SpCas9 recognition.

Methods
Plasmid cloning
Unless stated otherwise, cloning was performed using the Hieff Clone One Step Cloning Kit (YEASEN). The DNA fragments of AcrIIA1, AcrIIA2, AcrIIA4, AcrIIA5-D1126, AcrIIA5, hA3A, hA3Bctd, hA3Gctd, evoEc48, evoTf1, La, Npu intein, viral nucleocapsid protein, MLH1-SB, and sgRNA scaffolds of SpCas9 were synthesized by GenScript. PCR was performed using Phanta Max Super-Fidelity DNA polymerase (Vazyme). Site-directed mutagenesis of was performed using Mut Express II Fast Mutagenesis Kit V2 (Vazyme).

The DNA fragments of hA3A, hA3Bctd, and hA3Gctd were amplified and cloned into the backbone of pCMV-BE311 (Addgene #73021) to construct CBEs. The DNA fragment of Acrs were amplified and cloned into the backbone of pcDNA3.1(-). The DNA fragments of evoEc48 and evoTf1 were amplified and cloned into the backbone of pCMV-PE219 (Addgene #132775) to construct PE6a and PE6c. The SpRY was cloned from RTW502554 (Plasmid #140003) to construct nSpRY-PE2ΔRNaseH. AAV vectors were constructed based on the backbone of pAAV-EFS-CjCas9-eGFP-HIF1a55 (Plasmid #137929). The sgRNA scaffolds of SpCas9 were cloned into pUC57 under the U6 promoter amplified from the PX459 v2.0 plasmid. Protospacer oligos were phosphorylated by PNK (Thermo Fisher Scientific) and cloned into sgRNA expression plasmids by T4 DNA Ligase (Thermo Fisher Scientific). The pegRNA 3’ extension sequences were added in sgRNA expressing plasmids by PCR. The amino acid sequences or nucleotide sequences were list in Supplementary Data 1 and Supplementary Data 2. All newly generated plasmids used in this study are available from the corresponding author upon reasonable request.

Culture condition and transfection
HEK293T cells (ATCC CRL-3216), HeLa cells (ATCC CCL-2), and U2OS cells (ATCC HTB-96) were kept in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% (vol/vol) fetal bovine serum (FBS, Gibco) in 5% CO2 incubator at 37 °C. HepG2 cells were obtained from Procell Life Science & Technology Co., Ltd. (CL-0103) and were kept in HepG2 cell complete medium (Procell, CM-0103).

Cells were seeded into 48-well plates (Corning) at a density of 55,000 cells per well and transfected 18 h after plating. Transfection was performed using Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions. For each well, 500 ng of SpCas9, CBE, or PE expression plasmid (9753 bp for PE2), 200 ng of pegRNA or sgRNA expression plasmid (3200 bp), and a variable amount of AcrIIA5 expression plasmid (5869 bp) were co-transfected to achieve the desired molar ratio of AcrIIA5 relative to PE2. At a molar ratio of 0.1, the corresponding mass of AcrIIA5 plasmid is ~30 ng, while at a ratio of 3.0, it is ~900 ng. For PE3 conditions, the nicking sgRNA expression plasmid was also included at 200 ng per well. All plasmid ratios refer to molar amounts unless otherwise specified.

Production and transduction of AAV vectors
AAV vectors were packaged in AAV DJ capsids by OBiO Technology and validated using quantitative PCR. The vector titres were 5.82×1012, 1.02×1013, and 7.36×1012 v. g. /mL for AAV_PE_1, AAV_PE_2, and AAV_AcrIIA5, respectively. After diluting AAV_PE_1 and AAV_PE_2 to 5.00×1010 and 5.00×1010 titres in Opti-MEM I Reduced Serum Medium (Gibco), 10 µL of each was mixed and transduced into HEK293T cells that had been cultured in a 48-well plate to 50% confluence. AAV_AcrIIA5 was diluted to 3.00×1010 first and then serially diluted in 5-fold increments, then 10 μL of each was added to the wells containing HEK293T cells with AAV_PE_1 and AAV_PE_2, either immediately or 12 h later. Besides, an equal volume of medium without AAV_AcrIIA5 was simultaneously added to the control wells containing AAV_PE_1 and AAV_PE_2 for control.

mRNA production and LNP delivery
mRNA of PE2ΔRNaseH and AcrIIA5 were synthesized using the T7 High Yield RNA Synthesis Kit for Co-transcription (Yeasen) according to the manufacturer’s instructions. Briefly, plasmids harboring the coding sequence of PE2ΔRNaseH or AcrIIA5 were first constructed with an upstream inactivated T7 promoter, a 5’-untranslated region (UTR), and a Kozak sequence, as well as a downstream 3’-UTR. PCR was used to generate in vitro transcription templates by restoring the functional T7 promoter and appending a 115-nucleotide poly(A) tail downstream of the 3’ UTR. Then, linearized DNA templates containing the T7 promoter were used for in vitro transcription in a 20 µL reaction system, which included T7 RNA polymerase mix, transcription buffer, canonical and modified nucleotides (ATP, CTP, GTP, and N¹-methyl-pseudouridine-UTP), and a cap analog (GAG). The reaction was incubated at 37 °C for 3 h, and the reaction was treated with DNase I to remove the DNA template, and RNA was purified via lithium chloride precipitation. The RNA pellet was washed with cold ethanol, resuspended in RNase-free water, and quantified by UV spectrophotometry. RNA quality and integrity were assessed by agarose gel electrophoresis. Chemically synthesized pegRNAs (GenScript) were modified with 2’-O-methyl and phosphorothioate linkages at the first three and last three nucleotides, and were purified by high-performance liquid chromatography (HPLC).

mRNA and pegRNA was formulated into LNPs using the NeoLNP RNA transfection reagent (SDR8006, Scindy) according to the manufacturer’s instructions. Briefly, 400 ng mRNA and 200 ng pegRNA complex were diluted with RNA dilution buffer to a final concentration of 40 ng/μL, and an equal volume of NeoLNP working reagent was added. The mixture was gently pipetted and incubated at room temperature for 10 min to allow complex formation. The resulting mRNA-pegRNA-NeoLNP complexes were then diluted in culture medium to a final mRNA concentration of 20 ng/μL and used immediately for cell transfection. Complexes were applied to cells and incubated under standard culture conditions for 72 h prior to downstream analysis.

Extraction of genomic DNA
To extract HEK293T genomic DNA, cells were washed with PBS once and lysed in 100 µL DNA lysis buffer (10 mM Tris-HCl, pH 8.0, 5 mM EDTA, 0.1% SDS, 30 µg mL−1 proteinase K (YEASEN)) at 37 °C for 1 h. After heating at 80 °C for 20 min, the DNA template solution was stored at –80 °C.

Amplicon sequencing
The primers used to amplify the target regions are listed in Supplementary Data 3. 50 µL PCR with presupposed barcodes reactions were purified with TIANquick Maxi Purification Kit (TIANGEN). The PCR products were added with Illumina-compatible adapters and indices. Final amplicon libraries were sequenced with 150 bp paired-end reads on an Illumina MiSeq instrument, and the RAW data were analyzed by the CRISPResso256. The sgRNA-dependent off-target sites were investigated using Cas-OFFinder57. The primers used to amplify the off-target regions are also listed in Supplementary Data 3.

For samples in which the edit was a single base change, samples were aligned to the wild type amplicon in standard batch mode. To calculate editing efficiency, the base change was multiplied by an indel correction factor. Percent base changes came from the output file titled “Nucleotide_percentage_summary_around_sgRNA.txt”. The indel correction factor was obtained by dividing “reads aligned”/“reads aligned all amplicons” values in the output file titled “CRISPResso-Batch_quantification_of_editing_frequency.txt”. To calculate percent indels, “Discarded” was divided by “reads aligned all amplicons” in the same file.

For samples in which the edit was an insertion or deletion, CRISPResso2 was run in HDR batch mode. Percent editing was calculated by dividing the HDR-aligned reads/reads aligned all amplicons. Unintended indels were calculated by adding the “Discarded” reads from the reference-aligned sequences and the “Discarded” reads from the HDR-aligned sequences and then dividing that sum by “reads aligned all amplicons”. All of these values came from the “CRISPResso-Batch_quantification_of_editing_frequency.txt” output file.

All CRISPResso2 parameters are listed in Supplementary Data 4, and the editing efficiencies were listed in Source Data file.

Western blotting assay
Plasmids expressing PE2 or aPE2 (including the pegRNA) were transfected into HEK293T cells cultured in 24-well plates. After 48 h, cells were lysed using X-RIPA UltraMix (X-Blot), and 10 µL of lysate was mixed with 2.5 µL of 5× SDS loading buffer. After boiling, 10 µL of the mixture was loaded onto a 4–15% gradient SDS-PAGE gel. Proteins were separated by electrophoresis and transferred to a PVDF membrane. The membrane was cut at appropriate molecular weights and incubated with primary antibodies: anti-Cas9 (Genscript, A01935, clone 4A1, 1:500) and anti-GAPDH (Proteintech, 60004-1, clone 1E6D9, 1:1000), respectively. After incubation, the membranes were washed with TBST (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1% Tween-20) for three times and then incubated with HRP-conjugated goat anti-mouse IgG antibody (Proteintech, SA00001-1, 1:4000). After washing with TBST three times, the membranes were finally incubated with ECL substrate and imaged using an Amersham Imager 600.

Protein expressing and purification
The SpCas9 gene was cloned into a pET28a expression vector containing an N-terminal His6-tag. Site-directed mutagenesis was performed using the Mut Express II Fast Mutagenesis Kit V2 (Vazyme) to introduce the D10A and H840A substitutions. The resulting constructs were transformed into E. coli strain BL21(DE3) for protein expression. Bacterial cultures were grown in LB medium at 37 °C to the mid-log phase, then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated for 12 h at 18 °C. Cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris–HCl, pH 7.4; 300 mM NaCl; 20 mM imidazole; 10% glycerol; 5 mM β-mercaptoethanol) supplemented with 1 mM PMSF. Then the cells were disrupted by sonication and the lysate was centrifuged to remove cell debris. The supernatant was bound to Ni-NTA agarose (Genscript), and bound proteins were eluted via a linear gradient of imidazole (0–500 mM). The eluted protein was further purified using size-exclusion chromatography on a Superdex 200 26/60 column (GE Healthcare).

The synthetic AcrIIA5 gene and mutant genes were cloned into a pET28a vector with an N-terminal His6-tag and a maltose-binding protein (MBP) tag. The resulting constructs were transformed into E. coli strain BL21(DE3), and the His6-MBP-AcrIIA5 fusion proteins were expressed and purified following the procedure described above. The His6- and MBP-tags were cleaved using a tobacco etch virus protease (YEASEN). The AcrIIA5 were finally purified on a Superdex 200 26/60 column (GE Healthcare).

sgRNA Preparation and in vitro DNA cleavage assay
Synthetic DNA templates encoding T7 promoter-driven sgRNAs was obtained from Tsingke (5’-GATCACTAATACGACTCACTATAGGC CGCAGTGTTATCACTCAGTTTTAGAGCTAGAAA-3’). In vitro transcription was performed using a T7 RNA polymerase system (Thermo Scientific), followed by RNA extraction using phenol-chloroform and ethanol precipitation. The RNA pellet was resuspended in nuclease-free water and its integrity was confirmed by agarose gel electrophoresis.

For in vitro DNA cleavage assays, SpCas9, SpCas9 nickase, and sgRNA (each at 200 nM) were pre-incubated in NEB Buffer 3.1 at 37 °C for 10 min to assemble the RNP complex. AcrIIA5 was then added at varying concentrations (20–2000 nM) and incubated for an additional 10 min. Subsequently, 10 nM of pUC57 vector was added and the reaction was carried out at 37 °C for 2 h. To terminate the reaction, proteinase K was added and incubated at 37 °C for 15 min. The reaction products were mixed with loading dye and analyzed via 1.5% agarose gel electrophoresis.

PCSK9 editing and Dil-LDL uptake assay
HepG2 cells were seeded in 24-well plates and cultured for 12 h to reach ~60% confluence. Cells were then transfected with LNP-PE or LNP-aPE, as conducted in the “mRNA production and LNP delivery” section. Control group was treated with working buffer. After 84 h, the medium was replaced with 1 mL of fresh medium. 24 h later, 200 µL of the supernatant was collected for ELISA (JONLNBIO, JL19731) measurement of secreted PCSK9. At the same time, cells were incubated with 10 µg/mL Dil-LDL (Yiyuan Biotechnologies, YB-0011) at 37 °C for 4 h. Following incubation, cells were washed twice with PBS, trypsinized, collected into 1.5 mL EP tubes, washed three additional times with PBS, and was finally resuspended in PBS containing 0.5% BSA. For flow cytometry (Sony ID7000), 20,000 gated cells were analyzed using 549 nm excitation and 570 nm emission detection (ID7000 Acquisition and Analysis Software); mean fluorescence intensity (MFI) was used to represent Dil-LDL uptake (FlowJo 10.5.0). The gating strategy is exemplified in Supplementary fig. 10. The remaining cells were centrifuged and lysed with lysis buffer for genomic DNA extraction, PCR amplification, and deep sequencing to determine editing outcomes.

Statistics and reproducibility
The data were collected under randomized conditions. All statistical analyses were performed on three biologically independent experiments using GraphPad Prism 9 (GraphPad Software). The number of sample size was determined according to standard practice in similar studies and supported by previous publications, ensuring the reproducibility and robustness of the results. Error bars represent means ± s.e.m. For comparisons between control and treatments, P values were calculated using a two-tailed Student’s t-test. For comparisons among three groups in “PCSK9 editing and Dil-LDL uptake assay”, one-way ANOVA followed by Tukey’s multiple comparison test was applied. Structural visualization and analysis of AcrIIA5 were performed using PyMOL (The PyMOL Molecular Graphics System, Version 2.4, Schrödinger, LLC).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Chen et al. investigates the novel role of the anti-CRISPR protein AcrIIA5 in augmenting the efficiency and precision of prime editing (PE) systems. Prime editing, which facilitates targeted genomic modifications without inducing double-strand breaks, often results in unintended insertions and deletions. This study demonstrates that AcrIIA5 enhances PE activity while concurrently reducing these undesirable indels. The enhancement effect of AcrIIA5 is observed across various PE architectures (PE2 to PE6), different types of genomic edits (substitutions, insertions, deletions), and endogenous loci. Notably, AcrIIA5 does not increase the activity of Cas9 or base editors, suggesting a unique interaction mechanism with the PE system. Furthermore, the enhancement is position-dependent, indicating a specific binding mode between AcrIIA5 and Cas9. These findings underscore the potential application of Acr proteins in CRISPR/Cas systems beyond their traditional inhibitory functions, paving the way for more efficient and precise genome editing technologies.\n\nMajor comments:\n1. While I appreciate the study and its implications, the claims presented are often based on a single type of experiment that does not directly address the underlying mechanism. I am missing a biochemical and biophysical dissection and proof of the speculated mechanism on what AcrIIA5 does to RuvC that enhances prime editing. For example, is the improvement due to changes in cleavage kinetics or stabilization of the protein fold that makes it particularly amenable to efficient nicking? Additionally, I would like to see a split-function mutation analysis on Cas9 and AcrIIA5 to demonstrate that the effect is driven by their interaction.\n2. Since AcrIIA5 has been identified as a potent inhibitor of many Cas9 orthologues (Garcia et al.), I would like the authors to address whether the function of improving prime editing is also true for other Cas9 orthologues.\n2. I would like to see a biochemical experiment showing the effect of AcrIIA5 on both nuclease and nickase activity of Cas9.\n3. Can you experimentally address why AcrIIA5 has the most significant effect on PE2 and not on the more advanced versions like PE4 and PE5? I am particularly intrigued by the reverse effect observed with PE7.\n4. AcrIIA5 impacts PE2 to varying extents across different loci. Often, the effect is absent. Can you please explain the variation between loci? This raises concerns about the technology's applicability in real genome editing scenarios beyond the extensively studied HEK3 and HEK4 loci.\n5. A DNA repair profile analysis of indels generated in the presence or absence of AcrIIA5 is required to determine if there is a bias towards MMEJ or NHEJ. Additionally, please clarify the naming of the position of the installed base. This could help determine if the positional effect of AcrIIA5 on base conversion types is related to the prevention of recleavage by Cas9 after editing.\n5. I have serious doubts about the AAV experiments – Figure 5c shows only a modest effect after 12 hours. It makes me question whether this approach works effectively at all. Can you demonstrate a better in vivo application? Have you tried LNP delivery?\n\nMinor comments:\n1. Why is the improvement in the insertion and deletion of genomic fragments by AcrIIA5 only modest compared to substitutions?\n2. The figure presentation could be improved, such as in Figure 1b and others. What is the reason for plotting fold change on the same plots? This is confusing to the reader.\n3. In the introduction, the authors state that 'Guided by pegRNA, the PE2 protein binds to one strand of target DNA and nicks the opposite, PAM-containing strand.' This is not correct; the PE2 protein binds to both DNA strands.", "reviewer_2": "In this manuscript, the authors proposed and provided extensive data demonstrating that low doses of AcrIIA5 can enhance the editing efficiency of Prime Editing. To the best of my knowledge, this is the first study reporting Cas9 inhibitors AcrIIA5 to improve the editing efficiency of genome editing tool-PE, which is novel and interesting. In the field of CRISPR-Cas, Acr was originally discovered as an inhibitor of Cas9, and previous studies have also demonstrated that Acr can inhibit the editing efficiency of Cas9 and BE. Therefore, this study unexpectedly found that Acr can enhance the efficiency of PE, offering new insights for the design and optimization of Acr and PE.\n\nIn sum, I find this research to be novel and interesting. The manuscript is well-organized, the methods are sound, the data is detailed and provides strong support for the conclusions. the conclusions are reliable. It will attract widespread interest among researchers in the CRISPR field.\n\nHowever, I have a few suggestions for the authors.\n1. The study focuses on AcrII5 enhancing the efficiency of various PE constructions and reducing indel, but lacks an explanation of the mechanism.\n2. The authors compared the editing efficiency of Acr:PEplasmids at different ratios and found that a ratio of 0.1 yielded the best efficiency. However, the Materials and Methods section does not specify the exact amount of Acr plasmid added. I suggest that the authors provide a more detailed protocol to facilitate replication by other researchers. For example, in this study, 500 ng of PE plasmid and 200 ng of pegRNA plasmid were added per well, so does a 0.1 ratio of Acr mean 50 ng? A ratio of 3 would mean 1500 ng? And is the amount of nick gRNA also 200 ng?\n3. Could the authors provide Western blot data to demonstrate that the increase in editing efficiency is not due to changes in PE expression levels? At least, could they provide WB data for the 0.1 ratio condition in Fig. 1d?\n4. Since the PE system is too large to deliver easily, this is currently a major issue in the field. The authors used three AAVs to deliver the aPE system they developed; however, a three-AAV system is challenging to implement in practical therapeutic applications, and the efficiency increase in Fig. 5C is not significant. The authors should discuss the challenges of AAV delivery for the aPE system and potential solutions.\n5. Will the authors submit the plasmids constructed in this study to Addgene to facilitate use by interested researchers?\n6. The annotations in the main text does not correspond to the graph in Figure 1e, should correspond to Fig1d?\n7. There is no F1i mentioned in the Fig1.\n8. The study focuses on AcrII5 enhancing the efficiency of various PE constructions and reducing indel, but lacks an explanation of the mechanism.\n9. The study needs more application, such as animal model, to verify the effect of aPE.", "reviewer_3": "Thank you for the opportunity to review this manuscript entitled “Anti-CRISPR protein AcrIIA5 can enhance the activity and security of prime editing” by Chen, et al.\n\nIn this manuscript, the authors describe an approach for improving the efficiency of prime editing in HEK293T cells by co-expressing a phage-derived anti-CRISPR protein, AcrIIA5. In what likely began as an exploration of applying anti-CRISPR proteins to inhibit CRISPR-based gene editing tools, the authors came upon the surprising result that AcrIIA5, an anti-CRISPR protein with established Cas9 inhibitory activity, counterintuitively improved the editing efficiency of prime editing while also decreasing undesired indel byproducts. The effect appeared specific to prime editing and AcrIIA5, as AcrIIA5 inhibited the activity of Cas9 nuclease and base editors, and other anti-CRISPR proteins inhibited prime editing, as expected. The authors optimized the ratio of prime editing and AcrIIA5 components delivered to cells targeting a sentinel site, then characterized the system co-expressing AcrIIA5 with prime editing components (named aPE) on a variety of prime edit types and positions at different genomic loci. The authors found that the efficiency of single base substitution edits at the +1 through +4 positions improved with aPE (that is, substitutions within 4 bp of the PE-induced nick), but that substitution edits at the +5 and +6 position (which are notably the critical G bases of the PAM) decreased in efficiency as evaluated across 4 genomic target sites in HEK293T cells. The authors went on to evaluate aPE with several prime editing formats, prime editor protein variants and edit types, including PE3 (which incorporates a second nicking guide RNA), PE4 (which incorporates an MLH1dn protein for MMR inhibition), PE5 (nicking plus MLH1dn), PE6 variants, SpRY Cas9 variants, insertion edits and deletion edits. Modest improvements to editing efficiency were observed across these edits, except for deletions over 3-bp. The authors then applied aPE to install pathogenic variants in HEK293T cells with modest improvement in efficiency observed over standard PE. Delivery of the aPE system with AAV, however, did not lead to improved editing efficiency compared to a standard PE system, despite attempts to titrate the amount of the AcrIIA5 component. Several attempts were made to understand the origins of the AcrIIA5 improvements, including evaluating pegRNA features like the RTT and PBS length, although none of these revealed a feature influencing aPE activity. Lastly, the authors proposed a mechanistic model for how AcrIIA5 might improve prime editing efficiency.\n\nMajor comments\nThe authors cite prior publications proposing that AcrIIA5 (used in aPE) inhibits Cas9’s RuvC nuclease domain, which prime editing relies upon to nick the non-target DNA strand to generate a primer for initiating reverse transcription. Therefore, it is unexpected that AcrIIA5 would improve prime editing efficiency instead of inhibiting prime editing, particularly when AcrIIA5 inhibits Cas9 nuclease and base editing activity. The observation that AcrIIA5 can improve prime editing is therefore an interesting finding, and potentially a useful one, if it could be properly exploited.\n\nThe data presented in the manuscript show that the aPE system can improve prime editing efficiency in HEK293T cells in a somewhat site-dependent manner. In most cases, aPE provided modest benefits in efficiency and product purity. A critical finding is that the aPE system is detrimental when edits are made to the essential G bases of the PAM in the +5 and +6 positions (by either substitution or deletion). It has previously been demonstrated that PAM edits improve prime editing efficiency and decrease undesired indel byproducts, presumably because they prevent re-nicking of the prime-edited strand within the heteroduplex intermediate, which would promote reversion of the intended edit back to the original sequence or indel formation in the context of PE3. Moreover, there is a clear AcrIIA5 dose-dependence of the effect, suggesting it is deleterious to have too much AcrIIA5 and that tuning AcrIIA5 activity is important to achieve the desired outcome. It therefore seems that the simplest explanation for how AcrIIA5 affects prime editing (in a way that is consistent with its known activities) is that it decreases the effective Cas9 nicking activity enough so that re-targeting of the edited strand is slowed, but not so much that the initial necessary nicking activity is too weak. If PAM edits are made that prevent re-nicking on their own, then there is no benefit from AcrIIA5, which can then only weaken the initial nicking activity and decrease the overall editing efficiency. This also potentially explains the site-to-site variability that is observed, as different spacers likely have different intrinsic nickase properties.\n\nWith the above in mind, the following additional data and revisions would substantially strengthen the manuscript:\n1. To distinguish whether the observed decrease in aPE for +5 and +6 position edits is due to the distal nature of the edit or the edit being part of the PAM, the authors should evaluate more broadly the impact of AcrIIA5 co-expression on prime edits downstream of the PAM (position +7 and beyond), and proximal edits coupled with PAM-silencing edits (e.g., +1 and +5). There are existing pegRNAs reported in the literature that make edits at these positions which could be readily tested.\n2. The PE4 and PE5 systems used in this manuscript do not appear to represent the state-of-the-art systems, which co-express MLH1dn proteins separately from the PE protein. The fusion proteins used in this manuscript do not appear to improve prime editing efficiency as would be expected for PE4 and PE5 systems. As the interplay of re-nicking and MMR seem critical to understanding the potential utility of the aPE system, the experiments with PE4 and PE5 should be repeated so that the effects of MLH1dn are consistent with prior publications by separately expressing MLH1dn.\n3. The data presented in this manuscript appear to be entirely from HEK293T cells. The efficacy of aPE should be evaluated in several other mammalian cell types to characterize the effects in those contexts.\n4. Much of the data presented in the main figures are not essential to supporting the main conclusions of the manuscript (e.g., compatibility with different prime editor proteins in figure 2, the AAV experiment in figure 5, pegRNA features in figure 6). These would be more appropriate as supplementary figures.\n5. The mechanistic model appears somewhat speculative and would require further experimental data to support inclusion in this manuscript.\n\nMinor comments\n1. Most figures do not indicate the cell type used in the experiment. This information should be added either directly to the figures or to the legend.\n2. It appears the same data are presented in figure 1f and supplementary figure 2.\n3. In the description of quantitative results, relative editing or indel formation efficiency would be better represented as fold-changes as opposed to % changes, since % change could be interpreted as differences in %. For example, “AcrIIA5 reduced indels by 78.0% (aPE2) and 81.9% (aPE3) for PE2 and PE3, respectively” could be changed to 4.5-fold and 5.5-fold.\n4. Abbreviations, like RHA, need to be defined in the text when first used.\n5. When using the terminology AAV1, AAV2, and AAV3, there is confusion with serotype, so the naming of these constructs should be changed to avoid this potential confusion.\n6. Figures 5b and 5c are challenging to interpret, and the colored shading with inversion of the direction of the bars is not helpful. Recommend reformatting to a standard layout without shading red or green and using clear headers for the graphs conveying that the timepoints indicate when the AcrIIA5 AAV was added."}, "author_response": "Responses to Reviewer #1:\nThe manuscript by Chen et al. investigates the novel role of the anti-CRISPR protein\nAcrIIA5 in augmenting the efficiency and precision of prime editing (PE) systems. Prime\nediting, which facilitates targeted genomic modifications without inducing double-strand\nbreaks, often results in unintended insertionsand deletions.Thisstudydemonstratesthat\nAcrIIA5 enhances PE activity while concurrently reducing these undesirable indels. The\nenhancement effectofAcrIIA5 isobserved acrossvariousPE architectures(PE2to PE6),\ndifferenttypesofgenomicedits(substitutions,insertions,deletions),andendogenousloci.\nNotably, AcrIIA5 does not increase the activity of Cas9 or base editors, suggesting a\nunique interaction mechanism with the PE system. Furthermore, the enhancement is\nposition-dependent, indicatinga specificbinding mode betweenAcrIIA5 and Cas9.These\nfindings underscore the potential application of Acr proteins in CRISPR/Cas systems\nbeyond their traditional inhibitory functions, paving the way for more efficient and precise\ngenomeeditingtechnologies.\nMajor comments:\n1. While I appreciate the study and its implications, the claims presented are often\nbased on a single type of experiment that does not directly address the underlying\nmechanism. I am missing a biochemical and biophysical dissection and proof of\nthe speculated mechanism on what AcrIIA5 does to RuvC that enhances prime\nediting. For example, is the improvement due to changes in cleavage kinetics or\nstabilization of the protein fold that makes it particularly amenable to efficient\nnicking?Additionally, I would like to seea split-function mutation analysis on Cas9\nandAcrIIA5todemonstratethat theeffectisdrivenbytheirinteraction.\nResponse:Wesincerelythankyourinsightfulcommentsandconstructivesuggestions.In\nour original manuscript, we speculated thatAcrIIA5 might influence the recognition speed\nof the extended edited strand installed by the PE system in a position-dependent manner\nin our proposed mechanism. However, we realized that this hypothesis lacks solid\nexperimental support, and we now consider that a more plausible mechanism may exist\nbasedonournewly-addedexperimental results.\nThekeyissueliesinexplainingtheobservedphenomenonthataPE2typicallyexhibit\nhigher editing efficiency at the +1 to +3/+4 positions, but significantly lower efficiency at\nthe +5 or +6 positions. Considering that protein abundance might directly influence PE\noutcomes, we first examined the expression levels of PE2 protein in the presence or\n1\nabsence of AcrIIA5 using an additional Western blot at the HEK2 locus. The results\nshowed that aPE2 exhibited expression levels comparable to those of PE2 across +1 to\n+6 positions, indicating that AcrIIA5 does not interfere with PE2 protein expression\n(Supplementary Fig. 10 in the revised Supplementary Information). Therefore, the\nchanges in editing efficiency observed between PE and aPE systems are not attributable\ntodifferencesinproteinabundance.\nMW:\nkDa\nPE2\n250\n(nCas9-\nlinker-RT)\nPlasmids expressing PE2 or aPE2 (including the pegRNA) were transfected into\nHEK293T cells cultured in 24-well plates. After 48 hours, cells were lysed using X-RIPA\nUltraMix(X-Blot),and10 μLoflysatewasmixedwith2.5 μLof5×SDSloadingbuffer.After\nboiling, 10 μLof the mixture was loaded onto a 4–15% gradient SDS-PAGE gel. Proteins\nwereseparatedbyelectrophoresisandtransferredtoaPVDFmembrane.Themembrane\nwas cut at appropriate molecular weights and incubated with primary antibodies:\nanti-Cas9 (Genscript,A01935, clone 4A1, 1:500) and anti-GAPDH (Proteintech, 60004-1,\nclone 1E6D9, 1:1000), respectively. After incubation with HRP-conjugated secondary\nantibodiesandwashing,themembranewastreatedwithchemiluminescentsubstrateand\nimagedusinganAmershamImager600.\nNext, considering that the aPE system exhibited a sharp decrease in editing activity\nat the +5 and +6 positions, which correspond to the PAM sequence of SpCas9, we\nhypothesizedthateditswithinoroutsidethePAMregionmightdifferentiallyinfluenceaPE\nactivity. To test this hypothesis, we further evaluated the impact ofAcrIIA5 co-expression\nonPE2atPAM downstream positions,specificallyfrom+7to+14, attheHEK2locuswith\nadditional experiments (Fig. 5a,b in the revised Main text). Interestingly, the editing\nefficiency of aPE2 was consistently higher than that of PE2 across all positions in this\nregion.\n2\na b\nHEK2,+7 to +14, base conversion HEK2, +7 to +14, indels\nFig. 5a,b | Editing efficiencies and indels of base conversions installed by PE or\naPE2. a, Base conversion efficiencies at +7 to +14 positions at HEK2 locus. b, Indels\nformation at +7 to +14 positions at HEK2 locus. All data came from at least three\nindependentbiologicalreplicates.Barsrepresentmean ± s.d.\nBesides, we also newly examined aPE2-mediated insertions and deletions at\npositions downstream of the PAM sequence. The results showed that aPE2 consistently\nachieved higher activity than PE2 for these edits as well (Supplementary Fig. 11 in the\nrevisedSupplementaryInformation).\na -3 -1+1 +3 +5 +7 +9 +11+13+15 b HEK2, deletions c HEK2, indels for deletions\nHEK2 GACTGCGGGGCGGGCCAG\n+8 del GACTGCGGGG-GGGCCAG\n+9 del GACTGCGGGGC-GGCCAG\n+10 del GACTGCGGGGCG-GCCAG\n+11 del GACTGCGGGGCGG-CCAG\n+12 del GACTGCGGGGCGGG-CAG\n+13 del GACTGCGGGGCGGGC-AG\n+14 del GACTGCGGGGCGGGCC-G\nd e f\n-3 -1+1 +3 +5 +7 +9 +11+13+15 HEK2, insertions HEK2, indels for insertions\nHEK2 GACTGCGGGGCGGGCCAG\n+8 ins A GACTGCGGGGACGGGCCAG\n+9 ins A GACTGCGGGGCAGGGCCAG\n+10 ins A GACTGCGGGGCGAGGCCAG\n+11 ins A GACTGCGGGGCGGAGCCAG\n+12 ins A GACTGCGGGGCGGGACCAG\n+13 ins A GACTGCGGGGCGGGCACAG\n+14 ins A GACTGCGGGGCGGGCCAAG\nSupplementary Fig. 11 | Evaluation of PE2 and aPE2 editing activity on designed\ndeletionsandinsertionsattheHEK2locus.a,ReferencesequenceoftheHEK2target\nsite and representative single-nucleotide deletions ranging from +8 to +14 positions. b,\nEditing efficiencies for the designed deletions induced by PE2 and aPE2. c, Indels\nmeasured at the same deletion-targeting conditions. d, Reference sequence of the HEK2\ntarget site and representative single-nucleotide insertions (red) ranging from +8 to +14\npositions. e, Editing efficiencies for the designed insertions induced by PE2 and aPE2. f,\nIndels measured at the same insertion-targeting conditions. Positions are numbered\nrelative to the SpCas9 nick site. Data are shown as mean ±s.d. from three biological\nreplicates.\nPAM-silencing edits. When only PAM-proximal positions (+1 to +4) were edited, aPE2\nshowed higher efficiency than PE2 (Fig. 5c in the revised Main text). In contrast, at the\nPAM-silencing position (+5), the editing efficiency of aPE2 was lower than that of PE2.\nNotably, when PAM-proximal edits were introduced together with a PAM-silencing\nmutation, the editing efficiency of aPE2 was significantly reduced compared to PE2 (Fig.\n5d in the revised Main text). These new findings suggest that PAM-silencing edits\nmaybeakeyfactorinfluencing theeditingoutcome oftheaPEsystem.\nc d\nHEK2, single base conversions HEK2, double base conversions\nFig. 5c,d | Editing efficiencies of single and double base conversions installed by\nPEoraPE2.c,Singlebaseconversionefficienciesat+1to+5positionsatHEK2locus.d,\nThe conditional base conversions at +1, +2, +3, and +4 positions in the context of a\nsimultaneous +5 G-to-A conversion at the same locus. All data came from at least three\nindependentbiologicalreplicates.Barsrepresentmean ± s.d.\nTo further test this guess, we utilized SpG, an engineered variant of SpCas9 that\nrecognizes a relaxed N G N PAM, to construct an SpG-based PE2 system with\n+4 +5 +6\nexpanded targetable regions. We then evaluated the editing outcomes of both\nnSpCas9-PE2 (mainly N G G PAM and slightly N G A PAM) and nSpG-PE2 at the\n+4 +5 +6 +4 +5 +6\n+5 and +6 positions. Consistent with our previous findings, the editing efficiency of\nnSpCas9-aPE2 at both +5 and +6 positions was lower than that of nSpCas9-PE2.\nHowever,forthenSpG-basedsystem,aPE2exhibitedlowereditingefficiencythanPE2at\nthe +5 position (Fig. 5e in the revisedMain text), but higher efficiency at the +6 position\n(Fig.5fintherevisedMaintext).ThiscontrastingoutcomeobservedinthenSpG-based\nsystemisconsistentwithitsrecognitionoftheN\n+4\nG\n+5\nN\n+6\nPAM.Thesenew-addedresults\nfurther support the notion that PAM-silencing edits play a key role in modulating\ntheactivityoftheaPEsystem.\n4\ne f\nHEK2, +5 base conversions HEK2, +6 base conversions\nFig.5e,f|ComparisonofSpCas9-PE2andSpG-PE2 withorwithoutAcrIIA5.e,Base\nconversion efficiencies of+5 position at HEK2 locususing SpCas9-PE2(N G G PAM)\n+4 +5 +6\nor SpG-PE2 (N +4 G +5 N +6 PAM). f, Base conversion efficiencies of +6 position at HEK2\nlocus.All data came from at least three independent biological replicates. Bars represent\nmean ± s.d.\nBased on our newly-obtained experimental results, we updated our previous model to\nexplain the mechanism of the aPE system (Fig. 5g in the revised Main text). Previous\nstudies have demonstrated that PAM-silencing edits can enhance PE activity and reduce\nundesiredindelbyproducts,presumablybypreventingre-recognitionandre-nickingofthe\nedited strand (Anzalone et al. Nature 2019;576:149-157). Such re-nicking can lead to\nreversion of the desired edit or promote indel formation, especially in the context of PE3\nsystems. Inspired by these findings, we speculated that the activity of aPE might also be\ninfluenced by a similar prevention of re-nicking mechanism. Specifically, for\nPAM-proximal or distal edits,AcrIIA5 may suppress nSpCas9-H840Anicking activity just\nenough to slow re-targeting of the edited strand, without completely abolishing the initial\nnicking required for initiating the PE process. Moreover, once the intended mutations are\ninstalled, the edited targets may exhibit reduced binding affinity for the original\nnSpCas9-H840A-pegRNAcomplexduetosequencemismatcheswithinthespacerregion,\nmaking them more resistant to re-targeting and potentially further delaying re-nicking in\nthepresenceofAcrIIA5.Incontrast,forPAM-silencingedits,theinhibitionprovidedbythe\nPAM mutation itself may already be sufficient to prevent re-nicking. Thus, the additional\nsuppression fromAcrIIA5 could overly weaken the initial nicking activity, thereby reducing\ntheoveralleditingefficiency.\n5\ng\nPE permits re-nicking aPEmitigates re-nicking\nNTS extended NTS extended\nby RT by RT\nRepair Repair\nRe-nicking Re-nicking\nPE-mediated edit PE-mediated edit\nFig. 5g | Proposed mechanism for AcrIIA5 enhance PE activity. g, In the PE system\n(Left),theeditedstrandremainssusceptibletore-cleavagebyCas9aftertheinitialediting\nevent. In contrast, in the aPE system (Right), co-expression of AcrIIA5 partially inhibits\nCas9 activity, thereby reducing re-cleavage of the edited strand. nSpCas9 -H840A\nnickase (gray), initial nick site (red triangle), extended DNA strand from RT (red line),\nAcrIIA5(orange),RT(green),double-strandedDNA(blacklines)havebeenshown.\nForyoursuggestiontoinvestigatewhethertheaPEeffectisdrivenbyPEandAcrIIA5\ninteractionthroughthesplit-functionmutationanalysisonCas9andAcrIIA5,wefollowedit\nperformedsuchexperiments.\nAlthough no structural complex of SpCas9 and AcrIIA5 has been reported to date,\nprevious studies have identified two key regions through which AcrIIA5 inhibits SpCas9\n(An et al. Nucleic Acids Res. 2020;48:7584-7594; Garcia et al. Cell Rep.\n2019;29:1739-1746.e5). They include the positively charged N-terminal intrinsically\ndisordered region (IDR), and the loop between α3 and α4 along with the surrounding\nregion.WefirstconductedstepwisedeletionsoftheN-terminalIDR.Whileshortdeletions\ndid not significantly impact the function of AcrIIA5, the AcrIIA5 mutant exhibited a\nΔ7\nsignificantly reduced enhancement of PE2 activity compared to wild-type AcrIIA5,\nindicatingthattheN-terminalIDRplaysacriticalroleinmodulatingPE2activity(Fig.6ein\nt\nin the revised Main text). Next, we replaced the positively charged residues with alanine\ninside IDR. The AcrIIA5 and AcrIIA5 mutants showed a modest reduction in their\nKR RK\nability to enhance PE2 activity, whereas theAcrIIA5 mutant almost completely lost its\nRKR\nenhancing effect and even impaired PE2 editing efficiency (Fig. 6e in the revised Main\ntext).ThesefindingsareconsistentwithpreviousinvitroCas9cleavageassays(Anetal.\nNucleic Acids Res. 2020;48:7584-7594), which demonstrated that RKR mutations or\nextended deletions within the IDR abolish the inhibitory function of AcrIIA5, indicating\nthateffectiveCas9inhibition isessentialforitsenhancement ofPEactivity.\n6\ne\nN-terminal IDR mutants of AcrIIA5\n1 6 11 16 21\nWT MAYGKSRYNSYRKRSFNRSNKQRRE\nΔ2 M-YGKSRYNSYRKRSFNRSNKQRRE\nΔ2-3 M--GKSRYNSYRKRSFNRSNKQRRE\nΔ2-4 M---KSRYNSYRKRSFNRSNKQRRE\nΔ2-5 M----SRYNSYRKRSFNRSNKQRRE\nΔ2-6 M-----RYNSYRKRSFNRSNKQRRE\nΔ2-7 M------YNSYRKRSFNRSNKQRRE\nKR MAYGASAYNSYRKRSFNRSNKQRRE\nRKR MAYGKSRYNSYAAASFNRSNKQRRE\nRK MAYGKSRYNSYRKRSFNASNAQRRE\nf\nCleavage inactive mutants of AcrIIA5\n50 55 60 65 70\nWT DSAYKDFGKYEIRLSNHSADNKYHD\nD50A ASAYKDFGKYEIRLSNHSADNKYHD\nR62A DSAYKDFGKYEIALSNHSADNKYHD\nH66A DSAYKDFGKYEIRLSNASADNKYHD\nN70A DSAYKDFGKYEIRLSNHSADAKYHD\nH73A DSAYKDFGKYEIRLSNHSADNKYAD\nD74A DSAYKDFGKYEIRLSNHSADNKYHA\nDR ASAYKDFGKYEIALSNHSADNKYHD\nβ2 β3\ng Mutational screen of AcrIIA5-D1126 h\nHEK2, +2 G-to-A\nK8\nR62 R9\nR7\nH66 H73\nE29 N70 D74\nD31\nQ28 D49\nFig. 6e–h | Mutational analysis of AcrIIA5 and AcrIIA5-D1126 to investigate key\nregions involved in PE2 enhancement. e, Editing efficiencies and indels of AcrIIA5\nmutants with stepwise N-terminal deletions and positive-charge residue substitutions in\nthe IDR region, co-expressed with PE2. f, Editing efficiencies and indels of AcrIIA5\nmutants with point mutations in residues surrounding the α3–α4 loop, co-expressed with\nPE2. g, Mutational rescue screen of individual residues within the 18-amino-acid region\nthatdiffersbetweenAcrIIA5andAcrIIA5-D1126.Eachmutantwasco-expressedwithPE2\nand tested for the HEK2, +2 G-to-A edit. h, Structural model of AcrIIA5. Functionally\ncritical residues identified in e–g are highlighted in red and are clustered on one side of\nthe protein. Red dashed lines in e–g indicate the editing efficiency of PE2 in the absence\nof AcrIIA5 co-expression. All data came from at least three independent biological\nreplicates.Barsrepresentmean ± s.d.\nsubstitutionsinthisregion,andtheresultingmutantsshowedamarkeddecreaseinaPE2\nactivity(Fig.6fintherevisedMaintext).Interestingly,weobservedaconcurrentdecline\nin both editing efficiency and indel suppression when the activity of aPE2 was reduced,\nsuggesting that mutations weakening AcrIIA5’s inhibitory function compromise its dual\nrole in enhancing editing and minimizing byproducts of PE system (Supplementary Fig.\n14 in the revised Supplementary Information). In summary, our data confirm that\nmutationspreviouslyshowntodisruptAcrIIA5’sinhibitionofCas9inbothbiochemicaland\ncellularsettingsalso markedly reduced itsabilityto enhance PE activity.Thisparallel loss\nof function indicates that the enhancement of PE activity byAcrIIA5 fundamentally relies\non its inhibitory interaction with Cas9. Thus, although AcrIIA5 appears to act as an\nenhancerinthecontextofPE,itseffectultimatelystemsfromitscanonicalroleasaCas9\ninhibitor.\nAdditionally,toidentify residuescriticalfor thePE2-enhancingfunctionofAcrIIA5,we\nperformedsingle-pointreversionmutationsonAcrIIA5-D1126,whichdiffersfromwild-type\nAcrIIA5 by 18 amino acids. We aimed to determine whether specific mutations could\nrestore its ability to enhance PE2 activity. The results revealed that K28Q, K29E, and\nK31D were key mutations that significantly improved the activity ofAcrIIA5-D1126 toward\nwild-type AcrIIA5 levels (Fig. 6g in the revised Main text). When mapping all three\nfunctional regions onto the 3D structure of AcrIIA5, we found that they are spatially\nclustered on the same surface (Fig. 6h in the revised Main text). This observation\nsuggests that these residues may contribute to the interaction with Cas9 and its\nsubsequentinhibition.\nAs for SpCas9, to further investigate the key regions in Cas9 that mediate its\ninteraction withAcrIIA5, we sought to identify specific residues involved in this response.\nBoth our results (Fig. 6e,f in the revised Main text) and previous studies (An et al.\nNucleic Acids Res. 2020;48:7584-7594) have demonstrated that positively charged\nresiduesintheIDRregionofAcrIIA5arecriticalforitsinhibitoryactivity.Basedonthis,we\nhypothesized that AcrIIA5 may interact with negatively charged regions within the RuvC\ndomain through electrostatic forces. To test this, we generated alanine substitutions at a\nseries of candidate acidic residues in the RuvC domain, specifically aspartic acid (D) and\nglutamic acid (E), and evaluated PE2 editing efficiency with or without AcrIIA5..\nUnexpectedly, none of these mutations obviously affected the ability of AcrIIA5 to\nmodulate PE2 activity, suggesting that the tested residues are unlikely to be directly\n8\ninvolved in the interaction (Supplementary Fig. 15 in the revised Supplementary\nInformation). This finding indicates that the binding interface between AcrIIA5 and\nSpCas9ismorecomplexthaninitiallyanticipated,andfuturestudiesusinghigh-resolution\nstructural analysis or systematic mutagenesis will be needed to clarify the precise\nmechanismofAcrIIA5-Cas9recognition.\na\nTesting AcrIIA5-dependent activation of PE2 in RuvC-mutated SpCas9, HEK2, +2 G-to-A\nb\nSupplementary Fig. 15 | Evaluation of SpCas9 RuvC domain mutations on aPE2\nactivity. a,b, Assessment of PE2 editing efficiencies (a) and indels formation (b) using\nSpCas9 mutants with substitutions at negatively charged residues in the RuvC domain,\nwithorwithoutAcrIIA5.Alldatacamefromatleastthreeindependentbiologicalreplicates.\nBarsrepresentmean ± s.d.\n2.SinceAcrIIA5hasbeenidentified asapotentinhibitorofmanyCas9orthologues\n(Garciaetal.),Iwouldliketheauthorstoaddresswhetherthefunctionofimproving\nprimeeditingisalsotrueforotherCas9orthologues.\nResponse: We agree that assessing whether the beneficial effect of AcrIIA5 on PE\nextendstootherCas9orthologuesisanimportantissue.Toaddressthis,weattemptedto\nestablishPE2systemsbasedoncommonlyusednSt1Cas9andnNmeCas9(Garciaetal.\nCell Rep. 2019;29:1739-1746.e5) and co-expressed them with AcrIIA5. However, under\nourexperimentalconditionsandatthetestedtargetsites,theseorthologousPE2systems\nexhibited undetectable editing activity.As a result, we were unable to evaluate the impact\nof AcrIIA5 in these contexts. Nevertheless, given that AcrIIA5 is thought to enhance PE\nactivity by limiting re-nicking of the edited strand, it is conceivable that PE systems based\n9\nonotherCas9orthologsmaysimilarlybenefitfromAcrIIA5-mediatedenhancement.\n3. I would like to see a biochemical experiment showing the effect of AcrIIA5 on\nbothnucleaseandnickaseactivityofCas9.\nResponse: Thank you for your constructive suggestion and we designed a series of\nbiochemical experiments using Cas9 variants with distinct catalytic activities. Specifically,\nwe purified wild-type SpCas9, SpCas9-D10A (HNH-active, RuvC-inactive),\nSpCas9-H840A(RuvC-active,HNH-inactive),andAcrIIA5,andreconstitutedCas9-sgRNA\nribonucleoprotein (RNP) complexes with each Cas9 variant (Supplementary Fig. 12a,b\nintherevisedSupplementaryInformation).\na b\nT7 His6 SpCas9 T7 terminator\npet28a-SpCas9\nT7 His6 SpCas9-H840A T7 terminator\n160.5 kDa\npet28a-SpCas9-H840A\nT7 His6 SpCas9-D10A T7 terminator\npet28a-SpCas9-D10A\nTEV site\nT7 His6 MBP AcrIIA5 16.9 kDa\npet28a-MBP-AcrIIA5 T7 terminator\nc\nSpCas9 SpCas9-H840A SpCas9-D10A\nAcrIIA5 AcrIIA5 AcrIIA5\nM L1 L2 L3 0 0.1 1.0 10 0 0.1 1.0 10 0 0.1 1.0 10\nOC or\nLIN\nSC\nSupplementaryFig.12|Construction,purification,andinvitroDNAcleavageassay\nof SpCas9 variants in the presence of AcrIIA5. a, Schematic representation of\nexpression constructs used for producing SpCas9, SpCas9-H840A, SpCas9-D10A, and\nAcrIIA5proteins.b,SDS–PAGEanalysisofpurifiedproteins,showingexpectedmolecular\nweights of ~160.5 kDa for SpCas9 variants and ~16.9 kDa for AcrIIA5. c, Plasmid\ncleavage assays were performed using SpCas9, SpCas9-H840A (RuvC active), and\nSpCas9-D10A(HNH active) in the presence of increasing concentrations ofAcrIIA5. SC,\nLIN, and OC forms of pUC57 plasmid were separated on a 1.5% agarose gel and are\nshown. Lane 1 (L1) represent the input pUC57. The controls of LIN (L2) and OC (L3)\nplasmidweregeneratedbycatalyzedpUC57withEcoRIandNt.BspQI,respectively.Lane\nM contains a 1 kb DNA ladder. AcrIIA5 was added at molar ratios of 0, 0.1, 1.0, and 10\nrelativetoSpCas9oritsvariants.\npresence of increasing concentrations of AcrIIA5 (at molar ratios of 0, 0.1, 1.0, and 10\nrelative to Cas9). In the absence of AcrIIA5, wild-type SpCas9 mainly converted the\nsupercoiled (SC) plasmid into the linear (LIN) form. Similarly, SpCas9-D10A nicked the\ntargeted strand, producing predominantly open circular (OC) DNA, while SpCas9-H840A,\nwhich nicks the opposite strand, also generated OC products, albeit with slightly different\nefficiency. Upon titration of AcrIIA5, we observed a progressive reduction in\ncleavage/nicking activity for wild-type SpCas9 and SpCas9-H840A(Supplementary Fig.\n12cintherevisedSupplementaryInformation).Ata1:1molarratioofAcrIIA5toCas9,\nboth enzymes exhibited a considerable suppression of activity, with most DNAremaining\nin the SC form. This indicates effective inhibition of both double-strand cleavage and\nRuvC-mediated nicking. In contrast, SpCas9-D10A retained substantial nicking activity\neven at AcrIIA5 molar ratios of 0.1 and 1.0, and only at the highest ratio (10:1) did we\nobserve notable reduction in OC product formation. These results suggest that the RuvC\ndomainismoresusceptibletoAcrIIA5inhibitionthantheHNHdomain,stronglysupporting\nour hypothesis that AcrIIA5 fine-tunes PE activity by selectively dampening redundant\nre-nickingeventswhilepreservingtheinitialnickingrequiredtoinitiateprimeediting.\n4. AcrIIA5 impacts PE2 to varying extents across different loci. Often, the effect is\nabsent. Can you please explain the variation between loci? This raises concerns\nabout the technology's applicability in real genome editing scenarios beyond the\nextensivelystudiedHEK3andHEK4loci.\nResponse: Indeed, in our initial investigations, we observed that AcrIIA5 enhances PE\nefficiency to varying degrees across different target loci, and in certain cases, it even\nappears to reduce PE activity. This variation also prompted us to explore the underlying\nfactorsthatdeterminethelocus-dependenteffectofAcrIIA5onPEefficiency.\nAs mentioned above (Fig. 5g in the revised Main text), we hypothesized that aPE\nactivity may be associated with the initial DNAcleavage activity of Cas9. Specifically, we\nproposed that target sites with intrinsically high Cas9 cleavage activity are more likely to\ntolerateAcrIIA5-mediatedinhibition,astheystillpermittheinitialnickingeventrequiredfor\ninitiating PE. In this context, AcrIIA5 may help improve editing efficiency by delaying\nre-nicking and reducing unwanted repair events. In contrast, at sites with low inherent\nCas9 cleavage activity, AcrIIA5 may excessively preventing the initial nicking step and\nthereby abolishing PE activity. In other words, high Cas9 activity ensures that the\nessential nicking still occurs, while AcrIIA5 acts as a brake to prevent excessive or\n11\nmistimed cuts that could disrupt the editing process. Under this hypothesis, strong initial\nnickingactivitywoulddirectlyresultinhigheraPEefficiencycomparedtothePEsystem.\nHence, we sought to determine whether a correlation exists between Cas9 cleavage\nactivity and aPE performance. Given that in vitro Cas9 cleavage assays may not\naccurately reflect chromatin accessibility and other cellular factors influencing cleavage\nefficiencyinvivo,weturnedtoacell-basedpredictiveapproach.WeutilizedDeepSpCas9,\na deep learning model (Kim et al., Sci Adv. 2019; 5: eaax9249.) which predicts SpCas9\ncleavage activity across thousands of genomic loci in HEK293T cells. By inputting the\nsequences of our tested loci into this model, we obtained predicted cleavage scores\n(Supplementary Table 5 in the revised Supplementary Data), where higher scores\nindicatestrongerCas9activityincellularcontexts.\nBy correlating the DeepSpCas9 scores with the observed fold change in editing\nefficiency between aPE and PE systems (aPE:PE ratio), we found a strong association:\nhigher predicted SpCas9 activity scores generally linked to greater enhancement by\nAcrIIA5 (Fig. 5h in the revised Main text). Specifically, at target sites with DeepSpCas9\nscores above 50, aPE consistently outperformed PE. In contrast, at sites with scores\nbelow50,aPEoftenshowedreducedefficiencycomparedtoPE.\nTo further validate this observation, we selected an additional set of four loci with\npredicted DeepSpCas9 scores >50 and four with scores <50 (Supplementary Table 5 in\nthe revised Supplementary Data). The results showed that all four high-score loci\nexhibited enhanced editing efficiency with aPE compared to PE, whereas all four\nlow-scorelocishowed reduced aPE activitycompared to PE (Fig. 5i in the revisedMain\ntext). These findings support the conclusion that the performance of aPE is indeed\ninfluenced by initial Cas9 cleavage activity at the target locus, and that\nDeepSpCas9 scores can be prospectively used to guide locus selection, thereby\nimproving the predictability and applicability of aPE in practical genome editing\nscenarios.\nNevertheless, we also identified one outlier: the HEXA target site, which had a high\npredicted cleavage score (69.8) but still showed reduced aPE editing efficiency,\nsuggestingthatadditionalfactorsinfluenceaPEactivitybeyondinitialcleavageactivity.To\naddress this, we plan to evaluate aPE editing outcomes at tens of thousands of genomic\nloci to more comprehensively define the determinants of aPE activity. This large-scale\ninvestigation will lay the foundation for developing a deep learning model that integrates\nboth target sequence and pegRNA features, ultimately enabling more accurate and\n12\nefficientdeploymentofaPEsystemsindiversebiologicalandtherapeuticsettings.\nTable 5 h\nLocation Protospacer (20 bp) DeepSpCas9 score aPE2:PE2 fold change\nDMD, c.7098+1G>T GACCATTTGACGTTAAGGTA 50.876 8.207829034\nDMD, c.5287C>T GTGAAATGGCTGCAAATCGA 61.152 5.202734458\nNF1, c.4191del TGCTTCATACGGTGAGACAA 60.486 1.589104428\nNF1, c.1059del GTCCATGGTGGTTGATCTTA 14.631 1.580188616\nGJB1, c.415G>A GTGGACCTATGTCATCAGCG 69.157 1.274528366\nNF1, c.5749+332A>G AGGCAACATGGATACTTATG 44.590 1.137492929\nNPC2, c.58G>T GGCGCTCAGCACCGCTGCCC 48.589 0.99511896\nFBN1, c.2638G>A GCTTCCCCACGCAGCACCGA 59.520 0.981571664\nPAH, c.590T>A GCAAGCATGGGTTTTATACA 20.199 0.972163079\nNF1, c.2409+1G>C GTCAACTTTACAGACTTACC 20.398 0.903836572\nABCA4, c.2461T>C AGCAAGGCCTGGGGCTGCAG 48.866 0.621486766\nHEXA, c.1073+1G>A TCCTTCTACATCCAGACGTG 69.793 0.481343653\nHEK2 GAACACAAAGCATAGACTGC 52.554 2.602209322\nHEK3 GGCCCAGACTGAGCACGTGA 63.265 5.276914342\nHEK5 GAATACTAAGCATAGACTCC 55.890 3.873978846\nHEK6 CACACACACTTAGAATCTGT 42.465 2.869341909\ni\nLocation Protospacer (20 bp) DeepSpCas9 score\nTest1, +1 T-to-A GGACAGGCAGCATAGACTGT 71.555\nTest2, +1 T-to-A GAAGACCAAGGATAGACTGC 59.416\nTest3, +1 T-to-A GGCACTGCGGCTGGAGGTGG 54.311\nTest4, +1 T-to-A GTATTCACCTGAAAGTGTGC 50.162\nTest5, +3 C-to-G TCCCAGACATCATTCTTACC 35.832\nTest6, +1 T-to-A CCTCTCTCCGGAAGGGAAAA 35.359\nTest7, +1 T-to-A CTGGACTCTGGAATCCATTC 33.443\nTest8, +1 A-to-C TCACGGTTCGGGGGTATACA 26.699\nFig. 5h,i | Cas9 cleavage activity determines the position-dependent effect of\nAcrIIA5 on PE system. Table 5 (Top left), Summary of protospacer sequences,\nDeepSpCas9-predicted cleavage scores, and fold changes in editing efficiency (aPE:PE)\nacross 16 genomic loci. (Bottom left), Eight additional loci were selected for validation:\nfour with DeepSpCas9 scores >50 and four <50. Protospacer sequences and predicted\nscores are shown. h, Correlation between DeepSpCas9 scores and aPE:PE fold change.\nHigh Cas9 activity (score >50, orange dashed line) is generally associated with stronger\naPEenhancement.i,EditingefficienciesofPEandaPEsystemsattheselectedloci.aPE\noutperformedPEathigh-scoringsites(Test1–4),butwaslessefficientatlow-scoringsites\n(Test5–8). All data came from at least three independent biological replicates. Bars\nrepresentmean ± s.d.\nBesides,toassessthegeneralizabilityofaPE,wetesteditsperformanceinadditional\ncell lines, including HeLa and U2OS, using transfection (Fig. 4e,f in the revised Main\ntext). In both cases, aPE2 consistently outperformed PE2, suggesting that the aPE\nsystemhasbroadapplicabilityandrobustnessacrossdiversecellularcontexts.\ne PE2/aPE2 in HeLa cell f PE2/aPE2 in U2OS cell\nEditing efficiencies of PE2 and aPE2 were measured at two edits (HEK2, +2 G-to-Aand\nHEK3,+1T-to-A)inHeLa cells(e) andU2OScells(f).Cellsweretransfected withPE2or\naPE2 components, and editing outcomes were quantified by targeted amplicon\nsequencing. All data came from at least three independent biological replicates. Bars\nrepresentmean ± s.d.\n5. Can you experimentally address why AcrIIA5 has the most significant effect on\nPE2 and not on the more advanced versions like PE4 and PE5? I am particularly\nintriguedbythereverseeffectobservedwithPE7.\nResponse: Indeed, our results indicate that AcrIIA5 most significantly enhances the\nactivity of PE2, whereas its effect is reduced or even reversed in more advanced PE\nsystems such as PE4, PE5, and PE7. We guess this may stem from structural and\nexpression-related constraints inherent to these more complex architectures. In PE4 and\nPE5, the MMR inhibitor MLH1dn is co-expressed via a P2A self-cleaving peptide.\nHowever, the cleavage efficiency of the P2A sequence has been reported to be\nincomplete in HEK293T cells (Kim et al. PLoS One. 2011;6:e18556). This incomplete\ncleavage may lead to a subset of Cas9-RT fusion proteins retaining a covalently linked\nMLH1dn domain. Given that MLH1dn is a relatively large protein (752 aa, ~30 kDa), this\nresidual fusion could introduce steric hindrance that interferes with AcrIIA5’s interaction\nwith Cas9. Such interference may impair AcrIIA5’s ability to fine-tune Cas9-DNA\nbinding and dissociation kinetics, thereby diminishing its enhancing effect in PE4\nandPE5.\nToinvestigatewhetherthegeneticlinkagebetweenthePE2expressioncassette and\nMLH1dn via a P2Asequence affects the regulatory impact ofAcrIIA5, we constructed an\nadditional system in which MLH1dn is expressed from a separate plasmid. For clarity, we\nrefer to the original PE4 system as PE2+iMLH1dn (integrated MLH1dn) and the system\nwith independent MLH1dn expression as PE2+sMLH1dn (separate MLH1dn). We then\nevaluated editing efficiencies across the PE2–PE5 architectures under these modified\nconditions (Supplementary Fig. 3d in the revised Supplementary Information).\nConsistent with our earlier findings, aPE2 exhibited higher editing efficiency than\naPE2+iMLH1dn, andsimilarly,aPE3outperformed aPE3+iMLH1dn (Supplementary Fig.\n3e in the revised Supplementary Information). Strikingly, both aPE2+sMLH1dn and\naPE3+sMLH1dn showed even greater editing efficiencies than their respective aPE2 and\naPE3 counterparts. This result further supports our hypothesis that incomplete P2A\ncleavage in PE4 can lead to residual MLH1dn interfering withAcrIIA5-Cas9 interactions,\nthereby reducing its regulatory effect. Moreover, the improved performance of aPE\n14\nsystems when MLH1dn is delivered in trans highlights the modularity and extensibility of\ntheaPEplatform.\nd\nHEK2, +3 C-to-A\ne\nSupplementaryFig.3d,e|Co-expressionofMLH1dnmodulatestheeffectofAcrIIA5\non PE efficiency. d, Editing efficienciesfor the HEK2,+3 C-to-Ausing PE/aPE system, in\ncombination with MLH1dn delivered via P2Alinkage (iMLH1dn), oras a separate plasmid\n(sMLH1dn).Tomaintain consistent totalplasmidamountsacrossallexperimental groups,\npcDNA3.1(-) was used to equalize any differences in DNA dosage. e, Comprehensive\nediting efficiency comparison of PE2, PE3, PE4 and PE5 with or withoutAcrIIA5.All data\ncame from at least three independent biological replicates. Bars represent mean ± s.d.. P\nvalueswerecalculatedusingthetwo-tailedStudent’st-test.\nAsforPE7,ourpreviousdatashowedthatthepresenceofAcrIIA5reducesitsediting\nactivity. Given that the only difference between PE2 and PE7 is the addition of the La\nprotein,whichstabilizesthepegRNAbybindingits3′poly(U)tail,wehypothesizethatthis\ncomponentisakeyfactorinfluencingtheresponseofaPE7toAcrIIA5.\nAccording to previous reports (An et al. Nucleic Acids Res. 2020;48:7584-7594),\nAcrIIA5canbindtosgRNAinCas9complex,suggestingitmayalsointeractwithpegRNA.\nWespeculated thatAcrIIA5 andLaproteinmightinterferewitheachotheronthepegRNA\nscaffold. Besides, like iMLH1dn tested above, the La protein alone may also prevent the\nAcrIIA5-Cas9 interaction through steric hindrance. To investigate these possibilities, we\nengineered a system in which the poly(U) tail was removed by inserting a self-cleaving\nHDV ribozyme between the pegRNAand the poly(U) sequence (Supplementary Fig. 3g\nin the revised Supplementary Information). This ribozyme cleaves cotranscriptionally,\nproducing pegRNA transcripts lacking the poly(U) extension. We then compared the\n15\neditingefficienciesof PE2,PE7, aPE2, andaPE7 systemswithorwithout the poly(U)tail.\nWefirstobservedthataPE2withHDV-cleavedpegRNAexhibitedhighereditingefficiency\nthanthecorrespondingPE2control,confirmingthatAcrIIA5functionsindependentlyofthe\npoly(U)sequenceanddoesnotactthroughdirectbindingtoit(SupplementaryFig.3hin\nthe revised Supplementary Information). Next, we found that PE7 with HDV-cleaved\npegRNArestored editing activity to levels comparable to PE2, consistent with La protein\nrequiring poly(U) to exert its function. Notably, aPE7 with HDV-cleaved pegRNA also\nregained activity similar to aPE2, proving that the size of La protein alone can not\nprevent the AcrIIA5-Cas9 interaction through steric hindrance. Together, these\nresults support a refined model in which the poly(U) tail enables La protein binding to the\n3′ end of pegRNA, and this bound La protein indirectly interferes with AcrIIA5 function.\nThis spatial interference likely impairs AcrIIA5’s ability to fine-tune the Cas9-pegRNA\ncomplex, ultimately leading to the decreased editing efficiency observed in the aPE7\nsystem.\ng CMV SpCas9-H840A M-MLV RT NLS Terminator h\nPE2\nNLS Terminator\nCMV SpCas9-H840A M-MLV RT La\nPE7\nU6 pegRNA\nCMV AcrIIA5 NLS Terminator pegRNA polyT\nAcrIIA5 U6 pegRNA HDV\npegRNA\npolyT\n+HDV\nSupplementary Fig. 3g,h | Testing the influence of the poly(U) tail on the editing\nperformance of aPE2 and aPE7. g, Schematic representation of constructs used in this\nstudy. PE2 and PE7 both encode SpCas9-H840A fused to M-MLV RT. PE7 uniquely\nincludes the La protein downstream of the RT. AcrIIA5 is expressed separately under a\nCMVpromoter.TwotypesofpegRNAsweretested:thestandardpegRNAwitha3′poly(U)\ntailandamodifiedpegRNAcontainingaself-cleavingHDVribozymetoremovethepoly(U)\nextension cotranscriptionally. h, Editing efficiencies in PE and aPE systems using\npegRNA with or without the poly(U) tail. All data came from at least three independent\nbiologicalreplicates. Barsrepresent mean ± s.d.\n6. A DNA repair profile analysis of indels generated in the presence or absence of\nAcrIIA5 is required to determine if there is a bias towards MMEJ or NHEJ.\nAdditionally, please clarify the naming of the position of the installed base. This\ncould help determine if the positional effect ofAcrIIA5 on base conversion types is\nrelatedtothepreventionofrecleavagebyCas9afterediting.\nResponse: To determine if there is a bias towards MMEJ or NHEJ for PE/aPE, we first\nanalyzed the indels composition accompanying PE and aPE editing using the\nclass, observed at most loci such as the HEK3, +1 T-to-A edit in which indels are\npredominantly deletions, with minimal or no unintended insertions (Supplementary Fig.\n16aintherevisedSupplementaryInformation).Thesedeletionstendtooccurnearthe\nnick,involvingsmall nucleotide losses, consistent withclassical NHEJ repair. Notably, the\napplication of aPE markedly reduced the frequency of these deletions, indicating a\nsuppressiveeffectonNHEJ-mediatederrors.\na b\nUnintended deletions in HEK3, +1 T-to-A Unintended deletions in HEK2, +3 C-to-A, -21 nick\nSequence around nicking site Type PE2 aPE2 Sequence around nicking site Type PE2 aPE2\nWT ACTGAGCACGTGATGGCAGAGGAAAGGAAG / / / WT AAGCATAGACTGCGGGGCGGGCCAGCCTGA / / /\ndel#1 ACTGAGCAC-TGATGGCAGAGGAAAGGAAG NHEJ 0.13% 0.01% del#1 AAGCATAG--TGCGGGGCGGGCCAGCCTGA NHEJ 0.12% 0.06%\ndel#2 ACTGAGC---TGATGGCAGAGGAAAGGAAG NHEJ 0.09% none del#2 AAGCATA---TGCGGGGCGGGCCAGCCTGA NHEJ 0.06% 0.05%\ndel#3 AC--------TGATGGCAGAGGAAAGGAAG NHEJ 0.06% none del#3 AAGCATAGA-TGCGGGGCGGGCCAGCCTGA NHEJ 0.06% 0.01%\ndel#4 ACTGAGCA--TGATGGCAGAGGAAAGGAAG NHEJ 0.06% 0.01% del#4 A---------TGCGGGGCGGGCCAGCCTGA NHEJ 0.06% 0.01%\ndel#5 ACTGA----GTGATGGCAGAGGAAAGGAAG NHEJ 0.04% 0.01% del#5 AAGCATAGA---CGGGGCGGGCCAGCCTGA NHEJ 0.04% 0.01%\nc\nUnintended insertions in HEK2, +3 C-to-A, -21 nick\nSequence around nicking site Type PE2 aPE2\nWT AAGCATAGACTGCGGGGCGGGCCAGCCTGA / / /\nins#1 AAGCATAGACTGAGGGGCGGGCCAGCTGCGGGGCGGGCCAGCCTGA NHEJ 1.00% 0.30%\nins#2 AAGCATAGACTGAGTGCGGGGCGGGCCAGCCTGA NHEJ 0.91% 0.24%\nins#3 AAGCATAGACTGATGCGGGGCGGGCCAGCCTGA NHEJ 0.43% 0.07%\nins#4 AAGCATAGACTGAGGTGCGGGGCGGGCCAGCCTGA NHEJ 0.34% 0.08%\nins#5 AAGCATAGACTGAGGGGCGGTGCGGGGCGGGCCAGCCTGA NHEJ 0.22% 0.03%\nSupplementary Fig. 16a–c | DNA repair profile analysis of indels generated by PE\nandaPEsystem.a,UnintendeddeletionsanalysisofPE2andaPE2forHEK3,+1T-to-A\nedit.b,UnintendeddeletionsanalysisofPE2andaPE2forHEK2,+3C-to-A,-21nickedit.\nc,UnintendedinsertionsanalysisofPE2andaPE2forHEK2,+3C-to-A,-21nickedit.For\neach condition, the wild-type reference sequence and the top five most frequent indel\nvariants are shown. PAM sequences are highlighted in yellow and bold. The presumed\nDNA repair pathway responsible for each indel (NHEJ or MMEJ) and the corresponding\nfrequenciesinPE2andaPE2areindicated. “None”indicatesundetectablefrequency.\nThe second class, observed most prominently at the HEK2 site, shares a similar\ndeletion profile near the nick site, yet is characterized by a large number of unintended\ninsertions,aphenomenonnotcommonlyseenatotherloci(SupplementaryFig.16b,cin\nthe revised Supplementary Information). Importantly, aPE2 also significantly reduced\ntheseinsertions compared to PE2.Todetermine the possible originsof these unexpected\ninsertions,weexaminedtheirsequencecompositionandstructuralcontext.\nInterestingly,manyoftheseinsertionscorrespondtopartialorfullreverse-transcribed\nRTT sequences, which appear to have been directly inserted at the nick site without\nreplacing the original strand. This suggests that the reverse-transcribed strand, normally\nintended to displace and substitute the original DNA segment via 3′ flap resolution, may\noccasionally fail to do so (Supplementary Fig. 16d in the revised Supplementary\nInformation).\n17\nd\nAAGCATAGACTGCGGGGCGGGCCAGCCTGA\n││││││││││││││││││││││││││││││\nTTCGTATCTGACGCCCCGCCCGGTCGGACT\n1. SpCas9(H840A)-mediated nicking\n2. PBS annealing\n3. Reverse transcription initiation from RTT\nAAGCATAGAC TGCGGGGCGGGCCAGCCTGA\n││││││││││ ││││││││││││││││││││\nTTCGTATCTG ACGCCCCGCCCGGTCGGACT\n4. 3′ flap extension 4. Extended strand fails to displace original DNA\n5. Strand displacement 5. RT product captured by NHEJ and ligated into nick\nAAGCATAGACTGAGGGGCGGGCCA GCCTGA AAGCATAGAC TGCGGGGCGGGCCAGCCTGA\n││││││││││││ │││││││││││ ││││││ ││││││││││ ││││││││││││││││││││\nTTCGTATCTGACGCCCCGCCCGGT CGGACT TTCGTATCTG ACGCCCCGCCCGGTCGGACT\n6. Repair 6. Repair\nAAGCATAGACTGAGGGGCGGGCCAGCCTGA AAGCATAGACTGAGGGGCGGGCCAGCTGCGGGGCGGGCCAGCCTGA\n││││││││││││││││││││││││││││││ ││││││││││││││││││││││││││││││││││││││││││││││\nTTCGTATCTGACTCCCCGCCCGGTCGGACT TTCGTATCTGACTCCCCGCCCGGTCGACGCCCCGCCCGGTCGGACT\nSupplementary Fig. 16d | Schematic model of uncanonical PE-derived unintended\ninsertion. Following nicking by SpCas9(H840A), the PBS of pegRNA anneals to the\ntarget strand, and reverse transcription initiates from the RTT. In the canonical editing\npathway (left), the extended DNAstrand displaces the original sequence to form a 3’flap,\nwhich undergoes mismatch repair to install the intended edit. However, in some cases\n(right), the reverse-transcribed strand fails to displace the original DNA, potentially due to\nhigh GC content that stabilizes the original duplex. As a result, the extended RT product\nremains exposed and is instead recognized and ligated by the NHEJ pathway, producing\nunintendedinsertionscontainingfullorpartialRTTsequences.Reddenotestheextended\nDNAsequence synthesized from RTT; Green marks the original genomicsequence; Blue\nrepresents a scaffold-derived nucleotide occasionally incorporated beyond the RTT\nboundary(Antoniouetal.NatCommun.2025;16(1):3374);Light bluerepresentsthenick\nsiteinducedbynSpCas9-H840A.\nInstead, the exposed extended strand is likely recognized and ligated into the DNA\nbreak by NHEJ machinery. This is especially plausible given the GC-rich environment\nsurrounding the HEK2 site, which may stabilize the original strand and interfere with\nsuccessful flap displacement. Therefore, these insertion events are best explained by a\nfailure of strand displacement followed by NHEJ-mediated ligation of the RT product,\nrather than by MMEJ, which typically requires microhomology at the ligation junctions.\nThe ability of AcrIIA5 to suppress both deletion- and insertion-type byproducts suggests\nthat it may reduce recleavage or repair pathway engagement at an earlier step, thereby\nshiftingtherepairoutcomeawayfromundesiredindelsformationentirely.\nFollowing your suggestion on the naming of the position of the installed base, we\nhave carefully annotated the exact position of each installed base for all editing events\n18\nthroughout the revised manuscript and corresponding figures (Fig. 3a,f in the revised\nMain text and Supplementary Fig. 1a in revised Supplementary Information). These\npositional annotations are provided upon their first mention and are intended to facilitate\ntheinterpretationofpotentialposition-dependenteffectsofAcrIIA5.\na b c\n-3 -1+1 +3 +5 +7 +9 +11+13\nHEK3, deletions HEK3, deletions, indels\nHEK3reference ACGTGATGGCAGAGGA\n+1 del ACG-GATGGCAGAGGA\n+1 to +2 del ACG--ATGGCAGAGGA\n+1 to +3 del ACG---TGGCAGAGGA\n+1 to +4 del ACG----GGCAGAGGA\n+1 to +5 del ACG-----GCAGAGGA\n+1 to +6 del ACG------CAGAGGA\nd 6 edits at 4 targets, deletions e 6 edits at 3 targets, indels\nf g h\n-3 -1+1 +3 +5 +7 +9 +11+13\nHEK3, insertions HEK3, insertions, indels\nHEK3reference ACGTGATGGCAGAGGA\n+1 ins 1 nt ACGATGATGGCAGAGGA\n+1 ins 2 nt ACGCATGATGGCAGAGGA\n+1 ins 3 nt ACGTCATGATGGCAGAGGA\n+1 ins 4 nt ACGATCATGATGGCAGAGGA\n+1 ins 5 nt ACGCATCATGATGGCAGAGGA\n+1 ins 6 nt ACGTCATCATGATGGCAGAGGA\ni j\n6 edits at 4 targets, insertions 6 edits at 3 targets, indels\nFig. 3 | Tests of aPE performance in deletions and insertions. a, Alignment of the\nHEK3 reference sequence and representative deletion variants spanning from the +1\nposition to +6. Numbers above the reference indicate positions relative to the SpCas9\nnicking site. Dashed lines represent deleted nucleotides in each variant. b, Editing\nefficiency of PE and aPE2-induced deletions at HEK3 site. c, Indels formation of PE and\naPE2-induced deletions at HEK3 site. d, Comprehensive editing efficiency comparison of\nPE2 and aPE2-induced deletions for edits of HEK2, HEK3, HEK5, and HEK6. e,\nComprehensive indels comparison of PE2 and aPE2-induced deletions for edits of of\nHEK2, HEK3, HEK5, and HEK6. f, Alignment of the HEK3 reference sequence and\nrepresentativeinsertionvariantsat+1position,withinsertedsequencesrangingfrom1to\n6 nucleotides. Numbers above the reference indicate positions relative to the SpCas9\n19\nnicking site. Inserted nucleotides are shown in red for clarity. g, Editing efficiency of PE\nand aPE2-induced insertions at HEK3 site. h, Indels formation of PE and aPE2-induced\ninsertions at HEK3 site. i, Comprehensive editing efficiency comparison of PE2 and\naPE2-induced insertions for edits of HEK2, HEK3, HEK5, and HEK6. j, Comprehensive\nindelscomparisonofPE2andaPE2-inducedinsertionsforeditsofofHEK2,HEK3,HEK5,\nand HEK6. All data came from at least three independent biological replicates. Bars\nrepresentmean ± s.d.Pvalueswerecalculatedusingthetwo-tailedStudent’st-test.\na\nCBEs\n1 2 3 4 5 6 7 8 91011121314151617181920 PAM\nHEK3 reference 5’ GGGGCCCCCCAAGGAACCTTGGAAGGCCAACCGGTTGGAATTGGGGCCAAGGAAGGGGAA 3’\n+1+2+3+4+5+6+7+8+9+10\nSpCas9 PE\nb c d e\nhA3A-CBE, HEK3 hA3Bctd-CBE, HEK3 hA3Gctd-CBE, HEK3 mAPOBEC1-CBE, HEK3\nSupplementary Fig. 1 | Schematic annotation of the HEK3 site and the editing\nefficiency of various CBEs. a, Schematic representation of the HEK3 target site\nshowingtherelativeactivitywindowsforCBEs(red)andPE(blue),withtheSpCas9PAM\nsequencehighlightedinpurpleandthenicksiteindicatedbythegreenarrow.b–d,Editing\nefficiencyofvariousCBEs.hA3Bctd-CBE(b),hA3Gctd-CBE(c),andmAPOBEC1-CBE(d)\nwere tested at HEK3 target. All CBEs were co-delivered with vector or vector containing\nAcrIIA5. All data came from at least three independent biological replicates. Bars\nrepresentmean±s.d.Pvalueswerecalculatedusingthetwo-tailedStudent’st-test.\n7. I have serious doubts about the AAV experiments – Figure 5c shows only a\nmodest effect after 12 hours. It makes me question whether this approach works\neffectively at all. Can you demonstrate a better in vivo application? Have you tried\nLNPdelivery?\nResponse: We acknowledge that the observed enhancement in editing at the 12-hour\ntime point was modest, with aPE2 exhibiting a 1.33-fold increase in editing efficiency\ncompared to PE2. We hypothesized that during protein assembly, the split PE2 protein\nmay be more susceptible toAcrIIA5 inhibition. We then speculated that further optimizing\nthetimingofAcrIIA5deliverycouldpotentiallyenhanceeditingefficiency,butitmaynotbe\nadecisivestrategy.\nAs you suggested, lipid nanoparticle (LNP) delivery could be a promising alternative,\nasitenablesthedirectdeliveryoffull-lengthmRNAtranscriptswithouttheneedforprotein\nsplitting. However, due to current technical and resource limitations, we have not yet\n20\nconducted in vivo experiments.Alternatively, we attempted to validate the LNP approach\ninvitrousingHEK293Tcells.Weinvitrotranscribedfull-lengthPE2ΔRNaseHandAcrIIA5\nmRNAs and encapsulated them with pegRNAin LNPs for co-delivery into HEK293Tcells\n(Fig. 4g in the revised Main text). Our results demonstrated that LNP-mediated delivery\nof PE2ΔRNaseH mRNA alone resulted in an intended editing efficiency of 7.5% at the\nHEK3, +1 T-to-Aedit (Fig. 4h in the revised Main text). When co-delivered withAcrIIA5\nmRNAatmolarratios of0.5and 0.1relativeto PE2ΔRNaseH,theeditingefficiencyofthe\naPE2 system increased to 11.1% (1.48-fold of PE2ΔRNaseH) and 13.5% (1.80-fold of\nPE2ΔRNaseH), respectively, representing a notable enhancement compared to\nPE2ΔRNaseH alone. Moreover, the frequency of undesired indels was consistently lower\nintheaPE2ΔRNaseHgroupsthaninthePE2ΔRNaseH-onlygroup.Theseresultssuggest\nthat LNP-based delivery of the full-length aPE system is both feasible and effective,\nenhancingitstherapeutic potential.\ng h LNP delivery of PE2/aPE2\nmRNA-PE2ΔRNaseH\nmRNA-AcrIIA5\npegRNA LNP\nLNP-PE2 LNP-aPE2\nFig. 4g,h | Evaluation of LNP-mediated delivery of PE2 and aPE2. g, Schematic\nillustration of LNP formulations used for delivering PE components. mRNA encoding\nPE2ΔRNaseH or aPE2ΔRNaseH (co-expressing AcrIIA5) and pegRNA were\nencapsulated into LNPs for cellular delivery. h Editing outcomes in HEK293T cells\nfollowing LNP-mediated delivery of PE2 or aPE2 at varying ratios of AcrIIA5 mRNA\n(0.5-foldand0.1-foldrelativetoPE2mRNA).Thepercentageofintendededitsandindels\nwas measured by amplicon sequencing. All data came from at least three independent\nbiologicalreplicates. Barsrepresent mean ± s.d.\nMinor comments:\n1. Why is the improvement in the insertion and deletion of genomic fragments by\nAcrIIA5onlymodestcomparedtosubstitutions?\nResponse: As we previously observed, PAM-silencing edits significantly reduce aPE\nactivity. Compared to single-nucleotide substitutions, insertions and deletions inherently\nalter the length or structure of the target DNAsequence, which may more strongly affect\n21\nthePAMregionandsurroundingelements.Thisstructuralcomplexitycouldlimittheability\nof AcrIIA5 to enhance PE editing efficiency, resulting in a more modest improvement in\ninsertions and deletions compared to base substitutions. This analysis has also been\nincludedintheDiscussionsection.\n2. The figure presentation could be improved, such as in Figure 1b and others.\nWhatisthe reason forplotting fold change onthe sameplots? This is confusing to\nthereader.\nResponse: We are sorry for the confusion caused by plotting the fold change and\nabsolutevaluesonthesameplots.WehaverevisedtheFigure1bassuggested.Besides,\nwehavealsorevisedallrelevantfiguresbyremovingthesecondaryY-axisandpresenting\nthe data in a clearer and more straightforward format to improve readability.Additionally,\nother presentations have been improved as well. We hope these modifications will\nfacilitatebetterinterpretationoftheresults.\n3. In the introduction, the authors state that 'Guided by pegRNA, the PE2 protein\nbinds to one strand of target DNAand nicks the opposite, PAM-containing strand.'\nThisisnotcorrect;thePE2proteinbindstobothDNAstrands.\nResponse: We have revised the sentence as follows, “Guided by pegRNA, the PE2\nproteinbindsthetargetDNA,andnicksthePAM-containingstrand”.\nIn this manuscript, the authors proposed and provided extensive data demonstrating that\nlowdosesofAcrIIA5canenhancetheeditingefficiencyofPrimeEditing.Tothebestofmy\nknowledge, this is the first study reporting Cas9 inhibitors AcrIIA5 to improve the editing\nefficiency of genome editing tool-PE, which is novel and interesting. In the field of\nCRISPR-Cas,Acr was originally discovered as an inhibitor of Cas9, and previous studies\nhave also demonstrated that Acr can inhibit the editing efficiency of Cas9 and BE.\nTherefore, this study unexpectedly found that Acr can enhance the efficiency of PE,\nofferingnewinsightsforthedesignandoptimizationofAcrandPE.\nIn sum, I find this research to be novel and interesting.The manuscript is well-organized,\nthe methods are sound, the data is detailed and provides strong support for the\nconclusions. the conclusions are reliable. It will attract widespread interest among\nresearchersintheCRISPRfield.\nHowever,Ihaveafewsuggestionsfortheauthors.\n1. The study focuses on AcrII5 enhancing the efficiency of various PE\nconstructionsandreducingindel, butlacksanexplanation ofthemechanism.\nResponse: We sincerely thank your insightful comments.As you mentioned, we lack an\nexplanation of the aPE mechanism. In our earlier originally submitted manuscript, we\nspeculated that AcrIIA5 might influence the recognition speed of the extended edited\nstrand installed by the PE system in a position-dependent manner. However, we realized\nthat this hypothesis lacks solid experimental support, and we now consider that a more\nplausiblemechanismmayexistbasedonournewly-addedexperimental results.\nThe key issue lies in explaining the observed phenomenon that PE2 typically exhibit\nhigher editing efficiency at the +1 to +3/+4 positions, but significantly lower efficiency at\nthe +5 or +6 positions. Considering that the aPE system exhibited a sharp decrease in\nediting activity at the +5 and +6 positions, which correspond to the PAM sequence of\nSpCas9, we hypothesized that edits within or outside the PAM region might differentially\ninfluence aPE activity. To test this hypothesis, we further evaluated the impact ofAcrIIA5\nco-expression on PE2 at PAM downstream positions, specifically from +7 to +14, at the\nHEK2locuswithadditionalexperiments(Fig.5a,bintherevisedMaintext).Interestingly,\ntheeditingefficiencyofaPE2wasconsistentlyhigherthanthatofPE2acrossallpositions\ninthisregion.\n23\na b\nHEK2,+7 to +14, base conversion HEK2, +7 to +14, indels\nFig. 5a,b | Editing efficiencies and indels of base conversions installed by PE or\naPE2. a, Base conversion efficiencies at +7 to +14 positions at HEK2 locus. b, Indels\nformation at +7 to +14 positions at HEK2 locus. All data came from at least three\nindependentbiologicalreplicates.Barsrepresentmean ± s.d.\nBesides, we also examined aPE2-mediated insertions and deletions at positions\ndownstream of the PAM sequence. The results showed that aPE2 consistently achieved\nhigher activity than PE2 for these edits as well (Supplementary Fig. 11 in the revised\nSupplementaryInformation).\na -3 -1+1 +3 +5 +7 +9 +11+13+15 b HEK2, deletions c HEK2, indels for deletions\nHEK2 GACTGCGGGGCGGGCCAG\n+8 del GACTGCGGGG-GGGCCAG\n+9 del GACTGCGGGGC-GGCCAG\n+10 del GACTGCGGGGCG-GCCAG\n+11 del GACTGCGGGGCGG-CCAG\n+12 del GACTGCGGGGCGGG-CAG\n+13 del GACTGCGGGGCGGGC-AG\n+14 del GACTGCGGGGCGGGCC-G\nd e f\n-3 -1+1 +3 +5 +7 +9 +11+13+15 HEK2, insertions HEK2, indels for insertions\nHEK2 GACTGCGGGGCGGGCCAG\n+8 ins A GACTGCGGGGACGGGCCAG\n+9 ins A GACTGCGGGGCAGGGCCAG\n+10 ins A GACTGCGGGGCGAGGCCAG\n+11 ins A GACTGCGGGGCGGAGCCAG\n+12 ins A GACTGCGGGGCGGGACCAG\n+13 ins A GACTGCGGGGCGGGCACAG\n+14 ins A GACTGCGGGGCGGGCCAAG\nSupplementary Fig. 11 | Evaluation of PE2 and aPE2 editing activity on designed\ndeletionsandinsertionsattheHEK2locus.a,ReferencesequenceoftheHEK2target\nsite and representative single-nucleotide deletions ranging from +8 to +14 positions. b,\nEditing efficiencies for the designed deletions induced by PE2 and aPE2. c, Indels\nmeasured at the same deletion-targeting conditions. d, Reference sequence of the HEK2\ntarget site and representative single-nucleotide insertions (red) ranging from +8 to +14\npositions. e, Editing efficiencies for the designed insertions induced by PE2 and aPE2. f,\nIndels measured at the same insertion-targeting conditions. Positions are numbered\nrelative to the SpCas9 nick site. Data are shown as mean ±s.d. from three biological\nreplicates.\nPAM-silencing edits (Fig. 5c in the revised Main text). When only PAM-proximal\npositions (+1 to +4) were edited, aPE2 showed higher efficiency than PE2. In contrast, at\nthePAM-silencingposition(+5),theeditingefficiencyofaPE2waslowerthanthatofPE2.\nNotably, when PAM-proximal edits were introduced together with a PAM-silencing\nmutation, the editing efficiency of aPE2 was significantly reduced compared to PE2 (Fig.\n5d in the revised Main text). These findings suggest that PAM-silencing edits may\nbeakeyfactorinfluencing theediting outcomeoftheaPEsystem.\nc d\nHEK2, single base conversions HEK2, double base conversions\nFig. 5c,d | Editing efficiencies of single and double base conversions installed by\nPEoraPE2.c,Singlebaseconversionefficienciesat+1to+5positionsatHEK2locus.d,\nThe conditional base conversions at +1, +2, +3, and +4 positions in the context of a\nsimultaneous +5 G-to-A conversion at the same locus. All data came from at least three\nindependentbiologicalreplicates.Barsrepresentmean ± s.d.\nTo further test this guess, we utilized SpG, an engineered variant of SpCas9 that\nrecognizes a relaxed N G N PAM, to construct an SpG-based PE2 system with\n+4 +5 +6\nexpanded targetable regions. We then evaluated the editing outcomes of both\nnSpCas9-PE2 (mainly N G G PAM and slightly N G A PAM) and nSpG-PE2 at the\n+4 +5 +6 +4 +5 +6\n+5 and +6 positions. Consistent with our previous findings, the editing efficiency of\nnSpCas9-aPE2 at both +5 and +6 positions was lower than that of nSpCas9-PE2.\nHowever,forthenSpG-basedsystem,aPE2exhibitedlowereditingefficiencythanPE2at\nthe +5 position (Fig. 5e in the revisedMain text), but higher efficiency at the +6 position\n(Fig.5fintherevisedMaintext).ThiscontrastingoutcomeobservedinthenSpG-based\nsystemisconsistentwithitsrecognitionoftheN\n+4\nG\n+5\nN\n+6\nPAM.Thesenew-addedresults\nfurther support the notion that PAM-silencing edits play a key role in modulating\ntheactivityoftheaPEsystem.\n25\ne f\nHEK2, +5 base conversions HEK2, +6 base conversions\nFig.5e,f|ComparisonofSpCas9-PE2andSpG-PE2 withorwithoutAcrIIA5.e,Base\nconversion efficiencies of+5 position at HEK2 locususing SpCas9-PE2(N G G PAM)\n+4 +5 +6\nor SpG-PE2 (N +4 G +5 N +6 PAM). f, Base conversion efficiencies of +6 position at HEK2\nlocus.All data came from at least three independent biological replicates. Bars represent\nmean ± s.d.\nBased on our newly-obtained experimental results, we attempted to establish a model\ntoexplainthemechanismoftheaPEsystem(Fig.5gintherevisedMaintext).Previous\nstudies have demonstrated that PAM-silencing edits can enhance PE activity and reduce\nundesiredindelbyproducts,presumablybypreventingre-recognitionandre-nickingofthe\nedited strand. (Anzalone et al. Nature 2019;576:149-157). Such re-nicking can lead to\nreversion of the desired edit or promote indel formation, especially in the context of PE3\nsystems. Inspired by these findings, we speculated that the activity of aPE might also be\ninfluenced by a similar prevention of re-nicking mechanism (Fig. 5g in the revised\nMain text). Specifically, for PAM-proximal or distal edits, AcrIIA5 may suppress\nnSpCas9-H840A nicking activity just enough to slow re-targeting of the edited strand,\nwithout completely abolishing the initial nicking required for initiating the PE process.\nMoreover, once the intended mutations are installed, the edited targets may exhibit\nreduced binding affinity for the original nSpCas9-H840A-pegRNA complex due to\nsequence mismatches within the spacer region, making them more resistant to\nre-targeting and potentially further delaying re-nicking in the presence of AcrIIA5. In\ncontrast, for PAM-silencing edits, the inhibition provided by the PAM mutation itself may\nalready be sufficient to prevent re-nicking. Thus, the additional suppression fromAcrIIA5\ncouldoverlyweakentheinitialnickingactivity,therebyreducingtheoverallediting.\nBased on this model, we hypothesized that the performance of aPE is closely tied to\nthe intrinsic DNA cleavage activity of Cas9 at each tested target site. At loci with high\ncleavage activity, initial nicking is more likely to occur even in the presence of AcrIIA5,\nallowing the PE process to proceed efficiently. In such cases, AcrIIA5 may primarily\nfunction to suppress excessive re-nicking, thereby enhancing editing precision and\nefficiency. Conversely, at sites with low Cas9 activity, AcrIIA5 may overly inhibit nicking,\nultimatelyreducingeditingefficiency.\n26\ng\nPE permits re-nicking aPEmitigates re-nicking\nNTS extended NTS extended\nby RT by RT\nRepair Repair\nRe-nicking Re-nicking\nPE-mediated edit PE-mediated edit\nFig. 5g | Proposed mechanism for AcrIIA5 enhance PE activity. g, In the PE system\n(Left),theeditedstrandremainssusceptibletore-cleavagebyCas9aftertheinitialediting\nevent. In contrast, in the aPE system (Right), co-expression of AcrIIA5 partially inhibits\nCas9 activity, thereby reducing re-cleavage of the edited strand. nSpCas9 -H840A\nnickase (gray), initial nick site (red triangle), extended DNA strand from RT (red line),\nAcrIIA5(orange),RT(green),double-strandedDNA(blacklines)havebeenshown.\nTo examine this hypothesis, we sought to determine whether a correlation exists\nbetween Cas9 cleavage activity and aPE performance. Given that in vitro Cas9 cleavage\nassays may not accurately reflect chromatin accessibility and other cellular factors\ninfluencing cleavage efficiency in vivo, we turned to a cell-based predictive approach. We\nutilized DeepSpCas9, a deep learning model (Kim et al., Sci Adv. 2019; 5: eaax9249.)\nwhich predicts SpCas9 cleavage activity across thousands of genomic loci in HEK293T\ncells. By inputting the sequences of our tested loci into this model, we obtained predicted\ncleavage scores (Supplementary Table 5 in the revised Supplementary Data), where\nhigherscoresindicatestrongerCas9activityincellularcontexts.\nBy correlating the DeepSpCas9 scores with the observed fold change in editing\nefficiency between aPE and PE systems (aPE:PE ratio) (Fig. 5h in the revised Main\ntext), we found a strong association: higher predicted Cas9 activity scores generally\nlinked to greater enhancement by AcrIIA5. Specifically, at target sites with DeepSpCas9\nscores above 50, aPE consistently outperformed PE. In contrast, at sites with scores\nbelow50,aPEoftenshowedreducedefficiencycomparedtoPE.\nTo further validate this observation, we selected an additional set of four loci with\n27\npredicted DeepSpCas9 scores >50 and four with scores <50 (Supplementary Table 5 in\nthe revised Supplementary Data). As anticipated, all four high-score loci exhibited\nenhanced editing efficiency with aPE compared to PE, whereas all four low-score loci\nshowed reduced aPE activity compared to PE (Fig. 5i in the revised Main text). These\nfindingssupporttheconclusionthattheperformance ofaPEisindeedinfluencedbyinitial\nCas9 cleavage activity at the target locus, which is consistent with our model that strong\ninitial nicking allows editing to proceed even under partial inhibition by AcrIIA5, whereas\nweak cleavage may result in the loss of editing due to over-suppression. Furthermore,\nDeepSpCas9 scores can be prospectively used to guide target selection, thereby\nimprovingthepredictabilityandapplicabilityofaPEinpracticalgenomeeditingscenarios.\nTable 5 h\nLocation Protospacer (20 bp) DeepSpCas9 score aPE2:PE2 fold change\nDMD, c.7098+1G>T GACCATTTGACGTTAAGGTA 50.876 8.207829034\nDMD, c.5287C>T GTGAAATGGCTGCAAATCGA 61.152 5.202734458\nNF1, c.4191del TGCTTCATACGGTGAGACAA 60.486 1.589104428\nNF1, c.1059del GTCCATGGTGGTTGATCTTA 14.631 1.580188616\nGJB1, c.415G>A GTGGACCTATGTCATCAGCG 69.157 1.274528366\nNF1, c.5749+332A>G AGGCAACATGGATACTTATG 44.590 1.137492929\nNPC2, c.58G>T GGCGCTCAGCACCGCTGCCC 48.589 0.99511896\nFBN1, c.2638G>A GCTTCCCCACGCAGCACCGA 59.520 0.981571664\nPAH, c.590T>A GCAAGCATGGGTTTTATACA 20.199 0.972163079\nNF1, c.2409+1G>C GTCAACTTTACAGACTTACC 20.398 0.903836572\nABCA4, c.2461T>C AGCAAGGCCTGGGGCTGCAG 48.866 0.621486766\nHEXA, c.1073+1G>A TCCTTCTACATCCAGACGTG 69.793 0.481343653\nHEK2 GAACACAAAGCATAGACTGC 52.554 2.602209322\nHEK3 GGCCCAGACTGAGCACGTGA 63.265 5.276914342\nHEK5 GAATACTAAGCATAGACTCC 55.890 3.873978846\nHEK6 CACACACACTTAGAATCTGT 42.465 2.869341909\ni\nLocation Protospacer (20 bp) DeepSpCas9 score\nTest1, +1 T-to-A GGACAGGCAGCATAGACTGT 71.555\nTest2, +1 T-to-A GAAGACCAAGGATAGACTGC 59.416\nTest3, +1 T-to-A GGCACTGCGGCTGGAGGTGG 54.311\nTest4, +1 T-to-A GTATTCACCTGAAAGTGTGC 50.162\nTest5, +3 C-to-G TCCCAGACATCATTCTTACC 35.832\nTest6, +1 T-to-A CCTCTCTCCGGAAGGGAAAA 35.359\nTest7, +1 T-to-A CTGGACTCTGGAATCCATTC 33.443\nTest8, +1 A-to-C TCACGGTTCGGGGGTATACA 26.699\nFig. 5h,i | Cas9 cleavage activity determines the position-dependent effect of\nAcrIIA5 on PE system. Table 5 (Top left), Summary of protospacer sequences,\nDeepSpCas9-predicted cleavage scores, and fold changes in editing efficiency (aPE:PE)\nacross 16 genomic loci. (Bottom left), Eight additional loci were selected for validation:\nfour with DeepSpCas9 scores >50 and four <50. Protospacer sequences and predicted\nscores are shown. h, Correlation between DeepSpCas9 scores and aPE:PE fold change.\nHigh Cas9 activity (score >50, orange dashed line) is generally associated with stronger\naPEenhancement.i,EditingefficienciesofPEandaPEsystemsattheselectedloci.aPE\noutperformedPEathigh-scoringsites(Test1–4),butwaslessefficientatlow-scoringsites\n(Test5–8). All data came from at least three independent biological replicates. Bars\nrepresentmean ± s.d.\n2. The authors compared the editing efficiency of Acr:PEplasmids at different\nratios and found that a ratio of 0.1 yielded the best efficiency. However, the\nMaterials and Methods section does not specify the exact amount of Acr plasmid\nadded. I suggest that the authors provide a more detailed protocol to facilitate\nreplication by other researchers. For example, in this study, 500 ng of PE plasmid\nand200ngofpegRNAplasmidwereaddedperwell,sodoesa0.1ratioofAcrmean\n50 ng?Aratio of 3 would mean 1500 ng?And is the amount of nick gRNAalso 200\nng?\nResponse: In our experiments, we used a molar ratio to define the amount of AcrIIA5\nplasmid relative to the PE plasmid, rather than a mass-based ratio. Specifically, we used\n500 ng of PE2 expression plasmid (9,753 bp), 200 ng of pegRNA expression plasmid\n(3,200bp),andavariableamountofAcrIIA5expressionplasmid(5,869bp)toachievethe\ndesired molar ratios. At a 0.1 ratio, the corresponding amount of AcrIIA5 expression\nplasmidisapproximately30ng;ataratioof3.0,theamountisapproximately900ng.The\npegRNAplasmid, at 200 ng,provides amolar quantityapproximately 1.2timesthatofthe\nPE2 expression plasmid and is thus in sufficient excess. For PE3-based conditions, the\nnicking gRNA expression plasmid was also used at 200 ng per well. To address this\npotentialconfusion,wehaverevisedthe“Cultureconditionandtransfection”sectioninthe\nMethodssectiontoprovideclarification.\n3. Could the authors provide Western blot data to demonstrate that the increase in\nediting efficiency is not due to changes in PE expression levels? At least, could\ntheyprovideWBdataforthe0.1ratioconditioninFig.1d?\nResponse: We examined the expression levels of PE2 protein in the presence or\nabsence ofAcrIIA5 using Western blot analysis at the HEK2 locus (Supplementary Fig.\n10intherevisedSupplementaryInformation).TheresultsshowedthataPE2exhibited\nexpression levels comparable to those of PE2 across +1 to +6 positions, indicating that\nAcrIIA5 does not interfere with PE2 protein expression. Therefore, the changes in editing\nefficiency observed between PE and aPE systems are not attributable to differences in\nproteinabundance.\nMW:\nkDa\nPE2\n250\n(nCas9-\nlinker-RT)\nPlasmids expressing PE2 or aPE2 (including the pegRNA) were transfected into\nUltraMix(X-Blot),and10 μLoflysatewasmixedwith2.5 μLof5×SDSloadingbuffer.After\nboiling, 10 μLof the mixture was loaded onto a 4–15% gradient SDS-PAGE gel. Proteins\nwereseparatedbyelectrophoresisandtransferredtoaPVDFmembrane.Themembrane\nwas cut at appropriate molecular weights and incubated with primary antibodies:\nanti-Cas9 (Genscript,A01935, clone 4A1, 1:500) and anti-GAPDH (Proteintech, 60004-1,\nclone 1E6D9, 1:1000), respectively. After incubation with HRP-conjugated secondary\nantibodiesandwashing,themembranewastreatedwithchemiluminescentsubstrateand\nimagedusinganAmershamImager600.\n4. Since the PE system is too large to deliver easily, this is currently a major issue\ninthefield.TheauthorsusedthreeAAVstodelivertheaPEsystemtheydeveloped;\nhowever, a three-AAV system is challenging to implement in practical therapeutic\napplications, and the efficiency increase in Fig. 5C is not significant. The authors\nshould discuss the challenges of AAV delivery for the aPE system and potential\nsolutions.\nResponse: In our experiments, only modest enhancement was observed when AcrIIA5\nwas co-delivered with split PE2 via AAV delivery. We hypothesized that the slow\nreconstitutionofthesplitPE2proteinmayhavedelayeditsactivity,whileearlyexpression\nof AcrIIA5 might have interfered with this process. To address this, we delayed the\ndelivery of AcrIIA5 by 12 h. This adjustment led to a slight improvement in editing\nefficiency, reaching 3.73% compared to 2.80% in the PE2-only group. However, the\nenhancement remained limited, suggesting that the requirement for protein reassembly\ncontinues to constrain the performance of the system in AAV contexts. We then\nspeculated that further optimizing the timing ofAcrIIA5 delivery could potentially enhance\neditingefficiency,butitmaynotbeadecisivestrategy.\nTo overcome this limitation, we turned to an alternative delivery method using lipid\nnanoparticles (LNP). Unlike AAVs, LNPs enable direct cytoplasmic delivery of full-length\nmRNAs, eliminating the need for protein splitting and reconstitution. We first in vitro\ntranscribed full-length PE2ΔRNaseH and AcrIIA5 mRNAs and encapsulated them with\npegRNA in LNPs for co-delivery into HEK293T cells (Fig. 4g in the revised Main text).\nOur results demonstrated that LNP-mediated delivery of PE2ΔRNaseH mRNA alone\nresulted in an intended editing efficiency of 7.5% at the HEK3, +1 T-to-Aedit (Fig. 4h in\nthe revised Main text). When co-delivered withAcrIIA5 mRNAat molar ratios of 0.5 and\n0.1 relative to PE2ΔRNaseH, the editing efficiency of the aPE2 system increased to\n11.1% (1.48-fold of PE2ΔRNaseH) and 13.5% (1.80-fold of PE2ΔRNaseH), respectively,\nrepresenting a notable enhancement compared to PE2ΔRNaseH alone. Moreover, the\n30\nfrequencyofundesiredindelswasconsistentlylowerintheaPE2ΔRNaseHgroupsthanin\nthe PE2ΔRNaseH-only group. These results suggest that LNP-based delivery of the\nfull-lengthaPEsystemisbothfeasibleandeffective,enhancingitstherapeuticpotential.\ng h LNP delivery of PE2/aPE2\nmRNA-PE2ΔRNaseH\nmRNA-AcrIIA5\npegRNA LNP\nLNP-PE2 LNP-aPE2\nFig. 4g,h | Evaluation of LNP-mediated delivery of PE2 and aPE2. g, Schematic\nillustration of LNP formulations used for delivering PE components. mRNA encoding\nPE2ΔRNaseH or aPE2ΔRNaseH (co-expressing AcrIIA5) and pegRNA were\nencapsulated into LNPs for cellular delivery. h Editing outcomes in HEK293T cells\nfollowing LNP-mediated delivery of PE2 or aPE2 at varying ratios of AcrIIA5 mRNA\n(0.5-foldand0.1-foldrelativetoPE2mRNA).Thepercentageofintendededitsandindels\nwas measured by amplicon sequencing. All data came from at least three independent\nbiologicalreplicates. Barsrepresent mean ± s.d..\n5. Will the authors submit the plasmids constructed in this study to Addgene to\nfacilitateusebyinterestedresearchers?\nResponse: Yes, we fully intend to deposit the key plasmids constructed in this study to\nAddgene upon acceptance of the manuscript. We are happy to share our tools with\nresearchcommunity.\n6. The annotations in the main text does not correspond to the graph in Figure 1e,\nshouldcorrespondtoFig1d?\nResponse: The citation in the main text was indeed incorrect. The described data\ncorrespond to Figure 1d, not Figure 1e. We have corrected the figure reference in the\nrevisedmanuscript.\n7. ThereisnoF1imentionedintheFig1.\nResponse:Wehavereviseditinthemanuscript.\n8. The study needs more application, such as animal model, to verify the effect of\naPE.\nResponse: We fully agree that validation in animal models would provide stronger\n31\nsupport for the translational potential of the aPE system. However, due to current\ntechnical and resource limitations, we have not yet conducted in vivo experiments. While\ninvivo application remains a goal for future work, we believe our currentresults provide a\nstrongfoundationfortheapplicationpotentialofaPE.\nFirst, we have demonstrated that the activity of aPE is closely associated with the\ninitial cleavage activity of Cas9 at the target locus. Using DeepSpCas9, we showed that\nsites with higher predicted Cas9 activity yield more pronounced improvements of aPE.\nThis enables rational selection of efficient target sites and improves predictability across\ndifferentgenomiccontexts(Fig. 5hintherevisedMaintext).\nSecond, we have observed that aPE consistently enhances editing efficiency across\ndiverse edit types, including base substitutions, insertions, and deletions. These\nenhancements are especially notable at non–PAM-silencing targets, indicating that the\nsystem is broadly effective beyond specific sequence contexts (Fig. 5a–f in the revised\nMaintextandSupplementaryFig. 11intherevisedSupplementaryInformation).\nThird, we have developed an mRNA-based delivery platform for aPE using LNP.\nAlthough this approach has so far been validated only in cultured cells, it may directly\naddresses the delivery challenges of large aPE tool and laysthe groundwork for potential\nadaptationtoinvivosystems(Fig. 4g,hintherevisedMaintext).\nFourth, to assess the generalizability of aPE across different cellular contexts, we\nhave tested it in HeLa and U2OS cells using plasmid transfection. Targeted amplicon\nsequencing at HEK2, +2 G-to-A(Fig. 4e in the revised Main text) and HEK3, +1 T-to-A\n(Fig. 4f in the revised Main text) demonstrated that aPE2 consistently achieved higher\neditingefficiencieswhilemaintainingcomparableorlowerindelscomparedtoPE2inboth\ncell lines. These results indicate that the aPE system retains its enhanced performance\nacross different cellular backgrounds, highlighting its broad applicability and potential for\ndiverse biomedical applications. Together, our results have proven that aPE was highly\neffective in more applications than previously recognized, and we are actively pursuing\nanimal-basedexperimentsinfuturestudies.\ne PE2/aPE2 in HeLa cell f PE2/aPE2 in U2OS cell\nEditing efficiencies of PE2 and aPE2 were measured at two edits (HEK2, +2 G-to-Aand\nHEK3,+1T-to-A)inHeLa cells(e) andU2OScells(f).Cellsweretransfected withPE2or\naPE2 components, and editing outcomes were quantified by targeted amplicon\nsequencing. All data came from at least three independent biological replicates. Bars\nrepresentmean ± s.d.\nIn this manuscript, the authors describe an approach for improving the efficiency of prime\neditinginHEK293Tcellsbyco-expressingaphage-derivedanti-CRISPRprotein,AcrIIA5.\nIn what likely began as an exploration of applying anti-CRISPR proteins to inhibit\nCRISPR-based gene editing tools, the authors came upon the surprising result that\nAcrIIA5, an anti-CRISPR protein with established Cas9 inhibitory activity,\ncounterintuitively improved the editing efficiency of prime editing while also decreasing\nundesired indel byproducts. The effect appeared specific to prime editing andAcrIIA5, as\nAcrIIA5 inhibited the activity of Cas9 nuclease and base editors, and other anti-CRISPR\nproteins inhibited prime editing, as expected. The authors optimized the ratio of prime\nediting and AcrIIA5 components delivered to cells targeting a sentinel site, then\ncharacterized the system co-expressing AcrIIA5 with prime editing components (named\naPE) on a variety of prime edit types and positions at different genomic loci. The authors\nfound that the efficiency of single base substitution edits at the +1 through +4 positions\nimproved with aPE (that is, substitutions within 4 bp of the PE-induced nick), but that\nsubstitution edits at the +5 and +6 position (which are notably the critical G bases of the\nPAM) decreased in efficiency as evaluated across 4 genomic target sites in HEK293T\ncells. The authors went on to evaluate aPE with several prime editing formats, prime\neditorprotein variants andedittypes, includingPE3 (which incorporatesa second nicking\nguide RNA), PE4 (which incorporates an MLH1dn protein for MMR inhibition), PE5\n(nicking plus MLH1dn), PE6 variants, SpRY Cas9 variants, insertion edits and deletion\nedits. Modest improvements to editing efficiency were observed across these edits,\nexceptfordeletionsover3-bp.TheauthorsthenappliedaPEtoinstallpathogenicvariants\nin HEK293T cells with modest improvement in efficiency observed over standard PE.\nDelivery of the aPE system withAAV, however, did not lead to improved editing efficiency\ncompared to a standard PE system, despite attempts to titrate the amount of theAcrIIA5\ncomponent. Several attempts were made to understand the origins of the AcrIIA5\nimprovements, including evaluating pegRNA features like the RTT and PBS length,\nalthough none of these revealed a feature influencing aPE activity. Lastly, the authors\nproposedamechanisticmodelforhowAcrIIA5mightimproveprimeeditingefficiency.\nMajor comments\nThe authors cite prior publications proposing that AcrIIA5 (used in aPE) inhibits Cas9’s\nRuvCnucleasedomain,whichprimeeditingrelianceupontonickthenon-targetDNAstrand\n34\nto generate a primer for initiating reverse transcription. Therefore, it is unexpected that\nAcrIIA5 would improve prime editing efficiency instead of inhibiting prime editing,\nparticularly when AcrIIA5 inhibits Cas9 nuclease and base editing activity. The\nobservation thatAcrIIA5 can improve prime editing is therefore an interesting finding, and\npotentiallyausefulone,ifitcouldbeproperlyexploited.\nThe data presented in the manuscript show that the aPE system can improve prime\neditingefficiencyinHEK293Tcellsinasomewhatsite-dependent manner.Inmostcases,\naPE provided modest benefits in efficiency and product purity.Acritical finding is that the\naPEsystemisdetrimentalwheneditsaremadetotheessentialGbasesofthePAMinthe\n+5 and +6 positions (by either substitution or deletion). It has previously been\ndemonstrated that PAM edits improve prime editing efficiency and decrease undesired\nindel byproducts, presumably because they prevent re-nicking of the prime-edited strand\nwithin the heteroduplex intermediate, which would promote reversion of the intended edit\nbacktotheoriginalsequenceorindelformationinthecontextofPE3.Moreover,thereisa\nclearAcrIIA5dose-dependenceoftheeffect,suggestingitisdeleterioustohavetoomuch\nAcrIIA5 and that tuning AcrIIA5 activity is important to achieve the desired outcome. It\ntherefore seems that the simplest explanation for how AcrIIA5 affects prime editing (in a\nway that is consistent with its known activities) is that it decreases the effective Cas9\nnickingactivityenoughsothatre-targetingoftheeditedstrandisslowed,butnotsomuch\nthat the initial necessary nicking activity is too weak. If PAM edits are made that prevent\nre-nickingontheirown,thenthereisnobenefitfromAcrIIA5,whichcanthenonlyweaken\nthe initial nicking activity and decrease the overall editing efficiency. This also potentially\nexplains the site-to-site variability that is observed, as different spacers likely have\ndifferentintrinsicnickaseproperties.\nWith the above in mind, the following additional data and revisions would substantially\nstrengthenthemanuscript:\n1. To distinguish whether the observed decrease in aPE for +5 and +6 position\nedits is due to the distal nature of the edit or the edit being part of the PAM, the\nauthors should evaluate more broadly the impact of AcrIIA5 co-expression on\nprime edits downstream of the PAM (position +7 and beyond), and proximal edits\ncoupled with PAM-silencing edits (e.g., +1 and +5). There are existing pegRNAs\nreported in the literature that make edits at these positions which could be readily\ntested.\nResponse: We sincerely appreciate your insightful comments, which have provided us\n35\nwithvaluableguidanceandinspiration.Asyousuggested,wefirstlyevaluatetheimpactof\nAcrIIA5 co-expression on PE2 downstream of the PAM. Specifically, we investigated the\nPE2/aPE2 editing outcome from +7 to +14, at the HEK2 locus (Fig. 5a,b in the revised\nMaintext).Interestingly,theeditingefficiencyofaPE2wasconsistentlyhigherthanthatof\nPE2acrossallpositionsinthisregion.\na b\nHEK2,+7 to +14, base conversion HEK2, +7 to +14, indels\nFig. 5a,b | Editing efficiencies and indels of base conversions installed by PE or\naPE2. a, Base conversion efficiencies at +7 to +14 positions at HEK2 locus. b, Indels\nformation at +7 to +14 positions at HEK2 locus. All data came from at least three\nindependentbiologicalreplicates.Barsrepresentmean ± s.d.\nBesides, we also examined aPE2-mediated insertions and deletions at positions\ndownstream of the PAM sequence. The results showed that aPE2 consistently achieved\nhigher activity than PE2 for these edits as well (Supplementary Fig. 11 in the revised\nSupplementaryInformation).\na -3 -1+1 +3 +5 +7 +9 +11+13+15 b HEK2, deletions c HEK2, indels for deletions\nHEK2 GACTGCGGGGCGGGCCAG\n+8 del GACTGCGGGG-GGGCCAG\n+9 del GACTGCGGGGC-GGCCAG\n+10 del GACTGCGGGGCG-GCCAG\n+11 del GACTGCGGGGCGG-CCAG\n+12 del GACTGCGGGGCGGG-CAG\n+13 del GACTGCGGGGCGGGC-AG\n+14 del GACTGCGGGGCGGGCC-G\nd e f\n-3 -1+1 +3 +5 +7 +9 +11+13+15 HEK2, insertions HEK2, indels for insertions\nHEK2 GACTGCGGGGCGGGCCAG\n+8 ins A GACTGCGGGGACGGGCCAG\n+9 ins A GACTGCGGGGCAGGGCCAG\n+10 ins A GACTGCGGGGCGAGGCCAG\n+11 ins A GACTGCGGGGCGGAGCCAG\n+12 ins A GACTGCGGGGCGGGACCAG\n+13 ins A GACTGCGGGGCGGGCACAG\n+14 ins A GACTGCGGGGCGGGCCAAG\nSupplementary Fig. 11 | Evaluation of PE2 and aPE2 editing activity on designed\ndeletionsandinsertionsattheHEK2locus.a,ReferencesequenceoftheHEK2target\nsite and representative single-nucleotide deletions ranging from +8 to +14 positions. b,\nmeasured at the same deletion-targeting conditions. d, Reference sequence of the HEK2\ntarget site and representative single-nucleotide insertions (red) ranging from +8 to +14\npositions. e, Editing efficiencies for the designed insertions induced by PE2 and aPE2. f,\nIndels measured at the same insertion-targeting conditions. Positions are numbered\nrelative to the SpCas9 nick site. Data are shown as mean ±s.d. from three biological\nreplicates.\nSubsequently, we examined the combined effects of PAM-proximal and\nPAM-silencing edits. When only PAM-proximal positions (+1 to +4) were edited, aPE2\nshowed higher efficiency than PE2 (Fig. 5c in the revised Main text). In contrast, at the\nPAM-silencing position (+5), the editing efficiency of aPE2 was lower than that of PE2.\nNotably, when PAM-proximal edits were introduced together with a PAM-silencing\nmutation, the editing efficiency of aPE2 was significantly reduced compared to PE2 (Fig.\n5d in the revised Main text). These findings suggest that PAM-silencing edits is a\nkeyfactorinfluencing theeditingoutcome oftheaPEsystem.\nc d\nHEK2, single base conversions HEK2, double base conversions\nFig. 5c,d | Editing efficiencies of single and double base conversions installed by\nPEoraPE2.c,Singlebaseconversionefficienciesat+1to+5positionsatHEK2locus.d,\nThe conditional base conversions at +1, +2, +3, and +4 positions in the context of a\nsimultaneous +5 G-to-A conversion at the same locus. All data came from at least three\nindependentbiologicalreplicates.Barsrepresentmean ± s.d..\n2. The PE4 and PE5 systems used in this manuscript do not appear to represent\nthe state-of-the-art systems, which co-express MLH1dn proteins separately from\nthe PE protein. The fusion proteins used in this manuscript do not appear to\nimprove prime editing efficiency as would be expected for PE4 and PE5 systems.\nAstheinterplay ofre-nicking andMMRseemcritical tounderstanding the potential\nutility of the aPE system, the experiments with PE4 and PE5 should be repeated so\nthat the effects of MLH1dn are consistent with prior publications by separately\n37\nexpressingMLH1dn.\nResponse: Indeed, our results indicate that AcrIIA5 most significantly enhances the\nactivity of PE2, whereas its effect is reduced or even reversed in more advanced PE\nsystems such as PE4, PE5. We guess this phenomenon may stem from structural and\nexpression-relatedconstraintsinherenttothesemorecomplexarchitectures.\nIn PE4 and PE5, the MMR inhibitor MLH1dn is co-expressed via a P2Aself-cleaving\npeptide. However, the cleavage efficiency of the P2A sequence has been reported to be\nincomplete in HEK293T cells (Kim et al. PLoS One. 2011;6:e18556). This incomplete\ncleavage may lead to a subset of Cas9-RT fusion proteins retaining a covalently linked\nMLH1dn domain. Given that MLH1dn is a relatively large protein (752 aa, ~30 kDa), this\nresidual fusion could introduce steric hindrance that interferes with AcrIIA5’s interaction\nwith Cas9. Such interference may impair AcrIIA5’s ability to fine-tune Cas9-DNA\nbinding and dissociation kinetics, thereby diminishing its enhancing effect in PE4\nandPE5.\nToinvestigatewhetherthegeneticlinkagebetweenthePE2expressioncassette and\nMLH1dn via a P2Asequence affects the regulatory impact ofAcrIIA5, we constructed an\nadditional system in which MLH1dn is expressed from a separate plasmid. For clarity, we\nrefer to the original PE4 system as PE2+iMLH1dn (integrated MLH1dn) and the system\nwith independent MLH1dn expression as PE2+sMLH1dn (separate MLH1dn). We then\nevaluated editing efficiencies across the PE2–PE5 architectures under these modified\nconditions(SupplementaryFig.3dintherevisedSupplementaryInformation).\nd\nHEK2, +3 C-to-A\ne\nSupplementaryFig.3d,e|Co-expressionofMLH1dnmodulatestheeffectofAcrIIA5\n38\non PE efficiency. d, Editing efficienciesfor the HEK2,+3 C-to-Ausing PE/aPE system, in\ncombination with MLH1dn delivered via P2Alinkage (iMLH1dn), oras a separate plasmid\n(sMLH1dn).Tomaintain consistent totalplasmidamountsacrossallexperimental groups,\npcDNA3.1(-) was used to equalize any differences in DNA dosage. e, Comprehensive\nediting efficiency comparison of PE2, PE3, PE4 and PE5 with or withoutAcrIIA5.All data\ncame from at least three independent biological replicates. Bars represent mean ± s.d.. P\nvalueswerecalculatedusingthetwo-tailedStudent’st-test.\nConsistent with our earlier findings, aPE2 exhibited higher editing efficiency than\naPE2+iMLH1dn, andsimilarly,aPE3outperformed aPE3+iMLH1dn (Supplementary Fig.\n3e in the revised Supplementary Information). Strikingly, both aPE2+sMLH1dn and\naPE3+sMLH1dn showed even greater editing efficiencies than their respective aPE2 and\naPE3 counterparts. This result further supports our hypothesis that incomplete P2A\ncleavage in PE4 can lead to residual MLH1dn interfering withAcrIIA5-Cas9 interactions,\nthereby reducing its regulatory effect. Moreover, the improved performance of aPE\nsystems when MLH1dn is delivered in trans highlights the modularity and extensibility of\ntheaPEplatform.\n3. The data presented in this manuscript appear to be entirely from HEK293T cells.\nThe efficacy of aPE should be evaluated in several other mammalian cell types to\ncharacterizetheeffectsinthose contexts.\nResponse: To explore whether the improved performance of the aPE system extends to\nother cellular contexts, we tested it in additional human cell lines, specifically HeLa and\nU2OS, by transfecting them with either PE2 or aPE2 systems. Targeted amplicon\nsequencing at HEK2, +2 G-to-A(Fig. 4e in the revised Main text) and HEK3, +1 T-to-A\n(Fig. 4f in the revised Main text) demonstrated that aPE2 consistently achieved higher\nintended editing efficiencies while maintaining comparable or lower indel frequencies\ncompared to PE2 in both cell lines. These results indicate that the aPE system retains its\nenhanced performance across different cellular backgrounds, highlighting its broad\napplicabilityandpotentialfordiversebiomedicalapplications.\ne PE2/aPE2 in HeLa cell f PE2/aPE2 in U2OS cell\nEditing efficiencies of PE2 and aPE2 were measured at two edits (HEK2, +2 G-to-Aand\nHEK3,+1T-to-A)inHeLa cells(e) andU2OScells(f).Cellsweretransfected withPE2or\naPE2 components, and editing outcomes were quantified by targeted amplicon\nsequencing. All data came from at least three independent biological replicates. Bars\nrepresentmean ± s.d.\n4. Much of the data presented in the main figures are not essential to supporting\nthe main conclusions of the manuscript (e.g., compatibility with different prime\neditorproteinsinfigure2,theAAVexperimentinfigure5,pegRNAfeaturesinfigure\n6).Thesewouldbemoreappropriateassupplementary figures.\nResponse:Asyousuggested,inordertoimprovethefocusandclarityofthemainfigures,\nwehavemoved thedataoncompatibilitywithdifferentPEproteins(Fig. 2intheoriginally\nsubmitted manuscript), the AAV delivery experiment (Fig. 5 in the originally submitted\nmanuscript), and pegRNAfeature analysis (Fig. 6 in the originally submitted manuscript)\ntotheSupplementaryInformationofrevisedmanuscript.\n5. Themechanisticmodelappearssomewhatspeculativeandwouldrequirefurther\nexperimentaldatatosupportinclusion inthismanuscript.\nResponse: We sincerely appreciate your advice. Our previous experiments, including\nedits downstream of the PAM (Fig. 5a,b in the revised Main text and Supplementary\nFig. 11 in the revised Supplementary Information) and combinations of PAM-silencing\nand non-PAM modifications (Fig. 5c,d in the revised Main text), revealed that\ndisruptionofthePAMsequence isakeydeterminantofaPEactivity.\nTo further test this guess, we utilized SpG, an engineered variant of SpCas9 that\nrecognizes a relaxed N G N PAM, to construct an SpG-based PE2 system with\n+4 +5 +6\nexpanded targetable regions. We then evaluated the editing outcomes of both\nnSpCas9-PE2 (mainly N G G PAM and slightly N G A PAM) and nSpG-PE2 at the\n+4 +5 +6 +4 +5 +6\n+5 and +6 positions. Consistent with our previous findings, the editing efficiency of\nnSpCas9-aPE2 at both +5 and +6 positions was lower than that of nSpCas9-PE2.\nHowever,forthenSpG-basedsystem,aPE2exhibitedlowereditingefficiencythanPE2at\nthe +5 position (Fig. 5e in the revisedMain text), but higher efficiency at the +6 position\n(Fig.5fintherevisedMaintext).ThiscontrastingoutcomeobservedinthenSpG-based\nsystem is consistent with its recognition of the N +4 G +5 N +6 PAM. These results further\nsupport the notion that PAM-silencing edits play a key role in modulating the\nactivityoftheaPEsystem.\n40\ne f\nHEK2, +5 base conversions HEK2, +6 base conversions\nFig.5e,f|ComparisonofSpCas9-PE2andSpG-PE2 withorwithoutAcrIIA5.e,Base\nconversion efficiencies of+5 position at HEK2 locususing SpCas9-PE2(N G G PAM)\n+4 +5 +6\nor SpG-PE2 (N +4 G +5 N +6 PAM). f, Base conversion efficiencies of +6 position at HEK2\nlocus.All data came from at least three independent biological replicates. Bars represent\nmean ± s.d.\nNow that we have obtained notable results, we attempted to establish a model to\nexplain the mechanism of the aPE system. Previous studies have demonstrated that\nPAM-silencing edits can enhance PE activity and reduce undesired indel byproducts,\npresumably by preventing re-recognition and re-nicking of the edited strand. (Anzalone et\nal.Nature2019;576:149-157).Suchre-nickingcanleadtoreversionofthedesirededitor\npromote indel formation, especially in the context of PE3 systems. Inspired by these\nfindings, we speculated that the activity of aPE might also be influenced by a similar\npreventionofre-nicking mechanism(Fig.5gintherevisedMaintext).\ng\nPE permits re-nicking aPEmitigates re-nicking\nNTS extended NTS extended\nby RT by RT\nRepair Repair\nRe-nicking Re-nicking\nPE-mediated edit PE-mediated edit\nFig. 5g | Proposed mechanism for AcrIIA5 enhance PE activity. g, In the PE system\n(Left),theeditedstrandremainssusceptibletore-cleavagebyCas9aftertheinitialediting\nevent. In contrast, in the aPE system (Right), co-expression of AcrIIA5 partially inhibits\nCas9 activity, thereby reducing re-cleavage of the edited strand. nSpCas9 -H840A\nnickase (gray), initial nick site (red triangle), extended DNA strand from RT (red line),\nAcrIIA5(orange),RT(green),double-strandedDNA(blacklines)havebeenshown.\nnicking activity just enough to slow re-targeting of the edited strand, without completely\nabolishing the initial nicking required for initiating the PE process. Moreover, once the\nintendedmutationsareinstalled,theeditedtargetsmayexhibitreducedbindingaffinityfor\nthe original nSpCas9-H840A-pegRNA complex due to sequence mismatches within the\nspacer region, making them more resistant to re-targeting and potentially further delaying\nre-nicking in the presence of AcrIIA5. In contrast, for PAM-silencing edits, the inhibition\nprovided by the PAM mutation itself may already be sufficient to prevent re-nicking.Thus,\nthe additional suppression from AcrIIA5 could overly weaken the initial nicking activity,\ntherebyreducingtheoverallefficiency.\nBased on this model, we hypothesized that the performance of aPE is closely tied to\nthe intrinsic DNA cleavage activity of Cas9 at each tested target site. At loci with high\ncleavage activity, initial nicking is more likely to occur even in the presence of AcrIIA5,\nallowing the PE process to proceed efficiently. In such cases, AcrIIA5 may primarily\nfunction to suppress excessive re-nicking, thereby enhancing editing precision and\nefficiency. Conversely, at sites with low Cas9 activity, AcrIIA5 may overly inhibit nicking,\nultimatelyreducingeditingefficiency.\nTo examine this hypothesis, we sought to determine whether a correlation exists\nbetween Cas9 cleavage activity and aPE performance. Given that in vitro Cas9 cleavage\nassays may not accurately reflect chromatin accessibility and other cellular factors\ninfluencing cleavage efficiency in vivo, we turned to a cell-based predictive approach. We\nutilized DeepSpCas9, a deep learning model (Kim et al., Sci Adv. 2019; 5: eaax9249)\nwhich predicts SpCas9 cleavage activity across thousands of genomic loci in HEK293T\ncells. By inputting the sequences of our tested loci into this model, we obtained predicted\ncleavage scores (Supplementary Table 5 in the revised Supplementary Data), where\nhigherscoresindicatestrongerCas9activityincellularcontexts.\nBy correlating the DeepSpCas9 scores with the observed fold change in editing\nefficiency between aPE and PE systems (aPE:PE ratio), we found a strong association:\nhigher predicted Cas9 activity scores generally linked to greater enhancement byAcrIIA5\n(Fig. 5h in the revised Main text). Specifically, at target sites with DeepSpCas9 scores\nabove 50, aPE consistently outperformed PE. In contrast, at sites with scores below 50,\naPEoftenshowedreduced efficiencycomparedtoPE.\nTo further validate this observation, we selected an additional set of four loci with\npredicted DeepSpCas9 scores >50 and four with scores <50 (Supplementary Table 5 in\n42\nthe revised Supplementary Data). As anticipated, all four high-score loci exhibited\nenhanced editing efficiency with aPE compared to PE, whereas all four low-score loci\nshowed reduced aPE activity compared to PE (Fig. 5i in the revised Main text). These\nfindings support the conclusion that the performance of aPE is indeed influenced\nby initial Cas9 cleavage activity at the target locus, which is consistent with our\nmodel that strong initial nicking allows editing to proceed even under partial\ninhibition by AcrIIA5, whereas weak cleavage may result in the loss of editing due\nto over-suppression. Furthermore, DeepSpCas9 scores can be prospectively used to\nguide target selection, thereby improving the predictability and applicability of aPE in\npracticalgenomeeditingscenarios.\nTable 5 h\nLocation Protospacer (20 bp) DeepSpCas9 score aPE2:PE2 fold change\nDMD, c.7098+1G>T GACCATTTGACGTTAAGGTA 50.876 8.207829034\nDMD, c.5287C>T GTGAAATGGCTGCAAATCGA 61.152 5.202734458\nNF1, c.4191del TGCTTCATACGGTGAGACAA 60.486 1.589104428\nNF1, c.1059del GTCCATGGTGGTTGATCTTA 14.631 1.580188616\nGJB1, c.415G>A GTGGACCTATGTCATCAGCG 69.157 1.274528366\nNF1, c.5749+332A>G AGGCAACATGGATACTTATG 44.590 1.137492929\nNPC2, c.58G>T GGCGCTCAGCACCGCTGCCC 48.589 0.99511896\nFBN1, c.2638G>A GCTTCCCCACGCAGCACCGA 59.520 0.981571664\nPAH, c.590T>A GCAAGCATGGGTTTTATACA 20.199 0.972163079\nNF1, c.2409+1G>C GTCAACTTTACAGACTTACC 20.398 0.903836572\nABCA4, c.2461T>C AGCAAGGCCTGGGGCTGCAG 48.866 0.621486766\nHEXA, c.1073+1G>A TCCTTCTACATCCAGACGTG 69.793 0.481343653\nHEK2 GAACACAAAGCATAGACTGC 52.554 2.602209322\nHEK3 GGCCCAGACTGAGCACGTGA 63.265 5.276914342\nHEK5 GAATACTAAGCATAGACTCC 55.890 3.873978846\nHEK6 CACACACACTTAGAATCTGT 42.465 2.869341909\ni\nLocation Protospacer (20 bp) DeepSpCas9 score\nTest1, +1 T-to-A GGACAGGCAGCATAGACTGT 71.555\nTest2, +1 T-to-A GAAGACCAAGGATAGACTGC 59.416\nTest3, +1 T-to-A GGCACTGCGGCTGGAGGTGG 54.311\nTest4, +1 T-to-A GTATTCACCTGAAAGTGTGC 50.162\nTest5, +3 C-to-G TCCCAGACATCATTCTTACC 35.832\nTest6, +1 T-to-A CCTCTCTCCGGAAGGGAAAA 35.359\nTest7, +1 T-to-A CTGGACTCTGGAATCCATTC 33.443\nTest8, +1 A-to-C TCACGGTTCGGGGGTATACA 26.699\nFig. 5h,i | Cas9 cleavage activity determines the position-dependent effect of\nAcrIIA5 on PE system. Table 5 (Top left), Summary of protospacer sequences,\nDeepSpCas9-predicted cleavage scores, and fold changes in editing efficiency (aPE:PE)\nacross 16 genomic loci. (Bottom left), Eight additional loci were selected for validation:\nfour with DeepSpCas9 scores >50 and four <50. Protospacer sequences and predicted\nscores are shown. h, Correlation between DeepSpCas9 scores and aPE:PE fold change.\nHigh Cas9 activity (score >50, orange dashed line) is generally associated with stronger\naPEenhancement.i,EditingefficienciesofPEandaPEsystemsattheselectedloci.aPE\noutperformedPEathigh-scoringsites(Test1–4),butwaslessefficientatlow-scoringsites\n(Test5–8). All data came from at least three independent biological replicates. Bars\nrepresentmean ± s.d.\nMinor comments\n1. Most figures do not indicate the cell type used in the experiment. This\ninformationshouldbeaddedeitherdirectlytothefiguresortothelegend.\nResponse:Wehaveaddedtheinformationtothefigurelegendsassuggested.\n43\n2. Itappearsthesamedataarepresentedinfigure1fandsupplementaryfigure2.\nResponse:WehaveremovedSupplementaryFigure2toavoidduplication.\n3. In the description of quantitative results, relative editing or indel formation\nefficiency would be better represented as fold-changes as opposed to % changes,\nsince % change could be interpreted as differences in %. For example, “AcrIIA5\nreduced indels by 78.0% (aPE2) and 81.9% (aPE3) for PE2 and PE3, respectively”\ncouldbechangedto4.5-foldand5.5-fold.\nResponse: We have revised the description of relative editing and indel efficiencies\nthroughoutthemanuscript toreportfold-changesinsteadofpercentagereductions.\n4. Abbreviations,likeRHA,needtobedefinedinthetextwhenfirstused.\nResponse: We have defined RHAand all other abbreviations upon their first mention in\nthetext,ensuringthatalltermsareclearlyexplained.\n5. When using the terminology AAV1, AAV2, and AAV3, there is confusion with\nserotype, so the naming of these constructs should be changed to avoid this\npotentialconfusion.\nResponse: To avoid confusion with AAV serotypes, we have revised the naming of our\nconstructs previously referred to as AAV1, AAV2, and AAV3. They are now renamed as\nAAV_PE_1, AAV_PE_2 and AAV_AcrIIA5, respectively. These changes have been\nappliedconsistentlythroughouttherevisedmanuscriptandSupplementaryInformation.\n6. Figures 5b and 5c are challenging to interpret, and the colored shading with\ninversion of the direction of the bars is not helpful. Recommend reformatting to a\nstandard layout without shading red or green and using clear headers for the\ngraphsconveyingthatthetimepointsindicatewhentheAcrIIA5AAVwasadded.\nResponse:Wehavereformattedthegraphsasyousuggested(SupplementaryFig.7in\ntherevisedSupplementaryInformation).\na b AAV was added at 0 h, HEK3, +1 T-to-A\nN-Npu intein\nAAV_PE_1 ITR EF-1α N-SpCas9-H840A (1024 aa) NLS Te IT rm R inator\n4376 bp\nC-Npu intein linker\nNLS Terminator AAV was added at 12 h, HEK3, +1 T-to-A\nAAV_PE_2 ITR U6epegRNACMV M-MLV RT∆RH ITR\n4833 b2p\nC-SpCas9-H840A (344 aa)\nAAV_AcrIIA5 ITR CMV AcrIIA5 NLS Te I r T m R inator\n1831 bp\nSupplementary Fig. 7 | AAV delivery of aPE. a, Schematic representation of AAV\n44\nvectors containing N-PE2ΔRNaseH (AAV_PE_1), C-PE2ΔRNaseH and epegRNA\n(AAV_PE_2), orAcrIIA5 (AAV_AcrIIA5). b,c, Editing efficiencies and indels forAAV-based\nHEK3, +1 T-to-A edit. All cells were co-delivered with AAV_PE_1+2 which can generate\ncompact PE2ΔRNaseH with epegRNA.TheAAV_AcrIIA5 expressingAcrIIA5 were added\nafter0h(b),or12h(c)atdifferentconcentrations.\nI appreciate that the authors have revisited their proposed mechanism and provided a\nmore plausible explanation for AcrIIA5’s effect on prime editing, supported by new\nexperimental data. The additional mechanistic studies, including the mutational analyses\nand expanded biochemical experiments, offer much greater confidence in the model and\naddress most of my initial concerns regarding empirical support for the hypotheses.\nI also recognize the effort to experimentally address many of the issues raised in my initial\nreview, leading to a more compelling narrative.\n1. That said, I remain concerned about the overall editing efficiencies achieved in\nyour system. The prime editing activities reported here appear substantially lower\ncompared to those described in several recent manuscripts in the field. As such,\nwhile the mechanistic insights are interesting, the low efficiency raises questions\nabout the practical utility of the approach.\nResponse: We sincerely thank you for your thoughtful and constructive comments. We\nacknowledge that partial PE efficiencies observed in our study were somewhat lower than\nthose reported for several recent state-of-the-art PE variants. However, it is important to\nnote that even when using the same cell type like HEK293T, differences in cell state, such\nas cellular vitality, passage number, and culture conditions, as well as variations in\ntransfection methods, can substantially influence the apparent activity of PE systems.\nWe therefore believe that a direct head-to-head comparison of aPE with other advanced PE\nsystems under identical cellular conditions are more reasonable, as this can provide the\nmost reliable assessment of their intrinsic performance. To enable such a clear\ncomparison, we constructed and tested totally seven engineered PE systems that had\nbeen reported prior to August 1, 2025 in HEK293T cells as following.\nAccording to the reported PE protein engineering, we constructed four advanced PE\nvariants with point mutations or additional functional elements (Supplementary Fig. 5a in\nthe revised Supplementary Information):\n(1) PE_Y18, obtained by directed evolution in yeast (Nat Commun. 2024;15(1):2092);\n(2) PEmax**, generated by introducing the functional mutations L435K and V233M\ninto the M-MLV RT of PEmax (Nat Biotechnol. 2025;43(4):539-544);\n(3) PE2_NC, in which a viral nucleocapsid protein (NC) with nucleic acid chaperone\n1\nactivity was fused to PE2 (Nat Biotechnol. 2022;40(9):1394-1402);\n(4) PE2_SB, in which a de novo-designed MLH1 small binder (MLH1-SB) was\nincorporated to inhibit the MMR pathway, thereby functioning in a manner similar to the\nPE4 architecture (Cell 2025; doi:10.1016/j.cell.2025.07.010).\nHEK2, +2 G-to-A HEK2, +12 C-to-A HEK3, +1 T-to-A\nNLS NLS\nCMV nSpCas9-H840A M-MLV RT\nPE2\nNLS NLS\nCMV nSpCas9-H840A M-MLV RT\nPE_Y18\nA259D K445T\nNLS NLS\nCMV nSpCas9-H840A M-MLV RT\nPEmax**\nE221K N394K V223ML435K\nNLS NLS\nCMV nSpCas9-H840A NC M-MLV RT\nPE2_NC\nNLS\nCMV nSpCas9-H840A M-MLV RTMCA\nPE2_SB\nNLS\n2\n0 10 20 30 0 5 10 15 0 5 10 15 20\nHEK2, +2 G-to-A, indels\nPE\n4 aPE\n3\n)%\n( slednI 2\n1\n)%( \nslednI\n3\n2\n1\nHEK2, +12 C-to-A, indels\nPE2 PE_Y18 PEmax** PE2_NC PE2_SB\n)%( \nslednI\n3\n2\n1\na\nEditing efficiencies (%)\nb\nHEK3, +1 T-to-A, indels\n)%( \nsledni\ntuohtiw\ngnitidE\n30\n20\n10\n0\n-4 P=4.4×10\nPE2\n-3 P=2.7×10\nPE_Y18\n-3 P=1.1×10\nPEm ax**\nPE-4\nP=6.3×10\nPE2_NC\naPE-4\nP=3.1×10\nPE2_SB\nPE\naPE\nc d\nPE aPE\n3\n)%\n( slednI 2\n1\ne NLS polyA NLS polyA\nCMV nSpCas9-H840A M-MLV RT CMV nSpCas9-H840A M-MLV RT Orip EBNA1\ncPE2 epiPE2\nf g\nHEK2, +2 G-to-A, HEK2, +12 C-to-A, HEK3, +1 T-to-A,\nHEK2, +2 G-to-A HEK2, +12 C-to-A HEK3, +1 T-to-A indels indels indels\nPE PE PE PE PE PE\n)%10 aPE )%12 aPE )%15 aPE 4 aPE 1.0 aPE 3 aPE\n( sledni\ntuohtiw\ngnitidE\n8\n6\n4\n2\n0\n( sledni\ntuohtiw\ngnitidE\n8\n4\n0\n( sledni\ntuohtiw\ngnitidE\n1\n5\n0\n0 )%\n( slednI\n3\n2\n1\n0\n)% \n( slednI 0\n0\n.\n.\n5\n0\n)% \n( slednI\n2\n1\n0\ncPE2 epiPE2 cPE2 epiPE2 cPE2 epiPE2 cPE2 epiPE2 cPE2 epiPE2 cPE2 epiPE2\nSupplementary Fig. 5 | Evaluation of aPE strategy across multiple advanced PE\nvariants. a, Schematic diagrams of PE2 and four engineered PE variants: PE_Y18,\nPEmax**, PE2_NC, and PE2_SB. Editing efficiencies were evaluated with or without\nAcrIIA5 at three representative edits (HEK2, +2 G-to-A; HEK2, +12 C-to-A; and HEK3, +1\nT-to-A). b, Indels formation at the three edits for PE variants with or without AcrIIA5. c,d,\nComprehensive editing efficiencies (c) and indels (d) comparison of PE variants with or\nwithout AcrIIA5 for edits of (a,b). e, Schematic diagrams of cPE2 and epiPE2. f, g,\nEditing efficiencies (f) and indels formation (g) of cPE2 and epiPE2 with or without\nAcrIIA5, and HEK293T cells were cultured for 6 days after transfection. All data came from\nat least three independent biological replicates. Bars represent mean ± s.d. P values were\ncalculated using the two-tailed Student’s t-test.\nWe then evaluated the editing efficiencies (Supplementary Fig. 5a in the revised\nSupplementary Information) and unintended indels (Supplementary Fig. 5b in the\nrevised Supplementary Information) of these variants with or without AcrIIA5 at three\nedits (HEK2, +2 G-to-A; HEK2, +12 C-to-A; and HEK3, +1 T-to-A). The comprehensive\nanalysis showed that all four reported PE variants exhibited improved activity compared to\nPE2 (Supplementary Fig. 5c in the revised Supplementary Information). Among them,\nPE2_SB showed the highest activity, with an average editing efficiency of 8.7%, which\nwas 1.46-fold higher than that of PE2 (6.0%). By comparison, aPE2 reached 12.6%,\nwhich was 1.44 times that of PE2_SB, highlighting its robust activity. Notably, the addition\nof AcrIIA5 also led to a significant increase in the efficiencies of all the above advanced\nPE systems. Specifically, the editing activities of aPE_Y18, aPEmax**, aPE2_NC, and\naPE2_SB were enhanced to 2.04-fold, 2.08-fold, 2.12-fold, and 2.16-fold, respectively,\ncompared with their corresponding PE systems (Supplementary Fig. 5c in the revised\nSupplementary Information). Meanwhile, the indels caused by these tested PE variants\nwith AcrIIA5 were significantly decreased (Supplementary Fig. 5d in the revised\nSupplementary Information).\nRecently, Han et al. reported the (5) epiPE2 system, which can introduce additional\nmethylation on the edited DNA region and thereby improves PE outcomes (Adv Sci.\n2025;12(17):e2417790). We then compared cPE2 and epiPE2 (a generous gift from the\nJinxue Ruan group) with or without AcrIIA5 (Supplementary Fig. 5e in the revised\nSupplementary Information). The results showed epiPE2 consistently outperformed\ncPE2, and both systems were markedly enhanced by AcrIIA5 at the three tested edits\n(Supplementary Fig. 5f in the revised Supplementary Information). In addition,\nAcrIIA5 substantially reduced unintended indels in both editors at the same time\n(Supplementary Fig. 5g in the revised Supplementary Information).\nIn addition to engineering the PE protein itself, modifications to the pegRNA have also\nbeen shown to improve the performance of PE systems (Supplementary Fig. 6a in the\nrevised Supplementary Information). For examples in reported paper as following:\n(6) Li et al. converted the unstable G:A pair in the pegRNA scaffold into a C:G pair,\ngenerating the apegRNA, which enhanced PE activity (Nat Commun. 2022;13(1):1669);\n(7) Fei et al. developed a mismatched pegRNA (mpegRNA) strategy, in which\ndeliberate mismatches are introduced into the pegRNA protospacer, thereby\nsimultaneously increasing editing efficiency and reducing indels formation (Nat Commun.\n2025;16(1):139).\nU6 Spacer gRNA-Scaffold RTT+PBS\nwtpegRNA polyT\nU6 Spacer gRNA-Scaffold RTT+PBS\napegRNA polyT\nU6 Spacer gRNA-Scaffold RTT+PBS\nmpegRNA polyT\n4\n0 5 1 0 1 5 2 0 2 5 0 5 1 0 1 5 0 5 1 0 1 5 2 0\na\n)%\n( slednI 4\n3\n2\n1\n0\nw\nP E 2\naP E 2\ntpegR N A apegR N A m pegR N A\nHEK2, +2 G-to-A, indels HEK2, +12 C-to-A, indels\n)%\n( slednI 2.0\n1.5\n1.0\n0.5\n0.0\nw\nP E 2\naP E 2\ntpegR N A apegR N A m pegR N A\nHEK3, +1 T-to-A, indels\n)%\n( slednI 6\n4\n2\n0\nw\nP E 2\naP E 2\ntpegR N A apegR N A m pegR N A\nb\n)%\n( sledni\ntuohtiw\ngnitidE\n30\n20\n10\n0\n-4 P=4.4×10\nw tpegR N A\n-4 P=2.6×10\napegR N A\nP E 2\n-4 P=7.0×10\nm pegR N A\naP E 2\n)%\n( slednI 8\n6\n4\n2\n0\n-6 P=6.0×10\nw tpegR N A\n-2 P=4.3×10\napegR N A\nP E 2\n-2 P=3.8×10\nm pegR N A\naP E 2\nPE2\naPE2\nEditing efficiencies (%)\nc d\nSupplementary Fig. 6 | Evaluation of aPE strategy with engineered pegRNAs. a,\nSchematic diagrams of wtpegRNA, apegRNA, and mpegRNA. Editing efficiencies were\nevaluated with PE2 or aPE2 at three representative edits (HEK2, +2 G-to-A; HEK2, +12\nC-to-A; and HEK3, +1 T-to-A) b, Indels formation at the three edits for PE2 and aPE2\ncombined with different pegRNAs. c,d, Comprehensive comparison of editing efficiencies\n(c) and indels formation (d) across pegRNA types in the context of PE2 or aPE2. All data\ncame from at least three independent biological replicates. Bars represent mean ± s.d. P\nvalues were calculated using the two-tailed Student’s t-test.\nWe therefore tested these two reported engineered pegRNAs in combination with\nPE2 and aPE2 (Supplementary Fig. 6a,b in the revised Supplementary Information).\nThe results showed that the editing efficiencies of apegRNA-PE2 and mpegRNA-PE2\nwere improved to 1.16-fold and 1.15-fold, respectively, compared with wtpegRNA-PE2\n(Supplementary Fig. 6c in the revised Supplementary Information). Comparatively,\nwtpegRNA-aPE2 exhibited 1.83-fold and 1.85-fold higher activity than apegRNA-PE2 and\nmpegRNA-PE2, respectively. When combined with aPE2, apegRNA and mpegRNA\ndemonstrated stronger activity, achieving 13.8% and 15.4%, respectively, which were\n1.99-fold and 2.25-fold higher than their corresponding PE2 systems. Importantly,\nmpegRNA-PE2 markedly reduced indels (0.29%) compared with wtpegRNA-PE2 (1.81%)\n(Supplementary Fig. 6d in the revised Supplementary Information). Even under these\nconditions, mpegRNA-aPE2 showed a substantial decrease in indels (0.16%), nearly half\nthat of mpegRNA-PE2, underscoring the critical role of AcrIIA5 in improving editing\nspecificity.\nDuring the analysis of our sequencing data and the preparation of this response,\nChauhan VP et al. reported an advanced PE system carrying nSpCas9 mutations\n(K848A+H982A) that prompt the nicked-end degradation of the competing 5’ strand after\nPE editing, thereby significantly reducing unintended indels formation (Nature 2025,\ndoi:10.1038/s41586-025-09537-3, published online on September 17, 2025). Since\nAcrIIA5 has enhanced the editing activity and reduced the indels across nearly all point\nmutation-based PE variants we previously tested (Supplementary Fig. 4b and\nSupplementary Fig. 5 in the revised Supplementary Information), as well as the\nhighly specific mpegRNA-PE2 system (Supplementary Fig. 6 in the revised\nSupplementary Information), we speculate that AcrIIA5 could also further improve the\nperformance of the newest precise PE system.\nIn conclusion, our data demonstrated that aPE2 outperformed several state-of-the-art\nPE systems in terms of activity and specificity. More importantly, we demonstrated that\nAcrIIA5 can enhance both the efficiency and precision of the advanced PE systems\nincorporating point mutations, additional protein elements, or epigenetic modifications,\nwhile also improving the performance of engineered pegRNA-based systems. Collectively,\nthese findings indicated that the aPE strategy developed here has broad applicability\nacross improved PE architectures and holds considerable translational promise. These\nresults have also been discussed and incorporated on page 12 in the revised manuscript.\n2. Furthermore, I feel the translational potential of the work would be significantly\nstrengthened by demonstrating robust and efficient editing at disease-relevant loci\nin cell types that are more representative of therapeutic targets. For example,\ncomparing PE vs aPE when targeting PCSK9 in hepatocytes using LNP delivery\ncould greatly enhance the therapeutic relevance. In summary, the revised\nmanuscript demonstrates clear progress in mechanistic understanding and\nvalidation of this system but I still question the broader utility of the approach.\nResponse: As suggested, we evaluated the performance of PE and aPE at the\ntherapeutically relevant locus PCSK9 in hepatocyte-derived cell line HepG2, which has\nalso been widely used to assess the base editors-meditaed editing of PCSK9 (Nat\nBiotechnol. 2021;39(8):949-957; Cell Res. 2025, doi:10.1038/s41422-025-01164-x), using\nLNP delivery method. We previously found that aPE was more likely to achieve favorable\nediting outcomes at the target sites with a high DeepSpCas9 socre (usually >50). Based\n5\non this, we screened potential sites within the PCSK9 locus and selected the\nhPCSK9-intron 1 site (TCCACCGCTGCGCCAAGGTG, DeepSpCas9 score=58.575) for\nevaluation (Fig. 4h in the revised Main Text). We then applied LNP-PE\n(mRNA-PE2ΔRNaseH) and LNP-aPE (mRNA-AcrIIA5:mRNA-PE2ΔRNaseH=0.1:1) to\nintroduce a +1 G-to-A substitution, thereby disrupting the canonical GT splice donor site of\nintron 1 to AT. The results showed that LNP-aPE achieved markedly higher editing\nefficiency (24.7%) compared to LNP-PE (8.9%), while maintaining low indels frequencies\n(Fig. 4i in the revised Main Text). Functionally, the precise +1 G-to-A edit introduced by\nLNP-aPE led to a significant reduction in secreted PCSK9 protein levels in the cell\nsupernatant, as measured by ELISA, showing a 32.6% decrease compared with the\ncontrol group and a 21.28% decrease relative to LNP-PE treatment (Fig. 4j in the revised\nMain Text). In parallel, flow cytometry was used to assess cellular uptake of fluorescently\nlabeled LDL particles (Dil-LDL). Compared with the control group, LNP-PE treatment\nincreased Dil-LDL internalization by 8.5%, whereas LNP-aPE enhanced Dil-LDL uptake\nby 18.0%, proving that LNP-aPE-mediated editing of PCSK9 can effectively restore the\ncapacity for LDL metabolism in HepG2 cells (Fig. 4k in the revised Main Text).\n6\n5\n3\n’-\n’-\nC│G\nE\nA│T\nx\nC│G\no\nC│G\nn\nT│A\nG\nh PA C\n│ │\nT G\nT\nC S C G\n│ │\nG C\nIn tro n 1\nK 9 -in tro n C T G C G C C\n│ │ │ │ │ │ │\nG A C G C G G\nA\n1A A\n│ │\nT T\nG\n+ 1 G T G\n│ │ │\nC C A\nG│C\nE x\nP A C G\n│ │\nG C\no\nMG│C\nn\nG│C\n-5\n’\n’\nControl LNP-PE LNP-aPE\n)%\n( egnahc\ndetacidni\nhtiw\nsdaeR\n30\n20\n10\n0.2\n0.1\n0.0\n+1 G -to-A indels\nControl LNP-PE LNP-aPE\nh i j )Lm/gp(\n9KSCPh\ndeterceS\n900\n600\n300\nP=0.0187\nk\nControl LNP-PE LNP-aPE\nekatpu\nLDL-liD\nevitaleR\n1.5\n1.0\n0.5\n0.0\nP=0.0003 P=0.0077\nP=0.0135\nFig. 4h–k | Evaluation of LNP-delivered aPE at the therapeutically relevant PCSK9\nlocus. h, Schematic of the hPCSK9-intron 1 target site, which is boxed in red dashed\nlines. i, Editing efficiencies (+1 G-to-A) and indels frequencies caused by LNP-PE and\nLNP-aPE in HepG2 cells. j, Secreted PCSK9 protein levels measured by ELISA following\nediting. k, Cellular uptake of Dil-LDL was quantified by flow cytometry, and values were\nnormalized to the control group. All data came from at least three independent biological\nreplicates. HepG2 cells were seeded in 24-well plates and cultured for 12 h to reach ~60%\nconfluence. Cells were then transfected with LNP-PE or LNP-aPE. Control group was\ntreated with buffer. After 84 h, the medium was replaced with 1 mL of fresh medium. 24 h\nlater, 200 µL of the supernatant was collected for ELISA (JONLNBIO, JL19731)\nmeasurement of secreted PCSK9. At the same time, cells were incubated with 10 µg/mL\nDil-LDL (Yiyuan Biotechnologies, YB-0011) at 37°C for 4 h. Following incubation, cells\nwere washed twice with PBS, trypsinized, collected into 1.5 mL EP tubes, and washed\nthree additional times with PBS. For flow cytometry, 20,000 gated cells were analyzed\nusing 549 nm excitation and 570 nm emission detection; mean fluorescence intensity\n(MFI) was used to represent Dil-LDL uptake. The remaining cells were lysed with lysis\nbuffer for genomic DNA extraction, PCR amplification, and deep sequencing to determine\nediting outcomes. Bars represent mean ± s.d. P values were determined using one-way\nANOVA followed by Tukey’s multiple comparisons test.\nTogether, these results showed that the LNP-aPE system can efficiently disrupt\nPCSK9 splicing and attenuate its function in HepG2 cells. Although HepG2 cells are not\nfully representative of the in vivo situation, they have been widely used to investigate\ncholesterol metabolism-related pathways and thus provide a meaningful model for\nevaluating PCSK9 function (EBioMedicine 2021;65:103250; Atherosclerosis\n2008;201(2):266-273). Considering the well-established role of PCSK9 in\nhypercholesterolemia (Signal Transduct Target Ther. 2024;9(1):13), these results highlight\nthe ability of aPE to achieve precise and clinically relevant editing at disease-related loci\nand conditions, supporting its translational potential. These results have also been\ndiscussed and incorporated on page 19 in the revised manuscript.\nIn summary, our additional experiments systematically compared aPE2 with multiple\nadvanced PE variants and demonstrated consistent improvements in both activity and\nspecificity across diverse architectures. Moreover, AcrIIA5 markedly enhanced the\nperformance of these systems, underscoring the broad applicability of the aPE strategy.\nImportantly, validation at the therapeutically relevant PCSK9 locus in HepG2 cells using\nLNP delivery further supported its translational potential. Taken together, these findings\nestablish aPE as an efficient and generally applicable platform for potent prime editing.\nResponses to Reviewer #2:\n1. The authors have addressed most of my concerns, while the specific and detailed\nmechanism regarding \"Proposed mechanism for AcrIIA5 to enhance PE activity\" has not\nbeen clearly experimentally explained. It is recommended to mechanism of this models in\nthe revised manuscript.\nResponse: We sincerely thank you for your constructive comment. In our model (Fig. 5g),\nthe conventional PE system activity is hindered by re-nicking of the edited strand caused\nby SpCas9-H840A, whereas in the aPE system, AcrIIA5 partially inhibits Cas9 activity,\nthereby reducing re-nicking of the newly edited strand while still permitting the prime\nediting process, ultimately enhancing the editing efficiency.\n7\n8\ng\nP E p e r m it s r e - n\nN T S e x te n d e d\nb y R T\nR e -n ic k in g\nic k in\nR e p a\nP E -m\ng\nir\ne d ia te d e d it\na P E m it ig a t e s r e\nN T S e x te n d e\nb y R T\nR e -n ic k in g\n- \nd\nn ic k in\nR e p a ir\nP E -m e\ng\nd ia te d e d it\nFig. 5g | Proposed mechanism for AcrIIA5 enhance PE activity. g, In the PE system\n(Left), the edited strand remains susceptible to re-cleavage by Cas9 after the initial editing\nevent. In contrast, in the aPE system (Right), co-expression of AcrIIA5 partially inhibits\nCas9 activity, thereby reducing re-cleavage of the edited strand. nSpCas9-H840A nickase\n(gray), initial nick site (red triangle), extended DNA strand from RT (red line), AcrIIA5\n(orange), RT (green), double-stranded DNA (black lines) have been shown.\nAs reported by Anzalone et al. (Nature 2019; 576: 149–157), re-binding and\nre-nicking of the edited strand could compromise PE outcomes, thus highlighting the\nimportance of determining the extent to which AcrIIA5 suppresses the nicking\nactivity of SpCas9-H840A. To this point, we performed in vitro cleavage assays to\nevaluate whether AcrIIA5 could inhibit Cas9 and its nickase variants, and we found that\nthese results can experimentally provide strong evidence supporting our model\n(Supplementary Fig. 12 in the revised Supplementary Information).\na b\nT7 His6 SpCas9 T7 terminator\npet28a-SpCas9\nT7 His6 SpCas9-H840A T7 terminator\n160.5 kDa\npet28a-SpCas9-H840A\nT7 His6 SpCas9-D10A T7 terminator\npet28a-SpCas9-D10A\nTEV site\nT7 His6 MBP AcrIIA5 16.9 kDa\npet28a-MBP-AcrIIA5 T7 terminator\nc\nSpCas9 SpCas9-H840A SpCas9-D10A\nAcrIIA5 AcrIIA5 AcrIIA5\nM L1 L2 L3 0 0.1 1.0 10 0 0.1 1.0 10 0 0.1 1.0 10\nOC or\nLIN\nSC\nSupplementary Fig. 12 | Construction, purification, and in vitro DNA cleavage assay\nof SpCas9 variants in the presence of AcrIIA5. a, Schematic representation of\nexpression constructs used for producing SpCas9, SpCas9-H840A, SpCas9-D10A, and\nAcrIIA5 proteins. b, SDS–PAGE analysis of purified proteins, showing expected molecular\nweights of ~160.5 kDa for SpCas9 variants and ~16.9 kDa for AcrIIA5. c, Plasmid\ncleavage assays were performed using SpCas9, SpCas9-H840A (RuvC active), and\nSpCas9-D10A (HNH active) in the presence of increasing concentrations of AcrIIA5. SC,\nLIN, and OC forms of pUC57 plasmid were separated on a 1.5% agarose gel and are\nshown. Lane 1 (L1) represent the input pUC57. The controls of LIN (L2) and OC (L3)\nplasmid were generated by catalyzed pUC57 with EcoRI and Nt.BspQI, respectively. Lane\nM contains a 1 kb DNA ladder. AcrIIA5 was added at molar ratios of 0, 0.1, 1.0, and 10\nrelative to SpCas9 or its variants.\nBased on our obtained experimental results, upon titration of AcrIIA5, we observed a\nprogressive reduction in cleavage/nicking activity for SpCas9-H840A, which supports that\nAcrIIA5 can indeed inhibit SpCas9-H840A activity. We also found that this inhibition is\nincomplete: even at a 10:1 molar ratio of AcrIIA5 to SpCas9-H840A, the nicked OC\nplasmid still remained. This finding is precisely consistent with our model, in which such\n“soft” inhibition is sufficient to prevent extensive re-nicking of the newly edited NTS,\nwhile still permitting residual nicking of unedited DNA strands to enable prime editing.\nIn conclusion, the results of the in vitro assays provide a more detailed mechanistic\nexplanation of how AcrIIA5 enhances prime editing outcomes by mitigating undesired\nre-nicking without abolishing the initial nicking event required for prime editing. As\nrecommended, we have added these descriptions to experimentally explain our model in\nthe second paragraph of page 27 in the revised manuscript.\n9"}]

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