A combinatorial synthetic strategy for developing genome-editing protein-delivery agents targeting mouse retina

Domain: neuroscience
Published: 2026-02-07
Editorial outcome: minor_revision

Abstract
CRISPR/Cas9-based gene-editing technologies offer promise for treating inherited retinal diseases (IRDs), however safe and efficient ocular delivery of precision editors remains challenging. To address this challenge, we report a class of Coomassie brilliant blue (CBB)-derived lipidoids that bind and deliver proteins. Subretinal injection of Cre complexed with these lipidoids into mT/mG mice leads to robust recombination in the retinal pigment epithelium and photoreceptors. We employ the CBB-lipidoid platform to deliver adenine base editor (ABE) ribonucleoproteins (RNP). Incorporating CBB lipidoids into liposomes improves delivery efficiency. CBB11 stands out for facilitating precise in vivo ABE-mediated gene editing. Delivery of liposome-CBB11-RNP complexes results in a 120-fold increase in base editing compared to RNP alone and restores the scotopic ERG b-wave response in the rd12 mouse model. These results demonstrate the potential of CBB-augmented, liposome-RNP systems for therapeutic gene editing in the eye, paving the way for single-dose precision medicines to treat IRDs.

Introduction
The future of medicine is increasingly being shaped by advances in genome editing, which promises to revolutionize the treatment of genetic diseases, cancers, and various other conditions. Building on the initial discoveries of the CRISPR-Cas9 system,1 the transformative technologies of base editing2,3 and prime editing4 offers solutions for nearly all disease-causing mutations, in addition to opening up new avenues for enhancing genetic traits and studying complex biological systems. However, realizing the therapeutic potential of genome editing requires safe, efficient delivery of these gene editors to specific cells and tissues.

Currently, three primary modalities are utilized for delivering editors to target cells in vivo: viral vectors, non-viral mRNA formulations, and direct protein-delivery systems. Viral vectors, such as adeno-associated viruses (AAVs), are widely used due to their versatility and high efficiency in delivering genetic material into cells; however, they carry risks of immunogenicity and potential genomic integration5. Furthermore, these vectors can only package approximately 5 kb of genetic material, which is insufficient to accommodate large transgenes within a single AAV genome, but splitting the cargo into multiple AAVs limit the transduction efficiency6–8. Direct delivery of mRNA via non-viral vectors, including lipid nanoparticles (LNPs) and polymer-based systems, offers a safer alternative by avoiding many of the limitations of AAVs. There is currently rapid development of mRNA-delivery approaches to enable delivery to a variety of tissues,9–11 but efficient genome editing in this context has remained challenging in tissues other than liver12,13. Direct delivery into cells of pre-assembled editor proteins and guide-RNAs as ribonucleoprotein (RNP) complexes represents an emerging approach that conceptually provides transient and controlled activity, reducing the risk of off-target effects and immunogenicity14–16. Furthermore, protein delivery systems can be engineered to enhance specificity toward the tissue of interest, and thus improve the overall precision of genome-editing therapies17.

Despite the promise of tissue-specific delivery of pre-assembled, therapeutic Cas9 RNPs, including base editors (BEs) or prime editors (PEs), their large size, overall negative charge, and lack of a specific protein-transduction mechanism pose challenges that must be overcome. Various systems have been explored for the delivery of BE- and PE-RNPs, including liposomes,18 micelles,19 polymer-based systems,20 dendrimers,21 and nanoparticles22,23. Lipid-based systems offer notable advantages. The size and composition of lipid-based vehicles can be easily tailored to carry editors and RNA, and these vehicles often rely on electrostatic interactions between positively charged lipids and negatively charged nucleic acids. As there are non naturally occurring positively charged bilayer-forming lipids, researchers have leveraged design principles from liposome research to synthesize cationic-lipid molecules. The first commercial cationic liposome-based transfection product, Lipofectin, was introduced in 1988 to deliver plasmids into cells in vitro24. Since then, further development has resulted in numerous ionizable/permanently ionized lipids with multiple functionalities like Lipofectamine and MC3,25 and delivery of large RNPs has been reported using lecithin-based liposomes26 and ionizable LNPs27. Critically, several LNP formulations have obtained approval from the US Food and Drug Administration (FDA), most notably the branched lipid compositions (including SM102 and ALC-0315), which were approved for delivery of the mRNA COVID-19 vaccines28. Both in the case of mRNA and RNPs, negative charges on the RNA nucleotides provide a driving force to bind the ionizable lipids that facilitate the encapsulation of the cargo.

We improve the existing genome-editing toolkit by developing a safe and efficient protein delivery system. To achieve this, we design a methodology based on Coomassie Brilliant Blue (CBB) lipidoids, whereby the CBB moiety strongly binds proteins, and the lipid moiety interacts with the cell membrane to enable uptake of the protein cargo by the cell. We rapidly synthesize libraries of CBB derivatives via click chemistry and sulfonamide conjugation. We evaluate the ability of these CBB derivatives to enhance the delivery of genome-editor proteins (Cre) and RNPs (adenine base editors, ABEs) in vitro and in vivo. After initial success, we further refine our approach by incorporating the CBB lipidoid into liposomes to achieve efficient editing in an in vivo inherited retinal disease (IRD) mouse model, rd12. Our approach is distinct from LNPs, as we form the CBB liposomes without protein and use them as neutral, aqueous suspension to complex the cargo in neutral pH and without organic solvents. Our CBB lipidoids engage in CBB-protein interactions, and thus can be used for the delivery of all types of proteins, including recombinases (Cre, Bxb1), and genome-editing RNPs (zinc finger nucleases, TALENs), and genome-editing RNPs (Cas9, ABE, PE, and others). We maximize the potential of CBB liposomes for RNP delivery by incorporating ionizable lipids to achieve synergistic complexing of the RNPs via CBB-protein and ionizable lipid-guide RNA interactions. Incorporating CBB into liposomes enhances RNP-mediated genome editing, allowing to reach clinically significant recovery of visual function and editing in the genome. This outcome affirms that specialized delivery agents, including our CBB lipidoids, are essential for achieving the RNP-mediated editing efficiency necessary for further clinical translation.

Results
Design and synthesis of coomassie-derived lipidoids
Lipidoids have been developed as key components of lipid-based particles to facilitate membrane penetration, cargo release and endosomal escape via receptor mediated endocytosis, interactions with the lipid bilayer, and phase transitions in the acidifying endosome29. However, they do not offer specific interactions with proteins. To enable the interactions between the lipids and proteins, we incorporated a protein-binding domain into lipidoids, as shown in Fig. 1. Our first-generation CBB compounds began with commercially available CBBG-250 (CBB22), which was functionalized through the coupling of amides or esters of N-methyl-1,3-diaminopropane (K1) or PEG (K3) to CBB, utilizing phosphorus trichloride to form the amino-sulfonamide or sulfonic ester (see Supplementary Fig. 1 and Synthesis of CBB intermediates and lipidoids in Supplementary Information). This intermediate was then coupled to diverse lipid and non-lipid tails of various lengths and degrees of saturation. For example, CBB1 contained a cholesteryl moiety, and CBB3 contained docosahexaenoic acid (DHA). CBB5 and CBB16 were isolated as byproducts during silica gel chromatographic purification of CBB3 and CBB15. CBB1, CBB3, CBB6, and CBB13 were further methylated to produce the lipidoids CBB2, CBB4, CBB7, and CBB14 with the positively charged linker K2. CBB21 was also coupled to the C-terminus of a cell-penetrating peptide (CPP5, sequence KLPVM), to yield CBB18 and CBB19. Upon long-term storage in DMSO, we noted oxidation of CBB11, resulting in CBBox11. Mass spectrometry of separated impurities indicated that CBBox11 comprised two isomers, likely representing hydroxylation events on the polyunsaturated fatty acid or the tertiary amine (Supplementary Fig. 2).

We also developed an alternative pathway to generate protein-binding lipid derivatives of CBB. We reasoned that since our first-generation CBB derivatives eliminated the sulfonic-acid functionality of CBB, retention of this group would enhance protein-CBB interactions, as CBBG-250 binds to proteins through hydrophobic interactions with aromatic residues (Phe, Tyr, Trp) and electrostatic interactions between the anionic sulfonate and basic amino acids (His, Lys, Arg)30. We created a CBB derivative, CBBZ0, with a propargyl ether functionality that can be linked by click chemistry to lipid/lipidoid azides. The construction of the click library began with a family of bifunctional linkers, to which lipidoids were joined via Michael addition reactions with acrylates, as illustrated in Supplementary Fig. 3. The alcohol functionality of the bifunctional linker was then converted to an azide via mesylation and displacement with sodium azide. Finally, the CBBZ compounds were assembled via click reactions, creating the CBB-triazole library consisting of compounds CBBZ1-10.

In vitro delivery of Cre by coomassie-derived lipidoids
First, we used a small genome-editing protein, Cre recombinase, to assess the CBB lipidoid-mediated intracellular protein delivery. We used a HEK293-loxP-GFP-RFP color-switching reporter cell line (CS) that constitutively expresses GFP, while an upstream stop codon prevents expression of RFP. Cre recombinase activity in the nucleus triggers the excision of the floxed GFP-stop sequence, and RFP expression replaces GFP (Fig. 2a)15. Nine compounds (CBB2, 7, 11, 12, 13, 14, 18, 19, Z1) were selected for the titration of the [CBB] to [Enzyme] ratio. The CBB lipidoids were dissolved in DMSO (1 volume) and complexed at room temperature with Cre recombinase (19 volumes) at a 2:1, 4:1, 8:1 or 16:1 molar ratio before delivery (Supplementary Fig. 4a). The enzymatic activity of cytosolic Cre was evaluated by fluorescence microscopy according to the extent of GFP to RFP conversion, which indicated that Cre recombinase intracellular delivery was dependent on the CBB-lipidoids concentration, and the dose-response relationship showed that the effect was generally saturated at a ratio 8:1 for most compounds. Accordingly, in the overall screening of the CBB lipidoids, the [CBB]/[Enzyme] ratio was set to 8:1, Lipofectamine 3000 (LF) served as a positive-control vehicle, and free Cre protein was used alongside non-treated cells as a negative control. Compounds were considered positive hits if the ratio of red-to-green (RFP/GFP) quantified by flow cytometry (CBB compounds) or fluorescence microscopy (CBBZ compounds) was two-fold higher than in the cells treated with free Cre (Fig. 2b, panel “C”). Six compounds from the amide coupled library (CBB6, CBB11, CBB14, CBB18, CBB19, and CBB21) demonstrated the ability to enhance delivery of Cre (Fig. 2b, c). The CBBZ approach was tested with C12 and C16 aliphatic chains present in CBB12 and CBB6, respectively, but it did not improve the ability of CBB to deliver Cre compared to amide-linked CBB compounds (Fig. 2d, C12: Z1, Z2, Z6, Z10; C16: Z3, Z7, Z8). Although SM102 enabled efficient delivery of ABE-RNPs in our previous study15, neither of the CBBZ compounds, in which the branched 9-heptadecyl lipid moiety of SM102 or the entire SM102 moiety were attached to CBB via a triazole ring, displayed protein-delivery ability (Fig. 2d, Z4, Z5, Z9). We noted the formation of a substantial blue precipitate immediately following the complexation of Cre with any of the CBBZ compounds, more pronounced than with amide-linked CBB compounds. This precipitation may have resulted from stronger protein binding of the CBBZ compounds than the CBB compounds, due to conservation of the CBB sulfonic acids in the CBBZ compounds, followed by colloidal aggregation. Despite this outcome, the robust click-based synthetic methods described here may hold significant promise for expanding the chemical space around lipidoids with protein-binding domains in the future. The Cre recombinase and CBB compounds were tolerated well by the CS cells, as shown by flow cytometric exclusion of DAPI (Supplementary Fig. 5a).

The highest GFP-to-RFP conversion, 58.2 ± 2.8%, was achieved for Cre complexed with CBB14, which contained butyrate tails. Methylation of the ionizable tertiary-amine linker K1 to the positively ionized quaternary ammonium K2 was important for the protein trafficking activity of CBB14, as CBB13, a precursor with the same butyrate tails but with the K1 linker, did not consistently deliver Cre activity into the reporter cells. Alkylation of other CBB compounds that had lipid chains containing cholesterol (CBB1), DHA (CBB3), or palmitate (CBB6), to CBB2, CBB4, and CBB7, respectively, did not improve in vitro delivery of Cre. Notably, CBB21, a CBBG-250 amide without lipid moieties, delivered Cre recombinase into reporter cells and triggered a pronounced GFP-to-RFP conversion. Although we previously found that delivery of Cre recombinase fused to CPP5 was negligible in vitro and in vivo15, here, delivery of a CPP5-CBB conjugate (CBB19) was robust, producing up to 22.4 ± 0.6% GFP-to-RFP conversion. CBB18, a combination of CPP5 and TFA, further increased the color conversion to 49.0 ± 3.6%, suggesting synergistic effects between cell-penetrating peptides and CBB. The leading CBB compounds for Cre recombinase delivery, CBB14, 18, 19 and 21, may have a greater positive charge under neutral aqueous conditions, and have less hydrophobic lipid moieties. CBB11-Cre complex consisted of homogenous particles whose size was approximately 1440 nm, whereas CBB14-Cre consisted of two molecular species whose diameters were approximately 167 and 2130 nm (Supplementary Fig. 6). While both CBB compounds aggregated with Cre into large complexes, the presence of smaller species in CBB14-Cre may explain the enhanced Cre delivery activity. Overall positive charge and lower hydrophobicity may be beneficial for Cre delivery, despite the pronounced aggregation. Lower hydrophobicity may increase long-term colloidal stability of the complexes. In our hands, highly hydrophobic CBB1 and CBB2, that were previously proposed as general protein-delivery vehicles via an endocytosis-independent tagging-penetrating mechanism31, did not deliver Cre into the reporter cells.

In vivo delivery of Cre
After in vitro nomination of CBB lead compounds, Cre recombinase was delivered with the in vitro hits CBB6, CBB14, CBB11, and CBBox11, or Cre recombinase alone, into the eyes of mT/mG Cre reporter mice via subretinal injection. Analogous to the reporter cell line described above, the mT/mG mouse is a global fluorescence-reporter strain for monitoring Cre recombinase activity, but with the inverse color change. The Rosa26 locus in mT/mG mice constitutively expresses membrane-localized tdTomato; however, an upstream polyadenylation signal stops the expression of eGFP (Fig. 3a). Upon excision of the floxed tdTomato cassette by Cre recombinase, eGFP expression replaces tdTomato. This red-to-green color switching in mouse tissues, including the retina, can be non-invasively monitored by two-photon microscopy of intact eyes. In control mouse eyes, both the retinal pigment epithelium (RPE) and the photoreceptors exhibited uniform tdTomato expression (Fig. 3b). The injection of 20 µM Cre recombinase (850 ng in 1 µl) in buffer with 7.25% DMSO resulted in minimal eGFP expression in the RPE and photoreceptor layers. Injection of 20 µM Cre complexed with 160 μM (208 ng) CBB14 increased eGFP expression in the RPE, producing up to 90% red-to-green color switching (Fig. 3b and Supplementary Fig. 7). CBB6, CBB11, and CBB14 also mediated delivery of Cre into photoreceptors, with CBB14 achieving up to 20% tdTomato-to-eGFP conversion in photoreceptors (Fig. 3b and Supplementary Fig. 7). Notably, we observed that in vitro, the GFP-to-RFP conversion mediated by CBB11 was moderate, while in vivo CBB11 enhanced Cre-delivery-triggered eGFP expression in both outer and inner segments of the photoreceptors, with up to 30% red-to-green conversion. Although in vitro delivery of Cre recombinase mediated by CBBox11, an oxidation product of CBB11, was marginal, it boosted in vivo eGFP expression exclusively in RPE cells to a level comparable to that mediated by CBB14.

In vitro delivery of ABE-RNP
After confirming the ability of CBB to deliver Cre in vitro and in vivo, we next investigated the potential for CBB-lipidoids to transfer CRISPR genome editors. We used the adenine base editor (ABE) ABE8e as a model. To this end, we used our NIH/3T3 fluorescent reporter rd12 cell line32 that contains mCherry and eGFP separated by a 204-bp Rpe65 cDNA fragment containing the nonsense rd12 mutation (c.130C > T; p.R44X) (Fig. 4a). In this cell line, eGFP expression is suppressed by a stop codon, which can be corrected to an Arg codon by adenine base editing. After the editing, both mCherry and eGFP are expressed, appearing as yellow in merged fluorescent microscopic images. ABE8e RNP was prepared as previously described32 and applied to the reporter cell lines with or without delivery vehicles. When complexed with Lipofectamine 3000, ABE-RNPs successfully induced eGFP expression, whereas ABE-RNPs incubated with DMSO alone enabled only marginal conversion of the reporter cells (Fig. 4b). We titrated the ABE RNP with selected CBB compounds (CBB2, 7, 11, 12, 13, 14, 18, 19, Z1) and found that higher concentrations of CBB compounds may better facilitate RNP delivery (Supplementary Fig. 4b). However, at higher concentrations we also observed pronounced precipitation of CBB compounds and decided to maintain the CBB:RNP ratio of 8:1 to prevent complications such as clogging of flow cytometric fluidics or injection syringes. We then screened our CBB compounds for ABE RNP delivery ability and found that among CBB1-22 and CBBZ1-Z7, consistent eGFP expression in reporter cells was only achieved by CBB3 (3.9 ± 0.4%) and CBB11 (28.6 ± 0.7%) (Fig. 4b–d). The complexing of ABE-RNPs with CBB lipidoids with 10% DMSO also did not affect the deaminase activity (Fig. 4e–g)15. The common feature of CBB3 and CBB11 are unsaturated lipid domains, DHA (CBB3) and linoleate (CBB11), that contain multiple unsaturated C-C bonds. This may increase the fluidity of the lipid layer and promote fusion of the colloids with the cell membrane. Similarly to Cre, the flow cytometric DAPI exclusion assay showed that the ABE RNP-CBB complexes were tolerated by the rd12 reporter cells (Supplementary Fig. 5b).

We found that the CBB-RNP complexes have poor colloidal stability. Precipitation occurred at high concentrations or upon prolonged incubation, and was associated with a decrease in delivery efficiency. The colloidal instability of CBB11 could have hindered the uptake of ABE-RNPs by the cells or interfered with editing. To improve the distribution of CBB11 in an aqueous environment, we formulated liposomes (Lipo1/3/5) containing, in molar ratio, 5% CBB11, 50% ionizable lipids (either SM102, ALC-0315, or MC3), 10% 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 33.5% cholesterol, and 1.5% 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), and a control liposome (Lipo2/4/6) without CBB11 (Fig. 5a). The composition of Lipo2 (50% SM102, 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG2000) resembled the lipid nanoparticles described in our previous work32. We noted an improvement of colloidal properties of CBB11 after incorporating it into the liposomes. Upon buffer exchange, free CBB11 was absorbed by a size-exclusion chromatography resin, whereas liposome-bound CBB11 was recovered in the eluate (Supplementary Fig. 8). Dynamic light-scattering measurements indicated that the two groups of liposomes were highly monodisperse (polydispersity index = 0.16/0.19/0.17, and 0.19/0.20/0.15 for Lipo1/3/5 and Lipo2/4/6, respectively), with an average hydrodynamic diameter of 140-155 nm and 155-161 nm, respectively. In contrast, the particle size of CBB11 in the TAS buffer ranged from 400-500 nm (Fig. 5b and Supplementary Fig. 9a–d). We also did not observe precipitation of ABE-RNP with either Lipo1/3/5 or Lipo2/4/6. The cryo-TEM studies on Lipo1 revealed that it consists of unilamellar vesicles with an aqueous core, and that the ABE-RNP is prone to be recruited to the surface of the liposome (Fig. 5e and Supplementary Fig. 10). Furthermore, the presence of ABE-RNP dramatically changed the zeta potential on the Lipo1 surface (a net decrease of 10.16 mV), but marginally affected the zeta potential on the Lipo2 surface (a net decrease of 0.64 mV) (Fig. 5d). No ABE-RNP was observed to be incorporated into Lipo1. The ABE-RNP complexed with Lipo1 remained accessible to Proteinase K (Supplementary Fig. 11). Thus, the protein binding occurred on the surface of Lipo1, and was intensified by CBB11. As expected, this enhanced liposome-protein interaction boosted intracellular protein delivery. Compared to our positive control Lipofectamine 3000, Lipo1 and Lipo2 increased intracellular Cre delivery to CS reporter cells 2.4 and 2.2-fold, respectively, indicated by increased GFP to RFP conversion visualized with fluorescence microscopy (Fig. 5c). At 40 nM RNP, Lipo1 increased the efficiency of ABE8e-RNP delivery into the rd12-reporter cells, as monitored by eGFP fluorescence, 10.5-, 2.9-, and 1.3-fold when compared to CBB11, Lipo2, and LF, respectively (Fig. 5f–i). The RNP delivery enhancement mediated by CBB11 did not depend on the ionizable lipid, as we observed similar enhancement with liposomes made with ALC-0315 and MC3 (Fig. 5g, h and Supplementary Fig. 9e), and with liposomes formulated without ionizable lipids (Supplementary Fig. 12). With 25 nM RNP, the eGFP fluorescence relative to mCherry in reporter cells treated with Lipo3 was 4.4-fold higher than with Lipo4, and the eGFP expression in cells treated with Lipo5 was 31.9-fold higher than with Lipo6 under the same conditions (Fig. 5g, h). We also investigated the delivery of ABE-RNPs into TIGER HEK cells, where functional tdTomato expression is induced by precise A to G editing of the mutated tdTomato at position 7 of the protospacer (Supplementary Fig. 13a)33. With 25 nM ABE8e RNP, Lipo1 induced approximately 2-fold higher red fluorescence than our positive control, Lipofectamine 3000. Higher doses of ABE8e led to decreased red fluorescence, originating from bystander editing at of neighboring adenine A9 that lead to the expression of tdTomato with a missense mutation that decreases its fluorescence (Supplementary Fig. 13a, b). The use of a more precise ABE8e N108Q34 led to enhanced rescue of tdTomato fluorescence at a broader range of concentrations (Supplementary Fig. 13c). The incorporation of CBB and the ionizable lipid in the liposomes leads to the creation of a broadly applicable macromolecular delivery system optimized for use with ABE RNP. Separately, CBB and ionizable lipids specifically engage the ABE protein and guide RNA, respectively, as shown by the delivery activity of ionizable-lipid-only and CBB-only liposomes built on a scaffold of phospholipid-cholesterol-PEG liposome. Use of CBB and ionizable lipids leads to synergistic engagement of ABE RNP, as demonstrated by the increased potency of Lipo1/3/5 versus Lipo2/4/6. The dispersion of the CBB-RNP complexes likely is achieved by the embedding of the lipid chains of the CBB lipidoid within the liposomes.

Protein delivery by CBB-liposomes in vivo
Based on the promising results with Lipo1 and Lipo2 in vitro, we assessed liposome-mediated delivery of Cre recombinase to the retina. Lipo1 and Lipo2 were mixed with Cre recombinase to a final concentration of 3.2 mM total lipids and 20 µM Cre. In the Lipo1-Cre complex, the ratio of CBB11 to RNP was set at 8:1. The resulting mixtures were delivered to the eyes of mT/mG-reporter mice via subretinal injections, and tdTomato to eGFP conversion was monitored by two-photon microscopy (Supplementary Fig. S7)32. Both Lipo1 and Lipo2 selectively delivered Cre into RPE cells, triggering color change with similar efficiency. In contrast to free CBB11, Lipo1 that contained 5% CBB11 did not deliver Cre to photoreceptors, but to RPE (Fig. 3b and Supplementary Fig. 7). A possible explanation of this change in CBB11-delivery specificity is that liposome endocytosis was mediated by receptors such as low-density lipoprotein receptor-related protein 1 (LRP1) whose expression levels in RPE are much higher than in photoreceptors35.

Finally, we investigated the non-viral delivery of ABE-RNPs via our liposome formulations for therapeutic genome editing. To assess the delivery of ABE8e-RNPs, we utilized the rd12 mouse model of Leber congenital amaurosis. These mice harbor a nonsense mutation in Rpe65, which effectively eliminates RPE65 expression within the RPE. Without the enzyme to regenerate the visual chromophore 11-cis-retinal, the visual cycle is non-functional, and the electroretinography (ERG) waveform representing the photoresponse to dim-light stimulus is negligible. Effective editing of the Rpe65 gene restores functional RPE65 expression, the visual cycle, and in turn, induces the ERG response36–38.

To assess the editing capabilities of ABE8e-RNPs in conjunction with our CBB lipidoids, we measured restoration of functional vision mediated by several delivery vehicles. Two weeks after subretinal injections of 20 µM ABE-RNPs in these vehicles, rd12 mice were dark-adapted overnight, and their scotopic ERG responses were recorded (Fig. 6a). We did not observe an ERG-flash response in untreated rd12 mice, while mice treated with ABE-RNPs in TAS buffer or in TAS with 7.25% DMSO regained recordable ERG responses. The scotopic b-wave amplitudes in response to a 0.5 cd s m-2 stimulus were 45.8 ± 12.9 μV and 50.6 ± 33.9 μV, respectively. CBB11 and CBB14 complexed to ABE8e performed marginally better than untagged RNPs (60.8 ± 14.9 μV and 58.5 ± 37.4 μV, respectively). These results indicate that our observations in vitro with ABE-RNPs with CBB11, or in vivo with Cre recombinase delivery by CBB14, are not directly translatable to ABE-RNP delivery in vivo. This disconnect could originate from the previously observed propensity of CBB-RNP complexes to aggregate, which, together with the intrinsic propensity of ABE to dimerize15,39,40 might exacerbate the CBB-induced oligomerization and complicate RNP delivery to a larger extent than observed for Cre. Consistent with in vitro observations for the CBB-RNP complexes, the liposome formulations outperformed the non-liposomal CBB formulations. The restoration of ERG responses was robust, especially when CBB11-containing Lipo1 was used. The b-wave amplitudes were measured at 227 ± 69 μV for Lipo1, and 95.3 ± 62.5 μV for Lipo2. Notably, the best example of Lipo1-mediated editing achieved a b-wave amplitude of 353 μV, estimated to be 76% of the ERG response of wild-type mice. These results support the proposed synergy between nucleic acid engagement by the liposome and protein engagement by the CBB that enables the therapeutic effect of our RNP formulation.

We further analyzed the outcomes of the phenotypic rescue by performing retinoid analysis of eyes treated with ABE-RNP complexed with Lipo1 or Lipo2. After 24 h of dark adaptation, 11-cis-retinal levels reached approximately 40 and 16 pmol/eye for Lipo1 and Lipo2, respectively, estimated to be 10.5% and 4.2% of the level for wild-type mice (Fig. 6b). We also demonstrated precise editing of the rd12 mutation in the DNA and transcripts isolated from RPE samples obtained from the mice whose ERG measurements are reported in Fig. 6a. Next-generation sequencing revealed up to 9.9% total adenine editing in Rpe65 genomic amplicons (Fig. 6c), which was correlated with up to 28.8% sequence editing of the Rpe65 transcripts (Fig. 6d) in eyes treated with Lipo1. Strikingly, although ABE8e has the propensity to deaminate multiple adenosines within its activity window even upon transient delivery via eVLPs,38 in this case, when delivered in complex with Lipo1, we recorded precise A-to-G corrections by ABE8e up to 5.0% in gDNA sequences (Figs. 6e) and 11.7% in transcripts (Fig. 6f). Precisely edited gDNA sequences in mice injected with ABE-RNPs delivered by Lipo1 were 127-, 20-, 50-, 9.7-, and 6.5-fold higher than those with TAS, DMSO, CBB11, CBB14, and Lipo2, respectively. These findings indicate that the benefits of augmenting liposomes with CBB11 were corroborated by improved visual response and increased editing of the genome.

We also compared the ERG responses and genomic editing of rd12 mice treated with Lipo1-ABE8e-RNPs to the analogous measurements for mice treated with ABE8e-eVLP described previously (Fig. 6a, c, e)5. The ABE8e-RNP carried by eVLPs achieved 21.6 ± 6.6% total genomic editing, outperforming lentiviral delivery (11.6 ± 4.5%). Although the genome editing by Lipo1-ABE8e-RNP in this study (5.5 ± 3.3%) is less efficient, both the precise genomic correction rate (2.7 ± 1.5%) and ERG response (227 ± 69 μV) in rd12 mice treated with Lipo1-ABE8e-RNPs are almost 2.2- and 2.3-fold greater, respectively, than those in mice treated with eVLP-ABE8e-RNPs. We attribute this increase of vision-function recovery in rd12 mice to a more transient gene editing in RPE cells by the Lipo1-ABE8e-RNP system, effecting more precise correction. Furthermore, off-target editing analysis by CIRCLE-seq revealed A to G substitutions at background levels in the rd12 mice treated with Lipo1-ABE8e-RNPs, comparable to those observed in untreated animals (Fig. 6g).

To evaluate the safety of the ABE-RNP delivery to the retina mediated by Lipo1, we also administrated ABE-RNP-Lipo1 complex to WT mice (Supplementary Fig. 14) via subretinal injection. Since the oxidation of CBB11 might induce epoxides in the liposome, only freshly formulated Lipo1 was applied to the WT mice. Different batches of Lipo1 showed similar morphology and comparable ABE-RNP delivery efficiency to rd12 reporter cell lines (Supplementary Fig. 14a, b). In vivo retinal imaging by optical coherence tomography (OCT) indicated no substantial alterations to the retinal structure of WT mice administrated the complex of RNP (20 μM) and Lipo1. We noticed the presence of hyperreflective spots visible in the vitreous, indicating possible inflammation41, which could be ameliorated by lowering the RNP concentration (Supplementary Fig. 14c). The ERG response and the generation of the visual chromophore, 11-cis retinal, of WT mice treated with Lipo1 and RNP (10 μM) were also comparable to those of untreated WT mice (Supplementary Fig. 14d, e). In parallel experiments in which rd12 mice were treated with the same batch of Lipo1 and RNP complex, subretinal injections of 1 μl of Lipo1 with 10 μM RNP achieved optimal ERG response and visual chromophore recovery (Supplementary Fig. 14d, e), comparable to those of rd12 mice treated with Lipo1-ABE-RNP (20 μM) (Fig. 6). These results suggested that optimal vision-function rescue requires a balance between the biological tolerance of the cells and the editing efficiency of the gene editors.

Methods
Animals
The C57BL/6J (”WT”, JAX 000664), B6(A)-Rpe65 rd12/J (rd12) (JAX 005379), and B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP) Luo/J (mTmG) (JAX 007676) mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). mT/mG mice were crossed with BALB/cJ albino mice (JAX 000651) to establish an albino mT/mG line used in this study. The mice of both sexes between 4–13 weeks of age with a body weight of 15–25 grams were used for the experiments (see Source data). The mice were housed in the vivarium at the University of California, Irvine, where they were maintained on a normal mouse chow diet and a 12 h light (<10 lux)/12 h dark cycle. The temperature ranged from 75-76 °F, humidity from 30-40%, and animals were given food and water ad libitum. No sex-based stratification or sex-based analysis was performed in this study. Both male and female mice were used. The research applies to both sexes. The functional readout is electroretinography (ERG), and there are no documented differences in ERG responses between male and female mice48. We did not find literature evidence of the influence of sex on genome editing in mice. Here, the cohorts are too small to conduct sex-based analysis.

Mammalian cell culture
HEK293-loxP-GFP-RFP cells (referred to as “CS cells” GenTarget Inc., San Diego, CA, USA, SC018-Bsd), reporter-rd12 cells (generated previously in Palczewski laboratory)15 and TIGER HEK cells (generated previously in Palczewski laboratory)33 were maintained in DMEM/F-12 medium with GlutaMAX supplement (Thermo Fisher, Waltham, MA, USA, 10565018), or in DMEM with glutamine (Thermo Fisher, 11965092), both supplemented with 10% fetal bovine serum (FBS) (Genesee Scientific, San Diego, CA, USA, 25-514H) and optional 100 U ml-1 penicillin-streptomycin (Thermo Fisher, 15140122) (complete medium) in a humidified incubator at 37 °C, 5% CO2.
The cells were rapidly thawed from liquid nitrogen storage and passaged at least twice before use in experiments. For protein-delivery experiments, the cells were seeded 24 h prior to the experiment in 24-, 48-, and 96-well plates to reach 50–70% confluence (approximately 100,000, 50,000 and 25,000 cells per well, respectively, for the CS cell line; and 50,000, 24,000 and 10,000 cells per well, respectively, for rd12 reporter cells).

General experimental details for chemical synthesis
All reactions were performed in oven-dried glassware, under an inert atmosphere with exclusion of moisture from reagents and solvents. Reagents were used as supplied. Liquid chromatography was performed using flash chromatography on silica gel (230-400 mesh), on a CombiFlash NextGen 300+ system, using eluting solvent (reported as a V:V-ratio mixture). Analytical thin layer chromatography (TLC) was performed on 0.25 mm glass-backed Silicycle 60 F254 plates. Visualization of the developed chromatogram was accomplished with UV light (254 nm). Detailed procedures and analytical data are in the Supplementary Information.

Mass spectrometry
The Coomassie compound solution in methanol (Fisher Scientific) was diluted to approximately 5 µM and centrifuged at 17,000 g for 15 min. Five microliters of the supernatant were injected into a Vanquish HPLC system coupled with a Q-Exactive Mass spectrometer (Thermo Fisher Scientific, Waltham, MA). The separation was carried out on a BEH C18 Colum (1.7 µm, 2.1 × 50 mm, Waters, Milford, MA) using a mobile phase consisting of 0.1% formic acid (Thermo Fisher) in water (A) and 0.1% formic acid and 50% 2-propanol (Thermo Fisher) in acetonitrile (B) at a flow rate of 300 µl min−1 with a gradient of 30% B at 0-1 min, 30-100% B at 1–5 min and 100% B at 5–8.5 min. The normalized collision energy (NCE) was set at 40. The data was extracted using Xcalibur 1.4 (Thermo Fisher Scientific, Waltham, MA) and processed with FreeStyle 1.8 P2 (Thermo Fisher Scientific) and GraphPad Prism 10 (GraphPad, San Diego, CA).

Nuclear magnetic resonance
1H and 13C-NMR spectra were recorded on a Bruker Ascend Avance III HDTM spectrometer operating at 400 MHz, 500 MHz, or 600 MHz. Chemical shifts reported in δ units (part per million (ppm)) with reference to the residual solvent peak CD3OD (Millipore Sigma) (δ 4.87, 3.33) for 1H and (δ 49.15) for 13C spectra. Bruker TopSpin 4.4.1 software was used to collect the data, and Mnova 14.3.1 was used to analyze the results. NMR data are presented in the following order: chemical shift, peak multiplicity (s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, m = multiplet), coupling constant (in Hz).

Expression and purification of Cre recombinase
Plasmid encoding Cre recombinase for expression in Escherichia coli was a gift from Niels Geijsen (Addgene plasmid #62730; http://n2t.net/addgene:62730; RRID:Addgene_62730). The plasmid sequences were viewed using Snapgene 8.2.1 software. The Cre plasmid was transformed into Escherichia coli BL21 star (DE3) bacteria (Thermo Fisher Scientific, C601003) and plated on LB agar (Millipore Sigma) plates, with 50 µg ml−1 kanamycin (Goldbio, St. Louis, MO, USA, K-120-SL25); selected clones were grown in Terrific Broth (Thermo Fisher Scientific, 22711-022) containing 50 µg ml−1 kanamycin, overnight at 37 °C with mixing at 190 rpm. The production cultures were inoculated with overnight cultures and incubated at 37 °C with mixing at 190 rpm. After A600nm of the culture reached 0.5, protein expression was induced with 0.5 mM IPTG (Goldbio, I2481C25), and incubation was continued at 20 °C for 16 h. The cells were harvested by centrifugation at 7000 × g at 4 °C for 10 min, and the pellets were stored at −80 °C until used further for protein purification. Cre recombinase was kept on ice or refrigerated at 4–8 °C throughout the purification procedure.
The cell pellet from a 1.5-liter culture was thawed in a room-temperature water bath, resuspended in Cre-lysis buffer (50 mM Na phosphate (Fisher Scientific) pH 7.4, 1 M NaCl (Millipore Sigma), 1 complete EDTA-free protease inhibitor cocktail tablet (Millipore Sigma, St. Louis, MO, USA, COEDTAF-RO) per 50 ml buffer), and lysed by sonication (125-W pulses (5 secon/5 sec off) for 10 min total) or by French press (3 passes at up to 15,000 psi). The lysate was centrifuged at 48,500 × g for 15 min, and the supernatant was incubated with 1 ml of a suspension of Ni-Sepharose High Performance beads (Cytiva, Uppsala, Sweden, 17526801) in a rotating mixer for 1 h. The resin was centrifuged at 500 × g for 5 min, washed with 40 ml of the Cre-wash buffer (25 mM Na phosphate pH 7.4, 500 mM NaCl), centrifuged again, and then packed in a Tricorn 5/50 (Cytiva) column connected to a Bio-Rad DuoFlow system (Bio-Rad, Hercules, CA, USA) with Bio-Rad Bio-Logic DuoFlow 5.30 software, which was perfused at 0.5 ml min-1. The resin was washed with Cre-wash buffer (20 ml, or until a stable absorbance baseline (280 nm) was observed); then, the proteins were eluted with 30 ml of a continuous gradient of the Cre-elution buffer (25 mM Na phosphate, pH 7.4, 500 mM NaCl, and 0–500 mM imidazole (Fisher Scientific)). The fractions containing Cre recombinase that were identified by SDS-PAGE were concentrated and subjected to size-exclusion chromatography on a Superdex 200 Increase 10/300 GL column (Cytiva, 28990944), or a HiLoad 16/600 Superdex 200pg column (28989335), with P500G buffer (Na phosphate 20 mM pH 7.4, NaCl 500 mM, glycerol (Fisher Scientific) 20% (v/v)) as the mobile phase. Fractions containing pure Cre recombinase were concentrated, snap frozen in liquid nitrogen, and stored at −80 °C.

Expression and purification of ABE
E. coli BL21 star (DE3) bacteria (Thermo Fisher Scientific, C601003) were transformed with the ABE8e SpCas9NG (referred to as ABE8e) expression plasmid described previously,15 and grown overnight on Luria broth (LB)-agar plates containing 25 µg ml-1 kanamycin; single clones were used to inoculate starter cultures in Terrific broth (TB, Thermo Fisher) containing 25 µg ml−1 kanamycin, and grown overnight. One and a half liters of TB containing 25 µg ml−1 kanamycin was inoculated with 10 ml of starter culture, and the culture was grown at 37 °C with mixing at 190 rpm, until it reached an A600nm of 1.5. Then, the culture was cooled in an ice-water slurry, and protein expression was induced with 0.8% (w/v) rhamnose (Goldbio, R-105-250). The proteins were expressed at 17 °C with mixing of the culture at 190 rpm for 16–24 h. The bacteria were harvested by centrifugation at 5000 × g, 10 min, 4 °C, and the pellets were stored at −80 °C.
All protein purification steps were conducted in a cold room (~4-8 °C), or on ice. The thawed bacterial pellet from a 1.5-liter culture was homogenized with a 40 ml Dounce homogenizer in lysis buffer (100 mM Bis-Tris Propane (Millipore Sigma), pH 8.0, 1 M NaCl, 20% glycerol (v/v), 5 mM TCEP (Hampton Research), 1 cOmplete Ultra EDTA-Free protease inhibitor tablet per 40 ml of the buffer), and lysed by sonication with a Qsonica Q125 sonicator (125 W) with a 1/8-inch microtip at 100% amplitude for a total of 20 min (intermittent pulses: 5 secon; 5 sec off). The lysate was clarified at 4 °C by centrifugation at 48,500 × g for 10 min. ABE protein was captured on a 3 ml TALON metal-affinity resin (Takara Bio, San Jose, CA, USA, 635502). The resin was then washed with 100 ml of the lysis buffer without inhibitors, and with 10 ml of ABE wash buffer (100 mM Bis-Tris Propane, pH 8.0, 500 mM NaCl, 20% glycerol (v/v), 1 mM TCEP). The proteins were eluted with the wash buffer supplemented with 150 mM imidazole.
In a second step, ABE was purified by immunoaffinity chromatography using a 1D4 resin (CNBr-activated Sepharose 4B (Cytiva, 17043001), with immobilized 5–10 mg ml−1 1D4 antibody purified in house). The 1D4 resin (4 ml) was packed in a DWK Life Sciences Kimble™ Kontes™ Flex Column™. The column was equilibrated with ABE wash buffer. ABE was loaded by gravity flow. The column was washed with at least 40 ml of the wash buffer at 0.5 ml min−1, and then ABE was eluted with 1 mg ml−1 1D4 peptide in the wash buffer at approximately 1 ml h−1. Fractions containing ABE were pooled, concentrated, and further purified to remove contaminating nucleic acids and aggregates by size exclusion chromatography on a Superdex 200 Increase 10/300 GL column, or a Superdex 200 16/60 Prep Grade column. The protein was eluted with ABE storage buffer (HEPES 10 mM (Goldbio), pH 7.0, 500 mM NaCl, 20% glycerol). The fractions containing pure ABE were concentrated using Amicon Ultra centrifugal filters with 30-kDa MWCO membranes (Merck, UFC903024), sterilized by passage through 0.22-μm filters, quantified using UV absorption at 280 nm, divided into aliquots, and snap frozen in a chilled metal block for storage at -80 °C. Protein purity was assessed using SDS-PAGE in hand-cast Tris-glycine-SDS discontinuous gels with 4% acrylamide in the stacking gel (pH 6.8) and 10% acrylamide in the resolving gel (pH 8.8), with 2.7% cross-linker (acrylamide:bis-acrylamide ratio of 37.5:1, Bio-Rad 1610158). The samples were mixed with 4x-concentrated Laemmli sample buffer (Bio-Rad 1610747) and supplemented with 50 mM dithiothreitol (DTT, Millipore Sigma D9779), denatured at 70 °C for 10 min and centrifuged before applying the supernatant onto the gel. The protein concentration was quantified using UV absorption spectroscopy by measuring absorbance at 280 nm with a Nanodrop ND-1000 spectrophotometer (software version 3.8.1). Separation of contaminating nucleic acids was followed by monitoring the A260nm / A280nm ratio; a ratio < 0.60 was used as an indication of protein free from nucleic acids. A typical A260nm / A280nm value for purified ABE was 0.55. Extinction coefficients of the constructs were estimated using the ProtParam tool (https://web.expasy.org/protparam/).

Delivery of Cre recombinase in vitro
The CS cells were plated on 48-well treated tissue-culture plates in complete medium with 10% FBS. For each sample, Cre recombinase was first complexed for 15 min at room temperature with CBB compound in 12.5 µl of OptiMEM medium (Thermo Fisher, 31985070), at final concentrations of 10 µM of Cre enzyme and 20–160 µM of CBB compound. The mixture was then diluted with OptiMEM medium (Thermo Fisher, 31985070) to a final volume of 250 µl and applied to cells. After incubation for 3 h at 37 °C, 5% CO2, the medium was exchanged with DMEM medium (Thermo Fisher, 10566016) containing 10 % FBS, and the cells were incubated for an additional 24 h. For imaging, the medium was changed to FluoroBrite DMEM (Thermo Fisher, A1896701), and the cells were imaged using a Keyence BZ-X810 microscope with optical filters for GFP and Texas Red. Keyence BZ-X800 Viewer 1.1.1.3 was used to collect the images, and Keyence BZ-X800 Analyzer 1.1.1.2 and XnView 2.51.4 were used to analyze the images. Adobe Illustrator 28.4.1 was used to prepare the figures.

Flow cytometry
Cells were washed with PBS (Thermo Fisher, 10010023), detached with 0.05% trypsin (Thermo Fisher Scientific, 25300054), transferred to a 96-well round-bottom plate, and centrifuged at 180 × g for 5 min at room temperature. Centrifuged cells were washed with a FACS buffer (PBS with 2% FBS, 100 U ml-1 penicillin-streptomycin (Fisher Scientific, 15-140-122)), centrifuged again and resuspended in the FACS buffer with 1 µg ml-1 DAPI (Thermo Fisher, 62248). The cells were analyzed using a Novocyte Quanteon (Agilent) flow cytometer with Pacific Blue (445/45 nm), FITC (530/30 nm), and PE (586/20 nm) optical filters, with NovoExpress 1.6.2 software. Cells were gated on forward and side scatter, and single cells, and analyzed for viability via DAPI exclusion. The gating strategies are outlined in Supplementary Fig. 15.

ABE activity assay
Synthetic 60-bp-long DNA oligonucleotides (Millipore Sigma, see Supplementary Data 1) labelled with fluorescein on the strand undergoing deamination were annealed at a 1:1 ratio in 10 mM Tris pH 8.0, 50 mM NaCl and 1 mM EDTA (Millipore Sigma) by incubation at 95 °C for 5 min and subsequent slow cooling to 20 °C. End-modified sgRNA (Supplementary Data 1) was ordered from IDT Technologies, with 2′-O-methyl groups on the first three and last three nucleotides, and the first and last three phosphodiester bonds were replaced with phosphorothioate bonds. The guide RNAs were dissolved in nuclease-free water at 37 °C for 15 min at 500 rpm in a thermomixer and folded by incubation at ~75 °C for 5 min, followed by slow cooling. Prepared nucleic acids were quantified using UV absorption spectroscopy by measuring absorbance at 260 nm. Accordingly, an A260nm value of 1.0 corresponded to a DNA concentration of 50 µg ml−1 or an RNA concentration of 40 µg ml−1. DNA was stored at −20 °C and RNA at −80 °C. For the ABE enzymatic assay, ABE ribonucleoprotein was assembled by incubation with 1.5-fold molar excess of sgRNA in a reaction buffer (20 mM Bis-Tris propane, pH 7.5, 100 mM KCl (Fisher Scientific), 2.5 mM MgSO4 (Fisher Scientific), 2 mM DTT, 5% (v/v) glycerol) for 15 min at room temperature. Additional 10% (w/v) sucrose (Millipore Sigma) was used when RNPs were assembled above 4 µM. Then, ABE was diluted to 1 µM with the reaction buffer, preheated at 37 °C and added with 15 ng of DNA substrate (0.02 µM final). The deamination was conducted for 10-60 min for ABE8e in the absence/presence of 4- to 16-fold of CBB22, 11, 14. The 20 µl reactions were quenched by addition of 30 µl of water preheated to 95 °C and incubated for 2 min at 95 °C. After cooling, the mixtures were treated with 1 µl of RNase A (20 mg ml−1, Millipore Sigma) and 1 µl of proteinase K (20 mg ml−1, Viagen Biotech 501-PK) for 15 min at room temperature; then DNA products were purified using an Oligo Clean and Concentrator kit (Zymo Research, D4061). Purified DNA was nicked with 5 units of Endonuclease V (EndoV, NEB) for 2-3 h at 37 °C, after which the reaction was quenched by addition of TriTrack DNA-loading dye (1× final) (Thermo Fisher, R1161) and incubation at 95 °C for 2 min. The cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis with urea (Urea-PAGE) in Bio-Rad MiniProtean continuous hand cast 15% acrylamide gels in Tris-borate-EDTA (TBE; Bio-Rad, 1610770) with 7 M urea and 5% crosslinker (acrylamide:bis-acrylamide ratio of 19:1; Bio-Rad, 1610144). The voltage was controlled to maintain at least 42 °C in the electrophoresis chamber. Imaging was done using the ChemiDoc MP system (Bio-Rad) and analyzed with ImageLab 6.1.0 software (Bio-Rad).

Delivery of ABE in vitro
ABE-RNP was assembled by incubation of up to 20 µM ABE8e with 1.1-fold molar excess of chemically modified, heat-refolded, synthetic sgRNA (IDT and Genscript, see Supplementary Data 1) in OptiMEM medium with 10% (w/v) sucrose for at least 15 min at room temperature. The sgRNA had stabilizing modifications as described49. Next, ABE-RNP was diluted in TAS buffer (20.1 mM Tris, 4.3 mM sodium acetate (Fisher Scientific), pH 7.3, sucrose 10% (v/v)) and added to CBB-lipidoids at 1:2 to 1:16 ratios of enzyme:compound, 5%/7.25% (v/v) final DMSO (Millipore Sigma, D2650-100ML), 10 µM final RNP, and incubated for a further 15 min (Figs. 4 or 5h) (Fig. 5f, i) at room temperature. The mixtures were applied on the cells in a 48-well plate that had been washed with OptiMEM, at a concentration of 0.1 µM ABE in OptiMEM, without added sucrose. After 3 h, the medium was exchanged with 250 µl of complete medium.
To assemble complexes of ABE-RNP with liposomes (Lipo1-Lipo8), 10 µM ABE-RNP was diluted with TAS buffer and added to concentrated liposomes to achieve 1 µM ABE-RNP with 8-fold excess of CBB-11 (Lipo1/3/5/7) or equivalent amount of lipids (Lipo2/4/6/8) and incubated for 15 min at room temperature. Next, ABE-RNP complexed with liposomes was diluted ten-fold with preheated complete cell culture medium, serially diluted and applied onto the cells in 96-well plate, 100 µl per well.
To assemble complexes of ABE-RNP with Lipofectamine 3000 (LF, Fisher Scientific L3000001), 0.5 µl of LF per well of a 48-well plate, or 0.2 µl of LF per well of a 96-well plate, were diluted to 12.5 µl and 5 µl per well, respectively, with OptiMEM containing 10% (w/v) sucrose; the diluted ABE-RNPs were then added to the diluted lipids and incubated for 15 min at room temperature. A lipoplex of ABE RNP with Lipofectamine in a volume of 25 µl was added to the cells with 225 µl fresh complete medium in a 48-well plate, or 10 µl was added to the cells with 90 µl medium in a 96-well plate.
In all assays, cell culture medium was replaced 24 hours, and the analyses were done 48 h after applying ABE. The cells were imaged using a Keyence BZ-X810 microscope with optical filters for GFP and Texas Red after a medium change to Fluorobrite DMEM with 1% FBS, equilibrated for at least 15 min in CO2 incubator. Activities of ABE in the rd12 reporter cell line were estimated by comparing ratios of intensities of GFP and mCherry. Activities of ABE in the TIGER HEK reporter cell line were estimated by calculating the intensity of tdTomato in areas covered with cells using ImageJ/Fiji 1.54i.

Subretinal injections and delivery of Cre recombinase in vivo
Cre proteins complexed with CBB lipidoids (as above) were diluted in OptiMEM to 20 µM. First, the mice were anesthetized by an intraperitoneal injection of a cocktail consisting of 20 mg ml−1 ketamine (MWI, 120495) and 1.60 mg ml–1 xylazine (MWI, 008679) in PBS at a dose of 100 mg kg−1 of ketamine and 8 mg kg−1 of xylazine, and their pupils were dilated by topical administration of 1% tropicamide ophthalmic solution (Akorn, 17478-102-12) and 10% phenylephrine ophthalmic solution (MWI Animal Health, 054243). The corneas were hydrated with GenTeal Severe Lubricant Eye Gel (0.3% Hypromellose, Alcon). Subretinal injections were performed using an ophthalmic surgical microscope (Zeiss). An incision was made through the cornea adjacent to the limbus at the nasal side, using a 27-gauge needle. A 34-gauge blunt-end needle (World Precision Instruments, NF34BL-2), connected to an RPE-KIT (World Precision Instruments, RPE-KIT) with SilFlex tubing (World Precision Instruments, SILFLEX-2), was inserted through the corneal incision while avoiding the lens, and advanced into the subretinal space. Each mouse received a 1-μl injection per eye, and volume and rate were controlled with a UMP3 Ultra Micro Pump (World Precision Instruments, UMP3-4). After surgery, the mice were placed on a heating pad, and anesthesia was reversed with intraperitoneal atipamezole (2.5 mg kg−1, MWI Animal Health #032800). Triple antibiotic ophthalmic ointment (neomycin, polymyxin, and bacitracin, AmerisourceBergen 044525) was administered to the cornea to promote recovery.

Two-photon imaging of RPE and photoreceptor transfection in mouse eyes
After euthanasia of the mice, their intact enucleated eyes were submerged in room temperature PBS. Pulsing IR light from a Ti:sapphire laser (Coherent, Vision S, Santa Clara, CA; tunable between 690 and 1050 nm) was set to 950 nm and attenuated in a controlled, variable manner with an electro-optic modulator (EOM). To image and spectrally separate GFP and tdTomato signals, two de-scanned spectral detectors were used with their detection bandwidths set to 490–545 nm for GFP, and 590–680 nm for tdTomato. A 1.0 NA 20x Leica objective was used for the imaging50,51. Maximum projection images were generated from the collected Z-stack function of using LAX 4.7.0 and the Image J analyze particle function was used to quantify transfected cells.

Delivery of ABE in vivo
ABE was complexed with 1.1-fold excess of sgRNA in water with 10% (w/v) final sucrose at a protein concentration of 32 µM for 15 min at room temperature, diluted into TAS buffer, and kept at room temperature until injection. Right before injection, ABE RNP in TAS was added to concentrated CBB-lipidoid in DMSO or CBB-liposome in TAS, mixed by rapid pipetting, incubated with the reagents for at least 10 min at room temperature, and injected subretinally into rd12 or WT mice. The remaining reagents were then applied onto rd12 reporter cells in DMEM with 10% FBS in a 96-well plate (Fig. 5f, i).

RPE dissociation, genomic DNA and RNA, and lysate preparation
Mouse eyes were dissected under a light microscope to separate the posterior eye cup (containing RPE, choroid, and sclera) from the retina and anterior segment. Each posterior eye cup was immediately immersed in RLT Plus (Qiagen). RPE, choroid, and scleral cells were detached from the posterior eye cup by gentle pipetting, followed by removal of the remaining posterior eye cup. Cells were then processed for genomic DNA and RNA using the AllPrep DNA/RNA Micro kit according to manufacturer instructions (Qiagen 80284).

Electroretinography (ERG)
Prior to ERG recording, mice were dark adapted overnight. Under a safety light, mice were anesthetized by isoflurane (MWI, 502017) inhalation, and their pupils were dilated with topical administration of 1% tropicamide ophthalmic solution (Akorn; 17478-102-12) and 10% phenylephrine ophthalmic solution (MWI Animal Health #054243), followed by hypromellose (Akorn; 9050-1) for hydration. The mouse was placed on a heated 37 °C Diagnosys Celeris rodent-ERG device (Diagnosys LCC, Lowell, MA, USA). Ocular stimulator electrodes were placed on the corneas, the reference electrode was positioned subdermally between the ears, and a ground electrode was placed in the rear leg. The eyes were stimulated with a green-light stimulus of -0.3 log cd s m-2 (peak emission 544 nm, bandwidth ∼160 nm). The responses for 10 stimuli with an inter-stimulus interval of 10 sec were averaged, and the a- and b-wave amplitudes were acquired from the averaged ERG waveform. Data were analyzed with Espion 6.61.12 software (Diagnosys LLC).

Retinoid analysis
Mice were dark adapted for 24 h before eye enucleation. Eyes were homogenized in 1 ml of a 10 mM Na phosphate buffer (pH 8.0), containing 50% methanol (v/v) (Sigma–Aldrich; 34860-1L-R) and 100 mM hydroxylamine (Sigma–Aldrich; 159417-100G). After 15 min incubation at room temperature, 2 ml of 3 M NaCl was added. The resulting sample was extracted twice with 3 ml of ethyl acetate (Fisher Scientific; E195-4). Then, the combined organic phase was dried in vacuo and reconstituted in 300 µl hexanes. Extracted retinoids (100 µl) were separated on a normal-phase HPLC column (Zorbax Sil; 5 µm; 4.6 mm × 250 mm; Agilent Technologies) connected to an Agilent Infinity 1260 HPLC system equipped with a diode-array detector. Separation was achieved with a mobile phase of 0.6% ethyl acetate in hexane (Fisher Scientific; H302-4) at a flow rate of 1.4 ml min−1 for 17 min, followed by a step increase to 10% ethyl acetate in hexane for an additional 25 min. Retinoids were detected by monitoring absorbance at 325 nm and 360 nm, using Agilent ChemStation 11 software.

Liposome formulation
A mixture of CBB11 (0.25 µmol), ionizable lipid (2.5 µmol), DSPC (0.5 µmol, Avanti Polar Lipids), cholesterol (1.675 µmol, Millipore Sigma) and DMG-PEG2000 (0.075 µmol, Broad Pharm, San Diego, CA, USA) in ethanol/chloroform was dried with a gentle flow of nitrogen. The resulting blue film was resuspended in TAS buffer (500 µl), and sonicated for 30 min to form liposomes Lipo1, Lipo3, and Lipo5 that contained ionizable lipids SM-102 (Broad Pharm), ALC-0315 (Avanti Polar Lipids), and MC3 (Broad Pharm), respectively. Liposomes without CBB11 (Lipo2, Lipo4, Lipo6) contained additional cholesterol in place of CBB11, 1.925 µmol in total. Liposomes without ionizable lipid (Lipo7 with CBB11 and Lipo8 without CBB11) contained additional DSPC (1 µmol total in both Lipo7 and Lipo8) and cholesterol (3.675 µmol total in Lipo7, 3.925 µmol total in Lipo8) instead.

Size-exclusion chromatography
Free CBB11, Lipo1 and Lipo2 were diluted in TAS buffer to an equivalent of 80 µM CBB11 in 100 µl. Then, the samples were applied onto PD-10 desalting columns (Cytiva, 17-0851-01) equilibrated with TAS buffer, and resolved by adding 250-µl aliquots of TAS buffer with manual fractionation. The fractions were transferred to a clear-bottom 96-well plate and scanned for Coomassie fluorescence using a Bio-Rad ChemiDoc MP imager with Bio-Rad ImageLab Touch 2.4 software. The columns were also scanned for Coomassie fluorescence and photographed.

Particle-sizedistribution analysis
Particle-size distribution was measured using a Malvern Zetasizer Advance Nano instrument (Malvern Panalytical, Malvern, UK) with Malvern ZSXplorer 3.30 software. Freshly dialyzed liposomes (20 µl) were diluted to 200 µl with TAS buffer and subjected to measurement of particle-size distribution in triplicate. The particle size distributions were processed by the accompanying software to calculate average particle diameter and polydispersity index (PdI).

Cryogenic transmission electron microscopy (Cryo-TEM)
Cryo-TEM specimen was prepared by plunge-freezing in liquid ethane on a Vitrobot Mark IV (Thermo Fisher Scientific). Five microliters of the sample were applied on a lacey carbon coated grid after glow discharging in a Leica Sputter Coater ACE200 (Leica Microsystems). Excess liquid was removed with blotting of 3.0 seconds under chamber temperature of 8 °C and 100% humidity. The grids were then clipped and transferred into a Glacios TEM (Thermo Fisher Scientific) for examination. The microscope was operated at 200 kV, and images were recorded on a CETA camera (Thermo Fisher Scientific) under low-dose conditions to minimize the radiation damage on the specimen. EPU 2.11.1.11 REL (Thermo Fisher Scientific) was used to collect the data.

Next-generation sequencing
The genomic context of Rpe65 rd12 ABE editing is shown in Supplementary Fig. 16. Complementary DNA (cDNA) was synthesized from RNA with the High-Capacity RNA-to-cDNA kit (Thermo Fisher 4387406), according to manufacturer’s instructions. 0.5–1 µl of the isolated genomic DNA or cDNA was used as input for the first of two PCR reactions (PCR1). Genomic loci were amplified in PCR1, using Phusion Plus polymerase (Thermo Fisher Scientific F631S). PCR1 primers are listed in Supplementary Data 1. PCR1 was performed as follows: 98 °C for 30 sec; 30 cycles at 98 °C for 10 sec, 67 °C for 20 sec, and 72 °C for 30 sec; 72 °C for 5 min. PCR1 products were confirmed on a 2% agarose gel. One microliter of PCR1 was used as input for PCR2 to install Illumina barcodes. PCR2 was conducted for nine cycles of amplification using a Phusion HS II kit (Life Technologies). Following PCR2, samples were pooled, and gel-purified in a 1% agarose gel using a Qiaquick Gel Extraction Kit (Qiagen). Library concentration was quantified using the Qubit High-Sensitivity Assay Kit (Thermo Fisher Scientific). Samples were sequenced on an Illumina MiSeq instrument (paired-end read, read 1: 200–280 cycles, read 2: 0 cycles), using an Illumina MiSeq 300 v2 Kit (Illumina) and analyzed using Illumina MiSeq Reporter 2.6.

High-throughput sequencing data analysis
Sequencing reads were demultiplexed using the MiSeq Reporter 2.6 software (Illumina) and analyzed using CRISPResso2 2.2.7 as previously described52,53. Batch analysis mode (one batch for each unique amplicon and sgRNA combination analyzed) was used in all cases. The prompt is presented in the Supplementary Information. Reads were filtered according to minimum average quality score (Q > 60) prior to analysis. The following quantification window parameters were used: -w 20 -wc -3. Base editing efficiencies are reported as the percentage of sequencing reads containing a given base conversion at a specific position. Prism 10 (GraphPad) was used to generate the plots.

Statistics & reproducibility
No sample sizes were pre-determined. Animals were treated by litter cohorts (5-9 animals per litter, 1-2 eyes per animal). Sample sizes were 2-3 animals per group. In vitro cell studies included 2-4 replicates per experiment. Mice with failed subretinal injections were excluded on the basis of no recordable ERG a- or b-wave by a blinded observer. This exclusion criteria were pre-established. In vitro experiments were replicated 2 to 4 times independently. All animal experiments were replicated at least four times independently. In all studies, samples and animals were assigned randomly to treatment groups. Deep sequencing and ERG analysis were conducted blind. Other experiments were not conducted blindly.
No sex-based stratification or sex-based analysis was performed in this study. GraphPad Prism 10 was used to analyze the data. The data were tested for normality, and parametric one-way ANOVA was applied for datasets that passed the normality tests. The data that did not pass the normality test were analyzed with non-parametric ANOVA.

Ethics statement
All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine (protocol AUP-24-073), and were conducted in accordance with the NIH guidelines for the care and use of laboratory animals, and with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_2": "This manuscript reports the development of a new lipidoid using Coomassie Brilliant Blue (CBB) as a building block and explores its application for protein delivery, including Cre recombinase and adenine base editors (ABEs). The authors further demonstrate in vivo gene editing in mouse eyes, suggesting a potential therapeutic approach for LCA-related diseases. Functional assessments, including ERG responses and 11-cis-retinal levels, indicate improvement in mice treated with the Lipo1 formulation (SM102 doped with 10% CBB11, along with other helper lipids) compared to those treated with the Lipo2 formulation (SM102 without CBB11). On- and off-target editing events were also analyzed using NGS. Overall, the study is well executed and the data are convincing. However, my main concern lies in whether the novelty and significance of the work meet the publication standards of Nature Communications.\n\nThe rationale for developing a CBB-based lipidoid is to leverage its protein-binding ability to facilitate complexation and delivery. However, the data indicate that CBB alone is unstable, forms aggregates with proteins, and cannot be used independently for protein delivery. Instead, a conventional LNP formulation—comprising SM102, ALC-0315, or MC3 as the ionizable lipid, together with co-lipids—is still required for efficient protein (including ABE) delivery. It is well established that such LNP systems can deliver RNPs, and while the addition of CBB may enhance delivery efficiency (as shown in Fig. 6a–f), it also significantly increases particle size from ~100 nm to several hundred nanometers (Fig. 5b), suggesting reduced LNP/RNP stability. This instability could pose challenges for future clinical translation.\n\nIf CBB11 merely serves as an additional component to fine-tune LNP delivery efficiency, similar strategies have already been demonstrated, such as in the SORT technology developed by Daniel Siegwart’s group. Therefore, unless the authors can demonstrate that CBB11 forms a stable formulation with RNPs capable of achieving efficient in vivo delivery without relying on other ionizable lipids (e.g., SM102 or related molecules), the overall impact and novelty of this work appear limited.", "reviewer_4": "Please see the attached file for comments", "reviewer_5": "After evaluating the concerns raised by reviewer #3, the author’s response was sufficient to address all the relevant points. The article will be ready for publication after the minor corrections indicated below:\n\nFigure 9S panels d and e, along with f and g, require statistical descriptions and annotations to be presented. The absence of this information significantly weakens the figure's impact and prevents evaluation of the authors' claims regarding the safety and efficacy of their new Lipo1 batch. Please add the statistical test description, or at least the p-value, to the legend, and significance annotations to the graphs.\n\nFigure 9S panel c: how was mild inflammation observed in the OCT images? Please, describe.\n\nMinor errors in typing:\nLines 154, 232, 276: change “florescence” to “fluorescence”.\nLine 277: change “efeective” to “effective”."}, "author_response": "Point-by-Point Response to Reviewers:\n[Our original responses are abbreviated.]\nReviewer #2\nReviewer Comments 2.1. Overall, the study is well executed and the data are convincing. However,\nmy main concern lies in whether the novelty and significance of the work meet the publication\nstandards of Nature Communications.\n\nThe rationale for developing a CBB-based lipidoid is to leverage its protein-binding ability to facilitate\ncomplexation and delivery. However, the data indicate that CBB alone is unstable, forms aggregates\nwith proteins, and cannot be used independently for protein delivery. Instead, a conventional LNP\nformulation—comprising SM102, ALC-0315, or MC3 as the ionizable lipid, together with co-lipids—is\nstill required for efficient protein (including ABE) delivery. It is well established that such LNP systems\ncan deliver RNPs, and while the addition of CBB may enhance delivery efficiency (as shown in Fig.\n6a–f), it also significantly increases particle size from ~100 nm to several hundred nanometers (Fig.\n5b), suggesting reduced LNP/RNP stability. This instability could pose challenges for future clinical\ntranslation.\nResponse 2.1: We agree that CBB alone is not a suitable standalone vehicle for RNPs, as it forms\nlarger colloids and can aggregate with protein. Accordingly, our in vivo strategy uses CBB-doped\nliposomes (Lipo1-6), which present the CBB moiety at the bilayer surface to engage proteins while\nionizable lipids engage the guide RNA, an intended mechanism supported both by cryo-TEM (RNP\non the surface) and by a marked zeta-potential shift upon RNP addition.\nSize/stability. The reviewer’s size interpretation stems from CBB11 alone (400–500 nm). In contrast,\nCBB11-doped liposomes are ~140-160 nm and highly monodisperse, and we observed no RNP-\ninduced precipitation with Lipo1-6. Thus, adding CBB does not enlarge the liposomes to “several\nhundred nanometers” nor indicate reduced stability in our system.\nFunctional benefit and novelty. CBB-liposomes outperform control liposomes in vitro and in vivo.\nBeyond efficacy, this platform is not a conventional LNP; the promiscuous protein-binding domain\n(CBB) is the key design element, enabling surface recruitment of diverse proteins/RNPs rather than\nrelying solely on electrostatics or encapsulation.\nTranslational considerations. Our current liposome sizes already are in the 140–160 nm window\ntypical for clinically advanced nanoparticles, and size can be further tuned by standard formulation\nparameters, if needed. Importantly, this liposomal (non-microfluidic) assembly avoids low pH and high\n1\nethanol used in some RNP-LNP processes that can denature editors, and it improves precise editing\nrelative to our prior SM102-LNP ( 0.30% precise gDNA with LNP vs. ~2.7% with CBB-liposomes;\n~9-fold increase). Taken together, CBB-liposomes address the instability seen with CBB alone,\n∼\nmaintain clinically appropriate size and monodispersity, and deliver meaningful functional gains,\nthereby supporting both novelty and translational potential.\nRevision 2.1: To address this comment, we added the following sentences to Paragraph 5 of the\ndiscussion:\n“An additional practical determinant is particle size. Dynamic light scattering measurements indicated\nthat CBB11/Cre and CBB14/Cre form ~1.4–1.5 µm assemblies, with a significant fraction of CBB14-\n‑ ‑\nCre forming smaller, 160-nm particles, whereas CBB11 alone forms ~400–500 nm colloids;\nimportantly, CBB11 doped liposomes (Lipo1–6) are ~140–160 nm and highly monodisperse.\nMicrometer scale complexes are unlikely to diffuse broadly within the neural retina or traverse ocular\n‑\nbarriers from systemic delivery, whereas systemic delivery of the smaller, narrowly distributed\n‑\nliposomal formulations would be effective. Notably, despite their large hydrodynamic diameters,\nCBB11 and CBB14 still delivered Cre via subretinal injection.”\nReviewer Comments 2.2. If CBB11 merely serves as an additional component to fine-tune LNP\ndelivery efficiency, similar strategies have already been demonstrated, such as in the SORT\ntechnology developed by Daniel Siegwart’s group. Therefore, unless the authors can demonstrate\nthat CBB11 forms a stable formulation with RNPs capable of achieving efficient in vivo delivery\nwithout relying on other ionizable lipids (e.g., SM102 or related molecules), the overall impact and\nnovelty of this work appear limited.\nResponse 2.2: Our approach is not equivalent to SORT and its impact does not depend on eliminating\nionizable lipids. CBB does more than “fine-tune” delivery; it introduces a general protein-binding capture\nmodality that synergizes with ionizable lipids to efficiently deliver RNPs. To clarify the reliance of our CBB\nliposomes on ionizable lipids, we formulated DSPC-cholesterol-PEG liposomes that do not contain\nionizable lipids, with and without CBB11 (Lipo7 and Lipo8, respectively), and compared their RNP\ndelivery efficiency side-by-side with Lipo1 and Lipo2 that contain SM102. Liposomes that did not contain\nCBB11 or SM102 (Lipo8) did not aid ABE-RNP delivery into the rd12 reporter cells, as the eGFP\nfluorescence was similar as for ABE-RNP without additional reagents. Liposomes free from SM102 but\nsupplemented with CBB11 (Lipo7) delivered ABE-RNP with modest efficiency. Lipo2 (with SM102,\nwithout CBB11) also delivered the RNP, but we again observed the highest ABE-RNP delivery efficiency\nwith Lipo1 that contained both CBB11 and SM102. Our experimental results support our conclusion that\nCBB and ionizable lipids work together to achieve optimal delivery efficiency of ABE-RNP, highlighting\n2\nthe novelty and significance of this platform. This is further corroborated by our strong in vivo results,\nwhere we achieved healthier ERG responses and more precise editing compared to eVLP and lentivirus.\nImportantly, CBB and ionizable lipids work together to achieve efficient RNP delivery – CBB by binding\nABE protein, and ionizable lipids by binding guide RNA. We added the results obtained for ionizable-\nlipid-free liposomes to the new Supplementary Figure S9 and integrated the results within the\nmanuscript:\n“The RNP delivery enhancement mediated by CBB11 did not depend on the ionizable lipid, as we\nobserved similar enhancement with liposomes made with ALC-0315 and MC3 (Fig. 5g, h, & Fig. S6e),\nand with liposomes formulated without ionizable lipids (Fig. S9).”\nFig S9. Synergistic engagement of ABE-RNP by CBB liposomes. (a) Fluorescence microscopic\nquantification of delivery of ABE8e-RNP into rd12 reporter cells mediated by liposomes with CBB11\n(Lipo1, Lipo7) and SM102 (Lipo1, Lipo2). Lipofectamine 3000 (LF) was used as a positive control; buffer\n(TAS) as a negative control. Four biological replicates, mean ± S.D. (b) Representative fluorescence\nmicroscopic images of rd12 reporter cells treated with ABE8e-RNP complexed with liposomes. Scale\nbar, 200 µm.\nHow our platform differs from SORT. SORT is an mRNA/DNA LNP strategy that adds a fifth lipid to\ntune biodistribution via surface charge/composition. It does not address protein capture/display. In\ncontrast, our system presents a Coomassie-based, promiscuous protein-binding domain (CBB) on a lipid\nbilayer to recruit protein or RNP cargo at the vesicle surface under mild, neutral conditions. Thus, SORT\nmodifies where nucleic-acid LNPs go; our platform changes what and how cargo is loaded and delivered.\nStability and size. Whereas CBB alone forms heterogeneous colloids, CBB-doped liposomes are ~140–\n160 nm, exhibit a low-PdI, and do not precipitate upon RNP loading. ζ-potential shifts and cryo-TEM are\nconsistent with RNP association at the vesicle surface, and the complexes drive robust in vivo editing\nand ERG recovery (Fig. 5d–e; Fig. 6). Functionally stable performance in vivo, together with the absence\nof precipitation in formulation, addresses the reviewer’s stability concern.\nTranslational advantage. Unlike low-pH/ethanol LNP assembly, which can compromise editor integrity,\nour neutral, ethanol-free mixing preserves RNP activity and can be prepared from two stable\n3\ncomponents immediately prior to use (analogous to standard transfection reagents).\nThese features of our CBB liposome delivery system are presented in the introduction and discussion.\nRevised introduction:\n“After initial success, we further refined our approach by incorporating the CBB lipidoid into liposomes to\nachieve efficient editing in an in vivo inherited retinal disease (IRD) mouse model. Our approach is\ndistinct from LNPs, as the CBB liposomes are formed without protein and are used as a neutral, aqueous\nsuspension to complex the cargo in neutral pH and without organic solvents. Our CBB lipidoids engage\nin specific CBB-protein interactions, and thus they can be used for delivery of all types of proteins, with a\npotential to perform genome editing using recombinases (Cre, Bxb1), genome-editing proteins (zinc\nfinger nucleases, TALENs), and genome-editing RNPs (Cas9, ABE, PE, and others). We maximized the\npotential of CBB liposomes for RNP delivery by incorporating ionizable lipids to achieve synergistic\ncomplexing of the RNPs via CBB-protein and ionizable lipid-guide RNA interactions. Incorporating CBB\ninto liposomes enhanced RNP-mediated genome editing, allowing to reach clinically significant recovery\nof visual function and editing in the genome. This outcome reinforces our guiding principle that\nspecialized delivery agents, including our novel CBB lipidoids, are essential for achieving the RNP-\nmediated editing efficiency necessary for further clinical translation.”\nRevised discussion:\n“Several of the CBB-lipidoids substantially enhanced the delivery of both Cre recombinase and ABE\nproteins to mammalian cells in vitro and in vivo. Particularly striking was the high efficacy observed when\nthese compounds were formulated into ionizable cationic liposomal particles to improve the colloidal\nstabilities of the CBB-protein complexes. We designed our CBB liposomes with a goal of maintaining\nenzymatic activity of ABE RNP by minimizing denaturation. This imposed a requirement to avoid acidic\npH and ethanol, both of which are crucial for achieving efficient encapsulation of nucleic acids and RNPs\nwithin the LNPs. As a first step, we prepared lipid films containing all the lipid constituents: liposome\nscaffold of DSPC, cholesterol, and DMG-PEG 2000, ionizable lipid (SM102, ALC-0315 or MC3) to bind\nguide RNA, and CBB11 to bind protein. Next, instead of dissolving the film in ethanol and complexing the\nRNP in microfluidic system, we formed the liposomes in our mild dispersant, TAS buffer, and made\nliposome-RNP complexes by mixing in neutral, aqueous environment of TAS. This procedure, which\nresembles traditional transfection, led to adsorption of the RNP on the liposome surface. This enzyme-\nfocused approach led to striking editing efficiencies and clinically relevant restoration of vision in mice.\nFor instance, Lipo1 containing 5% CBB11 achieved up to 9.9% genomic and 28.8% transcript-level edits\nof the Rpe65 mutation in rd12 mice, a correction sufficient to restore vision to an estimated 76% of the\nwild-type ERG response (Fig. 6). These results surpass those reported for virus (AAV, LV) or virus-like\nparticle (VLP) delivery systems.34, 35, 40-42”\nReviewer 4 Comment: Firstly, I would like to compliment the authors on the amount of effort that they\nhave shown in addressing the original comments of Reviewer 1. Especially the addition of multiple in\nvivo experiments is laudable. I will address all author comments per point below. However, there are\na few comments that have not been fully addressed / resolved, as discussed below.\nResponse to Reviewer 4 Comment: We are very grateful for the reviewer’s comments and time\ninvested in our manuscript, undoubtedly leading to improvement. We have eliminated all comments\nwhere the reviewer approved our changes to the manuscript. We address the new comments below.\nOriginal Reviewer Comment 1.1: All in vitro results were done at a single concentration of the\nCBB derivatives and no dose-response curves are given… The optimal dose therefore needs to be\nestablished for each reagent, separately.\nOriginal Response 1.1: Our previous one-point analysis was based on optimized conditions, and we\nwould argue still valid. Nevertheless, we performed dose-response analyses as recommended (see\nFig. S2). In the revised manuscript, we now provide full in vitro dose-response curves (1–8 µM CBB)\nfor the nine CBB derivatives (CBB2, 7, 11, 12, 13, 14, 18, 19, and Z5) with Cre recombinase, or (0.2–\n1.6 µM CBB) with ABE8e-RNP, now included.\nReviewer Comment 4.1: Originally, reviewer 1 was somewhat vague in their wording, and their\nrequest could either be interpreted as a request to provide a dose-response curve in terms of the\nabsolute amount that would be used for transfection, or an optimization of CBB-to-cargo ratio. Here,\nthe authors opted to focus on a ratio optimization for all CBBs, which I agree is the best (and most\nrelevant) answer to the final statement by reviewer 1 “The optimal dose therefore needs to be\nestablished for each reagent, separately.” It is furthermore additionally appreciated that this was done\nfor both Cre recombinase and ABE. As such, I feel that these experiments are suitable to address the\ncomment of Reviewer 1.\nHowever, in this case the authors have performed the experiment twice (n=2). Whereas the data show\nhigh reproducibility and a robustness in the observed patterns, I would like to point out the following:\nPlease be aware that the Nature Portfolio checklist contains the following statement/guideline: “We\nstrongly discourage deriving statistics from technical replicates or less than 3 biological replicates,\nunless there is a clear scientific justification for why providing this information is important.”\nEspecially considering these experiments are in vitro assays, I would unfortunately request the\nauthors to perform this experiment once more, unless (as is in line with the Nature Portfolio\nguidelines) “there is a clear scientific justification for why providing this information is important”.\nResponses and Revisions Reviewer 4.1. Thank you for the comment. We repeated the titrations for\nCre and ABE with four replicates. In this new experiment, Cre delivery efficiency was comparable,\nwhereas ABE was this time not efficiently delivered into the rd12 reporter cells by CBB12. We replaced\nthe Figure S2 with the new data presented as separate data points.\nFig S2. Concentration dependence of the in vitro delivery of gene editors mediated by\nrepresentative CBB compounds. (a) Quantification by fluorescence microscopic imaging of GFP-to-\nRFP conversion in CS cells. Cells were treated with Cre recombinase (0.5 µM) complexed with CBB\ncompounds at increasing concentrations (1-8 µM). The cells are green at baseline and red after editing.\nData are presented as mean ± S.D.; n = 2. (b) Quantification by fluorescence microscopic imaging of the\nintensity ratio of eGFP to mCherry in rd12-reporter cells treated with ABE-RNPs (100 nM) complexed\nwith CBB compounds at increasing concentrations (0.2-1.6 µM). Data are presented as mean ± S.D.; n =\n2.\nCurrent version:\nFig S2. Concentration dependence of the in vitro delivery of gene editors mediated by\nrepresentative CBB compounds. (a) Quantification by fluorescence microscopic imaging of GFP-to-\nRFP conversion in CS cells. Cells were treated with Cre recombinase (0.5 µM) complexed with CBB\ncompounds at increasing concentrations (1-8 µM). The cells are green at baseline and red after editing.\nMean ± S.D., 4 biological replicates. (b) Quantification by fluorescence microscopic imaging of the area\nratio of eGFP to mCherry in rd12-reporter cells treated with ABE-RNPs (100 nM) complexed with CBB\ncompounds at increasing concentrations (0.2-1.6 µM). Data are presented as mean ± S.D.; n = 4\nbiological replicates.\nOriginal Reviewer Comment 1.2: The in vivo data focus on base editing results, but not so much on\ntoxicity and off-target editing… I would at least expect to see some evidence that toxicity to the retina\nis absent or limited at the tested concentrations.\nOriginal Response 1.2: To evaluate the safety of the ABE-RNP delivery to the retina mediated by Lipo1,\nwe also administrated ABE-RNP-Lipo1 complex to WT mice (Fig. S9) via subretinal injection. Since the\noxidation of CBB11 might induce epoxides in the liposome, only freshly formulated Lipo1 was\nadministered to the WT mice. Different batches of Lipo1 showed similar morphology and comparable\nABE-RNP delivery efficiency to rd12 reporter cell lines (Fig. S9a, b). The Optical Coherence Tomography\n(OCT) imaging indicated no substantial alterations to the retinal structure of WT mice administered the\ncomplex of RNP (20 μM) and Lipo1; except mild inflammation, which could be ameliorated substantially\nby lowering the RNP concentration (Fig. S9c). The ERG response and the generation of the visual\nchromophore, 11-cis retinal, of WT mice treated with Lipo1 and RNP (10 μM) were also comparable to\nthose of untreated WT mice (Fig. S9d, e). In parallel experiments in which rd12 mice were treated with\nthe same batch of Lipo1 and RNP complex, the subretinal injections of 1 μl of Lipo1 with 10 μM RNP\nachieved an optimal ERG response and visual chromophore recovery (Fig. S9d, e), comparable to those\nof rd12 mice treated with Lipo1-ABE-RNP (20 μM) (Fig. 6). These results suggest that optimal vision-\nfunction rescue requires a balance between the biological tolerance of the cells and the editing efficiency\nof the gene editors.\nOff-target editing–Targeted deep sequencing of the ten highest-ranked off-target sites (identified\nusing Cas-OFFinder) shows a background-level A•T→G•C substitution rate comparable to that of\nuntreated animals. Additional data on off-target analyses are now included.\nReviewer Comment 4.2: Once more, I feel that the authors have chosen suitable experiments to\naddress the points raised by reviewer 1. Whereas n=2 replicates for Fig S9B, I feel that this is justified\nin this case due to the robustness of the data, as well as the ethical implications to minimize the\namount of animals. Data is clear, methods are described well, conclusions in the main text are\nappropriate.\nOne request: I do not have access to the Supplementary Tables, so I’m not sure if all OT genomic\nsequences / analysis sequences are listed. As such, I would like to request to please make sure to\ninclude the analyzed Off-targeted sequence (+genomic location), as well as potential relevant\nprimer/probe sequences used for OT analysis (either to the methods section, or the Supplementary\nfiles).\nResponses and Revisions Reviewer 4.2: Thank you for the comment. We have included the\nprotospacer and guide RNA sequences, as well as genetic loci, in Supplementary Table 1. The\nsequencing data were deposited in the Sequencing Read Archive.\nReviewer Comment 1.3: Some claims are made in the discussion that are not covered by the data\npresented (toxicity, RNP encapsulation, immunogenicity). Better to remove such unsubstantiated claims.\nOriginal Response and Revision 1.3: Thank you for bringing up this important point. We have\nremoved unsubstantiated statements regarding toxicity (see Response 1.2), immunogenicity, and full\n\"encapsulation\". All remaining claims are now directly supported by new data (toxicity, off-target\nanalysis) or by published references cited in the Discussion.\nReviewer Comment 4.3: The changes are appreciated. Indeed, published references can be used to\nsupport claims on safety as well. In line with this, I would request adding a reference that shows\ndecreased bystander editing upon short-term ABE delivery (ie. as RNP) to the statement “In general, the\ndelivery of ABE-RNPs mediated by CBB-liposomes could be considered as a more transient mode of\ndelivery of gene-editing tools, consistent with decreased bystander editing”.\nAlso, please add a reference to the statement “small particles would better penetrate the retina and\nmake the therapeutic agent available for cellular uptake”.\nResponses and Revisions Reviewer 4.3: Thank you for the comment. In our previous study, we found\nthat ABE8e-RNP delivery in vitro via Lipofectamine 3000 or lipid nanoparticles led to more precise\nediting than plasmid delivery,1 (Figure 4, new panel “o,” shown below). In another study, we found that\nABE delivered as RNP encapsulated into virus-like particles (eVLPs) led to lower bystander and off-\ntarget editing than viral delivery via AAV and LV in vivo.2 We added the references to the manuscript.\nRegarding the tissue permeation, small protein GCAP1 whose diameter was 6.5 – 8.7 nm, permeated\nthe retina more efficiently than GCAP1 complexed with liposomes (size range 150-170 nm).3 Our own\ndata do not provide sufficient evidence to support the statement of enhanced penetration of the retina by\nsmaller particles; therefore, we removed that statement.\n[Figure Redacted]\n“Fig. 4 | Lipid nanoparticle delivery of ABE RNPs. …. o, Next-generation sequencing analysis\nof ABE-editing outcomes in the cells with cDNA encoding Rpe65 rd12, treated with ABE delivered on a\nplasmid with Lipofectamine 3000 (L3k) or as LNP. Three analytical replicates, mean ± S.D.”1\nReviewer Comment 1.4: From Cre recombinase to base editors. A logical step in between could have\nbeen CRISPR-Cas9 based gene correction. Did you try this as well?\nOriginal Response 1.4: Thank you for the suggestion. Cas9 in the eye causes severe off-target\n1\neffects and apoptosis. Therefore, we deliberately applied our CBB strategy to an adenine base\neditor (ABE) rather than to a Cas9 nuclease, since ABEs offer superior therapeutic promise. This\nchoice is further validated by recent clinical successes, most notably a personalized ABE-based\ngene therapy administered to an infant patient, which underscores the clinical relevance of base\n2\nediting over nuclease-driven approaches.\nReviewer Comment 4.4: This is a valid explanation, and as reviewer 1 did not request any additional\ndata or experiments this answer suffices. However, readers may from outside the ocular delivery field\nmay have similar questions. As such, I would request that this is briefly explained/addressed in the\ndiscussion as well.\nResponse and Revision 4.4: We added the following sentences to the Discussion, paragraph 2:\n“While CRISPR-Cas9 nuclease RNPs could be viewed as a logical intermediate between Cre and base\neditors, we prioritized ABE for in vivo studies because in comparison with Cas9 nuclease, ABE has the\nadvantage of catalyzing single-base transitions without creating double strand breaks, with equivalent\npotential for RNP delivery as Cas9. The molecular interactions that we engage to deliver ABE RNP are\napplicable to other RNPs, and thus we envision that our CBB liposomes are applicable to other CRISPR\nnucleases and precise editors as well.”\nReviewer Comment 1.5: CBB dyes have denaturing capacities. Do you see any reduction in activity\nof Cre recombinase or base editors in the presence (without encapsulation)?\nOriginal Response 1.5: Coomassie Brilliant Blue (CBB) dyes indeed show higher affinity for\npartially unfolded proteins (e.g., Triton-X100 increases binding of Coomassie G250 in the Bradford\n3\nassay ), yet decades of successful non-denaturing affinity-chromatography with structurally similar\ncompatible with native enzyme function (Fig 4e). The data below indicate that CBB11 and CBB14 do\nnot affect the activity of ABE.\nReviewer Comment 4.5: Regarding the schematic of Figure 4e: the ABE8e editing window tends to\nspan between spacer nucleotides 3/4 – 8. As such, would the following underlined A not also be\n9\nedited? GACCGTCAGAGGAGACTACA. Please check, and if indeed so, correct in the schematic.\nResponse and Revision 4.5: Thank you for pointing this out. In fact, adenines at positions 3 and 8 are\noften edited alongside the target adenine at position 6. The figure has been corrected to reflect this fact.\nFigure 4. Delivery of ABE-RNPs to rd12-reporter cells, mediated by CBB lipidoids. (…) (e)\nSchematic diagram of the ABE-activity assay. EndoV = endonuclease V. Blue, SpCas9 PAM; red, target\nbase; orange, bystander base; arrowhead, nick site; FAM, fluorescein. (f) Representative urea-PAGE-gel\nimage of products of ABE deamination cleaved by EndoV. The gel was imaged with a fluorescein filter. N,\nno enzyme; P, positive control: 60 bp DNA with inosine in the middle; A, ABE-RNP in TAS; D, ABE-RNP\nin TAS with10% DMSO. Numbers indicate molar excess of the CBB compound versus ABE. (g)\nFluorescence quantification of substrate and products of deamination and EndoV cleavage. Two\nindependent samples per data point were assayed in parallel.\nReviewer Comment 1.11: In all formulations DMSO is present at ~10%. Dialysis could have easily\nremoved it… Why not done?\nOriginal Response 1.11: We removed the solvent by dialyzing the complex of ABE-RNP and CBB11\nin TAS buffer with 10% DMSO through a 7000 MWCO membrane. The CBB11 amount in the\nresulting solution was quantified by LC-MS. The results indicated that most of the CBB11 (95.3%) was\nlost, suggesting that the CBB binding to protein is reversible, which helps support our stated goal of\nidentifying molecules that would possess transient non-specific protein binding to assist in delivery. It\nshould also be noted that DMSO is tolerated by the eye, and DMSO is present in some formulations.\nclinical applications. We have added a sentence to the Results conveying more clearly the utility and\nsafety of including DMSO in the formulation.\nReviewer Comment 4.11: Once more, this fully addresses the point raised by Reviewer 1. However,\nas other readers may have similar questions, and it appears that not removing DMSO is of pivotal\nimportance for the stability and downstream applications of these formulations, this information seems\nof high importance to the readers as well. Especially as it may not be directly clear that currently\nDMSO is therefore a component of the final formulation, which is of high relevance for potential\napplications.\nWhereas additional experiments / data that shows stability of specific CBBs are not required to\ndiscuss this issue, it is a potential limitation that should be discussed. Please add a section to the\ndiscussion section that underlines the importance of DMSO in the discussion. (indeed, this is not\nproblematic for a variety of clinical formulations, but this could of course also be added to the\ndiscussion if the authors would desire to do so).\nResponse and Revision 4.11: Thank you for the suggestion. We have added the following text to\nthe discussion:\n“The CBB-RNP complex contained 7.25% DMSO, which was well tolerated by ABE and as such\nmay be further explored as a vehicle for protein delivery.”\nReviewer Comment 1.13: What are the size of these CRE/CBB complexes?\nOriginal Response and Revision 1.13. Dynamic light scattering indicated that the size of the\nCre/CBB11 complex is 1400 nm and the size of the Cre/CBB14 complex is 1500 nm. These values\nare now included in the Result section.\nReviewer 4.13: Thank you for including this new data. As these particles are this large, this has\nimplications for potential suitability for specific in vivo applications, which should be discussed.\nResponse and Revision 1.13: Thank you for the comment. We cannot draw conclusions about\nimplications of charge and hydrophobicity of the Cre-CBB complexes on the in vivo efficacy, as both\nCBB11 and CBB14 effectively delivered Cre in vivo despite the higher hydrophobicity of CBB11. We\nrephrased this part of the Results section for clarity and added the following section to the discussion:.\n“An additional practical determinant is particle size. Dynamic light scattering measurements indicated\nthat CBB11/Cre and CBB14/Cre form ~1.4–1.5 µm assemblies, with a significant fraction of CBB14-\n‑ ‑\nCre forming smaller, 160-nm particles, whereas CBB11 alone forms ~400–500 nm colloids;\nimportantly, CBB11 doped liposomes (Lipo1–6) are ~140–160 nm and highly monodisperse.\nMicrometer scale complexes are unlikely to diffuse broadly within the neural retina or traverse ocular\n‑ 11 ‑\nbarriers from systemic delivery, whereas systemic delivery of the smaller, narrowly distributed\n‑\nliposomal formulations would be effective. Notably, despite their large hydrodynamic diameters,\nCBB11 and CBB14 still delivered Cre via subretinal injection.”\nOriginal Reviewer Comment 1.15: Please show experimental evidence that CBB11 is in fact\nincorporated into the liposomes.\nOriginal Response 1.15: We have performed experiments related to the solubility of CBB11 and the\nliposome (Lipo 1). Unconjugated CBB11 is mostly insoluble, but when mixed with lipids it is retained\nin the suspension (see below). Please also see relevant response to Comment 3.2.\nResponse Figure 2: CBB11 (40 µM) in TAS with 10% DMSO or Lipo1 (containing 40 µM CBB11) in\nTAS was dialyzed overnight against TAS buffer, followed by centrifugation at 5000 rpm for 5 min. The\nCBB11 in the supernatant was quantified via LC-MS. The data suggest that formulation with the\nliposome prevented CBB11 aggregation. Revisions in Discussion part:\nParticularly striking was the high efficacy observed when these compounds were formulated into\nionizable cationic liposomal particles to improve the colloidal stabilities of the CBB-protein complexes.\nThese facts indicated that the protein binding on the surface of Lipo1 was intensified by CBB11. As\nexpected, this enhanced liposome-protein interaction boosted intracellular protein delivery.\nComments Reviewer 4.15 Whereas the comparison of a control lipid to CBB11 in context of\nliposomes in Fig 5D is an interesting experiment, it does not fully answer the question of reviewer 1.\nThe question specifically states “: Please show experimental evidence that CBB11 is in fact\nincorporated into the liposomes”. However, the data shown in Figure 5d could also be explained by\ndirect nanocomplexation of ABE8e-RNP with CBB11. Response Figure 2 is a more conclusive / direct\nread-out for liposomal incorporation (especially since it’s LC-MS based analysis, which is a more\ndirect read-out).\nTo answer this question as the authors appear to have access to LC-MS for formulation analysis, I\nwould request that the authors incorporate data to the manuscript that directly confirms CBB11\nintegration to answer this comment.\nGeneral comment, based on these replies: please avoid the use of RPM in methods section, as the\nrcf (g) can differ dramatically based on the rotor diameter. Please check throughout the manuscript\nthat all mentions of centrifugation speeds are shown in rcf (g), and not in RPM.\nResponse and Revision 4.15: Thank you for your comment. To directly answer the question of\nincorporation of CBB11 into the liposomes, we decided to perform gel filtration. We used PD10 desalting\ncolumns with a Sephadex G-25 resin. We traced the elution of CBB11 by fluorescence. We found\nfluorescence signals originating from CBB at elution volume (3-3.5 ml) corresponding to Lipo1 (Fig.\nR1a). As expected, we did not observe CBB fluorescence at the elution volume for Lipo2 (negative\ncontrol without CBB11) or in the eluate for the column containing uncomplexed CBB11. Visually, we\nfound that free CBB11 was retained in the column (Fig. R1b-3), and fluorescence scans of the columns\nconfirmed the retention of the free CBB11 in the column (Fig. R1c-3).\nFigure R1: Gel filtration of CBB-liposomes. (a) Coomassie fluorescence scan of a 96-well plate\ncontaining samples collected from the PD10 desalting column. V = elution volume. (b) A photograph of\nE\nthe PD10 desalting columns after the gel filtration. (c) Coomassie fluorescence scan of the PD10\ndesalting columns after the gel filtration.\nResponse to General comment: We corrected the revolutions per minute to g throughout the manuscript,\nwhere applicable. In some instances (sample dissolution in bench top thermomixer, microbial shakers)\nwe kept rpm, a customary measure of mixing speed.\nReviewer Comment 1.18: P9L299: ‘encapsulation within the particle…’ if you refer to a new method,\nclarify or remove.\nOriginal Response and Revision 1.18: We now clarify that ABE-RNP predominantly adsorbs to,\nrather than being fully encapsulated by, the lipid corona; the text has been reworded accordingly.\nReviewer Comment 4.18: Thank you for explaining this. This is now in line with the TOC graphic.\nHowever, if possible, please make sure to include a bit more clarity to the TOC graphical abstract:\nright now it is implied that the H+ is present in the cytosol, rather than the endocytosis pathway.\nResponse and Revision 1.18: Thank you for the advice. We expanded the TOC graphic to present the\nconcept of endosomal escape triggered by acidification of the endosome. The TOC graphic has been\nmodified accordingly, and the new version is included in the revised manuscript.\ndelivery of genome-editing ribonucleoproteins (RNPs) mediated by Coomassie Brilliant Blue\n(CBB) liposomes. The fundamental design principles for a CBB-containing delivery agent include: 1) the\nCBB protein-binding domain; 2) an ionizable-linker domain; and 3) a lipid or lipidoid domain. The CBB\ncompounds are formulated into liposomes comprising ionizable lipid (orange), phospholipid (grey),\ncholesterol (red) and PEG lipid (light blue); and bind RNP on the liposome surface in aqueous, neutral\nenvironment. The RNP-liposome complex is absorbed by the cells via an endocytosis pathway.\nSubsequent protonation of the ionizable CBB-delivery agents and ionizable lipids releases the tagged\nproteins from the particles, enabling their intracellular transport to the nucleus to edit the targeted DNA\nsequence.\nReviewer Comment 5.1: After evaluating the concerns raised by reviewer #3, the author’s response\nwas sufficient to address all the relevant points. The article will be ready for publication\nafter the minor corrections indicated below:\nFigure 9S panels d and e, along with f and g, require statistical descriptions and annotations to be\npresented. The absence of this information significantly weakens the figure's impact and prevents\nevaluation of the authors' claims regarding the safety and efficacy of their new Lipo1 batch. Please add\nthe statistical test description, or at least the p-value, to the legend, and significance annotations to the\ngraphs.\nResponse and Revision 5.1: Thank you for the time and effort. These suggestions have clearly\nimproved the manuscript. Specific to Figure S9 (now Figure S11), the text has been corrected and\nstatistical test descriptions added, as shown below.\nFigure S11. Evaluation of the safety and efficacy of a new batch of Lipo1. Four-week-old wild type\n15\n(blue) and rd12 mice (green) were administered ABE8e-RNPs with/without fresh Lipo1 via subretinal\ninjections. (a) The particle size of the fresh Lipo1 preparation (green) was 148 nm, with PDI = 0.23; this\nwas similar to that of the stored sample (yellow), size =140 nm, PDI = 0.16. (b) Fluorescence\nmicroscopic quantification of the ratio of eGFP to mCherry for rd12 reporter cells, treated with the fresh\nLipo1-RNPs (100 nM). Lipofectamine 3000 (LF, purple) served as positive control, and ABE8e-RNPs\ndelivered without vehicles (A, gray) served as negative control; [CBB]/[RNP] ratio = 8:1. Data are\npresented as mean ± S.D., n = 2 biological replicates. (c) Representative OCT images of WT mice\ntreated with 20 µM ABE-RNPs alone (R20), with the complex of 10 µM RNPs plus Lipo1 containing 80\nµM CBB11 (L80R10), with Lipo1 containing 160 µM CBB11 (L160), or with the complex of 20 µM RNPs\nwith Lipo1 containing 160 µM CBB11 (L160R20); WT mice without treatment served as control (WT N).\nThe injection volume was 1 µl. (d) b-wave amplitude quantification of ERG responses of WT mice whose\nOCT scans are shown in panel c; data are presented as mean ± S.D., n =6 eyes. Ordinary one-way\nANOVA with Šídák's multiple comparisons test, **, P < 0.01. (e) Quantification of 11-cis-retinal levels in\nWT mice whose ERG responses are presented in panel d; data are presented as mean ± S.D., n = 5-6\neyes. Kruskal-Wallis test with Dunn's multiple comparisons test, **, P < 0.01. (f) Quantification of b-wave\namplitudes of ERG responses of rd12 mice treated with 0.5 µl of L80R10 (light green); 1 µl of L80R10\n(green); or 1 µl of L160R20 (dark green). Untreated WT mice (WT N, black) and rd12 mice (rd12 N, gray)\nserved as positive and negative controls, respectively; data are presented as mean ± S.D., n = at least 4\neyes. Ordinary one-way ANOVA with Šídák's multiple comparisons test, **, P < 0.01, ****, P < 0.0001.\n(g) Quantification of 11-cis-retinal levels in rd12 mice whose ERG responses are presented in panel f;\ndata are presented as mean ± S.D., n = at least 5 eyes. Kruskal-Wallis test with Dunn's multiple\ncomparisons test *, P < 0.05.\nReviewer Comment 5.2: Figure 9S panel c: how was mild inflammation observed in the OCT images?\nPlease, describe.\nResponse and Revision 5.2: The text has been revised and referenced as follows.\n“The optical coherence tomography (OCT) imaging indicated no substantial alterations to the retinal\nstructure of WT mice administrated the complex of RNP (20 μM) and Lipo1. We noticed the presence of\nhyperreflective spots visible in the vitreous, indicating possible inflammation,1 which could be ameliorated\nby lowering the RNP concentration (Fig. S1c)”\nReviewer Comment 5.3: Minor errors in typing:\nLines 154, 232, 276: change “florescence” to “fluorescence”.\nLine 277: change “efeective” to “effective”.\nResponse and Revision 5.3: The text has been corrected.\nREFERENCES\n1. Holubowicz, R. et al. Safer and efficient base editing and prime editing via ribonucleoproteins\ndelivered through optimized lipid-nanoparticle formulations. Nat Biomed Eng9, 57-78 (2025).\n2. Banskota, S. et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic\nproteins. Cell185, 250-265 e216 (2022).\n16\n3. Asteriti, S. et al. Recombinant protein delivery enables modulation of the phototransduction\ncascade in mouse retina. Cell Mol Life Sci80, 371 (2023).\n4. Liu, H., Zhu, Y., Li, M. & Gu, Z. Precise genome editing with base editors. Med Rev (2021)3, 75-84\n(2023).\n5. Xu, W., Zhang, S., Qin, H. & Yao, K. From bench to bedside: cutting-edge applications of base editing\nand prime editing in precision medicine. J Transl Med22, 1133 (2024).\n6. Hoang, C. et al. Application of Dimethyl Sulfoxide as a Therapeutic Agent and Drug Vehicle for Eye\nDiseases. J Ocul Pharmacol Th37, 441-451 (2021).\n17"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have done a good job in addressing the reviewers' concerns. the manuscript is significantly improved. i recommend its acceptance for publication.", "reviewer_4": "Please see the attached file for my comments."}, "author_response": "Reviewer #4\nOnce more, I’d like to compliment the authors on their efforts to address all comments. With these current\nrevisions, I feel that the concerns of reviewer 1 have been sufficiently addressed (see below – comments\nto revisions are highlighted in yellow). However, I do have one request for one minor revision: given the\nvalue and clarity of the results shown in figure R1, I feel that these data belong in the Supplementary\nInformation file, rather than solely as data presented to the reviewers. Furthermore, I have pointed out\ntwo small language suggestions for the figure legend of S2. Once these minor revisions are adjusted, I\nwould recommend the acceptance of this manuscript for publication.\nThank you for your compliment. Our responses are below in blue and italic font.\nReviewer Comment 4.1: Originally, reviewer 1 was somewhat vague in their wording, and their\nrequest could either be interpreted as a request to provide a dose-response curve in terms of the\nabsolute amount that would be used for transfection, or an optimization of CBB-to-cargo ratio. Here,\nthe authors opted to focus on a ratio optimization for all CBBs, which I agree is the best (and most\nrelevant) answer to the final statement by reviewer 1 “The optimal dose therefore needs to be\nestablished for each reagent, separately.” It is furthermore additionally appreciated that this was done\nfor both Cre recombinase and ABE. As such, I feel that these experiments are suitable to address the\ncomment of Reviewer 1.\nHowever, in this case the authors have performed the experiment twice (n=2). Whereas the data show\nhigh reproducibility and a robustness in the observed patterns, I would like to point out the following:\nPlease be aware that the Nature Portfolio checklist contains the following statement/guideline: “We\nstrongly discourage deriving statistics from technical replicates or less than 3 biological replicates,\nunless there is a clear scientific justification for why providing this information is important.”\nEspecially considering these experiments are in vitro assays, I would unfortunately request the\nauthors to perform this experiment once more, unless (as is in line with the Nature Portfolio\nguidelines) “there is a clear scientific justification for why providing this information is important”.\nResponses and Revisions Reviewer 4.1. Thank you for the comment. We repeated the titrations for\nCre and ABE with four replicates. In this new experiment, Cre delivery efficiency was comparable,\nwhereas ABE was this time not efficiently delivered into the rd12 reporter cells by CBB12. We replaced\nthe Figure S2 with the new data presented as separate data points.\nFig S2. Concentration dependence of the in vitro delivery of gene editors mediated by\nrepresentative CBB compounds. (a) Quantification by fluorescence microscopic imaging of GFP-\ntoRFP conversion in CS cells. Cells were treated with Cre recombinase (0.5 µM) complexed with CBB\ncompounds at increasing concentrations (1-8 µM). The cells are green at baseline and red after editing.\nData are presented as mean ± S.D.; n = 2. (b) Quantification by fluorescence microscopic imaging of the\n1\nintensity ratio of eGFP to mCherry in rd12-reporter cells treated with ABE-RNPs (100 nM) complexed\nwith CBB compounds at increasing concentrations (0.2-1.6 µM). Data are presented as mean ± S.D.; n =\n2.\nCurrent version:\nFig S2. Concentration dependence of the in vitro delivery of gene editors mediated by\nrepresentative CBB compounds. (a) Quantification by fluorescence microscopic imaging of GFP-\ntoRFP conversion in CS cells. Cells were treated with Cre recombinase (0.5 µM) complexed with CBB\ncompounds at increasing concentrations (1-8 µM). The cells are green at baseline and red after editing.\nMean ± S.D., 4 biological replicates. (b) Quantification by fluorescence microscopic imaging of the area\nratio of eGFP to mCherry in rd12-reporter cells treated with ABE-RNPs (100 nM) complexed with CBB\ncompounds at increasing concentrations (0.2-1.6 µM). Data are presented as mean ± S.D.; n = 4\nbiological replicates.\nWith these new data, this point has been sufficiently addressed. Please make the following corrections in\nthe figure legend: “GFP-toRFP conversion” should be “GFP-to-RFP”. Furthermore “the cells are green at\nbasaeline and red after editing” is too colloquial in its phrasing – please include “fluorescence in this\nphrasing.\nThank you for accepting our revision. This Figure is now Supplementary Figure 4. We changed the axis\nlabel accordingly, and we changed the phrasing of the figure legend to “The cells emit green\nfluorescence at baseline and red fluorescence after editing.”.\nOriginal Reviewer Comment 1.15: Please show experimental evidence that CBB11 is in fact\nincorporated into the liposomes.\nOriginal Response 1.15: We have performed experiments related to the solubility of CBB11 and the\nliposome (Lipo 1). Unconjugated CBB11 is mostly insoluble, but when mixed with lipids it is retained\nin the suspension (see below). Please also see relevant response to Comment 3.2.\nTAS was dialyzed overnight against TAS buffer, followed by centrifugation at 5000 rpm for 5 min. The\nCBB11 in the supernatant was quantified via LC-MS. The data suggest that formulation with the liposome\nprevented CBB11 aggregation. Revisions in Discussion part:\nParticularly striking was the high efficacy observed when these compounds were formulated into\nionizable cationic liposomal particles to improve the colloidal stabilities of the CBB-protein complexes.\nThese facts indicated that the protein binding on the surface of Lipo1 was intensified by CBB11. As\nexpected, this enhanced liposome-protein interaction boosted intracellular protein delivery.\nComments Reviewer 4.15 Whereas the comparison of a control lipid to CBB11 in context of\nliposomes in Fig 5D is an interesting experiment, it does not fully answer the question of reviewer 1.\nThe question specifically states “: Please show experimental evidence that CBB11 is in fact\nincorporated into the liposomes”. However, the data shown in Figure 5d could also be explained by\ndirect nanocomplexation of ABE8e-RNP with CBB11. Response Figure 2 is a more conclusive / direct\nread-out for liposomal incorporation (especially since it’s LC-MS based analysis, which is a more\ndirect read-out).\nTo answer this question as the authors appear to have access to LC-MS for formulation analysis,\nI would request that the authors incorporate data to the manuscript that directly confirms CBB11\nintegration to answer this comment.\nGeneral comment, based on these replies: please avoid the use of RPM in methods section, as the\nrcf (g) can differ dramatically based on the rotor diameter. Please check throughout the manuscript\nthat all mentions of centrifugation speeds are shown in rcf (g), and not in RPM.\nResponse and Revision 4.15: Thank you for your comment. To directly answer the question of\nincorporation of CBB11 into the liposomes, we decided to perform gel filtration. We used PD10 desalting\ncolumns with a Sephadex G-25 resin. We traced the elution of CBB11 by fluorescence. We found\nfluorescence signals originating from CBB at elution volume (3-3.5 ml) corresponding to Lipo1 (Fig.\nR1a). As expected, we did not observe CBB fluorescence at the elution volume for Lipo2 (negative\ncontrol without CBB11) or in the eluate for the column containing uncomplexed CBB11. Visually, we\nfound that free CBB11 was retained in the column (Fig. R1b-3), and fluorescence scans of the columns\nconfirmed the retention of the free CBB11 in the column (Fig. R1c-3).\nFigure R1: Gel filtration of CBB-liposomes. (a) Coomassie fluorescence scan of a 96-well plate\ncontaining samples collected from the PD10 desalting column. V = elution volume. (b) A photograph of\nE\nthe PD10 desalting columns after the gel filtration. (c) Coomassie fluorescence scan of the PD10\ndesalting columns after the gel filtration.\nthis proof is currently not included in the manuscript. Given the value of these data to support the general\nconclusions of this manuscript, and the clear results of this assay, I feel that these data belong, at the\nvery least, in the Supplementary Info of the manuscript. Given how clear the results are, I feel that the\ncurrent figure would suffice. However, if the authors wish to include a graph showing fluorescence per\nfraction instead of an image of the 96-well plate, this is equally acceptable.\nThank you for accepting our experiment. We included this result as a new Supplementary Figure 8. We\nincorporated the result in the manuscript, updated the figure numbering and Methods section.\nSupplementary Figure 8: Size-exclusion chromatography of CBB-liposomes. (a) Coomassie fluorescence\nscan of a 96-well plate containing samples collected from PD10 desalting columns. V = elution volume. (b,c)\ne\nA photograph (b) and a Coomassie fluorescence scan (c) of the PD10 desalting columns after the size-\nexclusion chromatography.\n“We noted an improvement of colloidal properties of CBB11 after incorporating it into the liposomes.\nUpon buffer exchange, free CBB11 was absorbed by a size-exclusion chromatography resin, whereas\nliposome-bound CBB11 was recovered in the eluate (Supplementary Fig. 8).”\nround was not reviewed by the same reviewer as the initial “Reviewer 1” from the previous round of peer\nreview. As reviewer number 1 was no longer able to comment on this manuscript, I was requested as a\nreplacing reviewer to evaluate the responses provided to the original comments of reviewer 1, with the\nfollowing request: “We ask that no new concerns be raised unless they are critical to the validity of the\narguments made in the manuscript”.\nFirstly, I would like to compliment the authors on the amount of effort that they have shown in addressing\nthe original comments of Reviewer 1. Especially the addition of multiple in vivo experiments is laudable.\nI will address all author comments per point below. However, there are a few comments that have not\nbeen fully addressed / resolved, as discussed below. To maintain clarity, my comments will be in blue,\nthe comments from the original reviewer will remain shown in red.\nReviewer #1\nReviewer Comment 1.1: All in vitro results were done at a single concentration of the CBB derivatives\nand no dose-response curves are given… The optimal dose therefore needs to be established for each\nreagent, separately.\nResponse 1.1: Our previous one-point analysis was based on optimized conditions, and we would argue\nstill valid. Nevertheless, we performed dose-response analyses as recommended.\nRevision: In the revised manuscript, we now provide full in vitro dose-response curves (1–8 µM CBB)\nfor the nine CBB derivatives (CBB2, 7, 11, 12, 13, 14, 18, 19, and Z5) with Cre recombinase, or (0.2–1.6 µM\nCBB) with ABE8e-RNP, now included as Fig. S2.\nFigure S2. Concentration dependence of the in vitro delivery of gene editors mediated by\nrepresentative CBB compounds. (a) Quantification by fluorescence microscopic imaging of GFP-to-\nRFP conversion in CS cells. Cells were treated with Cre recombinase (0.5 µM) complexed with CBB\ncompounds at increasing concentrations (1-8 µM). The cells are green at baseline and red after editing.\nintensity ratio of eGFP to mCherry in rd12-reporter cells treated with ABE-RNPs (100 nM) complexed\nwith CBB compounds at increasing concentrations (0.2-1.6 µM). Data are presented as mean ± S.D.; n =\n2.\nRevisions in Results part:\nIn Vitro Delivery of Cre by Coomassie-Derived Lipidoids\nNine compounds (CBB2, 7, 11, 12, 13, 14, 18,19, Z1) were selected for the titration of the [CBB] to\n[Enzyme] ratio. The CBB lipidoids were dissolved in DMSO (1 volume) and complexed at room\ntemperature with Cre recombinase (19 volumes) at a 2:1, 4:1, 8:1 or 16:1 molar ratio before delivery\n(Fig. S2a). The enzymatic activity of cytosolic Cre was evaluated by florescence microscopy according to\nthe extent of GFP to RFP conversion, which indicated that Cre recombinase intracellular delivery was\ndependent on the CBB-lipidoids concentration, and the dose-response relationship showed that the\neffect was generally saturated at a ratio 8:1 for most compounds.\nIn Vitro Delivery of ABE-RNP\nThe titration of [CBB2, 7, 11, 12, 13, 14, 18,19, Z1] to [ABE-RNP] with florescence microscopy also\nindicated that higher concentrations of CBB compounds facilitated RNP delivery (Fig. S2b). In the broad\nscreening of CBB compounds, the ratio of [CBB]/[Enzyme] was kept at 8:1 to decrease the blue\nprecipitation during flow cytometry measurement.\nRevisions in Methods part:\nDelivery of Cre recombinase in vitro\nat final concentrations of 10 M of Cre enzyme and 20-160 M of CBB compound.\nDelivery of ABE in vitro\nNext, ABE-RNP was diluted in TAS buffer (20.1 mM Tris, 4.3 mM sodium acetate, pH 7.3, sucrose 10%\n(v/v)) and added to CBB-lipidoids at 1:2 to 1:16 ratios of enzyme:compound, 5%/7.25% (v/v) final DMSO,\n10 µM final RNP, and incubated for a further 15 min (Fig. 4) or 5 h (Fig. 5 f, i) at room temperature.\nOriginally, reviewer 1 was somewhat vague in their wording, and their request could either be\ninterpreted as a request to provide a dose-response curve in terms of the absolute amount that\nwould be used for transfection, or an optimization of CBB-to-cargo ratio. Here, the authors opted\nto focus on a ratio optimization for all CBBs, which I agree is the best (and most\nrelevant) answer to the final statement by reviewer 1 “The optimal dose therefore needs to be\nestablished for each reagent, separately.” It is furthermore additionally appreciated that this was done\nfor both Cre recombinase and ABE. As such, I feel that these experiments are suitable to address\nthe comment of Reviewer 1.\nHowever, in this case the authors have performed the experiment twice (n=2). Whereas the data\nshow high reproducibility and a robustness in the observed patterns, I would like to point out the\nfollowing:\nPlease be aware that the Nature Portfolio checklist contains the following statement/guideline:\n“We strongly discourage deriving statistics from technical replicates or less than 3 biological\nreplicates, unless there is a clear scientific justification for why providing this information is\nimportant.”\nEspecially considering these experiments are in vitro assays, I would unfortunately request the\nauthors to perform this experiment once more, unless (as is in line with the Nature Portfolio\nguidelines) “there is a clear scientific justification for why providing this information is important”.\nReviewer Comment 1.2: The in vivo data focus on base editing results, but not so much on toxicity and\noff-target editing… I would at least expect to see some evidence that toxicity to the retina is absent or\nlimited at the tested concentrations.\nResponse 1.2:\nToxicity: We have performed a new series of experiments investigating the effects of our molecules on\nwild-type mice (C57BL/6). Here we present OCT images of wild-type retinas at 7 days post-injection,\nfollowed by a functional ERG analysis at 14 days and retinoid analyses at 21 days that reflect the content\nof rhodopsin. The data indicates that the wild-type mice treated with the complex of a new batch of Lipo1\nwith RNP (10 µM) showed minimal changes to the retinal structure and function. The minor changes\nobserved are due to retinal surgery. The mouse eyeball has a diameter of ~3.4 mm and volume ~ 24\nmm3 (~ 24 µL); and the lens represents about 15-20% of the total eye volume. Mouse lenses are\nrelatively large compared to the eye size (compared to human proportions), making injection to subretinal\nspace challenging without disturbing the lens. Any perturbation of the lens would lead to cataract, and\nthe eye would be useless for imaging or physiological testing. Only skilled personnel did the injections,\nand no such gross effects were observed.\nRevision:\n(blue) and rd12 mice (green) were administered ABE8e-RNPs with/without new Lipo1 via subretinal\ninjections. (a) The particle size of the new Lipo1 preparation (green) was 148 nm, with PDI = 0.23; this\nwas similar to that of the old batch (yellow), size =140 nm, PDI = 0.16. (b) Quantification via fluorescence\nmicroscopy of the ratio of eGFP to mCherry for rd12 reporter cells, treated with the new Lipo1-RNPs\n(100 nM). Lipofectamine 3000 (LF, purple) served as positive control, and ABE8e-RNPs delivered without\n4\nvehicles (A, gray) served as negative control; [CBB]/[RNP] ratio = 8:1. Data are presented as mean ±\nS.D., n = 2 replicates. (c) Representative OCT images of WT mice treated with 20 µM ABE-RNPs alone\n(R20), with the complex of 10 µM RNPs plus Lipo1 containing 80 µM CBB11 (L80R10), with Lipo1\ncontaining 160 µM CBB11 (L160), or with the complex of 20 µM RNPs with Lipo1 containing 160 µM\nCBB11 (L160R20); WT mice without treatment served as control (WT N). The injection volume was 1 µl.\n(d) b-wave amplitude quantification of ERG responses of WT mice whose representative OCT scans are\nshown in panel c; data are presented as mean ± S.D., n = 6 eyes. (e) Quantification of 11-cis-retinal\nlevels in WT mice whose ERG responses are presented in panel d; data are presented as mean ± S.D.,\nn = 5-6 eyes. (f) Quantification of b-wave amplitudes of ERG responses of rd12 mice treated with 0.5 µl\nof L80R10 (light green); 1 µl of L80R10 (green); or 1 µl of L160R20 (dark green). Untreated WT mice\n(WT N, black) and rd12 mice (rd12 N, gray) served as positive and negative controls, respectively; data\nare presented as mean ± S.D., n = 6-8 eyes. (g) Quantification of 11-cis-retinal levels in rd12 mice whose\nERG responses are presented in panel f; data are presented as mean ± S.D., n = 5-7 eyes.\nRevisions in Results part:\nProtein Delivery by CBB-liposomes In Vivo\nTo evaluate the safety of the ABE-RNP delivery to the retina mediated by Lipo1, we also\nadministrated ABE-RNP-Lipo1 complex to WT mice (Fig. S9) via subretinal injection. Since the\noxidation of CBB11 might induce epoxides in the liposome, only freshly formulated Lipo1 was applied to the WT\nmice. Different batches of Lipo1 showed similar morphology and comparable ABE-RNP delivery\nefficiency to rd12 reporter cell lines (Fig. S9a, b). The Optical Coherence Tomography (OCT) imaging\nindicated no substantial alterations to the retinal structure of WT mice administrated the complex of RNP\n(20 μM) and Lipo1; except mild inflammation, which could be ameliorated substantially by lowering the\nRNP concentration (Fig. S9c). The ERG response and the generation of the visual chromophore, 11-cis\nretinal, of WT mice treated with Lipo1 and RNP (10 μM) were also comparable to those of untreated WT\nmice (Fig. S9d, e). In parallel experiments in which rd12 mice were treated with the same batch of Lipo1\nand RNP complex, the subretinal injections of 1 μl of Lipo1 with 10 μM RNP achieved an optimal ERG\nresponse and visual chromophore recovery (Fig. S9d, e), comparable to those of rd12 mice treated with\nLipo1-ABE-RNP (20 μM) (Fig. 6). These results suggested that optimal vision-function rescue requires a\nbalance between the biological tolerance of the cells and the editing efficiency of the gene editors.\nRevisions in Methods part:\nDelivery of ABE in vivo\nABE RNP in TAS was added to concentrated CBB-lipidoid in DMSO or CBB-liposome in TAS, mixed by\nrapid pipetting and incubated with the reagents for at least 10 min at room temperature, and injected\n5\nsubretinally into rd12 or WT mice. The remaining reagents were then applied onto rd12 reporter cells in\nDMEM with 10% FBS in a 96-well plate (Fig. 5f, i).\nOff-target editing–Targeted deep sequencing of the ten highest-ranked off-target sites (identified using\nCas-OFFinder) shows a background-level A•T→G•C substitution rate comparable to that of\nuntreated animals. Additional data on off-target analyses are now included.\nRevisions: These data are now included in the Results as an expanded Fig. 6g.\nFigure 6 (g) Off-target genome-editing by ABE-RNPs delivered with Lipo1 in vivo. DNA from the RPE of\nmice treated with 20 µM ABE-RNP complexed with Lipo1, and from untreated controls were subjected to\nhigh-throughput targeted-amplicon sequencing. Adenosine deamination at the on-target and off-target\nsites (OT) were analyzed using CRISPResso. N = 4 replicates.\nRevisions in Results part:\nProtein Delivery by CBB-liposomes In Vivo\nThe targeted deep sequencing of the ten highest-ranked off-target sites (identified using Cas-OFFinder)\nshows a background-level A to G substitution rate on these sites for the rd12 mice treated with Lipo1-\nABE8e-RNPs which is comparable to that of untreated animals (Fig. 6g).\nOnce more, I feel that the authors have chosen suitable experiments to address the points raised by\nreviewer 1. Whereas n=2 replicates for Fig S9B, I feel that this is justified in this case due to the\nrobustness of the data, as well as the ethical implications to minimize the amount of animals. Data is\nclear, methods are described well, conclusions in the main text are appropriate.\nOne request: I do not have access to the Supplementary Tables, so I’m not sure if all OT genomic\nsequences / analysis sequences are listed. As such, I would like to request to please make sure to\ninclude the analyzed Off-targeted sequence (+genomic location), as well as potential relevant\nprimer/probe sequences used for OT analysis (either to the methods section, or the Supplementary\nfiles).\npresented (toxicity, RNP encapsulation, immunogenicity). Better to remove such unsubstantiated claims.\nResponse and Revision 1.3: Thank you for bringing up this important point. We have removed\nunsubstantiated statements regarding toxicity (see Response 1.2), immunogenicity, and full\n\"encapsulation\". All remaining claims are now directly supported by new data (toxicity, off-target analysis)\nor by published references cited in the Discussion.\nThe changes are appreciated.\nIndeed, published references can be used to support claims on safety as well. In line with this, I would\nrequest adding a reference that shows decreased bystander editing upon short-term ABE delivery (ie. as\nRNP) to the statement “In general, the delivery of ABE-RNPs mediated by CBB-liposomes could be\nconsidered as a more transient mode of delivery of gene-editing tools, consistent with decreased\nbystander editing”.\nAlso, please add a reference to the statement “small particles would better penetrate the retina and\nmake the therapeutic agent available for cellular uptake”.\nReviewer Comment 1.4: From Cre recombinase to base editors. A logical step in between could have\nbeen CRISPR-Cas9 based gene correction. Did you try this as well?\nResponse 1.4: Thank you for the suggestion. Cas9 in the eye causes severe off-target effects and\napoptosis.1 Therefore, we deliberately applied our CBB strategy to an adenine base editor (ABE) rather\nthan to a Cas9 nuclease, since ABEs offer superior therapeutic promise. This choice is further validated\nby recent clinical successes, most notably a personalized ABE-based gene therapy administered to an\ninfant patient, which underscores the clinical relevance of base editing over nuclease-driven\napproaches.2\nThis is a valid explanation, and as reviewer 1 did not request any additional data or experiments this\nanswer suffices. However, readers may from outside the ocular delivery field may have similar questions.\nAs such, I would request that this is briefly explained/addressed in the discussion as well.\nReviewer Comment 1.5: CBB dyes have denaturing capacities. Do you see any reduction in activity of\nCre recombinase or base editors in the presence (without encapsulation)?\nResponse 1.5: Coomassie Brilliant Blue (CBB) dyes indeed show higher affinity for partially unfolded\nproteins (e.g. Triton-X100 increase binding Coomassie G250 in the Bradford assay 3), yet decades of\nsuccessful non-denaturing affinity-chromatography with structurely similar Cibacron Blue–based \"Blue\nSepharose\"4 demonstrate that such interactions are routinely compatible with native enzyme function.\nThe only adverse phenomenon we observed was formulation-dependent precipitation with CBB lipi\ndoids. The data below indicate that CBB11 and CBB14 do not affect the activity of ABE.\nred, target base; arrowhead, nick site; FAM, fluorescein. (f) Representative urea-PAGE-gel image of\nproducts of ABE deamination cleaved by EndoV. The gel was imaged with a fluorescein filter. N, no\nenzyme; P, positive control: 60 bp DNA with inosine in the middle; A, ABE-RNP in TAS; D, ABE-RNP in\nTAS with10% DMSO. Numbers indicate molar excess of the CBB compound versus ABE. (g)\nFluorescence quantification of substrate and products of deamination and EndoV cleavage. Two\nindependent samples per data point were assayed in parallel.\nRevisions: We have added the new figure to the manuscript\nRevisions in Results part:\nIn Vitro Delivery of ABE-RNP\nThe complexing of ABE-RNPs with CBB lipidoids with 10% DMSO did not deteriorate the deaminase\nactivity (Fig. 4e-g)5.\nRevision in Methods part:\nABE activity assay\nRegarding the schematic of Figure 4e: the ABE8e editing window tends to span between spacer\nnucleotides 3/4 – 8. As such, would the following underlined A not also be edited?\nGACCGTCAGAGGAGACTACA\nPlease check, and if indeed so, correct in the schematic.\nReviewer Comment 1.6: P3L84: Lipofectin should be lipofectamine or else describe the procedure of\nlipofection.\nResponse and Revision 1.6: We were unclear, and appreciate the comment. We agree that the two\nterms mean different things, and we have now used them correctly. In the introduction, we correctly\nmentioned “Lipofectin.”6 Also, we removed all the lipofectin in the results and methods part.\nRevision in Introduction part:\ndeliver plasmids into cells in vitro.7 Since then, further development has resulted in numerous\nionizable/permanently ionized lipids with multiple functionalities like Lipofectamine, MC3;8 and delivery of\nlarge RNPs has been reported using lecithin-based liposomes9 and ionizable lipid nanoparticles.10\nThank you, this fully addresses the point of the reviewer.\nReviewer Comment 1.7: P4L956... remove claim of potential safety about your library of CBB lipidoids\nas you only tested a few.\nResponse 1.7: We would like to clarify that one component of the CBB lipidoids, specifically G250, is\napproved for clinical use. However, we acknowledge that the entire set of CBB lipidoids will need to be\nthoroughly investigated for safety and efficacy before any potential clinical application.\nRevision: We have modified our statements regarding safety to better reflect the speculative nature of\nour statements regarding safety.\nRevision in Introduction part.\nWe sought to improve the existing genome editing toolkit by developing a safe and efficient protein\ndelivery approach.\nThank you, this fully addresses the point of the reviewer.\nReviewer Comment 1.8: Fig. 2. Only one concentration of CBB derivatives was tested… some might be\nactive at lower/higher concentrations.\nResponse and Revision 1.8: Thank you for this suggestion. The response to this inquiry is addressed in\nour response to Comment 1.1.\nThank you, this fully addresses the point of the reviewer.\nReviewer Comment 1.9: There does not seem to be any SAR in the tested derivatives...\nResponse 1.9: While we cannot yet provide a predictive model for the construction of an ideal CBB\nlipidoid to facilitate RNP delivery, the presented data provide a foundation for a SAR. Our work presents\na novel concept in gene delivery, and we are confident that reviewers will appreciate how extensive our\nstudies are at this point. The next step would be to focus on a few CBBs and work to generate a family of\nhighly related compounds to discover the key molecular determinants of facilitated delivery. We expect\nthat the current studies will inspire other laboratories also to build on this approach to improve delivery of\ngene-editing agents.\nRevisions: We added few sentences in the limitation section of the manuscript.\nRevision in Results part:\nsuggesting synergistic effects between cell-penetrating peptides and CBB. In general, the leading CBB\ncompounds for Cre recombinase delivery, CBB14, 18, 19 and 21, are more positively charged under\nneutral aqueous conditions, and their lipid moieties are less hydrophobic. Even though such properties of\nthese compounds do not substantially change the particle size of the respective CBB/Cre complexes\n(The particle diameter of CBB11/Cre is about 1440 nm and that of CBB14/Cre is around 1510 nm), they\nstill could improve the colloidal stabilities of the CBB/Cre complexes.\nThis, in combination with the final sections and the discussion on C16-C18 tails in the discussion section\nsufficiently addresses this point (moreover, the reviewer did not request any specific deliverables).\nReviewer Comment 1.10: P5L150 'did not enable Cre uptake...’ Wrong conclusion as you are not\nlooking at cellular uptake but Cre activity. Please change.\nResponse 1.10: Thank you for pointing out this inaccuracy. We agree that lack of evidence of Cre\nactivity in the reporter cells does not mean that Cre was not absorbed by the cells.\nRevision: Accordingly, we have changed the wording in the text from \"did not enable Cre uptake\" to \"did\nnot lead to detectable Cre-mediated recombination activity\" in the reporter cells.\nRevision in Results part:\nIn Vitro Delivery of Cre by Coomassie-Derived Lipidoids\nThe enzymatic activity of cytosolic Cre was evaluated by florescence microscopy according to the extent\nof GFP to RFP conversion, which indicated that Cre recombinase intracellular delivery was dependent on\nthe CBB-lipidoids concentration,\nThank you, this fully addresses the point of the reviewer.\nReviewer Comment 1.11: In all formulations DMSO is present at ~10%. Dialysis could have easily\nremoved it… Why not done?\nResponse 1.11: We removed the solvent by dialyzing the complex of ABE-RNP and CBB11 in TAS\nbuffer with 10% DMSO through a 7000 MWCO membrane. The CBB11 amount in the resulting solution\nwas quantified by LC-MS. The results indicated that most of the CBB11 (95.3%) was lost, suggesting that\nthe CBB binding to protein is reversible, which helps support our stated goal of identifying molecules that\nwould possess transient non-specific protein binding to assist in delivery. It should also be noted that\nDMSO is tolerated by the eye, and DMSO is present in some formulations. The solvent is quickly\nremoved through vitreous exchange, thus not presenting an issue even for clinical applications.\nRevision: We have added a sentence to the Results conveying more clearly the utility and safety of\nincluding DMSO in the formulation.\nRevision in Results part:\nThe complexing of ABE-RNP with CBB lipidoids with 10% DMSO did not deteriorate the deaminase\nactivity (Fig. 4e-g).\nOnce more, this fully addresses the point raised by Reviewer 1. However, as other readers may have\nsimilar questions, and it appears that not removing DMSO is of pivotal importance for the stability and\ndownstream applications of these formulations, this information seems of high importance to the readers\nas well. Especially as it may not be directly clear that currently DMSO is therefore a component of the\nfinal formulation, which is of high relevance for potential applications.\nWhereas additional experiments / data that shows stability of specific CBBs are not required to\ndiscuss this issue, it is a potential limitation that should be discussed. Please add a section to the\ndiscussion section that underlines the importance of DMSO in the discussion.\n(indeed, this is not problematic for a variety of clinical formulations, but this could of course also be\nadded to the discussion if the authors would desire to do so).\nReviewer Comment 1.12: P6 concentrations for subretinal injection of Cre recombinase do not match\nthose in the MandM section.\nResponse and Revision 1.12: Thank you. We corrected this mistake. Now, the concentrations in the\nMethods match the main text (Cre, 2 µg per µL; injection volume = 1 µL).\nThank you, this fully addresses the point of the reviewer.\nReviewer Comment 1.13: What are the size of these CRE/CBB complexes?\nResponse and Revision 1.13: Dynamic light scattering indicated that the size of the Cre/CBB11\ncomplex is 1400 nm and the size of the Cre/CBB14 complex is 1500 nm. These values are now included\nin the Result section.\nRevisions in Results part:\nIn Vitro Delivery of Cre by Coomassie-Derived Lipidoids\nEven though such properties of these compounds do not substantially change the particle size of the\nrespective CBB/Cre complexes (The particle diameter of CBB11/Cre is about 1440 nm and that of\nCBB14/Cre is around 1510 nm), they still could improve the colloidal stabilities of the CBB/Cre\ncomplexes.\nThank you for including this new data. As these particles are this large, this has implications for potential\nsuitability for specific in vivo applications, which should be discussed.\nbase editor.\nResponse 1.14: Cre-CBB and ABE-CBB are different entities. They dramatically differ in size, and their\ncellular pathways to the nucleus could be affected by their physicochemical properties. We have\ncollected new data in an attempt to understand these differences. A key issue is the relative solubility of\nthe two complexes; and we are actively investigating whether differential performance is attributable to\ncolloidal stability and net charge. Cre (38 kDa, pI ~ 9.6, net positive charge) forms a stable shell with\nCBB14, whereas the larger, anionic ABE-RNP (180 kDa protein and 33 kDa RNA) induces rapid\naggregation of CBB14 (See Response Figure 1, below).\nResponse Figure1. Soluble versus Precipitable CBB14 in the complexes of CBB14 with Cre or\nRNP after gentle centrifugation. Ten microliters of stock solutions of Cre or ABE-RNP (final 10 µM)\nwere mixed with a solution of CBB14 (final 80 µM) in cell-culture medium with 5% DMSO; then\ncentrifuged at 800 g for 10 min. The amounts of CBB14 in the supernatant and precipitate were analyzed\nvia LC-MS. The larger amount of CBB14 in the precipitate of the CBB-RNP complex suggests that the\nCBB-RNP complex has lower colloidal stability.\nRevision: We have added a brief description of this pilot experiment and its implication to the\nDiscussion, but these data are preliminary, and will be the focus of future work.\nRevisions in Discussion part:\nThe CBB lipidoids facilitated the cellular uptake and recombination event of the small positively charged\nprotein, Cre. Remarkable Cre-recombinase delivery mediated by CBB14 was achieved in vitro (Fig. 2c)\nand in vivo (Fig. 3b). The colloidal stability and protein-delivery efficiency of CBB compounds dropped\ndramatically when they were bound to the large, aggregation-prone and negatively charged ABE-RNP\n(Fig. 4c).\nReviewer Comment 1.15: Please show experimental evidence that CBB11 is in fact\nincorporated into the liposomes.\nResponse 1.15: We have performed experiments related to the solubility of CBB11 and the\nliposome (Lipo 1). Unconjugated CBB11 is mostly insoluble, but when mixed with lipids it is retained\nin the suspension (see below). Please also see relevant response to Comment 3.2.\nResponse Figure 2: CBB11 (40 µM) in TAS with 10% DMSO or Lipo1 (containing 40 µM CBB11) in TAS\nwas dialyzed overnight against TAS buffer, followed by centrifugation at 5000 rpm for 5 min. The\nCBB11 in the supernatant was quantified via LC-MS. The data suggest that formulation with the liposome\nprevented CBB11 aggregation.\nMoreover, solubilization of CBB11 is documented further by comparing the effects of Lipo 1 versus Lipo 2\n(see Fig. 5 d, below). Lipo1 is less negatively charged than Lipo2. The observed difference also\nindicates that CBB11 is in fact incorporated into the liposome membrane. This interpretation is further\nsupported by the fact that Lipo1 became much more negatively charged after complexing with RNP,\nindicating intensified protein binding to the liposome surface. We attribute the differences to the\nincorporation of CBB11 in the Lipo1 membrane.\nRevision: We have added a verbal description of these observations to the results.\nRevisions in Results part:\nIn Vitro Delivery of ABE-RNP\nincubation time of CBB11 with ABE-RNP (final concentration at 100 nM) increased from 15 min (Fig. 4c)\nto 5 h (Fig. 5d), accompanied by precipitation. Apparently, the colloidal instability of CBB11 hindered the\nuptake of ABE-RNPs by the cells, or possibly interfered with editing. To improve the distribution of CBB11\nin an aqueous environment, we formulated novel liposomes (Lipo1/3/5) containing, in molar ratio, 5%\nCBB11, 50% ionizable lipids (SM102, ALC-0315, MC3), 10% 1,2-distearoyl-sn-glycero-3-phosphocholine\n(DSPC), 33.5% cholesterol, and 1.5% 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000\n(DMG-PEG 2000); and a control liposome (Lipo2/4/6) without CBB11 (Fig. 5a).\nWe also did not observe precipitation of ABE-RNP with either Lipo1/3/5 or Lipo2/4/6.\nRevisions in Discussion part:\nParticularly striking was the high efficacy observed when these compounds were formulated into\nionizable cationic liposomal particles to improve the colloidal stabilities of the CBB-protein complexes.\nFigure 5 (d) The zeta potentials of Lipo1 (blue) and Lipo2 (yellow) in the absence (-, light color) or the\npresence (+, dark color) of ABE-RNP. The ratio of ABE-RNP to CBB11 is 1:8.\nRevisions in Results part:\nIn Vitro Delivery of ABE-RNP\nFurthermore, the presence of ABE-RNP dramatically changed the zeta potential on the Lipo1 surface (a\nnet decrease of 10.16 mV), but marginally affected the zeta potential on the Lipo2 surface (a net\ndecrease of 0.64 mV) (Fig. 5d).\nThese facts indicated that the protein binding on the surface of Lipo1 was intensified by CBB11.\nAs expected, this enhanced liposome-protein interaction boosted intracellular protein delivery.\nWhereas the comparison of a control lipid to CBB11 in context of liposomes in Fig 5D is an interesting\nexperiment, it does not fully answer the question of reviewer 1. The question specifically states “: Please\nshow experimental evidence that CBB11 is in fact\nincorporated into the liposomes”. However, the data\nshown in Figure 5d could also be explained by direct nanocomplexation of ABE8e-RNP with CBB11.\nResponse Figure 2 is a more conclusive / direct\nread-out for liposomal incorporation (especially since it’s LC-MS based analysis, which is a more\ndirect read-out).\nTo answer this question As the authors appear to have access to LC-MS for formulation analysis,\nI would request that the authors incorporate data to the manuscript that directly confirms CBB11\nintegration to answer this comment.\nGeneral comment, based on these replies: please avoid the use of RPM in methods section, as the\nrcf (g) can differ dramatically based on the rotor diameter. Please check throughout the manuscript\nthat all mentions of centrifugation speeds are shown in rcf (g), and not in RPM.\nReviewer Comment 1.16: It is unclear what is shown in Fig 5C… Please explain.\nResponse 1.16: Fig. 5c reports the liposome-facilitated Cre recombinase delivery into color-switching\ncells. The RFP/GFP conversion measured by fluorescence microscopy indicates the presence of Cre\nrecombination activity in the nucleus.\nFig. 5f, i report the liposome-facilitated ABE8e-RNP delivery into rd12 reporter cells. The discrepancy\nbetween Fig. 4 and Fig. 5e is due to the slow colloidal aggregation triggered by protein binding to the\ncolloids formed with CBB11 alone. The complexing time of CBB compound with RNP in Fig. 4 is 15 min\nand in Fig 5f, i is 5 h because the complex was first used for subretinal injection, then for the in vitro\nassay.\nRevision: More details are described under Methods\nRevisions in Methods parts:\nDelivery of ABE in vitro.\nNext, ABE-RNP was diluted in TAS buffer (20.1 mM Tris, 4.3 mM sodium acetate, pH 7.3, sucrose 10%\n(v/v)) and added to CBB-lipidoids at a 1:2 to 1:16 ratio of enzyme:compound, 5%/7.25% (v/v) final\nDMSO, 10 µM final RNP, and incubated for a further 15 min (Fig. 4) or 5 h (Fig. 5 f, i) at room\ntemperature.\nThank you for the explanation, and the extra information in the methods section.\nReviewer Comment 1.17: Fig 6: 20 µM ABE-RNP was dosed in vivo… 100x higher than in vitro.\nProvide rationale. Wouldn’t you expect toxicity? Show toxicology.\nResponse and Revision 1.17: The maximum volume that can be injected into a mouse eye is 1-2 µl.\nHowever, the subretinal space is highly restricted and prevents any dilution with the vitreous. In tissue\nculture, the cells are present as adhesion cells on the surface with large access to tissue culture medium,\n15\ntherefore requiring higher concentrations as our reagent is highly diluted and the ratio to the cells is\ndrastically lower than in vivo. In terms of toxicity, please see response to comment 1.2.\nThank you for the explanation.\nReviewer Comment 1.18: P9L299: ‘encapsulation within the particle…’ if you refer to a new method,\nclarify or remove.\nResponse and Revision 1.18: We now clarify that ABE-RNP predominantly adsorbs to,\nrather than being fully encapsulated by, the lipid corona; the text has been reworded accordingly.\nThank you for explaining this. This is now in line with the TOC graphic. However, if possible, please make\nsure to include a bit more clarity to the TOC graphical abstract: right now it is implied that the H+\nis present in the cytosol, rather than the endocytosis pathway.\nRevisions in Results part:\nProtein Delivery by CBB-liposomes In Vivo\nPrecisely edited gDNA sequences in mice injected with ABE-RNPs absorbed to the corona layer on\nLipo1 were 127-, 20-, 50-, 9.7-, and 6.5-fold higher than those with TAS, DMSO, CBB11,\nCBB14, and\nLipo2, respectively.\nThank you, this fully addresses the point of the reviewer.\nReviewer Comment 1.19: P10L318: ‘… CBB-lipidoids also showed reduced ocular toxicity… Where can\nI find these results?\nResponse and Revision 1.19: This comment is broadly addressed with the new toxicity data addressed\nin response to comment 1.2.\nThank you, this fully addresses the point of the reviewer.\nReviewer Comment 1.20\nP10L328: ‘We predict less immunogenic than a liposome without CBB…’ The reviewer argues the\nopposite. Please comment.\nResponse 1.20\nRevision: We have removed the speculative statement, and again refer to the response to comment\n1.2.\nThank you, this fully addresses the point of the reviewer.\n16\nenables genome editing in the retina. Mol Ther Nucleic Acids 35, 102349 (2024).\nhttps://doi.org/10.1016/j.omtn.2024.102349\nDisease. N. Engl. J. Med. 392, 2235–2243 (2025).\nhttps://doi.org/10.1056/NEJMoa2504747\nthe presence of detergents. Anal Biochem 178, 263–268 (1989).\nhttps://doi.org/10.1016/0003-2697(89)90636-2\nphosphofructokinase using Cibacron blue F3G-A. J Chromatogr 69, 209–214 (1972).\nhttps://doi.org/10.1016/s0021-9673(00)83103-9\nribonucleoproteins delivered through optimized lipid-nanoparticle formulations. Nat\nBiomed Eng 9, 57–78 (2025). https://doi.org/10.1038/s41551-024-01296-2\ntransfection. Proc. Natl. Acad. Sci. U. S. A. 86, 6077–6081 (1989).\nhttps://doi.org/10.1073/pnas.86.16.6077\nprocedure. Proc Natl Acad Sci U S A 84, 7413–7417 (1987).\nhttps://doi.org/10.1073/pnas.84.21.7413\ndelivery. Nat Rev Drug Discov 23, 709–722 (2024). https://doi.org/10.1038/s41573-024-\n00977-6\nsystem for treating type 2 diabetes. J Nanobiotechnology 17, 19 (2019).\nhttps://doi.org/10.1186/s12951-019-0452-8\n(2021). https://doi.org/10.1126/sciadv.aba1028\nOnce more, I’d like to compliment the authors on their efforts to address all comments. With these current\nrevisions, I feel that the concerns of reviewer 1 have been sufficiently addressed (see below – comments\nto revisions are highlighted in yellow). However, I do have one request for one minor revision: given the\nvalue and clarity of the results shown in figure R1, I feel that these data belong in the Supplementary\nInformation file, rather than solely as data presented to the reviewers. Furthermore, I have pointed out\ntwo small language suggestions for the figure legend of S2. Once these minor revisions are adjusted, I\nwould recommend the acceptance of this manuscript for publication.\nReviewer Comment 4.1: Originally, reviewer 1 was somewhat vague in their wording, and their\nrequest could either be interpreted as a request to provide a dose-response curve in terms of the\nabsolute amount that would be used for transfection, or an optimization of CBB-to-cargo ratio. Here,\nthe authors opted to focus on a ratio optimization for all CBBs, which I agree is the best (and most\nrelevant) answer to the final statement by reviewer 1 “The optimal dose therefore needs to be\nestablished for each reagent, separately.” It is furthermore additionally appreciated that this was done\nfor both Cre recombinase and ABE. As such, I feel that these experiments are suitable to address the\ncomment of Reviewer 1.\nHowever, in this case the authors have performed the experiment twice (n=2). Whereas the data show\nhigh reproducibility and a robustness in the observed patterns, I would like to point out the following:\nPlease be aware that the Nature Portfolio checklist contains the following statement/guideline: “We\nstrongly discourage deriving statistics from technical replicates or less than 3 biological replicates,\nunless there is a clear scientific justification for why providing this information is important.”\nEspecially considering these experiments are in vitro assays, I would unfortunately request the\nauthors to perform this experiment once more, unless (as is in line with the Nature Portfolio\nguidelines) “there is a clear scientific justification for why providing this information is important”.\nResponses and Revisions Reviewer 4.1. Thank you for the comment. We repeated the titrations for\nCre and ABE with four replicates. In this new experiment, Cre delivery efficiency was comparable,\nwhereas ABE was this time not efficiently delivered into the rd12 reporter cells by CBB12. We replaced\nthe Figure S2 with the new data presented as separate data points.\nFig S2. Concentration dependence of the in vitro delivery of gene editors mediated by\nrepresentative CBB compounds. (a) Quantification by fluorescence microscopic imaging of GFP-\ntoRFP conversion in CS cells. Cells were treated with Cre recombinase (0.5 µM) complexed with CBB\ncompounds at increasing concentrations (1-8 µM). The cells are green at baseline and red after editing.\nData are presented as mean ± S.D.; n = 2. (b) Quantification by fluorescence microscopic imaging of the\n1\nintensity ratio of eGFP to mCherry in rd12-reporter cells treated with ABE-RNPs (100 nM) complexed\nwith CBB compounds at increasing concentrations (0.2-1.6 µM). Data are presented as mean ± S.D.; n =\n2.\nCurrent version:\nFig S2. Concentration dependence of the in vitro delivery of gene editors mediated by\nrepresentative CBB compounds. (a) Quantification by fluorescence microscopic imaging of GFP-\ntoRFP conversion in CS cells. Cells were treated with Cre recombinase (0.5 µM) complexed with CBB\ncompounds at increasing concentrations (1-8 µM). The cells are green at baseline and red after editing.\nMean ± S.D., 4 biological replicates. (b) Quantification by fluorescence microscopic imaging of the area\nratio of eGFP to mCherry in rd12-reporter cells treated with ABE-RNPs (100 nM) complexed with CBB\ncompounds at increasing concentrations (0.2-1.6 µM). Data are presented as mean ± S.D.; n = 4\nbiological replicates.\nWith these new data, this point has been sufficiently addressed. Please make the following corrections in\nthe figure legend: “GFP-toRFP conversion” should be “GFP-to-RFP”. Furthermore “the cells are green at\nbasaeline and red after editing” is too colloquial in its phrasing – please include “fluorescence in this\nphrasing.\nReviewer Comment 4.2: Once more, I feel that the authors have chosen suitable experiments to\naddress the points raised by reviewer 1. Whereas n=2 replicates for Fig S9B, I feel that this is justified\nin this case due to the robustness of the data, as well as the ethical implications to minimize the\namount of animals. Data is clear, methods are described well, conclusions in the main text are\nappropriate.\nOne request: I do not have access to the Supplementary Tables, so I’m not sure if all OT genomic\nsequences / analysis sequences are listed. As such, I would like to request to please make sure to\ninclude the analyzed Off-targeted sequence (+genomic location), as well as potential relevant\nprimer/probe sequences used for OT analysis (either to the methods section, or the Supplementary\nfiles).\nprotospacer and guide RNA sequences, as well as genetic loci, in Supplementary Table 1. The\nsequencing data were deposited in the Sequencing Read Archive.\nThank you, this fully addresses my previous comment.\nReviewer Comment 4.3: The changes are appreciated. Indeed, published references can be used to\nsupport claims on safety as well. In line with this, I would request adding a reference that shows\ndecreased bystander editing upon short-term ABE delivery (ie. as RNP) to the statement “In general, the\ndelivery of ABE-RNPs mediated by CBB-liposomes could be considered as a more transient mode of\ndelivery of gene-editing tools, consistent with decreased bystander editing”.\nAlso, please add a reference to the statement “small particles would better penetrate the retina and\nmake the therapeutic agent available for cellular uptake”.\nResponses and Revisions Reviewer 4.3: Thank you for the comment. In our previous study, we found\nthat ABE8e-RNP delivery in vitro via Lipofectamine 3000 or lipid nanoparticles led to more precise\nediting than plasmid delivery,1 (Figure 4, new panel “o,” shown below). In another study, we found that ABE\ndelivered as RNP encapsulated into virus-like particles (eVLPs) led to lower bystander and offtarget\nediting than viral delivery via AAV and LV in vivo.2 We added the references to the manuscript.\nRegarding the tissue permeation, small protein GCAP1 whose diameter was 6.5 – 8.7 nm, permeated\nthe retina more efficiently than GCAP1 complexed with liposomes (size range 150-170 nm).3 Our own\ndata do not provide sufficient evidence to support the statement of enhanced penetration of the retina by\nsmaller particles; therefore, we removed that statement.\n[Figure Redacted]\n3\n“Fig. 4 | Lipid nanoparticle delivery of ABE RNPs. …. o, Next-generation sequencing analysis of\nABE-editing outcomes in the cells with cDNA encoding Rpe65 rd12, treated with ABE delivered on a\nplasmid with Lipofectamine 3000 (L3k) or as LNP. Three analytical replicates, mean ± S.D.”1\nThank you, this fully addresses my previous comment.\nReviewer Comment 1.4: From Cre recombinase to base editors. A logical step in between could have\nbeen CRISPR-Cas9 based gene correction. Did you try this as well?\nOriginal Response 1.4: Thank you for the suggestion. Cas9 in the eye causes severe off-target\neffects and apoptosis.1 Therefore, we deliberately applied our CBB strategy to an adenine base\neditor (ABE) rather than to a Cas9 nuclease, since ABEs offer superior therapeutic promise. This\nchoice is further validated by recent clinical successes, most notably a personalized ABE-based\ngene therapy administered to an infant patient, which underscores the clinical relevance of base\nediting over nuclease-driven approaches.2\nReviewer Comment 4.4: This is a valid explanation, and as reviewer 1 did not request any additional\ndata or experiments this answer suffices. However, readers may from outside the ocular delivery field\nmay have similar questions. As such, I would request that this is briefly explained/addressed in the\ndiscussion as well.\nResponse and Revision 4.4: We added the following sentences to the Discussion, paragraph 2:\n“While CRISPR-Cas9 nuclease RNPs could be viewed as a logical intermediate between Cre and base\neditors, we prioritized ABE for in vivo studies because in comparison with Cas9 nuclease, ABE has the\nadvantage of catalyzing single-base transitions without creating double strand breaks, with equivalent\npotential for RNP delivery as Cas9. The molecular interactions that we engage to deliver ABE RNP are\napplicable to other RNPs, and thus we envision that our CBB liposomes are applicable to other CRISPR\nnucleases and precise editors as well.”\nThank you for including this additional explanation to the manuscript.\nReviewer Comment 4.5: Regarding the schematic of Figure 4e: the ABE8e editing window tends to\nspan between spacer nucleotides 3/4 – 8. As such, would the following underlined A not also be\nedited? GACCGTCAGAGGAGACTACA. Please check, and if indeed so, correct in the schematic.\noften edited alongside the target adenine at position 6. The figure has been corrected to reflect this fact.\nFigure 4. Delivery of ABE-RNPs to rd12-reporter cells, mediated by CBB lipidoids. (…) (e)\nSchematic diagram of the ABE-activity assay. EndoV = endonuclease V. Blue, SpCas9 PAM; red, target\nbase; orange, bystander base; arrowhead, nick site; FAM, fluorescein. (f) Representative urea-PAGE-gel\nimage of products of ABE deamination cleaved by EndoV. The gel was imaged with a fluorescein filter. N,\nno enzyme; P, positive control: 60 bp DNA with inosine in the middle; A, ABE-RNP in TAS; D, ABE-RNP\nin TAS with10% DMSO. Numbers indicate molar excess of the CBB compound versus ABE. (g)\nFluorescence quantification of substrate and products of deamination and EndoV cleavage. Two\nindependent samples per data point were assayed in parallel.\nWith these revisions in Figure 4, my points on this topic have been fully addressed.\nReviewer Comment 1.11: In all formulations DMSO is present at ~10%. Dialysis could have easily\nremoved it… Why not done?\nOriginal Response 1.11: We removed the solvent by dialyzing the complex of ABE-RNP and CBB11\nin TAS buffer with 10% DMSO through a 7000 MWCO membrane. The CBB11 amount in the resulting\nsolution was quantified by LC-MS. The results indicated that most of the CBB11 (95.3%) was lost,\nsuggesting that the CBB binding to protein is reversible, which helps support our stated goal of\nidentifying molecules that would possess transient non-specific protein binding to assist in delivery. It\nshould also be noted that DMSO is tolerated by the eye, and DMSO is present in some formulations.\nclinical applications. We have added a sentence to the Results conveying more clearly the utility and\nsafety of including DMSO in the formulation.\nReviewer Comment 4.11: Once more, this fully addresses the point raised by Reviewer 1. However,\nas other readers may have similar questions, and it appears that not removing DMSO is of pivotal\nimportance for the stability and downstream applications of these formulations, this information seems\nof high importance to the readers as well. Especially as it may not be directly clear that currently\nDMSO is therefore a component of the final formulation, which is of high relevance for potential\napplications.\nWhereas additional experiments / data that shows stability of specific CBBs are not required to\ndiscuss this issue, it is a potential limitation that should be discussed. Please add a section to the\ndiscussion section that underlines the importance of DMSO in the discussion. (indeed, this is not\nproblematic for a variety of clinical formulations, but this could of course also be added to the\ndiscussion if the authors would desire to do so).\nResponse and Revision 4.11: Thank you for the suggestion. We have added the following text to the\ndiscussion:\n“The CBB-RNP complex contained 7.25% DMSO, which was well tolerated by ABE and as such may\nbe further explored as a vehicle for protein delivery.”\nThank you, this fully addresses my previous comment.\nReviewer Comment 1.13: What are the size of these CRE/CBB complexes?\nOriginal Response and Revision 1.13. Dynamic light scattering indicated that the size of the\nCre/CBB11 complex is 1400 nm and the size of the Cre/CBB14 complex is 1500 nm. These values\nare now included in the Result section.\nReviewer 4.13: Thank you for including this new data. As these particles are this large, this has\nimplications for potential suitability for specific in vivo applications, which should be discussed.\nResponse and Revision 1.13: Thank you for the comment. We cannot draw conclusions about\nimplications of charge and hydrophobicity of the Cre-CBB complexes on the in vivo efficacy, as both\nCBB11 and CBB14 effectively delivered Cre in vivo despite the higher hydrophobicity of CBB11. We\nrephrased this part of the Results section for clarity and added the following section to the discussion:.\n“An additional practical determinant is particle size. Dynamic lightscattering measurements indicated that\nCBB11/Cre and CBB14/Cre form ~1.4–1.5 µm assemblies, with a significant fraction of CBB14-Cre\nforming smaller, 160-nm particles, whereas CBB11 alone forms ~400–500 nm colloids; importantly,\ncomplexes are unlikely to diffuse broadly within the neural retina or traverse ocular barriers from systemic\ndelivery, whereas systemic delivery of the smaller, narrowly distributed liposomal formulations would be\neffective. Notably, despite their large hydrodynamic diameters, CBB11 and CBB14 still delivered Cre via\nsubretinal injection.”\nThank you, this fully addresses my previous comment.\nOriginal Reviewer Comment 1.15: Please show experimental evidence that CBB11 is in fact\nincorporated into the liposomes.\nOriginal Response 1.15: We have performed experiments related to the solubility of CBB11 and the\nliposome (Lipo 1). Unconjugated CBB11 is mostly insoluble, but when mixed with lipids it is retained\nin the suspension (see below). Please also see relevant response to Comment 3.2.\nResponse Figure 2: CBB11 (40 µM) in TAS with 10% DMSO or Lipo1 (containing 40 µM CBB11) in\nTAS was dialyzed overnight against TAS buffer, followed by centrifugation at 5000 rpm for 5 min. The\nCBB11 in the supernatant was quantified via LC-MS. The data suggest that formulation with the liposome\nprevented CBB11 aggregation. Revisions in Discussion part:\nParticularly striking was the high efficacy observed when these compounds were formulated into\nionizable cationic liposomal particles to improve the colloidal stabilities of the CBB-protein complexes.\nThese facts indicated that the protein binding on the surface of Lipo1 was intensified by CBB11. As\nexpected, this enhanced liposome-protein interaction boosted intracellular protein delivery.\nComments Reviewer 4.15 Whereas the comparison of a control lipid to CBB11 in context of\nliposomes in Fig 5D is an interesting experiment, it does not fully answer the question of reviewer 1.\nThe question specifically states “: Please show experimental evidence that CBB11 is in fact\nincorporated into the liposomes”. However, the data shown in Figure 5d could also be explained by\ndirect nanocomplexation of ABE8e-RNP with CBB11. Response Figure 2 is a more conclusive / direct\nread-out for liposomal incorporation (especially since it’s LC-MS based analysis, which is a more\ndirect read-out).\nTo answer this question as the authors appear to have access to LC-MS for formulation analysis,\nI would request that the authors incorporate data to the manuscript that directly confirms CBB11\nintegration to answer this comment.\nGeneral comment, based on these replies: please avoid the use of RPM in methods section, as the\nrcf (g) can differ dramatically based on the rotor diameter. Please check throughout the manuscript\nthat all mentions of centrifugation speeds are shown in rcf (g), and not in RPM.\nincorporation of CBB11 into the liposomes, we decided to perform gel filtration. We used PD10 desalting\ncolumns with a Sephadex G-25 resin. We traced the elution of CBB11 by fluorescence. We found\nfluorescence signals originating from CBB at elution volume (3-3.5 ml) corresponding to Lipo1 (Fig.\nR1a). As expected, we did not observe CBB fluorescence at the elution volume for Lipo2 (negative\ncontrol without CBB11) or in the eluate for the column containing uncomplexed CBB11. Visually, we\nfound that free CBB11 was retained in the column (Fig. R1b-3), and fluorescence scans of the columns\nconfirmed the retention of the free CBB11 in the column (Fig. R1c-3).\nFigure R1: Gel filtration of CBB-liposomes. (a) Coomassie fluorescence scan of a 96-well plate\ncontaining samples collected from the PD10 desalting column. V = elution volume. (b) A photograph of\nE\nthe PD10 desalting columns after the gel filtration. (c) Coomassie fluorescence scan of the PD10\ndesalting columns after the gel filtration.\nWith this experiment, I feel that the initial concern of Reviewer 1 has indeed been addressed. However,\nthis proof is currently not included in the manuscript. Given the value of these data to support the general\nconclusions of this manuscript, and the clear results of this assay, I feel that these data belong, at the\nvery least, in the Supplementary Info of the manuscript. Given how clear the results are, I feel that the\ncurrent figure would suffice. However, if the authors wish to include a graph showing fluorescence per\nfraction instead of an image of the 96-well plate, this is equally acceptable.\nResponse to General comment: We corrected the revolutions per minute to g throughout the manuscript,\nwhere applicable. In some instances (sample dissolution in bench top thermomixer, microbial shakers)\nwe kept rpm, a customary measure of mixing speed.\nThank you for these changes.\nReviewer Comment 1.18: P9L299: ‘encapsulation within the particle…’ if you refer to a new method,\nclarify or remove.\nrather than being fully encapsulated by, the lipid corona; the text has been reworded accordingly.\nReviewer Comment 4.18: Thank you for explaining this. This is now in line with the TOC graphic.\nHowever, if possible, please make sure to include a bit more clarity to the TOC graphical abstract:\nright now it is implied that the H+ is present in the cytosol, rather than the endocytosis pathway.\nResponse and Revision 1.18: Thank you for the advice. We expanded the TOC graphic to present the\nconcept of endosomal escape triggered by acidification of the endosome. The TOC graphic has been\nmodified accordingly, and the new version is included in the revised manuscript.\nFigure 1. Construction of complexes, and proposed mechanism(s) underlying intracellular\ndelivery of genome-editing ribonucleoproteins (RNPs) mediated by Coomassie Brilliant Blue\n(CBB) liposomes. The fundamental design principles for a CBB-containing delivery agent include: 1) the\nCBB protein-binding domain; 2) an ionizable-linker domain; and 3) a lipid or lipidoid domain. The CBB\ncompounds are formulated into liposomes comprising ionizable lipid (orange), phospholipid (grey),\ncholesterol (red) and PEG lipid (light blue); and bind RNP on the liposome surface in aqueous, neutral\nenvironment. The RNP-liposome complex is absorbed by the cells via an endocytosis pathway.\nWith these changes to the figure, my comment has been fully addressed.\n9"}]

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