Retinitis pigmentosa (RP [MIM:268000]), a subtype of inherited retinal dystrophies (IRDs), is one of the most common causes of blindness in industrialized countries, with an incidence of 1 in 1000 in North China (Xu et al., 2006). However, there are currently no reliable therapies available to cure or delay the disease progression of RP. In 2017, the US Food and Drug Administration approved Luxturna, an adeno-associated virus (AAV)-based gene therapy drug for RPE65-associated Leber’s Congenital Amaurosis (RPE65-LCA) (Russell et al., 2017; Smalley, 2017), which marked the start of gene therapy for IRDs (Lee et al., 2019).

Mutations in the human rhodopsin (RHO) gene are the most prominent genetic causes of autosomal dominant RP (adRP), accounting for about 30% of adRP cases (Iannaccone et al., 2006). However, different from the RPE65-LCA, which is caused by a loss-of-function mutation, RHO-adRP is mainly caused by a gain-of-function or dominant-negative mutation (Li et al., 2018; Mendes et al., 2005), thereby restricting the application of gene replacement therapy.

Many studies have been conducted regarding the treatment technique for RHO-adRP, such as allele-independent gene ablation. This technique includes silencing the expression of both the mutant RHO allele and its wild-type (WT) counterpart and simultaneously adding an exogenous WT RHO gene beyond the regulation of the retina in vivo (Mitra et al., 2018; Cideciyan et al., 2018; Tsai et al., 2018). Although these methods are applicable to RHO-adRP, many risks remain with the disruption of the WT allele. Recently, allele-specific methods have been used extensively to treat adRP (Meng et al., 2020). With the aim of allele-specific RHO gene downregulation at the RNA level using antisense oligonucleotides (ASO), short hairpin RNA, or RNA interference (Meng et al., 2020) can be achieved, some of which have been translated into clinical trials. However, repeated treatments, especially for subretinal injections, are bound to hamper therapeutic effectiveness. In comparison, allele-specific mutant DNA targeting approaches, specifically with the CRISPR/Cas9 system (Mali et al., 2013; Ran et al., 2015), which leads to permanent DNA modification, may be more promising. In fact, the CRISPR/Cas9 system has been used extensively to treat adRP caused by RHO-P23H, S334ter, P347S mutations, etc. This resulted in the delay in photoreceptor degeneration and rescue of retinal function in animal models (Latella et al., 2016; Li et al., 2018; Giannelli et al., 2018; Patrizi et al., 2021; Bakondi et al., 2016). However, these proof-of-concept studies are not suitable for direct translation into clinical practice. Therefore, there is currently no approved treatment for adRP.

The P23H mutation is the most common RHO mutation in North America, accounting for 10% of adRP cases due to a founder effect, while it has not been reported elsewhere, including Europe and Asia (Meng et al., 2020). Data gathered from our previous study (Liu et al., 2021) showed that some Chinese adRP patients carried RHO-T17M mutation. Several studies and data from the Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/ac/index.php) also prove that T17M (Sheffield, 1991; Krebs et al., 2010; Rakoczy et al., 2011) is one of the most common RHO mutations.

Previous in vitro and in vivo studies have shown that if the disease-causing allele has unique protospacer adjacent motif (PAM) sequences that are not present in the WT allele, or when the mutations are within the spacer region of the Cas9/single guide RNA (sgRNA) target site, the disease-causing allele may be discriminated from its WT counterpart. Therefore, the aim of selectively inactivating the mutant allele while leaving the WT ones functionally intact can be achieved (Burnight et al., 2017; Li et al., 2018; Giannelli et al., 2018; Patrizi et al., 2021). Non-homologous end joining (NHEJ) is the primary mechanism for double-strand breaks in terminally differentiated rod cells, whereas homologous recombination commonly occurs in mitotic cells such as induced pluripotent stem cells (iPSCs) or HEK293T (293T) (Chan et al., 2011). This difference in rod cells from mitotic cells emphasizes the significance and necessity of performing experiments in vivo, especially in humanized animal models. Unfortunately, allele-specific CRISPR/Cas9 experiments have not been performed in humanized animal models.

Hence, we developed an allele-specific gene-editing therapy to selectively target the human T17M (c. 50C>T) RHO mutant allele while leaving the WT RHO allele intact for the first time. Our in vitro experiments using 293T cells and patient-specific iPSCs demonstrated high nuclease specificity. Furthermore, a human RHO-knock-in (RHO humanized) mouse model (Liu et al., 2022) generated previously can simulate pathological changes in RHO-adRP, the retinal degeneration might be caused by any one of the five variants (T17M, G51D, G114R, R135W, and P171R, RHO-5m). Using this mouse model, our in vivo experiments demonstrated specific inactivation of the mutant RHO allele and a delay in photoreceptor degeneration for up to 11 months after treatment. Our study demonstrated a dose-dependent therapeutic effect in vivo. Unwanted off-target effects were not observed. Thus, our study clearly provides strong evidence to support further development of this therapeutic drug for patients with RHO-T17M-adRP.

There is currently an increasing number of gene-editing clinical trials to treat hereditary diseases such as sickle cell disease, β-thalassemia (Frangoul et al., 2021) and transthyretin amyloidosis (Gillmore et al., 2021), and even cancer (Stadtmauer et al., 2020). These published data demonstrated the safety and feasibility of the CRISPR/Cas9 system in vivo. Previously, scientists have contributed significantly to the treatment of RHO-adRP using the allele-specific CRISPR/Cas9 gene-editing strategy in iPSCs and animal models (Behnen et al., 2018; Bakondi et al., 2016), demonstrating the promising prospect of this approach in developing effective treatment for this disease. However, none of these methods can be directly applied in clinical practice. Herein, we developed an allele-specific gene-editing therapeutic for RHO c.50C>T (p.T17M) for the first time. SaCas9/17-Sg2 was highly active and specific to the human RHO target sequence. In vitro experiments using 293T cells and patient-specific iPSCs have demonstrated strong nuclease specificity. A single AAV2/8 containing the SaCas9 and 17-Sg2 could be directly used as a therapeutic drug for adRP caused by the RHO-T17M mutation. This potential application was experimentally tested by subretinal delivery of AAV2/8-EFS-SaCas9-U6-17-Sg2 into the RHO humanized mice. Analysis of the treated retinas clearly showed its long-term therapeutic effect (up to 11 mpi) including retinal function improvement and photoreceptor preservation in the heterozygous mutant humanized mice (Liu et al., 2022). Moreover, there was no unwanted off-target activities observed. Thus, our study makes a significant step toward the development of an effective drug for RHO-adRP.

Compared with allele-specific gene-editing therapy, allele-independent gene ablation and replacement approaches are more cost-effective and efficient. However, there are potential risks with the gene ablation and replacement strategy. First, prior studies show that there is a fine balance between insufficiency and toxicity of rhodopsin (Lewin et al., 2014). Changes in the rhodopsin level (either suppression of rhodopsin expression or overexpression) may alter the morphology of rod OS (ROS) and discs (Makino et al., 2012; Wen et al., 2009). It is difficult for the ablation and replacement approach to strike such a balance in vivo. Second, previous studies have indicated that Rho^-/- mice lose all of their rod photoreceptors extremely rapidly (approximately 3 months) (Humphries et al., 1997; Hobson et al., 2000), implying that disruption of both WT and mutant RHO alleles may aggravate the disease, when the exogenous augmentation of WT cannot rescue this disruption. Allele-specific silencing of mutant rhodopsin at the RNA or DNA levels can avoid the above problems. Although rhodopsin function is thought to be essential for the formation of ROS and phototransduction initiation (Gunkel et al., 2015), heterozygous rhodopsin-knockout mice showed little retinal degeneration, indicating that one copy of this gene is sufficient for normal retinal development and functions (Lem et al., 1999). In this respect, maximal therapeutic benefit is expected when the mutant allele is completely ablated, while the normal allele is functionally intact. However, many factors may affect therapeutic effects. For instance, ASO can deregulate mutant rhodopsin levels transcriptionally, but the degree to which the toxic protein must be reduced to eliminate any disease-causing effect is not known (Jun Wan, 2016). The presence of gain-of-function mutations and repeated subretinal injection may diminish the effectiveness of treatment. The CRISPR/Cas9 gene-editing strategy in mammalian cells can provide a means to delete or correct disease-causing mutations while leaving the gene under the control of its endogenous regulatory elements (Jinek et al., 2013; Mali et al., 2013; Burnight et al., 2018), making this approach also promising for the treatment of inherited diseases.

CRISPR/Cas9 gene-editing therapy is currently being advanced as the treatment of human IRDs (Maeder et al., 2019; Burnight et al., 2018). A study conducted by Maeder et al., 2019, shows that one of the complexities of drug development for gene editing is that it is human genome-specific. Herein, we used human cells and the RHO humanized mice to demonstrate that SaCas9/17-Sg2 could reach efficient and allele-specific gene editing, thereby improving the retinal functions and delaying photoreceptor cells loss. We also proved that SaCas9/17-Sg2 did not result in off-target activity using WGS. In vitro patient-derived iPSCs generated in this study offered as an ideal research material to validate gene-editing specificity and cutting efficiency at the DNA level. Moreover, 3D retinal organoids or rod cell precursors derived from iPSCs (Capowski et al., 2019) have the potential to be used to mimic disease phenotypes or for autologous transplantation (Assawachananont et al., 2014; Nozie, 2017; Santos-Ferreira et al., 2016) after gene editing. However, we must emphasize that the transfection and gene-editing efficiency in iPSCs are evidently lower than those of some commonly used cell lines (Burnight et al., 2017; Lin et al., 2014), which may be due to the significant cell death that probably results from an overwhelming cytosolic dsDNA-induced innate immune response (Sun et al., 2013; Kim et al., 2018), implying that the on-target and off-target effects in iPSCs might be underestimated.

The first factor that may raise safety concerns for allele-specific CRISPR/Cas9 drug development is the specificity of SaCas9/SgRNA (Maeder et al., 2019). This study demonstrated that SaCas9/17-Sg2 did not disrupt human WT RHO both in vitro and in vivo. In fact, a large number of allele-specific sgRNAs can be designed for disease-causing mutations in RHO (over 200 mutations, HGMD), such as the three sgRNAs for T17M. However, not all of them work well. A previous study proves theoretically and practically that the specificity of sgRNA depends on its seed sequence, which is a stretch of 8–14 nucleotide (nt) immediately upstream of the PAM. Even point mutations within the seed sequence can abolish cleavage by SpCas9 nucleases (Hsu et al., 2013; Jiang et al., 2013; Semenova et al., 2011), and spCas9 nuclease tolerates single-base mismatches in the PAM-distal region to a greater extent than in the PAM proximal region (Hsu et al., 2013; Jinek et al., 2012). Herein, the specificity of 17-Sg1 and -Sg2 are better than that of 17-Sg3, may due to the fact that the point mismatch (c.50C>T) is within the seed sequence of 17-Sg1 and -Sg2 but not within that of 17-Sg3 (Figure 1), showing that the targeting specificity of SaCas9/SgRNA is also dependent on the seed sequence. Previous studies have indicated that T17M rhodopsin proteins fail to fold correctly, are retained in the ER, and cannot easily reconstitute with 11-cis-retinal (Mendes et al., 2005; Behnen et al., 2018). Our experiments in Hela cells and 293T cells indicates that rhodopsin protein with T17M and five variants are retained in cytoplasm and the endoplasmic reticulum (Liu et al., 2022). Another factor which may raise safety concerns is that indels created by gene editing possibly result in new rhodopsin variants which may also exert toxic effect or dominant-negative on WT rhodopsin (Patrizi et al., 2021). In silico analysis and detailed in vitro experiments indicate that RHO-5m mRNA with these most frequent indels generated by gene editing near the N-terminal of RHO gene can be degraded due to the effect of NMD (Figure 6 and Figure 6—figure supplement 1). Work conducted by Luo et al., 2021, suggested that most of the truncated variants involving upstream of K296 are likely benign, as shown in Supplementary file 1c, all frameshift indels created by AAV-SaCas9/17-Sg2 are upstream of K296, thereby the truncated products after gene editing are benign. Experiments in 293T cells show that this insertion can lead to NMD of the RHO-5m mRNA, consisting with the above theory. These results support the potential safety of gene editing of the N-terminal domain of rhodopsin. Additionally, the preclinical results in NHP further confirmed the safety of the gene-editing drug.

Currently, there are few available animal models compared to the number of known RHO mutations (over 200). Animals with human gene knock-in allow us to study and model disease, and permit testing of the therapeutic effect of CRISPR/Cas9 in preclinical in vivo systems, without putting humans at risk (Hosur et al., 2017). The shortage of humanized animal models may hamper the application of CRISPR/Cas9 technologies from animals toward patients. In the past, several RHO humanized mouse models were bred to express mutant human rhodopsin in a heterozygous knockout mouse rhodopsin (Rho^+/-) or WT background (Lem et al., 1999; Chan et al., 2004; Olsson et al., 1992) by introducing human RHO genomic fragments that encompassed the entire transcriptional unit as well as its upstream and downstream flanking sequences. In contrast, our humanized models are generated by replacing the mouse Rho gene with the human RHO gene, but not interrupting the flaking sequencing of this gene. Previous studies (Mendes et al., 2005; Liu et al., 2021) and data from HGMD have reported that each of the five heterozygous mutations in RHO can cause adRP. Our study indicates (Liu et al., 2022) that the typical adRP phenotypes of Mut-Rho^wt/hum mice result from the RHO-5m allele, and the slow disease progression offers us the opportunity to observe therapeutic effects after treatment. Importantly, mice with different genotypes can be obtained from the intercross of these models according to our needs, thereby facilitating our in vivo experiments. The humanized mouse models combined with our strategy to selectively target the mutant RHO allele faithfully demonstrate their application prospects as well.

Recently, AAV has been considered an ideal choice for therapeutic gene delivery in the retina and has been confirmed to be promising for the delivery of CRISPR/Cas9 reagents (El Refaey et al., 2017; Yang et al., 2016). Besides Luxturna, many gene therapy clinical trials based on AAVs are ongoing (https://www.clinicaltrials.gov/), indicating the feasibility and safety of AAVs. However, AAVs have a relatively small packaging capacity (~4.7 kb), which limits the size and number of sequences that can be incorporated into a single virus (Burnight et al., 2018; Ran et al., 2015). To put all components into one AAV vector, the cytomegalovirus (CMV) enhancer promoter is replaced by the EFS promoter, and SaCas9, which is much smaller than SpCas9 (Ran et al., 2015), is chosen and packed in an AAV vector with its sgRNA. As long as 9 mpi, no detectable toxic effects in mouse retinas are identified at three different doses when comparing the retinal structures of treated and untreated mice. Gene-editing efficiencies herein are dose-dependent, yet the specificity of AAV-SaCas9/17-Sg2 is not affected by increasing AAV dose. However, indel analysis by Sanger sequencing in mouse retinas after treatment showed that a 49 bp insertion from the AAV inverted terminal repeat (ITR) sequence occurs at the cut site at a dose of 3×10⁹. Previous studies confirm that the majority of AAV genome insertions consist of an ITR sequence (Jarrett et al., 2017), which might be due to the combined properties of AAV ITR and its interaction with NHEJ host-cell machinery (McCullough et al., 2019; Young and Samulski, 2001). In this study, no AAV ITR insertions in treated retinas were detected in the 5×10⁸ and 1×10⁹ dose groups. We consider whether AAV dose will influence the possibility of insertions in question, and more research should be conducted to validate this hypothesis. At either DNA or RNA levels, the therapeutic effect in the retina at the 3×10⁹ dose was better than that at the 1×10⁹ dose. As shown in Figure 8—figure supplement 1, at 3 mpi, the retinal functions of untreated WT and Mut-Rho^wt/hum mice were significantly better than that of SaCas9/17-Sg2- and SaCas9/CTRL-treated mice, while the remarkable long-term therapeutic effect in SaCas9/17-Sg2-treated mice was detected, implying that: (a) compared to NHP and human eyes, the eye volume of mouse is relatively small, subretinal injection which is an invasive operation will result in retinal detachment, certainly leading to a damage to retinal functions. However, as times go on, retinal detachment will recover, exerting less effect on the retinal functions, indicating that the long-term therapeutic effect of drug should be observed. (b) In preclinical experiments or even clinical trials, one variable between treated group and control group (gene-editing drug with 17-Sg2 or not) is essential, to ensure the statistical results reliable. For instance, between untreated Mut-Rho^wt/hum mice and treated ones, there were two variables: (a) subretinal injection, which is an invasive operation, (b) the gene-editing drug including AAV-SaCas9/17-Sg2 or not. Previous studies (Mahfouz et al., 2011; Hsu et al., 2013) have indicated that enzymatic specificity and activity strength are often highly dependent on reaction conditions, high enzyme concentration will amplify off-target activity, and one potential strategy for minimizing nonspecific cleavage is to limit the enzyme concentration. Therefore, the balance between therapeutic efficiency and AAV dose of subretinal injection in vivo must be weighted. Since approximately 10% of functional foveal cone photoreceptors are sufficient for near-normal visual acuity, researchers hypothesize that correction of the CEP290 IVS26 mutation in at least 10% of foveal cones will lead to clinical benefit in patients (Maeder et al., 2019). The average cutting efficiencies in GFP+ retinas at 1×10⁹ or 3×10⁹ doses are more than 10%, and the long-term therapeutic effect in more treated mice should be evaluated to obtain more unbiased results.

In summary, this study demonstrates the safety and effectiveness of our allele-specific gene-editing therapy in vitro and in humanized mouse models, thereby offering a generalizable framework for the preclinical development of gene-editing medicine and future clinical trials.

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided in Source Data 1, 2 and 3.

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Decision letter after peer review:

Thank you for submitting your article "Allele-specific gene editing approach for vision loss restoration in RHO-associated Retinitis Pigmentosa" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Lois Smith as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

There are several concerns to be addressed:

1. Detailed description of the discovery phase of the sgRNAs.

2. Knockdown efficiency in photoreceptors at the cellular level in vivo.

3. Clarification of the choice of mouse mutants (RHO humanized mice instead of Rho-T17M mice).

4. Inclusion of two more controls (wild-type mice and untreated mutant mice).

5. Examination of other retinal cell types.

6. Evaluation of the toxicity of the vectors on mouse retinas.

Reviewer #1 (Recommendations for the authors):

Some additional in vivo experiments would greatly strengthen the study:

1. Show the pathology and progression rate of rod death in Rho-T17M mice (not Rho-5m) as a baseline.

2. Test the gene editing efficiency of these vectors in rods at the cellular level using T17M mice (not entire retina or non-specific retinal cells).

3. Check if the vectors rescue vision in T17M mice vision and determine the duration of improvement.

4. Perform histology on mouse retina that is treated with these vector to check for any toxicity.

Reviewer #2 (Recommendations for the authors):

Lines 81-82: RHO-T17M is uncommon in Chinese patients.

Line 183: Figure 3M is cited in the main text (line 183) but is absent in the Figure.

The legend of Figure 7: It lacks descriptions for panel C and D.

Lines 144-146, WB results showed that the expression of Rho in HEK293T cell lines transfected with 17-sg1 or sg2 plasmid was strongly reduced compared with RHOwt cells. Please provide the statistical analysis of the WB result.

Lines 178-180: The results of Hi-Tom sequence of SaCas9 and 17-Sg2-treated P1 iPSCs and P1 iPSCs colony were different. Please explain why the ipS and iPS clones in this study produce different indels. And state how the colonies from P1 iPS can further confirm the allele-specific cutting.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Allele-specific gene editing approach for vision loss restoration in RHO-associated Retinitis Pigmentosa" for further consideration by eLife. Your revised article has been evaluated by Lois Smith (Senior Editor) and Reviewing Editors.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

The current study identified SaCas9 guide RNA that is highly active and specific to human T17M RHO allele, evidenced in HEK293T cells and iPSCs in vitro, as well as RHO humanized mice. This therapeutic approach was supported with solid in vitro data and showed long-term beneficial effects with improved retinal function and preservation of photoreceptors in mutant humanized heterozygous mice. However, some of the key concerns raised in the previous review need to be addressed.

1. Editing efficiency in photoreceptors at cellular level in vivo. Please check the DNA or RNA of rods in situ, report the percentage of rods which were successfully edited by the vector.

2. Clarification of the choice of mouse mutants (RHO humanized mice instead of Rho-T17M mice). The authors need to include the following information regarding RHO humanized mice in the Introduction session: please change "humanized mice" to "hRho-knock-in". Please include the following statement "the retinal degeneration might be caused by any one of the five variants (T17M, G51D, G114R, R135W and P171R)." Please discuss and refer to the literature if each of the five mutations is pathogenic in heterozygous status. Also please include the following reviewer comment "as the five variants are in cis on one allele, gene editing on T17M alone would erase the effect of other four variants because such editing creates frameshift variants at the N-terminal region before the other four".

3. Please edit the language to tone down any overstatement. For example, in the sentence "Humanized animals are revolutionizing our ability to study and model disease and permit testing of the therapeutic effect of CRISPR/Cas9 in preclinical in vivo systems without putting humans at risk", the authors may consider revising as "Animals with human gene knock-in allow us to study and model disease and permit testing of the therapeutic effect of CRISPR/Cas9 in preclinical in vivo systems". The authors are encouraged to check through and edit the manuscript.

Essential revisions:

There are several concerns to be addressed:

1. Detailed description of the discovery phase of the sgRNAs.

Thanks for your suggestion. Presently, there are several websites (CHOPCHOP, Benchling, E-CRISP, etc.) which can be used to design sgRNA easily, herein, sgRNA were designed based on the Benchling website (https://www.benchling.com/). Every sgRNA design website will provide off-target data, these data will help us to evaluate the off-target sites of sgRNA. Herein, Off-target sites were obtained from the Benchling’s CRISPR Tool. For more detailed assessment of off target, off-target sites were identified using the Cas-OFFinder online tool (http://www.rgenome.net/cas-offinder), the detailed method is listed in ref. 1. This was presented in the Material and Methods on Page 31 Line 623 to 627.

ref. 1: Bae S, Park J, Kim JS. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014 May 15;30(10):1473-5. doi: 10.1093/bioinformatics/btu048.

2. Knockdown efficiency in photoreceptors at the cellular level in vivo.

Thanks for your suggestion. Thanks to the fact that RHO gene expressed in rods specially in retina, the mutant human rhodopsin mRNA levels could be evaluated using sequencing. The aim of allele-specific gene-editing treatment was to attenuate or ablate the expression of the mutant allele. Herein, we then determined whether the indels created by SaCas9/17-Sg2 at the DNA level could result in decreased mRNA levels of the RHO-5m allele. AAV-EGFP and AAV-SaCas9/17-Sg2 (1:1 mixture) were co-delivered into both eyes of the Rho^hum/m-hum mice. Rho^hum/m-hum mouse, a humanized mouse model carried a human RHO-WT allele and RHO-5m allele. Truncated cleaved products treated with T7E1 nuclease were observed in treated Rho^hum/m-hum retinas (Figure 7). We also employed Hi-Tom sequencing, Sanger sequencing of RT-PCR products to compare the amount of mRNA transcripts of RHO-WT and RHO-5m in Rho^hum/m-hum retinas at 3 mpi. The results indicated that the percentage of WT transcripts was statistically higher in treated retinas than that in untreated retinas (67.47 % vs. 48.51% (p<0.005) and 60.32 % vs. 42.66% (p<0.05), respectively), and the mutant transcript was statistically lower in treated retinas than that in untreated retinas (32.53 % vs. 51.49% (p<0.005) and 39.68 % vs. 57.34% (p<0.05), respectively, Figure 7C-7D), indicating the mRNA expression of RHO-5m decreased by 36.82% after treatment (Hi-Tom sequencing). This was presented in the Results on Page 16 Line 315 to 324.

3. Clarification of the choice of mouse mutants (RHO humanized mice instead of Rho-T17M mice).

Thanks for your suggestion.

1) Mutant RHO proteins cause disease via either a dominant negative or a toxic gain-of-function effect. The aim of this study is to evaluate whether can effectively treat human RHO-adRP by gene-editing knockout of the mutant protein. Humanized mice permitted permit testing of the therapeutic effect of CRISPR/Cas9 in preclinical in vivo systems, without putting humans at risk. See Figure 5A and Figure 5—figure supplement 1C, there was 8 mismatches between 17-Sg2 and the corresponding mouse Rho sequence, if we want to design sgRNA to target human mutant RHO gDNA, humanized mice were needed.

2) Available RHO humanized models are few compared to the number of known RHO mutations (more than 200), but it is time-consuming and costly to build humanized mice for each mutation. We want to develop a humanized mouse model having several mutations simultaneously to investigate the therapeutic effect of CRISPR/Cas9 for RHO-mediated adRP in preclinical in vivo systems. These five mutations in the humanized mouse model occur in the same human RHO allele, our study indicated the majority of indels created by the activity of SaCas9/17-Sg2 around T17M DNA sequence in Mut-Rho^wt/hum mice could lead to attenuation or ablation of the RHO-5m allele. Thereby, before the gene-editing treatment experiments, we custom designed and generated the humanized mouse models.

3) Our previous study (ref. 2) demonstrated that the pattern of retinal degeneration caused by mutant human rhodopsin was a typical rod-cone decay, thereby Mut-Rho^wt/hum mice were affected with adRP, mimicking human adRP phenotypes. In conclusion, at DNA level, this mouse model could be used to analyze gene-editing efficiency, at RNA level, it could be used to analyze whether the mutant human rhodopsin was knocked down, and it could be employed to evaluate whether its adRP phenotypes were rescued after treatment.

ref. 2: Liu X, Jia R, Meng X, Li Y, Yang L. Retinal degeneration in humanized mice expressing mutant rhodopsin under the control of the endogenous murine promoter. Exp Eye Res. 2022 Feb;215: 108893. doi: 10.1016/j.exer.2021.108893.

4. Inclusion of two more controls (wild-type mice and untreated mutant mice).

Thanks for your suggestion. We added these two control groups, see Figure 8—figure supplement 1. We added these sentences “Besides, the retinal functions of AAV-SaCas9/17-Sg2-treated Mut-Rho^wt/hum mice were better than that of untreated age-matched Mut-Rho^wt/hum mice, and had no significant difference when compared to that of untreated same-age WT mice (Figure 8—figure supplement 1B) (the Results on Page 17 Line 346 to 349).” and “ at 3 mpi, the retinal functions of untreated WT and Mut-Rho^wt/hum mice were significantly better than that of AAV-SaCas9/17-Sg2- and AAV-SaCas9/CTRL -treated mice, while the remarkable long-term therapeutic effect in AAV-SaCas9/17-Sg2- treated mice was detected, implying that: (a) compared to NHP and human eyes, the eye volume of mouse was relatively small, subretinal injection which was an invasive operation would result in retinal detachment, certainly leading to a damage to retinal functions. However, as times went on, retinal detachment would recover, exerting less effect on the retinal functions, indicating that the long-term therapeutic effect of drug should be observed. (b) In preclinical experiments or even clinical trials, one variable between treated group and control group (gene-editing drug with 17-Sg2 or not) was essential, to ensure the statistical results was reliable. For instance, between untreated Mut-Rho^wt/hum mice and treated ones, there were two variables: (a) subretinal injection, which is an invasive operation, (b) the gene-editing drug including AAV-SaCas9/17-Sg2 or not (the Discussion on Page 27-28 Line 555 to 568).

5. Examination of other retinal cell types.

Thanks for your suggestion. We added Figure 9—figure supplement 3 to Supplementary file. Protein kinase C (PKC-α) for rod bipolar cells, calbindin for horizontal cells, and CRALBP for RPE and Müller cells which span throughout the retina vertically, compared to untreated mice and AAV-SaCas9/CTRL-treated mice at 9 mpi and 11 mpi, more and healthier bipolar cells, horizontal cells and Müller cells were detected in AAV-SaCas9/17-Sg2-treated retinas. This was added to the Results on Page 18 Line 361 to 367.

6. Evaluation of the toxicity of the vectors on mouse retinas.

Thanks for your suggestion. First, the first factor that may raise safety concerns for allele-specific CRISPR/Cas9 drug development is the specificity of SaCas9/SgRNA (ref. 3). This study demonstrated that SaCas9/17-Sg2 did not disrupt human WT RHO both in vitro and in vivo (Figure 5-figure supplement 1) and no off-target effect (Figure 10). Second, up to 11 mpi, no detectable toxic effects in WT mouse retinas were identified when comparing the retinal structures of treated and untreated mice (Figure 4D). In addition, AAV-SaCas9/17-Sg2 was also delivered to subretinal space of nonhuman primate (NHP), ERG and HE staining indicated no toxicity of the vector (Figure 10—figure supplement 2).

ref. 3: Maeder ML, Stefanidakis M, Wilson CJ, et.al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat Med. 2019 Feb;25(2):229-233. doi: 10.1038/s41591-018-0327-9.

Reviewer #1 (Recommendations for the authors):

Some additional in vivo experiments would greatly strengthen the study:

1. Show the pathology and progression rate of rod death in Rho-T17M mice (not Rho-5m) as a baseline.

Thanks for your suggestion.

1) Mutant RHO proteins cause disease via either a dominant negative or a toxic gain-of-function effect. The aim of this study is to evaluate whether can effectively treat human RHO-adRP by gene-editing knockout of the mutant protein. Humanized mice permitted permit testing of the therapeutic effect of CRISPR/Cas9 in preclinical in vivo systems, without putting humans at risk. See Figure 5A and Figure 5—figure supplement 1C, there was 8 mismatches between 17-Sg2 and the corresponding mouse Rho sequence, if we want to design sgRNA to target human mutant RHO gDNA, humanized mice were needed.

2) Available RHO humanized models are few compared to the number of known RHO mutations (more than 200), but it is time-consuming and costly to build humanized mice for each mutation. We want to develop a humanized mouse model having several mutations simultaneously to investigate the therapeutic effect of CRISPR/Cas9 for RHO-mediated adRP in preclinical in vivo systems. These five mutations in the humanized mouse model occur in the same hRHO allele, our study indicated the majority of indels created by the activity of SaCas9/17-Sg2 around T17M DNA sequence in Mut-Rho^wt/hum mice could lead to attenuation or ablation of the RHO-5m allele. Thereby, before the gene-editing treatment experiments, we custom designed and generated the humanized mouse models.

3) Our previous study (ref. 2) demonstrated that the pattern of retinal degeneration caused by mutant human rhodopsin was a typical rod-cone decay, thereby Mut-Rho^wt/hum mice were affected with adRP, mimicking human adRP phenotypes. In conclusion, at DNA level, this mouse model could be used to analyze gene-editing efficiency, at RNA level, it could be used to analyze whether the mutant human rhodopsin was knocked down, and it could be employed to evaluate whether its adRP phenotypes were rescued after treatment.

ref. 2: Liu X, Jia R, Meng X, Li Y, Yang L. Retinal degeneration in humanized mice expressing mutant rhodopsin under the control of the endogenous murine promoter. Exp Eye Res. 2022 Feb;215: 108893. doi: 10.1016/j.exer.2021.108893.

2. Test the gene editing efficiency of these vectors in rods at the cellular level using T17M mice (not entire retina or non-specific retinal cells).

Thanks for your suggestion. Thanks to the fact that RHO gene expressed rods specially in retina, the mutant human rhodopsin mRNA levels could be evaluated using sequencing. The aim of allele-specific gene-editing treatment was to attenuate or ablate the expression of the mutant allele. Herein, we then determined whether the indels created by SaCas9/17-Sg2 at the DNA level could result in decreased mRNA levels of the RHO-5m allele. AAV-EGFP and AAV-SaCas9/17-Sg2 (1:1 mixture) were co-delivered into both eyes of the Rho^hum/m-hum mice. Rho^hum/m-hum mouse, a humanized mouse model carried a human RHO-WT allele and RHO-5m allele. Truncated cleaved products treated with T7E1 nuclease were observed in treated Rho^hum/m-hum retinas (Figure 7). We employed Hi-Tom sequencing, Sanger sequencing of RT-PCR products to compare the amount of mRNA transcripts of RHO-WT and RHO-5m in Rho^hum/m-hum retinas at 3 mpi. The results indicated that the percentage of WT transcripts was statistically higher in treated retinas than that in untreated retinas (67.47 % vs. 48.51% (p<0.005) and 60.32 % vs. 42.66% (p<0.05), respectively), and the mutant transcript was statistically lower in treated retinas than that in untreated retinas (32.53 % vs. 51.49% (p<0.005) and 39.68 % vs. 57.34% (p<0.05), respectively, Figure 7C-7D), indicating the mRNA expression of RHO-5m decreased by 36.82% after treatment (Hi-Tom sequencing). This was presented in the Results on Page 16 Line 315 to 324.

3. Check if the vectors rescue vision in T17M mice vision and determine the duration of improvement.

Thanks for your suggestion. Mutant RHO proteins cause disease via either a dominant negative or a toxic gain-of-function effect. The aim of this study is to evaluate whether can effectively treat human RHO-adRP by gene-editing knockout of the mutant protein. These five mutations in the humanized mouse model occur in the same human RHO allele, once the mutant allele around the T17M DNA sequence was cut by Cas9/sgRNA, the downstream sequence of the cut site would be abolished due the DNA NHEJ repair. Our results herein confirmed the above theory (Figure 6-7). That’s no doubt that our humanized models could be used gene-editing efficiency at DNA level. In addition, the previous study (ref. 5) has established a transgenic mouse that expresses both normal and mutant murine rhodopsin in rods, the mutant rhodopsin contains 3 amino acids substitutions (V20G, P23H and P27L) simultaneously and results in a slowly progressing rods and cones degeneration. The structural and functional defects of P23H mice closely mimic those in patients carrying the same mutation. Several previous studies (ref. 6-7) indicated that rhodopsin with T17M, P23H, etc. could result in late onset adRP in transgenic mouse models. Our previous study (ref. 2) demonstrated that the pattern of retinal degeneration caused by mutant human rhodopsin was a typical rod-cone decay, thereby Mut-Rho^wt/hum mice were affected with adRP, mimicking human adRP phenotypes. In conclusion, Mut-Rho^wt/hum mice can be used to evaluate the gene-editing efficiency, retinal functions improvement and photoreceptor preservation after gene-editing treatment. Administration of this therapeutic drug resulted in a long-term (up to 11 months after treatment) improvement of retinal function and preservation of photoreceptors in the mutant humanized heterozygous mice. Our study demonstrated a dose-dependent therapeutic effect in vivo.

ref. 2: Liu X, Jia R, Meng X, Li Y, Yang L. Retinal degeneration in humanized mice expressing mutant rhodopsin under the control of the endogenous murine promoter. Exp Eye Res. 2022 Feb; 215:108893. doi: 10.1016/j.exer.2021.108893.

ref. 5: Naash MI, Hollyfield JG, al-Ubaidi MR, Baehr W. Simulation of human autosomal dominant retinitis pigmentosa in transgenic mice expressing a mutated murine opsin gene. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5499-503. doi: 10.1073/pnas.90.12.5499.

ref. 6: Olsson, J. E., Gordon, 1. W., Pawlyk, B. S., Roof, D., Hayes, A., Molday, R. S., Mukai, S., Cowley, G. S., Berson, E. L., and Dryja, T. P., 1992, Transgenic mice with a rhodopsin mutation (Pr023His): a mouse model of autosomal dominant retinitis pigmentosa, Neuron 9:815-830.

ref. 7: Kunte MM, Choudhury S, Manheim JF, Shinde VM, Miura M, Chiodo VA, Hauswirth WW, Gorbatyuk OS, Gorbatyuk MS. ER stress is involved in T17M rhodopsin-induced retinal degeneration. Invest Ophthalmol Vis Sci. 2012 Jun 20;53(7):3792-800. doi: 10.1167/iovs.11-9235.

4. Perform histology on mouse retina that is treated with these vector to check for any toxicity.

Thanks for your suggestion. First, the first factor that may raise safety concerns for allele-specific CRISPR/Cas9 drug development is the specificity of SaCas9/SgRNA (ref. 3). This study demonstrated that SaCas9/17-Sg2 did not disrupt human WT RHO both in vitro and in vivo (Figure 5—figure supplement 1) and no off-target effect (Figure 10). Second, up to 11 mpi, no detectable toxic effects in WT mouse retinas were identified when comparing the retinal structures of treated and untreated mice (Figure 4D). In addition, AAV-SaCas9/17-Sg2 was also delivered to subretinal space of nonhuman primate (NHP), ERG and HE staining indicated no toxicity of the vector (Figure 10—figure supplement 2).

ref. 3: Maeder ML, Stefanidakis M, Wilson CJ, et.al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat Med. 2019 Feb;25(2):229-233. doi: 10.1038/s41591-018-0327-9.

Reviewer #2 (Recommendations for the authors):

Lines 81-82: RHO-T17M is uncommon in Chinese patients.

Thanks for your suggestion. In our previous study and clinical practice (Peking university third hospital and Beijing Tongren hosptial), several adRP families with many affected individuals had RHO-T17M mutation, and in HGMD (http://www.hgmd.cf.ac.uk/ac/index.php) database, at least 7 references reported this mutation, thereby, we indicated “some Chinese adRP patients carried RHO-T17M mutation”.

ref. 4: Liu X, Tao T, Zhao L, Li G, Yang L. Molecular diagnosis based on comprehensive genetic testing in 800 Chinese families with non-syndromic inherited retinal dystrophies. Clin Exp Ophthalmol. 2021 Jan;49(1):46-59. doi: 10.1111/ceo.13875.

Line 183: Figure 3M is cited in the main text (line 183) but is absent in the Figure.

Thanks for your suggestion. We have corrected this mistake, Figure 3M should be Figure 3L.

The legend of Figure 7: It lacks descriptions for panel C and D.

Thanks for your suggestion. We have corrected this mistake. This was presented in the Figure Legends on Page 59 Line 1219 to 1221.

Lines 144-146, WB results showed that the expression of RHO in HEK293T cell lines transfected with 17-sg1 or sg2 plasmid was strongly reduced compared with RHOwt cells. Please provide the statistical analysis of the WB result.

Thanks for your suggestion. We added the statistical analysis of the WB result to Figure 2, as illustrated in Figure 2E, densitometric analysis of immunoblots performed on RHOwt and RHO17 cells transfected with 17-Sg1 and Sg2 plasmid, respectively. This was presented in the Figure Legends on Page 56 Line 1156 to 1159.

Lines 178-180: The results of Hi-Tom sequence of SaCas9 and 17-Sg2-treated P1 iPSCs and P1 iPSCs colony were different. Please explain why the ipS and iPS clones in this study produce different indels. And state how the colonies from P1 iPS can further confirm the allele-specific cutting.

Thanks for your suggestion. In this study, iPSCs were obtained from UCs using the integration-free CytoTune 2.0 Sendai Reprogramming Kit (A16517, Thermo Fisher Scientific, MA, USA), the detailed protocol was provided by the manufacturer. Herein, sequencing results of treated P1 iPSCs was listed herein, P1 iPSCs colony was picked from P1 iPSCs, which were better shaped, well-conditioned cells, in order to validate the result and to obtain more objective result, experiment of P1 iPSCs colony was performed repeatedly, we just presented the objective results of DNA sequencing, just like qPCR results, if we want to get credible results, the experiment will be performed in triplicate.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

The current study identified SaCas9 guide RNA that is highly active and specific to human T17M RHO allele, evidenced in HEK293T cells and iPSCs in vitro, as well as RHO humanized mice. This therapeutic approach was supported with solid in vitro data and showed long-term beneficial effects with improved retinal function and preservation of photoreceptors in mutant humanized heterozygous mice. However, some of the key concerns raised in the previous review need to be addressed.

1. Editing efficiency in photoreceptors at cellular level in vivo. Please check the DNA or RNA of rods in situ, report the percentage of rods which were successfully edited by the vector.

Thanks for your suggestion.

As we know, CRISPR/Cas9 system can lead to permanent DNA modification because of endonuclease properties of Cas9, thereby it is essential to calculate the gene-editing efficiency at DNA level. While the aim of CRISPR/Cas9 gene-editing is to alter gene expression transcriptionally and translationally. Mutant RHO proteins cause disease via either a dominant negative or a toxic gain-of-function effect. So, it is ideal and promising to treat human RHO-adRP by gene-editing knockout of the mutant protein, thereby to increase WT and mutant RHO protein ratio. That is the core idea of this study.

1) Initially, to test the editing efficiency of rods at the cellular level in vivo, we tried to use Fluorescence activated Cell Sorting (FACS) technique to sort AAV-infected rods by labeling the cells with specific marker, then to calculate the gene editing efficiency. However, in practice, we have encountered a lot of problems, for instance, during FACS experiments, it was difficult to a) digest the entire retinal tissue into single cells, b) prevent excessive rods loss, and we could not obtain abundant gDNA from few sorted cells. Previously, several articles reported that under a fluorescent dissection microscope, GFP+ retinal areas which represented AAV-infected areas could be obtained (ref. 1~2). Herein, AAV-EGFP and AAV-SaCas9/17-Sg2 (1:1 mixture) were co-delivered into both eyes of the humanized mouse models. IF analysis indicated that GFP was expressed in the OS/IS and ONL of photoreceptor cells (Figure 4D, Figure 9A, Figure 9—figure supplement 1 and Figure 9—figure supplement 4), showing that SaCas9 and 17-Sg2 were also expressed in OS/IS and ONL, therefore, the gDNA of rods and cones in ONL would be edited.

Neural retina consists of nine layers as illustrated in ref.3. In this study, AAV infected in OS/IS and ONL mainly. Under a fluorescent dissection microscope, we flattened the neural retina (Figure 4C), the GFP+ areas were dissected and gene-editing efficiency in these areas were focused. While in fact, we collected all types of cells apart from rods and cones, including those cells without infection in other retinal layers. So, compared to the gene-editing efficiency in all types of cells (we calculated in this study), actual gene-editing efficiency in rods was expected to be higher.

2) As we said above, the aim of CRISPR/Cas9 gene-editing is to alter gene expression transcriptionally and translationally. To treat RHO-adRP, it is promising and important to increase the WT RHO mRNA level and decrease the mutant RHO mRNA level. Thanks to the fact that RHO gene only expressed in rods both transcriptionally and translationally, its encoding protein rhodopsin is the marker of rods and embedded at high density in the membrane of the rods outer segment (ROS) disks. At DNA level, gene-editing could occur in all types of AAV-infected retinal cells, while at mRNA and protein level, RHO expression could be attenuated or ablated only in rods. Fortunately, Hi-Tom sequencing result of RT-PCR products indicated that the mRNA expression of RHO-5m decreased by 36.82% after treatment. That is the editing efficiency or in rods (the targeted cell type) in situ at mRNA level in vivo.

3) Our previous study (ref. 4) reported that the mouse Rho gene contains 5 exons and shares a high homology at coding sequence (88.3% homology, Supplementary Figure S1) and amino acids sequence (94.8% homology, Supplementary Figure S2) with human RHO gene. That is a prerequisite for successful construction of our humanized mice (ref. 4).

Previously, we tried to use fluorescence in situ hybridization (FISH) technique to discriminate mouse and human RHO gene, or WT and mutant RHO gene after treatment. However, we could not obtain any appropriate probe due to their high homology at coding sequence. Most importantly, if the mutant RHO gene was not edited (control groups), the rods would die ultimately, the RHO expression would decrease, while the RHO expression decreased due to allele-specific gene-editing (SaCas9/17-Sg2-treated-groups, Figure 7C-E). Such decreased expression cannot be detected by FISH or WB, so the difference of gene ablation expression in control and treated groups cannot be counted effectively. Fortunately, Sequencing of RT-PCR products could help us to reach this target. And a previous reference (ref. 1) also used RT-PCR product sequencing to evaluate the mutant and WT RHO allele ratio after allele-specific gene-editing therapy.

4) Presently, commercial primary antibodies for rhodopsin are 4D2 antibody (antibody recognizing N-terminal of rhodopsin protein) and 1D4 antibody (antibody recognizing C-terminal nine amino acids of rhodopsin). However, both cannot discriminate mouse and human rhodopsin due to the high homology at amino acids sequence (94.8% homology). Our previous study (ref. 4) showed that in mutant humanized mice, mutant rhodopsin localized to RIS and cell bodies while WT rhodopsin should correctly be trafficked to ROS. The aim of allele-specific gene-editing treatment was to attenuate or ablate the expression of the mutant RHO. “As showed in Figure 9—figure supplement 1A and C, compared to CTRL groups, mutant rhodopsin in RIS and rods bodies (4D2, red) decreased significantly in SaCas9/17-Sg2-treated mouse retinas, indicating the mutant rhodopsin was ablated in rods (the targeted cell type), similar results were also detected at 9 mpi and 11 mpi (Figure 9—figure supplement 3)”. This was presented on the Results on Page 18 Line 364 to 370. The Figure legends was presented on Page 64 to 65 Line 1332 to 1340.

ref. 1: LI, P., KLEINSTIVER, B. P., LEON, M. Y., et al. 2018. Allele-Specific CRISPR-Cas9 Genome Editing of the Single-Base P23H Mutation for Rhodopsin-Associated Dominant Retinitis Pigmentosa. CRISPR J, 1, 55-64.

ref. 2: Benati D, Marigo V, Recchia A. CRISPR/Cas9 Gene Editing in vitro and in Retinal Cells in vivo. Methods Mol Biol. 2019;1834:59-74.

ref. 3: Power M, Das S, Schutze K, et al. Cellular mechanisms of hereditary photoreceptor degeneration – Focus on cGMP [J]. Prog Retin Eye Res, 2020, 74(100772).

ref. 4: Liu X, Jia R, Meng X, Li Y, Yang L. Retinal degeneration in humanized mice expressing mutant rhodopsin under the control of the endogenous murine promoter. Exp Eye Res. 2022 Feb;215: 108893. doi: 10.1016/j.exer.2021.108893.

2. Clarification of the choice of mouse mutants (RHO humanized mice instead of Rho-T17M mice). The authors need to include the following information regarding RHO humanized mice in the Introduction session: please change "humanized mice" to "hRho-knock-in". Please include the following statement "the retinal degeneration might be caused by any one of the five variants (T17M, G51D, G114R, R135W and P171R)." Please discuss and refer to the literature if each of the five mutations is pathogenic in heterozygous status. Also please include the following reviewer comment "as the five variants are in cis on one allele, gene editing on T17M alone would erase the effect of other four variants because such editing creates frameshift variants at the N-terminal region before the other four".

Thanks for your suggestion.

1) In this study, we emphasized the significance of humanized mouse models if we want to develop gene-editing medicine for human RP and explain why we generated and used RHO-5m humanized mouse (Introduction and Discussion). Herein, Rho^wt/hum mice, which was heterozygotes with a mouse Rho-WT allele and human RHO-WT allele, were used to evaluate gene editing specificity. Mut-Rho^wt/hum mice, which were heterozygotes with a mouse Rho-WT allele and human RHO-5m allele, were used to test cutting efficiency in vivo (Page 11 to 12 on Line 221 to 227). Rho^hum/m-hum mouse, a humanized mouse model carried a human RHO-WT allele and RHO-5m allele. They were used to test whether RHO-5m expression was reduced, and to test whether allele-specific gene editing could increase WT and mutant RHO protein ratio in vivo (Page 15 on Line 304 to 308).

2) We changed “RHO humanized mouse model” to “human RHO-knock-in mouse model”. And add the sentence “the retinal degeneration might be caused by any one of the five variants (T17M, G51D, G114R, R135W and P171R, RHO-5m)”. This was presented on the Introduction on Page 5 Line 103 to 106.

3) The sentence “Previous studies (Mendes et al., 2005, Liu et al., 2021) and data from HGMD have reported t that each of the five heterozygous mutations in RHO can cause adRP” was added to the Introduction (Page 26 Line 531 to 532).

4) The sentence “as the five variants are in cis on one allele, allele-specific gene editing on T17M alone would erase the effect of other four variants because such editing creates frameshift variants at the N-terminal region before the other four” was added. The sentence “The existence of the other four variants in the RHO coding region would not interrupt the allele specific gene editing for T17M” was deleted. This was presented on the Results on Page 15 Line 297 to 299.

3. Please edit the language to tone down any overstatement. For example, in the sentence "Humanized animals are revolutionizing our ability to study and model disease and permit testing of the therapeutic effect of CRISPR/Cas9 in preclinical in vivo systems without putting humans at risk", the authors may consider revising as "Animals with human gene knock-in allow us to study and model disease and permit testing of the therapeutic effect of CRISPR/Cas9 in preclinical in vivo systems". The authors are encouraged to check through and edit the manuscript.

Thanks for your suggestion. The entire manuscript has also been proofread again to correct any other mistakes and grammatical errors. The sentence “Humanized animals are revolutionizing our ability to study and model disease and permit testing of the therapeutic effect of CRISPR/Cas9 in preclinical in vivo systems without putting humans at risk” was replaced with “Animals with human gene knock-in allow us to study and model disease and permit testing of the therapeutic effect of CRISPR/Cas9 in preclinical in vivo systems”. This was presented on the Discussion on Page 25 Line 520 to 522.

Author details

1. Xiaozhen Liu

   1. Department of Ophthalmology, Third Hospital, Peking University, Beijing, China
   2. Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital,, Beijing, China

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Methodology, Writing – original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9893-1986

2. Jing Qiao

   Beijing Chinagene Co., LTD, Beijing, China

   Contribution

   Resources, Software, Formal analysis, Validation, Investigation, Writing – original draft, Project administration

   Competing interests

   is an employee of Beijing Chinagene Co.,LTD and was employeed by Beijing Chinagene Co.,LTD at the time this work was conducted. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed

3. Ruixuan Jia

   1. Department of Ophthalmology, Third Hospital, Peking University, Beijing, China
   2. Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital,, Beijing, China

   Contribution

   Data curation, Software, Formal analysis, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Fan Zhang

   Beijing Chinagene Co., LTD, Beijing, China

   Contribution

   Software, Formal analysis, Investigation, Methodology

   Competing interests

   is an employee of Beijing Chinagene Co.,LTD and was employeed by Beijing Chinagene Co.,LTD at the time this work was conducted. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed

5. Xiang Meng

   1. Department of Ophthalmology, Third Hospital, Peking University, Beijing, China
   2. Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital,, Beijing, China

   Contribution

   Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Yang Li

   Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

7. Liping Yang

   1. Department of Ophthalmology, Third Hospital, Peking University, Beijing, China
   2. Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital,, Beijing, China

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing - review and editing

   For correspondence

   alexlipingyang@bjmu.edu.cn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4239-228X

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