Mutation-independent genome editing

Abstract

The present invention is directed to a genome editing kit for targeted integration of an exogenous DNA in a target site of a large gene by non-homologous end joining (NHEJ) or by microhomology-mediated end joining (MMEJ), wherein said kit comprises donor nucleic acids for replacing the 5' and 3' portions of the target gene relative to the target site.

Description

[0001] MUTATION-INDEPENDENT GENOME EDITING

[0002] FIELD OF THE INVENTION

[0003] The present invention is directed to a system for gene editing of large genes by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) targeted integration of exogenous DNAs configured to reconstituting the entire coding sequence of the target gene. The invention is particularly suitable for the treatment of diseases due to mutations in large genes. Therefore, the invention is preferably directed to a system for genome editing of a large gene having a coding sequence of at least 4 kb.

[0004] BACKGROUND

[0005] Targeted genome editing restoring wild-type DNA sequences within an endogenous locus may represent a valid alternative to gene therapy for correcting disease-causing mutations.

[0006] Programmable site-specific nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR-associated Cas9 nucleases can be designed to target any gene of interest, inducing a double-strand break (DSB) in the gene, that can be then modified or repaired through insertion of exogenous DNA.

[0007] In particular, CRISPR / Cas system is programmed by a small guide RNA (gRNA) to generate the DSB in a sequence-specific manner, at any desired genomic locus that is followed by a protospacer-adjacent motif (PAM).

[0008] However, this approach is mutation-specific, which limits its pharmaceutical development in highly heterogenous diseases, due to different mutations in a gene.

[0009] Additionally, precise gene correction typically requires homology-directed repair (HDR) which is inefficient in post-mitotic, terminally differentiated neurons like photoreceptors.

[0010] A knock-in strategy defined as Homology -Independent Targeted Integration (HITI) (Suzuki K, et al. In vivo genome editing via the HITI method as a tool for gene therapy. Journal of Human Genetics. 2018 Feb;63(2):157-164) has been developed recently for gene editing in differentiated and proliferating cells, taking advantage of genome repair mediated by the Non-homologous end joining (NHEJ). NHEJ pathway is more efficient than HDR in mammalian species, while relying on the use of a donor template DNA flanked by two gRNA recognition sites that correspond to the intended target sites at the genomic locus. Upon co-delivery to cells of the donor template DNA, with the nuclease and the gRNA specific for the target locus where the intended template DNA should be incorporated, the genomic locus and the donor vector are simultaneously cleaved producing a double-strand break (DSB). NHEJ connects these DSB, resulting in the integration of the entire donor template into the targeted genomic locus. Besides classical NHEJ, microhomology-mediated end joining (MMEJ) has also emerged in the last two decades as another DNA repair pathway which is active during all phases of the cell cycle (Yanik M, et al. In vivo genome editing as a potential treatment strategy for inherited retinal dystrophies. Prog Retin Eye Res. 56:1-18, 2017). In MMEJ, microhomologies exist upstream and downstream of the DSB site on the two DNA strands. DSBs with microhomologies can result in annealing of the microhomologies and subsequent repair by MMEJ. This pathway can be also used to integrate donor DNAs if microhomologies exist between the target locus and the extremities of the donor template. This strategy is also called microhomology-dependent targeted integrations. (MITI).

[0011] Delivery of HITI and MITI strategies have the advantage of being mutation independent, as they allow replacement of entire portions of a target gene (or even of the entire gene).

[0012] However, a true mutation independent strategy is not available for those inherited diseases due to mutations in large genes, in particular for delivery through adeno-associated viruses (AAVs) because of the AAV vector cargo capacity, that is restricted to about 5 kb. In fact, AAV-mediated gene editing strategies of the prior art targeting large genes can at most deliver a donor template for substituting a portion of the target gene, requiring multiple gRNA-nuclease-donor template systems when mutations to be corrected are located in portions of the gene that are far from each other, e.g. farther than 4 kb.

[0013] A mutation-independent system for treating any mutation in large genes by AAV delivery of a donor template is not available and is highly desirable as AAVs are particularly efficient for in vivo applications, due to their wide tropism and ability to provide long-term transgene expression, and moreover have a high safety profile.

[0014] For instance, the retina is one of the most relevant target tissues of AAV-mediated gene therapy. However, common blinding conditions are due to mutations in large genes, such as Stargardt macular degeneration (STGD1) and Leber Congenital Amaurosis type 10 (LCA10).

[0015] Therefore, treatment of blinding conditions or other diseases due to mutations in large genes would benefit from a system for AAV-mediated gene editing which is completely mutation independent.

[0016] BRIEF DESCRIPTION OF THE INVENTION

[0017] The limitations of the prior-art are overcome by the present invention, providing a novel mutation-independent genome editing strategy suitable for the therapy of inherited diseases that are not addressable by conventional AAV-mediated gene replacement, being due to mutations in large genes, such as retinal degenerations. The approach is based on the delivery of a nuclease, preferably a CRISPR-Cas9 nuclease, to induce double strand breaks (DSBs) in an intron of a large gene, and to the induction of targeted integration of an exogenous DNA replacing the coding sequence (CDS) of the gene that is either upstream (5’ exogenous DNA) or downstream (3’ exogenous DNA) the DSB target site, wherein integration of the exogenous DNA reconstitutes the target gene’s coding sequence.

[0018] In particular, the invention is directed to a kit for gene editing of a target gene by targeted integration of an exogenous nucleic acid, i.e. an exogenous DNA, comprising:

[0019] a) a nuclease capable of introducing a double strand break (DSB) in a target site of a target gene, or a polynucleotide encoding thereof, said target site being an intron of the target gene;

[0020] b) a complementary strand oligonucleotide having sequence homologous to the target site sequence;

[0021] c.l) a first donor nucleic acid comprising a first knock-in cassette for reconstitution of the target gene, said first cassette comprising from 5’ to 3’:

[0022] - a promoter,

[0023] - an exogenous polynucleotide operably linked to said promoter and having sequence homologous to the portion of the target gene upstream the DSB (5’ exogenous polynucleotide), and

[0024] - a splicing donor signal;

[0025] and

[0026] c.2) a second donor nucleic acid comprising a second knock-in cassette for reconstitution of the target gene, said second cassette comprising:

[0027] - a splicing acceptor signal,

[0028] - an exogenous polynucleotide having sequence homologous to the portion of the target gene downstream the DSB (3’ exogenous polynucleotide), and

[0029] - a poly adenylation signal;

[0030] wherein each of said first and second knock-in cassettes is flanked, at both the 5’ and 3’, by target integration polynucleotides for NHEJ having sequence homologous to the target site sequence in inverted orientation (inverted targeting sequence), for HITI-based strategies, or is flanked, at both the 5’ and 3’, by homology arms polynucleotides for MMEJ having sequence of 5 to 25 base pairs (bp) in length (microhomology arms), for MITI-based strategies; and wherein the target gene has a coding sequence of at least 4 kb in length, and preferably not greater than 10 kb in length. In some embodiments, the microhomology arms are flanked by target integration polynucleotides having sequence homologous to the target site sequence; this advantageously leads to cutting of the donor nucleic acid at 5’ and 3’ of microhomology arms, favoring exposure of microhomology arms.

[0031] Preferably the 5’ and 3’ exogenous nucleic acids have respectively sequence homologous to the coding sequence of the 5’ and 3’ portions of the target gene. Targeted integration of the 5’ or 3’ exogenous DNA will lead to the generation of a hybrid transcript that derives from the fusion of the endogenous gene to the sequence of the exogenous DNA that replaces the mutated sequence (Fig. 1).

[0032] Thanks to the inclusion of appropriate splicing signals flanking the exogenous DNA (a splicing donor for the exogenous DNA replacing the 5’ portion of the gene or a splicing acceptor for the exogenous DNA replacing the 3’ portion), expression of the full-length wild-type transcript of the target gene is restored, independently of the accuracy of integration at the two extremities. The exogenous DNAs can then correct target gene portions that comprise any mutation by replacing them with a correct portion encoded by a sequence that can be easily accommodated into a single AAV vector. The strategy of the invention overcomes the important limitation of delivering the full-length gene to photoreceptors using the golden-standard AAV vectors. The 5’ and 3’ exogenous DNAs and the guide RNA- nuclease system are properly designed to correcting any mutation of a target gene with a single gRNA-nuclease system, coupled with a donor DNA providing the correct 5’ or the 3’ portion of the gene, depending on the mutation to be corrected.

[0033] Therefore, the kit of the invention is suitable for treating any mutation in a large gene, providing a genome editing system that allows a truly mutation-independent correction of large genes with a safe and efficient in vivo approach.

[0034] The donor nucleic acids comprising the 5 ’ and 3 ’ exogenous DNA are meant to be independently delivered to patients carrying mutations in, respectively, the 5’ or in 3’ portion of the gene, together with a gRNA-nuclease system that fits for all.

[0035] Therefore, the invention is also directed to a gene editing system for targeted integration of an exogenous nucleic acid in a target gene having a coding sequence of at least 4 kb in length, comprising:

[0036] a) a nuclease capable of introducing a double strand break (DSB) in a target site of a target gene, or a polynucleotide encoding thereof, said target site being an intron of the target gene; b) a complementary strand oligonucleotide having sequence homologous to the target site sequence; and

[0037] c) a donor nucleic acid for reconstitution of the target gene, said donor nucleic acid being:

[0038] c.l) a first donor nucleic acid comprising a first knock-in cassette for reconstitution of the target gene, said first cassette comprising from 5’ to 3’:

[0039] - a promoter,

[0040] - an exogenous polynucleotide operably linked to said promoter and having sequence homologous to the portion of the target gene upstream the DSB (5’ exogenous polynucleotide), and

[0041] - a splicing donor signal;

[0042] or

[0043] c.2) a second donor nucleic acid comprising a second knock-in cassette for reconstitution of the target gene, said second cassette comprising:

[0044] - a splicing acceptor signal,

[0045] - an exogenous polynucleotide having sequence homologous to the portion of the target gene downstream the DSB (3’ exogenous polynucleotide), and

[0046] - a poly adenylation signal;

[0047] wherein said first or second knock-in cassette is flanked at both the 5’ and 3’ by target integration polynucleotides for NHEJ having sequence homologous to the target site sequence and inverted (inverted targeting sequence), for HITI-based strategies, or by homology arms polynucleotides for MMEJ (microhomology arms) having sequence of 5 to 25 base pairs (bp) in length, for MITI-based strategies.

[0048] The invention also relates to vectors comprising one or more of the components of the gene editing kit, such as the donor nucleic acid, the nuclease and / or the complementary strand oligonucleotide, or comprising polynucleotides encoding thereof. The invention also relates to viral particles comprising said vectors, preferably AAV viral particles comprising AAV capsid proteins.

[0049] The invention also refers to a method of integrating an exogenous DNA into a target gene in a cell, comprising contacting the cell with a kit according to the invention comprising the first or the second donor nucleic acid. Preferably, said method is an in vitro or ex vivo method.

[0050] Further, the invention is directed to a gene edited cell, obtainable by the method of the invention or comprising the nuclease, the complementary strand oligonucleotide and either the first or the second donor nucleic acid of the invention, or comprising vectors for expressing the same, and to pharmaceutical formulations thereof, further comprising suitable pharmaceutically acceptable excipients.

[0051] In a further aspect, the present invention is also directed to said kit, vectors, cells, or pharmaceutical formulations thereof, for use as a medicament, preferably for use in the treatment of a genetic disease caused by mutations in the target gene.

[0052] The present invention is therefore also directed to a method of treatment of a genetic disease comprising administering to a subject in need thereof a therapeutic amount of the kit, vector, or cell of the invention, or of pharmaceutical formulations thereof.

[0053] The features and advantages of the present invention will become apparent from the following detailed description, from the embodiments provided by way of illustrative and non-limiting examples, and from the attached figures.

[0054] DESCRIPTION OF FIGURES

[0055] Fig- 1 exhibits a scheme of the mutation-independent genome editing by targeted integration, according to a preferred embodiment of the invention: A) targeted integration to correct mutations in the 5 ’-half of a target gene; B) targeted integration to correct mutations in the 3’-half of a target gene. Arrowheads above the target gene indicate the various exons of the gene, stars and rectangles indicate the PAM sequences and the gRNA annealing sites, respectively; DSB: double strand breaks; ITR: Adeno-associated virus inverted terminal repeats; CDS: coding sequence; Ex: exon; Prom: promoter; SD: splicing donor signal; SA: splicing acceptor signal.

[0056] Fig.2 shows the frequency of INDEL in the murine Abca4 locus generated by DSB using gRNA 2.

[0057] Fig- 3 shows a scheme of the AAV constructs used for in vitro experiments. CMV: ubiquitous cytomegalovirus promoter; SD: splicing donor signal. Arrows represent gRNA target sites flanking the donor DNA. HA 20bp boxes= homology arms in the MMEJ-based vector.

[0058] Fig- 4 shows efficient editing of Abca4 by NHEJ- and MMEJ-based donor DNAs according to preferred embodiments of the invention. (A) Schematic representation of the nested PCR and representative images of the PCR products to amplify 5’ and 3’ integration junctions in transfected HEPA1-6 cells. (B) qPCR analysis to evaluate Abca4 expression in transfected HEPA1-6 cells. (C) PCR product from cDNA from HEPA 1-6 cells transfected with NHEJ-based donor DNAs (upper panel) with primers annealing on the last exon of the donor DNA (ex21) and on the following exon from the endogenous genome (ex22). Sanger sequencing of the product (lower panel) confirms accuracy of splicing.

[0059] Fig- 5 shows efficient editing of Abca4 in Abca4’1’ mice by NHEJ- and MMEJ-based donor DNAs according to preferred embodiments of the invention. (A) Representative images of PCR products including 5’ and 3’ integration junctions from injected A bea1' eyes. (B) RNA scope with a probe annealing to Abca4 cDNA downstream of the integration site. Spots represent Abca4 mRNA in transduced photoreceptors, which otherwise do not express Abca4. (C) WB analysis on injected retinas confirming proper Abca4 protein expression. (D) Analysis of lipofuscin accumulation in the RPE of AAV-HITI / MITI injected eyes, relative to negative controls eyes. Scr: cells / eyes treated with Cas9 and scramble gRNA, as negative control Fig- 6 shows a human ABCA4 gene-specific genome editing approach according to preferred embodiments of the invention. (A) Efficiency of SpCas9 gRNAs in editing the human ABCA4 gene as assessed via quantitative TIDE analysis. (B) Schematic of the AAV constructs used for ABCA4 gene editing. CMV: ubiquitous cytomegalovirus promoter; SD: splicing donor signal. Arrows represent gRNA target sites flanking the donor DNA. HA 20bp boxes= homology arms in the MMEJ-based vector.

[0060] Fig- 7 shows efficiency of a human ABCA4 gene-specific genome editing approach according to preferred embodiments of the invention. (A) Representative images of PCR products including 5’ integration junctions from HEK293 cells transfected with the NHEJ- and MMEJ-based constructs. (B) Western blot analysis on HEK293 cells transfected with the NHEJ- and MMEJ-based constructs. Anti-3xflag antibody was used to detect full-length ABCA4 produced upon donor DNA integration. (C) Retinal organoid at day 231 of differentiation. Fully formed outer segments (OS) surrounding the organoid can be noted. ONL: outer nuclear layer. Presence of mature photoreceptors in an organoid at day 217 is confirmed by the positive staining via IF against the photoreceptor-specific rhodopsin (RHO) protein. (D) Representative images of PCR products including 5’ integration junctions from retinal organoids at 4 weeks post infection with AAVNHEJ-based constructs.

[0061] Fig. 8 shows: (A) a representative image of PCR products including 5’integration junctions after editing of human CEP290 gene by NHEJ-based genome editing approaches according to preferred embodiments of the invention; (B) the qPCR analysis evaluating expression of CEP290 in transfected cells (n=2).

[0062] DETAILED DESCRIPTION OF THE INVENTION

[0063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of cells, such as a population of cells.

[0064] The term “about” or “approximately” in relation to a numerical means, a range of values that fall within 10% greater than or less than the value. For example, about x means x± (10% * x). The term “gene editing” refers to the modification of the genome of a cell at a specific location to correct or alter a genetic sequence. The term “gene editing” refers in particular to a type of genetic engineering in which a nucleic acid is inserted, deleted or replaced in a cell. In accordance with the present invention, the term “gene editing” encompasses targeted disruption of a gene coding sequence and sequence substitution for in situ and targeted transgene insertion into a predetermined locus in the genome of a cell.

[0065] The term “gene editing” refers to editing a target site in the genome of a cell, e.g. by mediating disruption of the target site and / or correcting the target site. In accordance with the present invention, gene editing refers to disrupting a target site in the genome of a cell and replacing a genome portion with a corrective nucleic acid, with reconstitution of the target gene, preferably of the coding sequence of the target gene.

[0066] “Reconstitution of the target gene” means that the functionality of the target gene, disrupted by the DSB, is restored upon integration of the exogenous DNA. For instance, the exogenous DNA integrated in the target gene can reconstitute the open reading frame of the target gene, that was disrupted by the DSB.

[0067] In accordance with the present invention, the exogenous DNA has sequence “homologous” to the sequence of the target gene that is replaced, being preferably a sequence that differs from the sequence of the target gene in one or more mutations; e.g. when the target gene is a mutated gene compared to the wild-type gene and the exogenous DNA is not mutated; optionally the sequence of the exogenous DNA can be a codon-usage optimized sequence.

[0068] The term "vector" refers to a particle capable of delivering, and optionally expressing, one or more polynucleotides of interest into a host cell. Examples of vectors include, but are not limited to, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. The “vector” can be a cloning vector, suitable for propagation and for obtaining polynucleotides, gene constructs or expression vectors incorporated to several heterologous organisms. A vector is capable of transferring nucleic acid sequences to target cells, therefore also viral vectors, non-viral vectors, particulate carriers, and liposomes are included in the term “vector”. Typically, "vector construct", "expression vector" and "gene transfer vector" means any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0069] The term “recombinant plasmid” or “plasmid” refers to a small, circular, double- stranded, selfreplicating DNA molecule obtained through genetic engineering techniques capable of transferring genetic material of interest to a cell, which results in production of the product encoded by that said genetic material (e.g., a protein polypeptide, peptide or functional RNA) in the target cell. Furthermore, the term “recombinant plasmid” or “plasmid” also refers to a small, circular, double-stranded, self-replicating DNA molecule obtained through genetic engineering techniques used during the manufacturing of viral vectors as carriers of the recombinant vector genome.

[0070] The term “recombinant viral vector” or “viral vector” refers to an agent obtained from a naturally occurring virus through genetic engineering techniques capable of transferring genetic material (e.g., DNA or RNA) of interest to a cell, which results in production of the product encoded by that said genetic material (e.g., a protein polypeptide, peptide or functional RNA) in the target cell. Herein, the terms “vector transgene" or “recombinant vector transgene" refer to a transgene that is transferred to the recipient cell upon transduction. The term “viral vector” or “recombinant viral vector”, as used herein, also refers to the recombinant viral particles being a packaged viral vector, capable of binding to and entering recipient cells, delivering the vector transgene.

[0071] Viral vectors and viral particles are preferably derived from adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, integrase-defective lentiviral vectors and baculoviral vectors; more preferably in accordance with the present invention a viral vector or viral particle is an AAV viral vector or particle. The terms “nucleotide sequence” or “isolated nucleotide sequence” or “polynucleotide sequence” or “polynucleotide” or “isolated polynucleotide sequence” are interchangeably used herein and refer to a nucleic acid molecule, either DNA or RNA, containing deoxyribonucleotides or ribonucleotides respectively. The nucleic acid may be double stranded, single stranded, or contain portions of both double stranded and single stranded sequence. The terms "variant" refers to biologically active derivatives of the reference molecule that retain desired activity. In general, the term "variant" refers to molecules having a native sequence and structure with one or more additions, substitutions (generally conservative in nature) and / or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity, and which are "substantially homologous" to the reference molecule. In general, the sequences of such variants will have a high degree of sequence homology to the reference sequence, e.g., sequence homology of more than 50%, generally more than 60-70%, even more particularly 80-85% or more, such as at least 90-95% or more, when the two sequences are aligned. In accordance with the present invention, a variant of any biomolecule is a biomolecule that has a nucleic acid or aminoacidic sequence having a % of identity of 50%, 60%, 70%, 80%, 90%, 95%, or 99% to the wild-type nucleic acid or aminoacidic sequence and that retains the biological activity of the wild-type biomolecule. In preferred aspects, the term “variant” of a polynucleotide sequence is used herein to indicate a sequence having a % of identity of at least 90%, 95% or 99% to said polynucleotide sequence. In preferred aspects, the term “variant” of a polynucleotide sequence is used herein to indicate a sequence that is a codon-optimized sequence for expressing the biomolecule encoded by said sequence.

[0072] The terms “% sequence identity”, “% identity” or “% sequence homology” refer to the percentage of nucleotides or amino acids of a candidate sequence that are identical to the nucleotides or amino acids in the sequence of reference, after aligning the sequences to achieve the maximum % sequence identity. In a preferred embodiment, sequence identity is calculated based on the full length of two given sequences or on part thereof. The % sequence identity can be determined by any methods or algorithms established in the art, such as the ALIGN, BLAST and BLAST 2.0 algorithms and followings. Herein, the “% sequence identity”, “% identity” “or “% sequence homology” is calculated dividing the number of nucleotides or amino acids that are identical after aligning the sequence of reference and the candidate sequence, by the total number of nucleotides or amino acids in the sequence of reference and multiplying the result by 100. In accordance with degeneration of genetic code, variants include sequences where at least one base of the base sequence of a gene is replaced with a different type of base, without changing the amino acid sequence of the polypeptide expressed from the gene. Variants also include codon-optimized sequences and sequences comprising mutated or added nucleotides, e.g., for cloning needs. Codon optimization has previously been described in WO 1999 / 41397 and WO 2001 / 79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available. Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms. Variants also include sequences encoding fragments of any biomolecule, i.e., a shorter form of the biomolecule, such as a truncated form, that retains the biological activity of the wild-type biomolecule.

[0073] The terms “codify”, “coding” or “encoding” refer to the genetic code that determines how a nucleotide sequence is translated into a polypeptide or a protein. The order of the nucleotides in a sequence determines the order of amino acids along a polypeptide or a protein.

[0074] The term "transcriptional regulatory region" or “regulatory element, or region”, as used herein, refers to a nucleic acid fragment capable of regulating the expression of one or more genes. The regulatory regions of a polynucleotides of the invention may include a promoter, plus response elements, activator and enhancer sequences for binding of transcription factors to aid RNA polymerase binding and promote expression, and operator or silencer sequences to which repressor proteins bind to block RNA polymerase attachment and prevent expression.

[0075] The term "promoter" must be understood as a nucleic acid fragment that functions to control the transcription of one or more polynucleotides e.g. coding sequences, which is placed 5' upstream of the polynucleotide sequence(s), and which is structurally identified by the presence of a binding site for DNA dependent RNA polymerase, transcription initiation sites and, but not limited to, binding sites for transcription factors, repressors, and any other nucleotide sequences known in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A promoter is said to be operatively linked to a nucleotide sequence or to drive the expression of it when it can initiate transcription of said nucleotide sequence in an expression system using a gene construct comprising said promoter operably linked to a nucleotide sequence of interest using a suitable assay such a RT- qPCR or Northern blotting (detection of the transcript). The activity of said promoter may also be assessed at the protein level using a suitable assay for the encoded protein such as Western blotting or an ELISA. A promoter is said to be capable to initiate transcription if a transcript can be detected or if an increase in a transcript or protein level is found of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%, 1000%, 1500% or 2000% as compared to transcription using a construct which only differs in that it is free of said promoter.

[0076] The term "constitutive" promoter refers to a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is preferably regulated depending on physiological or developmental conditions. A "tissue-specific" promoter is preferably active in specific types of cells / tissues. A “ubiquitous” promoter may be defined as a promoter that is active in many or in any different tissue(s). A “strong promoter” is generally meant to indicate a promoter capable of inducing expression of high levels of a gene product under its control.

[0077] "Host cells," "cells", "cell lines," "cell cultures", “engineered cells” and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for gene modification include the original progeny of the original cell.

[0078] The terms “culture or culturing”, “growth or growing”, referred to cells, are used herein interchangeably and are meant to indicate maintenance of a cell population in vitro or ex vivo, preferably including expansion of the cell population. The term “incubating” or “incubation”, as referred herein to cells and agents according to the invention, means contacting a cell with an agent, e.g., a means for delivering a gene editing agent, and maintaining the contact for a time suitable for delivering the agent into the cell.

[0079] The terms "treatment", "treating", "treat" and the like, as used herein, refer to the administration of a compound, agent, composition or formulation of the invention to obtain a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease or control of disease progression. The terms "prevent," "preventing," and "prevention", as used herein, refer to inhibiting the inception or decreasing the occurrence of a disease in a subject. Prevention may be complete (e.g., the total absence of pathological cells in a subject) or partial. Prevention also refers to a reduced susceptibility to a clinical condition. Control of disease progression is understood as the achievement of the beneficial or desired clinical results that include, but are not limited to, reduction of the symptoms, reduction of the duration of the disease, stabilization of pathological states (specifically to avoid additional deterioration), delay of the progression of the disease, improvement in the pathological state, and remission (both partial and total). The control of progression of the disease also involves an extension of survival, compared with the expected survival if treatment is not applied.

[0080] In particular, in accordance with the present invention, the terms "treatment", "treating", "treat" and the like, as used herein, preferably refer to the administration of a compound, composition or formulation to cure, prevent, delay and / or control the clinical manifestations of a pathology. The term “effective amount” or “therapeutical amount” refers to a quantity of a composition, for example a gene editing agent(s) and / or a gene therapy composition, that can be sufficient to result in a desired activity upon introduction into a subject as disclosed herein. The effective amount can be provided to the target gene or cell. In some embodiments, the “effective amount” or “therapeutically effective amount” is the amount that is required to ameliorate the symptoms of a disease relative to an untreated patient. In some embodiments, an effective amount is the amount sufficient to introduce an alteration in a gene of interest in a cell (e.g., in vitro, ex vivo or in vivo). In some embodiments, an effective amount can be an amount to induce, when administered to a population of cells, a certain percentage of the population of cells to have a correction of a mutation or to bear a transgene. For example, in some embodiments, an effective amount can be the amount to induce, when administered to or introduced to a population of cells, installation of one or more intended nucleotide edits in the target gene.

[0081] The term “individual” or “subject” herein refers to a mammal, preferably human or non-human mammal, more preferably a human, or a mouse, rat, other rodents, rabbit, dog, cat, pig, cow, horse or primate. Those in need of treatment include those already inflicted as well as those in which prevention is desired (e.g., those with no symptoms but diagnosed with the genetic disorder, etc.).

[0082] Target site

[0083] The target site of the kit for gene editing of the invention is preferably within a target gene having a coding sequence of at least 4 kb in length, preferably having a coding sequence not greater than 10 kb in length, more preferably within a target gene having a coding sequence of from 4.5 to 9.5 kb, from 5 to 9 kb, from 6 to 8 kb, from 6.5 to 7.5 kb, in length.

[0084] The target site of the nuclease is within an intron of the target gene, preferably said intron being a central intron of the gene. The term “central intron” means that the gene portion that is upstream the central intron and the gene portion that is downstream the central intron have approximately the same length, in terms of base pairs (bp). Preferably the term “gene portion” is meant to indicate the coding sequence of the gene.

[0085] In preferred embodiments, the target gene is one of the genes listed in Table 1 that follows; preferred introns to be targeted for introducing the DSB are also indicated for each gene in the last column.

[0086]

[0087]

[0088] Table 1

[0089] Gene editing agents

[0090] The kit of the invention comprises gene editing agents consisting of:

[0091] a) a nuclease capable of introducing a double strand break (DSB) in the target site of the target gene, or a polynucleotide encoding thereof; and

[0092] b) a complementary strand oligonucleotide having sequence homologous to the target site sequence.

[0093] Nucleases suitable for gene editing of a target gene include zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease (Gaj, T. et al. (2013) Trends Biotechnol. 31: 397-405). Meganucleases (Silve, G. et al. (2011) Cur. Gene Ther. 11: 11-27).

[0094] Preferably, the nuclease of the kit of the invention is a “CRISPR / Cas” nuclease and the complementary strand oligonucleotide having sequence homologous to the target site sequence is a guide RNA (gRNA or sgRNA) selected to enable the nuclease to be targeted to a specific sequence in the target gene (van der Oost et al. (2014) Nat. Rev. Microbiol. 12: 479-92). Methods for the design of gRNAs are known in the art. Furthermore, fully orthogonal Cas9 proteins, as well as Cas9 / gRNA ribonucleoprotein complexes and modifications of the gRNA structure / composition to bind different proteins, have been recently developed to simultaneously and directionally target different effector domains to desired genomic sites of the cells (Esvelt et al. (2013) Nat. Methods 10: 1116-21), and are suitable for use in the invention.

[0095] More preferably said nuclease is a CRISPR nuclease selected from the group consisting of: Cas9, Cpf1, Casl2b (C2cl), Casl3a (C2c2), Cas3, Csfl, Casl3b (C2c6), and C2c3 or variants thereof such as SaCas9 or VQR-Cas9-HF1.

[0096] Preferably the gRNA has sequence complementary to a target sequence in the introns indicated in Table 1 for each gene. Preferably, the gRNA is selected within the list of gRNAs of Table 2, having sequence comprising, or consisting of, any one of SEQ ID NO: 21-23, targeting human ABCA4 gene or any one of SEQ ID NO: 27-29, targeting human CEP290 gene. Most preferably, the gRNA is a gRNA having sequence comprising, or consisting of, SEQ ID NO: 22, targeting human ABCA4 gene, or SEQ ID NO: 28, targeting human CEP290 gene.

[0097]

[0098] Table 2

[0099] Donor nucleic acid

[0100] The donor nucleic acid of the kit of the invention is a donor DNA, more preferably a double stranded donor DNA.

[0101] The first donor nucleic acid comprises a first knock-in cassette for reconstitution of the target gene by replacement of the portion of the target gene upstream the DSB (5’ portion) with an exogenous polynucleotide, preferably an exogenous DNA, having sequence homologous to said 5’ portion (5’ exogenous polynucleotide), and the second donor nucleic acid comprises a second knock-in cassette for reconstitution of the target gene by replacement of the portion of the target gene downstream the DSB (3’ portion) with an exogenous polynucleotide, preferably an exogenous DNA, having sequence homologous to said 3’ portion (3’ exogenous polynucleotide).

[0102] The 5’ and 3’ exogenous polynucleotides have preferably sequence homologous to the coding sequence of the 5’ and 3’ portions of the target gene.

[0103] Therefore, preferably the 5’ exogenous polynucleotides have sequence homologous to the coding sequence of the 5’ portion of the target gene comprising, or consisting of, exons 1 to N, wherein exon 1 is the first exon and exon N is the exon located before the target intron N, optionally further comprising at its 3’ end the portion of the target intron N that is upstream the DSB, and preferably the 3’ exogenous polynucleotides have sequence homologous to the coding sequence of the 3’ portion of the target gene comprising, or consisting of, exons Ni to exon to Nn, wherein exon Ni is the exon located after the target intron N and exon Nnis the last exon of the target gene’s coding sequence, optionally further comprising at its 5’ end the portion of the target intron N that is downstream the DSB.

[0104] As an example, when the target intron is intron 20 of a gene, the 5’ exogenous polynucleotides have sequence homologous to the coding sequence of the target gene comprising, or consisting of, exons 1 to 19 and the 3’ exogenous polynucleotides have sequence homologous to the coding sequence of the target gene comprising, or consisting of, exons 21 to the last exon of the gene. Preferably, each of the 5’ and 3’ exogenous polynucleotides have sequence of from 2 kb to 5 kb in length, more preferably of from 3 to 4 kb in length, being configured to replace the whole coding sequence of the 5’ or 3’ portions of the target gene, respectively upstream or downstream the DSB.

[0105] The first knock-in cassette further comprises a promoter upstream the 5’ exogenous DNA driving expression of the reconstituted gene or gene coding sequence, and a splicing donor signal downstream the 5’ exogenous DNA for reconstituting the gene or gene coding sequence with the endogenous 3’ portion of the gene.

[0106] The second knock-in cassette further comprises a splicing acceptor signal, upstream the 3’ exogenous DNA, for reconstituting the gene or gene coding sequence with the endogenous 5’ portion of the gene, and a poly adenylation signal, downstream the exogenous DNA.

[0107] Preferably, the sequence of the exogenous DNA of the donor template capable of reconstituting the target gene is a codon-usage optimized sequence, that it is not recognized and / or disrupted by the gene editing agent.

[0108] In some embodiments, the donor template does not comprise a reporter gene, e.g., a fluorescent reporter gene or an antibiotic resistance gene.

[0109] Preferably a vector, comprising a first donor DNA and a cassette for expressing the complementary strand oligonucleotide, comprises, from 5’ to 3’:

[0110] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0111] a first inverted targeting sequence;

[0112] a promoter driving expression of the 5’ exogenous polynucleotide,

[0113] the 5’ exogenous polynucleotide,

[0114] a splicing donor signal,

[0115] a second inverted targeting sequence;

[0116] a promoter driving expression of the complementary strand oligonucleotide

[0117] the complementary strand oligonucleotide, and

[0118] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence. Preferably a vector, comprising a first donor DNA and a cassette for expressing the complementary strand oligonucleotide, comprises, from 5’ to 3’:

[0119] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0120] left microhomology arm;

[0121] a promoter driving expression of the 5’ exogenous polynucleotide,

[0122] the 5’ exogenous polynucleotide,

[0123] a splicing donor signal,

[0124] a right microhomology arm;

[0125] a promoter driving expression of the complementary strand oligonucleotide the complementary strand oligonucleotide, and

[0126] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0127] Preferably a vector, comprising a first donor DNA and a cassette for expressing the complementary strand oligonucleotide, comprises, from 5’ to 3’:

[0128] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0129] a first targeting sequence;

[0130] left microhomology arm;

[0131] a promoter driving expression of the 5’ exogenous polynucleotide,

[0132] the 5’ exogenous polynucleotide,

[0133] a splicing donor signal,

[0134] a right microhomology arm;

[0135] a second targeting sequence;

[0136] a promoter driving expression of the complementary strand oligonucleotide the complementary strand oligonucleotide, and

[0137] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0138] Preferably a vector, comprising a second donor DNA and a cassette for expressing the complementary strand oligonucleotide, comprises, from 5’ to 3’:

[0139] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0140] a first inverted targeting sequence;

[0141] a splicing acceptor signal,

[0142] the 3’ exogenous polynucleotide,

[0143] a poly adenylation signal,

[0144] a second inverted targeting sequence; a promoter driving expression of the complementary strand oligonucleotide the complementary strand oligonucleotide, and

[0145] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0146] Preferably a vector, comprising a second donor DNA and a cassette for expressing the complementary strand oligonucleotide, comprises, from 5’ to 3’:

[0147] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0148] a left microhomology arm;

[0149] a splicing acceptor signal,

[0150] the 3’ exogenous polynucleotide,

[0151] a poly adenylation signal,

[0152] a right microhomology arm;

[0153] a promoter driving expression of the complementary strand oligonucleotide the complementary strand oligonucleotide, and

[0154] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0155] Preferably a vector, comprising a second donor DNA and a cassette for expressing the complementary strand oligonucleotide, comprises, from 5’ to 3’:

[0156] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0157] a first targeting sequence;

[0158] a left microhomology arm;

[0159] a splicing acceptor signal,

[0160] the 3’ exogenous polynucleotide,

[0161] a poly adenylation signal,

[0162] a right microhomology arm;

[0163] a second targeting sequence;

[0164] a promoter driving expression of the complementary strand oligonucleotide the complementary strand oligonucleotide, and

[0165] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0166] In some embodiments, the first donor nucleic acid comprises a first knock-in cassette for reconstitution of the target gene, said first knock-in cassette comprising from 5’ to 3’:

[0167] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence, a first inverted targeting sequence,

[0168] a promoter, preferably a ubiquitous promoter or a photoreceptor-specific promoter, more preferably a CMV promoter (SEQ ID NO: 48) or a GRK1 promoter (SEQ ID NO: 50), driving expression of the 5’ exogenous polynucleotide,

[0169] the 5’ exogenous polynucleotide,

[0170] a splicing donor signal (SEQ ID NO: 34),

[0171] a second inverted targeting sequence,

[0172] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0173] In some embodiments, the first donor nucleic acid comprises a first knock-in cassette for reconstitution of the target gene, said first knock-in cassette comprises from 5’ to 3’:

[0174] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0175] a first targeting sequence;

[0176] a left microhomology arm;

[0177] a promoter, preferably a ubiquitous promoter or a photoreceptor-specific promoter, more preferably a CMV promoter (SEQ ID NO: 48) or a GRK1 promoter (SEQ ID NO: 50), driving expression of the 5’ exogenous polynucleotide,

[0178] the 5’ exogenous polynucleotide,

[0179] a splicing donor signal (SEQ ID NO: 34),

[0180] a right microhomology arm;

[0181] a second targeting sequence;

[0182] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0183] In some embodiments, the second donor nucleic acid comprises a second knock-in cassette for reconstitution of the target gene, said second knock-in cassette comprising from 5’ to 3’:

[0184] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0185] a first inverted targeting sequence;

[0186] a splicing acceptor signal (SEQ ID NO: 35),

[0187] the 3’ exogenous polynucleotide,

[0188] a SV40 poly adenylation signal (SEQ ID NO: 46),

[0189] a second inverted targeting sequence;

[0190] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence. In some embodiments, the second donor nucleic acid comprises a second knock-in cassette for reconstitution of the target gene, said second knock-in cassette comprising from 5’ to 3’:

[0191] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0192] a first targeting sequence;

[0193] a left microhomology arm;

[0194] a splicing acceptor signal (SEQ ID NO: 35),

[0195] the 3’ exogenous polynucleotide,

[0196] a SV40 poly adenylation signal (SEQ ID NO: 46),

[0197] a right microhomology arm,

[0198] a second targeting sequence,

[0199] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0200] In some embodiments, the nuclease targets intron 20 of the hABCA4 gene, the complementary strand oligonucleotide is a gRNA having sequence comprising, or consisting of, SEQ ID NO: 22, and the first donor nucleic acid comprises a first knock-in cassette for reconstitution of the hABCA4 gene, said first knock-in cassette comprising from 5’ to 3’:

[0201] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence,

[0202] a first inverted targeting sequence,

[0203] a promoter, preferably a ubiquitous promoter or a photoreceptor-specific promoter, more preferably a CMV promoter (SEQ ID NO: 48) or a GRK1 promoter (SEQ ID NO: 50), driving expression of the 5’ exogenous polynucleotide,

[0204] the 5’ exogenous polynucleotide comprising hABCA4 coding sequence from ATG to exon 20 (NM_000350.3; bp 104-3153),

[0205] a splicing donor signal (SEQ ID NO: 34),

[0206] a second inverted targeting sequence,

[0207] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0208] In some embodiments, the nuclease targets intron 20 of the hABCA4 gene, the complementary strand oligonucleotide is a gRNA having sequence comprising, or consisting of, SEQ ID NO: 22, and the first donor nucleic acid comprises a first knock-in cassette for reconstitution of the hABCA4 gene, said first knock-in cassette comprises from 5’ to 3’:

[0209] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0210] a first targeting sequence;

[0211] a left microhomology arm of 8bp (SEQ ID NO: 36) or of 20bp (SEQ ID NO: 37); a promoter, preferably a ubiquitous promoter or a photoreceptor-specific promoter, more preferably a CMV promoter (SEQ ID NO: 48) or a GRK1 promoter (SEQ ID NO: 50), driving expression of the 5’ exogenous polynucleotide,

[0212] the 5’ exogenous polynucleotide comprising hABCA4 coding sequence from ATG to exon 20 (NM_000350.3; bp 104-3153),

[0213] a splicing donor signal (SEQ ID NO: 34),

[0214] a right microhomology arm of 8bp (SEQ ID NO: 38) or of 20bp (SEQ ID NO: 39); a second targeting sequence;

[0215] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0216] In some embodiments, the nuclease targets intron 20 of the hABCA4 gene, the complementary strand oligonucleotide is a gRNA having sequence comprising, or consisting of, SEQ ID NO: 22, and the second donor nucleic acid comprises a second knock-in cassette for reconstitution of the hABCA4 gene, said second knock-in cassette comprising from 5’ to 3’:

[0217] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0218] a first inverted targeting sequence;

[0219] a splicing acceptor signal (SEQ ID NO: 35),

[0220] the 3’ exogenous polynucleotide CDS of hABCA4 from the first bp of exon 21 to the last exon of hABCA4 (NM_000350.3; bp 3154-6919),

[0221] a SV40 poly adenylation signal (SEQ ID NO: 46),

[0222] a second inverted targeting sequence;

[0223] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0224] In some embodiments, the nuclease targets intron 20 of the hABCA4 gene, the complementary strand oligonucleotide is a gRNA having sequence comprising, or consisting of, SEQ ID NO: 22, and the second donor nucleic acid comprises a second knock-in cassette for reconstitution of the hABCA4 gene, said second knock-in cassette comprising from 5’ to 3’:

[0225] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0226] a first targeting sequence;

[0227] a left microhomology arm of 8bp (SEQ ID NO: 40) or of 20bp (SEQ ID NO: 41); a splicing acceptor signal (SEQ ID NO: 35),

[0228] the 3’ exogenous polynucleotide comprising the CDS of hABCA4 from the first bp of exon 21 to the last exon of hABCA4 (NM_000350.3; bp 3154-6919), a SV40 poly adenylation signal (SEQ ID NO: 46),

[0229] a right microhomology arm of 8bp (SEQ ID NO: 42) or of 20bp (SEQ ID NO: 43), a second targeting sequence,

[0230] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0231] In some embodiments, the nuclease targets intron 30 of the hCEP290 gene, the complementary strand oligonucleotide is a gRNA having sequence comprising, or consisting of, SEQ ID NO: 28, and the second donor nucleic acid comprises a second knock-in cassette for reconstitution of the hCEP290 gene, said second knock-in cassette comprising from 5’ to 3’:

[0232] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0233] a first inverted targeting sequence;

[0234] a splicing acceptor signal (SEQ ID NO: 35)

[0235] the 3’ exogenous polynucleotide comprising the CDS of hCEP29 from exon 31 to the end of the CDS (NM_025114.4; bp 3790-7653),

[0236] a Bgh poly adenylation signal (SEQ ID NO: 47),

[0237] a second inverted targeting sequence;

[0238] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0239] In some embodiments, the nuclease targets intron 30 of the hCEP290 gene, the complementary strand oligonucleotide is a gRNA having sequence comprising, or consisting of, SEQ ID NO: 28, and the second donor nucleic acid comprises a second knock-in cassette for reconstitution of the hCEP290 gene, said second knock-in cassette comprising from 5’ to 3’:

[0240] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0241] a first inverted targeting sequence;

[0242] a splicing acceptor signal (SEQ ID NO: 35)

[0243] the 3’ exogenous polynucleotide comprising the CDS of hCEP29 from exon 31 to the end of the CDS (NM_025114.4; bp 3790-7653),

[0244] a Bgh poly adenylation signal (SEQ ID NO: 47),

[0245] a second inverted targeting sequence;

[0246] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0247] Splicing Signals RNA splicing is a form of RNA processing in which a newly made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). During splicing, introns (non-coding regions) are removed, and exons (coding regions) are joined together. Within introns, a donor site (5' end of the intron), a branch site (near the 3' end of the intron) and an acceptor site (3' end of the intron) are required for splicing. The splice donor site includes an almost invariant sequence GU at the 5' end of the intron, within a larger, less highly conserved region. The splice acceptor site at the 3' end of the intron terminates the intron with an almost invariant AG sequence. Upstream (5'-ward) from the AG there is a region high in pyrimidines (C and U), or polypyrimidine tract. Further upstream from the polypyrimidine tract is the branchpoint.

[0248] Consensus sequences and frequencies of human splicing signals are described in Ma, S. L., et al., 2015. PLoS One, 10(6), p.e0130729.

[0249] Preferably, the splicing donor signal of the first knock in cassette of the first donor nucleic acid of the invention has sequence comprising or consisting of SEQ ID NO: 34, or a variant thereof. Preferably, the splicing acceptor signal of the second knock in cassette of the second donor nucleic acid of the invention has sequence comprising or consisting of SEQ ID NO: 35, or a variant thereof.

[0250] Promoter

[0251] In some embodiments, the promoter of the 5’ knock-in cassette of the invention is a ubiquitous promoter, such as a CMV promoter (SEQ ID NO: 48). In some embodiments, said promoter is a retinal specific promoter, such as a photoreceptor-specific promoter, preferably a photoreceptor-specific human G protein-coupled receptor kinase 1 (GRK1, SEQ ID NO: 50). Other suitable photoreceptor-specific promoters include: Interphotoreceptor retinoid binding protein promoter (IRBP), Rhodopsin promoter (RHO), vitelliform macular dystrophy 2 promoter (VMD2), Rhodopsin kinase promoter (RK).

[0252] Optionally the promoter comprises an enhancer sequence.

[0253] Promoters contemplated for use in the subject invention include, but are not limited to, native gene promoters or fragments thereof. Preferably, the nuclease expression is under the control of the same promoter driving expression of the exogenous nucleic acid.

[0254] Preferably, the complementary strand oligonucleotide, e.g. the gRNA targeting the target site, is expressed under the control of a humaU6 promoter.

[0255] HITI In some embodiments, the donor nucleic acid comprises, at both 5’ and 3’ of the knock-in cassette, target integration polynucleotides for NHEJ with the target gene, having sequence homologous to the target site sequence and in inverted orientation (inverted targeting sequence or HITI sequence). Inverted targeting sequence is then inverted with respect to the target sequence of the complementary strand oligonucleotide. Preferably, the complementary strand oligonucleotide is a gRNA and the inverted targeting sequence has sequence homologous to the gRNA target sequences.

[0256] The inverted targeting sequence preferably comprises a PAM sequence, preferably at its 3’.

[0257] MITI

[0258] In some embodiments, the donor nucleic acid comprises, at both 5’ and 3’ of the knock-in cassette, microhomology arms for MMEJ with the target gene. The 5’ microhomology arms (or “left” homology arm or left MITI sequence) of the donor template has a sequence that is homologous, preferably identical, to a sequence located 5' of the DSB in the target site, more preferably contiguous to the DSB, and the 3' homology arm (“right” homology arm or right MITI sequence) of the donor template has a sequence that is homologous, preferably identical, to a sequence located 3' of the DSB, more preferably contiguous to the DSB, in the target gene. Said left and / or right microhomology arms are preferably of 5 to 25 bp in length, more preferably 8 to 20 bp in length.

[0259] In some embodiments, the target gene is hABCA4 and the first and / or second donor nucleic acid comprises microhomology arms having sequence selected from SEQ ID NO: 36-43. Optionally the microhomology arms are flanked

[0260] Degradation Signal

[0261] In some embodiments the first donor nucleic acid comprises a degradation signal upstream the promoter, preferably being: CL1, CL2, CL6, CL9, CLIO, CL11, CL12, CL15, CL16, SL17, SMN, CIITA, ODc7, ecDHFR, PEST or a Mini ecDHFR degradation signal, more preferably being CL1 degradation signal or a variant thereof, having sequence comprising or consisting of a sequence having at least 80% homology to SEQ ID NO: 30, most preferably having sequence comprising or consisting of SEQ ID NO: 30.

[0262] Polyadenylation sequence

[0263] Preferably, the 3’ exogenous nucleic acid of the second donor nucleic acid of the invention is operably linked to a polyadenylation sequence. Therefore, the second knock-in cassette of the second donor nucleic acid of the invention preferably comprises a polyadenylation sequence downstream the 3’ exogenous nucleic acid.

[0264] A polyadenylation sequence typically comprises a polyadenylation signal, a polyadenylation site and a downstream element: the polyadenylation signal comprises the sequence motif recognized by the RNA cleavage complex; the polyadenylation site is the site of cleavage at which a poly-A tails is added to the mRNA; the downstream element is a GT-rich region which usually lies just downstream of the polyadenylation site, which is important for efficient processing.

[0265] In some embodiments, the polyadenylation sequence is a bovine growth hormone (Bgh) polyadenylation sequence (SEQ ID NO: 47) or an SV40 polyadenylation sequence (SEQ ID NO: 46); or a fragment thereof that retains the natural function of the polyadenylation sequence.

[0266] Vectors

[0267] The nuclease, the complementary strand oligonucleotide and / or a donor nucleic acids of the kit of the invention can be introduced in the cell as DNA encoding the same, or as RNA transcript thereof.

[0268] Therefore, the invention is also directed to vectors comprising one or more of a nucleic acid coding for the nuclease, the complementary strand oligonucleotide and a donor nucleic acids of the kit of the invention.

[0269] Preferably, a first vector comprises the first or second donor nucleic acid and the complementary strand oligonucleotide homologous to a targeting sequence and a second vector comprises the nucleic acid coding for the nuclease. Alternatively, a first vector comprises the first or second donor nucleic acid and a second vector comprises the complementary strand oligonucleotide homologous to a targeting sequence and the nucleic acid coding for the nuclease. As a further alternative, three vectors are provided: a first vector comprising the first or second donor nucleic acid, a second vector comprising the complementary strand oligonucleotide homologous to a targeting sequence and a third vector comprising the nucleic acid coding for the nuclease of the kit of the invention.

[0270] Preferably a vector, comprising a first donor DNA and a cassette for expressing the complementary strand oligonucleotide, comprises, from 5’ to 3’:

[0271] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0272] a first inverted targeting sequence;

[0273] a promoter driving expression of the 5’ exogenous polynucleotide, the 5’ exogenous polynucleotide,

[0274] a splicing donor signal,

[0275] a second inverted targeting sequence;

[0276] a promoter driving expression of the complementary strand oligonucleotide the complementary strand oligonucleotide, and

[0277] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0278] Preferably a vector, comprising a first donor DNA and a cassette for expressing the complementary strand oligonucleotide, comprises, from 5’ to 3’:

[0279] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0280] a first targeting sequence;

[0281] left microhomology arm;

[0282] a promoter driving expression of the 5’ exogenous polynucleotide,

[0283] the 5’ exogenous polynucleotide,

[0284] a splicing donor signal,

[0285] a right microhomology arm;

[0286] a second targeting sequence;

[0287] a promoter driving expression of the complementary strand oligonucleotide the complementary strand oligonucleotide, and

[0288] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0289] Preferably a vector, comprising a second donor DNA and a cassette for expressing the complementary strand oligonucleotide, comprises, from 5’ to 3’:

[0290] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0291] a first inverted targeting sequence;

[0292] a splicing acceptor signal,

[0293] the 3’ exogenous polynucleotide,

[0294] a poly adenylation signal,

[0295] a second inverted targeting sequence;

[0296] a promoter driving expression of the complementary strand oligonucleotide the complementary strand oligonucleotide, and

[0297] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence. Preferably a vector, comprising a second donor DNA and a cassette for expressing the complementary strand oligonucleotide, comprises, from 5’ to 3’:

[0298] an AAV 5 ’ -inverted terminal repeat (5 ’ -ITR) sequence;

[0299] a first targeting sequence;

[0300] a left microhomology arm;

[0301] a splicing acceptor signal,

[0302] the 3’ exogenous polynucleotide,

[0303] a poly adenylation signal,

[0304] a right microhomology arm;

[0305] a second targeting sequence;

[0306] a promoter driving expression of the complementary strand oligonucleotide

[0307] the complementary strand oligonucleotide, and

[0308] an AAV 3 ’ -inverted terminal repeat (3 ’ -ITR) sequence.

[0309] Particularly preferred vectors are those having sequence comprising SEQ ID NO: 1, 3, 5 and 7 or 52.

[0310] Preferably, said vectors are viral vectors, more preferably adeno-associated viral (AAV) vectors. Therefore, donor nucleic acids of the invention are preferably flanked by a 5 '-inverted terminal repeat (5'-ITR) sequence and 3 '-inverted terminal repeat (3'-ITR), said ITRs being derived from an AAV virus, preferably from an AAV of serotype selected from one or more of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 AAV9 and AAV 10 AAVSH19, AAVPHP. B, or a derivative thereof serotypes, more preferably of an AAV2 or AAV8 serotype.

[0311] Said viral vectors are preferably comprised in a viral particle, more preferably an AAV viral particle comprising capsid proteins of an AAV. Preferably, the viral particle comprises capsid proteins of an AAV of a serotype selected from one or more of the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 AAV9 and AAV 10, AAVSH19, AAVPHP. B or a derivative thereof, preferably from the AAV2 or AAV8 serotype.

[0312] Host cell

[0313] A further object of the invention is a gene edited cell, obtainable by the method of the invention or comprising the nuclease, the complementary strand oligonucleotide and either the first or the second donor nucleic acid of the invention, or comprising vectors for expressing the same. According to preferred embodiments of the present invention, the cell is a mammalian cell, more preferably, the cell is a human cell, most preferably a retinal cell, a lung cell, or a muscle cell.

[0314] Pharmaceutical composition

[0315] The invention is also directed to pharmaceutical formulations, comprising the nuclease, the complementary strand oligonucleotide and either the first or the second donor nucleic acid of the invention, or comprising vectors for expressing the same, and further comprising suitable pharmaceutically acceptable excipients.

[0316] The term "pharmaceutically acceptable excipient" refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any conventional type that may optionally be included in the formulations of the invention and that causes no significant adverse toxicological effects to the patient. A pharmaceutically acceptable excipient is essentially non-toxic to recipients at the employed dosages and concentrations and is compatible with other ingredients of the formulation. The number and the nature of the pharmaceutically acceptable excipients depend on the desired administration form.

[0317] Therapeutic applications

[0318] In a further aspect, the present invention is also directed to the kit, gene edited cell, vectors, viral particles, or pharmaceutical formulation of the invention, for use as a medicament, preferably for use in the treatment in the treatment of a disease caused by mutations in the target gene. Preferably said disease is an autosomal inherited diseases.

[0319] More preferably said disease is selected from: cone-rod dystrophy, Usher Syndrome (Type I), Stargardt Disease, Usher syndrome Type IF (USH1F) and DFNB23, Aland Islands eye disease. Night Blindness, Congenital Stationary, Type IE and Congenital Stationary Night Blindness. Neuroblastoma amplified sequence, Leber congenital amaurosis type 10, Retinitis Pigmentosa, Cystic Fibrosis, Alport Syndrome (Autosomal Recessive), Spastic Paraplegia 11 (SPG11), Spastic Paraplegia 15 (SPG15), Marfan Syndrome, Tuberous Sclerosis Complex, Neurofibromatosis Type 1, Congenital Muscular Dystrophy (LAMA2-related), Fanconi Anemia, Ataxia-Telangiectasia (A-T).

[0320] The present invention is then directed also to a method of treatment of a disease by gene editing, comprising administering to a subject in need thereof a therapeutic amount of the kit, gene edited cell, vectors, viral particles, or pharmaceutical formulation of the invention. A skilled worker would be able to determine appropriate dosage rates.

[0321] It should be understood that all the possible combinations of the preferred aspects of the present invention are also described, and therefore similarly preferred.

[0322] Examples of preferred embodiments of the present invention and analyses of their efficacy are provided below for illustrative and non-limiting purposes.

[0323] EXAMPLES MATERIALS AND METHODS

[0324] gRNAs

[0325] Guide RNAs used to edit mAbca4. hABCA4 and hCEP290 genes, shown in Table 3 were designed using the Benchling gRNA design tool (www.benchling.com), selecting the gRNAs with the best predicted on-target and off-target scores

[0326]

[0327] Table 3

[0328] Scramble gRNAs were designed to not align with any sequences in the mouse or human genome. gRNAs were then generated as forward or reverse oligonucleotides, annealed and cloned in the PX458 [pSpCas9 (BB)-2A-GFP] (Addgene #48138, SEQ ID NO: 31) plasmid encoding Streptococcus pyogenes Cas9 (SpCas9), as described by the Zhang lab (Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281-2308 (2013).

[0329] Generation of AAV plasmids

[0330] The plasmids used for AAV vectors production derived from the pTIGEM plasmid (Tornabene etal, Mol Ther Methods Clin Dev. 2021 Oct 19:23:448-459 that contains the inverted terminal repeats of AAV serotype 2.

[0331] All donor DNA fragments were generated by PCR amplification and cloned in pTIGEM backbone for further production of AAV vectors. Specifically:

[0332] -the 5’ donor nucleic acid for mAbca4 gene editing include: the CDS of mAbca4 from the ATG to the last bp of exon 21 (NM_007378.1; bp 6-3277), followed by the beginning of the following exon ((XM_036162859.1; bp 696-745), which act as splicing donor signal. Donor DNAs included either the ubiquitous cytomegalovirus (CMV) promoter, for the in vitro studies, or the photoreceptor-specific human G protein-coupled receptor kinase 1 (GRK1) promoter, for studies in Abca4' / _mice. Donor DNAs were flanked by the gRNAs target sequences, followed by the PAM, in inverted orientation as required for efficient HITI. Additionally, construct for MMEJ included 20bp-long homology arms (HAs, with left HA of sequence SEQ ID NO: 32; right HA of sequence SEQ ID NO: 33). Constructs were designed to include a 3xFLAG tag at the amino acidic position 590 of the CDS. The expression cassette of the selected gRNAs (i.e. mAbca4_gRNA), including the U6 promoter, was PCR-amplified and cloned in the plasmid including the above-described donor DNA, using the In-Fusion kit (Takara, Kusatsu, Japan). - the 5’ donor DNAs for hABCA4 editing include the CDS of hABCA4 from the ATG to the last bp of exon 20 (NM_000350.3; bp 104-3153), followed by a splicing donor signal (SEQ ID NO: 34). Donor DNAs included the ubiquitous cytomegalovirus (CMV) promoter and were flanked by the gRNAs target sequences, followed by the PAM, in inverted orientation as required for efficient HITI. Additionally, construct for MMEJ included either 8 or 20bp-long homology arms (with 8bp left HA of sequence SEQ ID NO: 36; 8bp right HA of sequence SEQ ID NO: 37; 20bp left HA of sequence SEQ ID NO: 38; 20bp right HA of sequence SEQ ID NO: 39). Constructs were designed to include a 3xFLAG tag at the amino acidic position 590 of the CDS. The expression cassette of the selected gRNAs (i.e. SpCas9_gRNAl), including the U6 promoter, was PCR-amplified and cloned in the plasmid including the above-described donor DNA, using the In-Fusion kit (Takara, Kusatsu, Japan).

[0333] - the 3 ’ donor DNA for hABCA4 editing include the CDS of hABCA4 from the first bp of exon 21 to the end of the CDS (NM_000350.3; bp 3154-6919), preceded by a splicing acceptor signal (SEQ ID NO: 35). Donor DNAs included the SV40 polyA and were flanked by the gRNAs target sequences, followed by the PAM, in inverted orientation as required for efficient HITI. Additionally, construct for MMEJ included either 8 or 20bp-long homology arms (8bp left HA of sequence SEQ ID NO: 40; 8bp right HA of sequence SEQ ID NO: 41; 20bp left HA of sequence SEQ ID NO: 42; 20bp right HA of sequence SEQ ID NO: 43). Constructs were designed to include a 3xFLAG tag at the C-terminal of the CDS. The expression cassette of the selected gRNAs (i.e. SpCas9_gRNAl), including the U6 promoter, was PCR-amplified and cloned in the plasmid including the above-described donor DNA, using the In-Fusion kit (Takara, Kusatsu, Japan).

[0334] - the 3’ donor DNA for hCEP290 editing include the CDS of hCEP290 from the first bp of exon 31 to the end of the CDS (NM_025114.4; bp 3790-7653), preceded by a splicing acceptor signal (SEQ ID NO: 35). Donor DNAs included the Bgh polyA (SEQ ID NO: 47) and were flanked by the gRNAs target sequences, followed by the PAM, in inverted orientation as required for efficient HITI. Constructs were designed to include a 3xFLAGtag at the C-terminal of the CDS.

[0335] For all donor DNA plasmids, versions where the gRNA expression cassette was replaced with the expression cassette for a scramble gRNA were also generated.

[0336] Cell culture and transfection

[0337] Hepal-6 (ATCC, CRL-1830) and HEK293 cells were maintained in DMEM containing 10% fetal bovine serum (FBS) and 2 mM L-glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Hepa 1-6 cell line were transfected with Effectene transfection reagent (QIAGEN, Milan, Italy) following manufacturer’s protocol. HEK293 cells were transfected using the calcium phosphate method.

[0338] For transfections aiming at assessing indel efficiency of each gRNA we transfected 2 pg of pCas9-gRNA plasmid. For studies evaluating genome editing efficiency of the developed approaches, we co-transfected 2 mg of donor DNA and 2 mg of pCas9 plasmids. Transfected cells were harvested 72 hours post-transfection and either EGFP+-FACS-sorted HEPA cells or unsorted HEK293 cells used for further analysis.

[0339] Evaluation of genome editing efficiency at the DNA level

[0340] DNA was extracted from cell pellets and mice retinas using either the (GeneArt Genomic Cleavage Detection Kit, Invitrogen) or the DNeasy Blood& Tissue kit (Qiagen, Germany) following manufacturer’s protocol.

[0341] For evaluation of gRNAs efficiency 50 to 200 ng of DNA extracted from Hepal-6 or HEK293 cells were used for PCR amplification of the region which encompasses the SpCas9 target site. PCR products were sequenced via Sanger sequencing (Eurofins Genomics) and quantification of INDELS was performed via TIDE analysis (Brinkman et al, 2014).

[0342] Nested PCRs to amplify the 5’ and 3’ integration junctions were performed using either 250 ng (for experiments on HEPA and HEK239 cells) or 1000 ng (for experiments on retinas) of DNA For the 5’ junction, PCRs were performed using forward primers annealing in the endogenous intron preceding the target site and reverse primers in the donor DNA. For the 3 ’junction, PCRs were performed using a forward primer annealing in the Donor DNA and a reverse primer annealing in the endogenous intron, downstream of the target site.

[0343] PCR products were either cloned into PCR-Blunt II-TOPO (Invitrogen) and single clones sequenced to confirm the identity of the PCR products or analyzed via NGS.

[0344] AAV vector production and characterization.

[0345] AAV vectors serotype 8 (AAV8) were produced by the TIGEM AAV Vector Core and InnovaVector srl by triple transfection of HEK293 cells followed by two rounds of CsCl2 purification61. For each viral preparation, physical titers (genome copies / ml) were determined by averaging the titer achieved by dot-blot analysis and by PCR quantification using TaqMan (Applied Biosystems, Carlsbad, California, USA).

[0346] Mice injection

[0347] Subretinal injections in Abca4- / - mice were carried out as previously described. Briefly, mice were anesthetized with an intraperitoneal injection of 10 pl / g of body weight of ketamine (10 mg / Kg) combined with medetomidine (1 mg / Kg), then AAV8 vectors were delivered subretinally via a trans-scleral trans-choroidal approach, as described by Liang et al. Eyes were injected with 1 pl of vector solution in the temporal -ventral side of the eye. AAV vectors for in vivo studies we produced from NHEJ- and MMEJ based plasmids under the control of the photoreceptor specific GRK1 -promoter. The SpCas9 included the photoreceptor-specific IRBP (InterPR retinoid-binding protein) promoter (Tornabene et al., Nature Communications 13 (1963), 2022). 2.2 × 109genome copies / eye of each vector (AAV. IRBP-SpCas9 and AAV.donor) were subretinally injected in either 4 week or 3 months-old Abca4- / -and Abca4+ / -mice. Mice were sacrificed at 1-7 months post injection.

[0348] RNA extraction and mAbca4 expression in HEPA cells

[0349] Total RNA was extracted using the RNeasy MiniKit (QIAGEN). RNA (250-1000 ng) was used as a template for LightCycler 480 SYBR Green I Master (Roche Molecular Systems, Inc.) according to the manufacturer’s instructions using the LightCycler 96 (Roche Molecular Systems, Inc.). Expression levels of mAbca4 were normalized vs. the corresponding housekeeping gene (GAPDH). The relative quantification analysis was done using the 2(−ΔΔCt) method.

[0350] RNAscope

[0351] Mouse eyes were fixed overnight in Davidson’s fixative (deionized water, 10% acetic acid, 20% formalin, 35% ethanol), dehydrated by serial passages in ethanol and then embedded in paraffin using Excelsior AS (ASHI, Italy) by the Advanced Histology Facility (Tigem, Italy). Ten-μm-thick sections were cut along the horizontal meridian, progressively distributed on slides and stained with Harris Hematoxylin and Eosin (Sigma-Aldrich). ABCA4 mRNA expression was determined using ISH with the fully automated RNAscope assay on the VENTANA BenchMark Ultra (Ventana Medical Systems, Roche) platform, sections were deparaffinized on the instrument, followed by target retrieval and protease treatment. Probes (RNAscope™ 2.5 VS Probe- Mm-Abca4-01, #474999, ACD) were then hybridized for 2h at 43°C followed by RNAscope amplification (RNAscope® VS Universal AP Reagent Kit #323250, ACD) and Red chromogenic detection using VS detection reagents (DISCOVERY mRNA Red Detection Kit #07099037001, Roche).

[0352] Protein analysis

[0353] Tissue was lysed in RIPA buffer (supplemented with protease inhibitors and 1 mM phenylmethyl sulfonyl fluoride) to extract proteins. After lysis, ABCA4 samples were separated by 6% SDS-polyacrylamide gel electrophoresis, followed by wet western blotting. Western blots were blocked with milk 5% and incubated with either anti-ABCA4 antibody (LS-C87292, LifeSpan BioSciences) or anit-3XFlag (A8592, Sigma Aldrich), followed by incubation with anti-dysferlin (MONX10795, MonosanXtra) or anti-filamin (4762S, Cell Signaling).

[0354] EXAMPLE 1 Testing Mutation-independent genome editing of the murine Abca4 gene in vitro

[0355] As a first exemplary target gene to investigate the efficiency of the mutation-independent genome editing approach of the invention, inventors planned to use the ABCA4 gene, since it recapitulates the above-mentioned major limiting features which the retinal gene therapy field has still to overcome. Mutations in ABCA4 are associated with Stargardt disease (STGD1), the most common form of inherited macular degeneration in humans. In the absence of a functional ABCA4 protein, as it occurs in STGD1 patients, improper recycling of the visual chromophore leads to an accumulation of toxic bisretinoid adducts both in the photoreceptors and in the RPE during the process of disc shedding and phagocytosis. Consequently, abnormal high levels of undigested byproducts, such as A2E, accumulate in lipofuscin granules in the RPE, triggering RPE-cell death and causing secondary photoreceptor degeneration. ABCA4 includes a large coding sequence (CDS) (6822 bp) and is highly polymorphic, with over 1200 disease-causing variants reported to date.

[0356] The most used model of STGD1 is the Abca4 knockout (Abca4- / -) mouse, generated by disrupting the promoter and the first protein-coding exon of the mAbca4 gene. Therefore Abca4- / - mouse can be considered as a model of STGD1 patients with mutations in the 5’ half of the gene.

[0357] gRNAs for SpCas9 targeting of the central region of the murine Abca4 gene have been identified through in silico analysis. To assess the editing efficiency, murine hepatocyte HEPA 1-6 cells were transfected with an expression cassette for gRNA2 (SEQ ID NO: 20, annealing in intron 21 of Abca4 gene) and a plasmid expressing SpCas9 fused to eGFP via a T2A signal (SEQ ID NO: 31), which allows quick identification of transfected cells.

[0358] Cells were harvested 72 hours (hrs) post-transfection and FACS-sorted to isolate EGFP+ cells (representative of those transfected with the Cas9-expressing plasmid). DNA was extracted and amplified by PCR with specific primers upstream and downstream the endogenous gRNA target site. Samples were sent to sequencing and data were analyzed by using the Tracking of Indels by Decomposition (TIDE) online tool (Brinkman, Chen, Amendola, & van Steensel, 2014). Evaluation of INDELS generation by TIDE analysis confirmed efficiency of gRNA 2, (52.5% of INDEL frequency, Fig. 2).

[0359] According to the gRNA site identified, donor nucleic acid constructs composed of the ubiquitous cytomegalovirus (CMV) promoter and the Abca4 coding sequence (CDS) from exon 1 until exon 21 were generated, as shown in Fig. 3. A flag tag was added in the Abca4 CDS (starting at amino acid position 590) in order to facilitate assessment of protein expression by WB. Furthermore, the region of the intron which precedes the target site, including the splicing donor signal, was included downstream of Abca4 CDS, so that upon proper targeted integration, the full-length endogenous intron is restored. This was meant to assure efficient splicing of the hybrid Abca4 mRNA composed by the donor vector derived-5’ half and the endogenous locus derived-3’ half. Donor DNAs sequences in these constructs were designed to be either directly flanked by gRNA 2 target sites, to allow targeted integration via the NHEJ repair pathway (5’ donor NHEJ, Fig. 3), or to be flanked by short homology arms (20bp) more internally to the gRNA 2 target site, to allow targeted integration via the MMEJ repair pathway (5’ donor_MMEJ, Fig. 3). The expression cassette for gRNA2 was included in the same AAV construct, downstream of the donor cassette (Fig. 3). In addition to the constructs including the expression cassette of gRNA2, plasmids versions including the expression cassette for a scramble gRNA, which do not cut in any site in the genome (NHEJ scramble and MMEJ scramble, respectively) were also generated to be used as negative control.

[0360] The efficiency of the genome editing approach in the murine Abca4 gene was initially assessed side-by-side, by transfection in HEPA 1-6 cells of both donor NHEJ and donor MMEJ constructs and of a plasmid encoding for SpCas9 fused to EGFP via a T2A peptide (SEQ ID NO: 31). Seventy-two hours post-transfection, EGFP+ cells were FACS-sorted and their DNA isolated to perform nested PCRs designed to amplify the putative junction points between the integrated donor DNA and the endogenous Abca4 locus. Successful amplification of the junction points in cells transfected with the donor DNA and the gRNA2 cassette, but not those treated with the donor DNA and a scramble-gRNA cassette, confirmed Cas9-mediated proper integration of the donor DNA at the expected site (Fig. 4A). Sequencing of PCR products including 5’ and 3’ integration junctions confirmed proper integration mediated by the MMEJ repair pathway, as well as less precise integrations through NHEJ. However, given that integration is designed to occur in an intron, both types of integration result in productive Abca4 overexpression. Indeed, both NHEJ- and MMEJ-based donor DNAs result in comparable levels of Abca4 expression in transfected HEPA1-6 cells, which otherwise only express background levels of this retinal specific gene (Fig. 4B).

[0361] Additionally, to confirm proper maturation and splicing in the hybrid mRNA formed by the donor DNA-derived 5’ half of Abca4 and locus-derived 3 ’half, the Abca4 mRNA across the splitting point was retrotranscribed and amplified. No evidence of either incorrect or alternative splicing was found (Fig. 4C).

[0362] EXAMPLE 2 Testing mutation-independent genome editing of the murine Abca4 gene in vivo

[0363] Next, the efficiency of both NHEJ- and MMEJ-mediated strategies was tested side-by-side in Abca4- / -mice. To this aim NHEJ- and MMEJ-donor constructs were generated comprising the exogenous DNAunder the control of the photoreceptor-specific GRK1 promoter (SEQ ID NO: 15 and 17, respectively), as well as a construct expressing Cas9 from the photoreceptor-specific IRBP promoter (SEQ ID NO: 19). These constructs were used to produce AAV8 vectors, which were subretinally injected in 1-3-month-old Abca4- / -mice (dose: 2.2xl09GC / vector / eye). Four to seven months post-injection the retinas were harvested and genome editing efficiency in photoreceptors was evaluated. Comparable levels of donor DNA templates were observed with both NHEJ- and MMEJ-based vectors in photoreceptors, as assessed by integration PCR (considering the number of positive eyes out of total eyes injected; Fig. 5 A) as well as by Abca4 expression analysis via RNA scope (Fig. 5B) and by Western Blot, WB analysis (Fig. 5C). NGS analysis of products including 5’ and 3’ integration junctions confirmed proper integration of both NHEJ- and MMEJ-based donor vectors, as well as less precise integrations of the MMEJ-based donor DNA template through NHEJ. Accordingly, preliminary analysis of lipofuscin accumulation, which occurs in Abca4- / - mice as in STGD1 patients, via electron microscopy (EM) in the RPE of Abca4- / - mice 4 to 6 months post-injection confirmed improvement in this retinal phenotype of affected animals injected with either NHEJ- and MMEJ-based vectors (Fig.5D).

[0364] EXAMPLE 3 Testing mutation-independent genome editing approaches for editing of the human ABCA4 gene in vitro.

[0365] A genome editing approach for editing of the human ABCA4 gene was then tested, exploiting either the NHEJ or MMEJ repair pathways. The central region of the hABCA4 gene was screened in silico and a gRNA for SpCas9-mediated editing of ABCA4, cutting in intron 20 (gRNA3, Seq ID NO: 23), was selected (Fig. 6A). Accordingly, two different donor DNA templates were generated to obtain integration of either the ABCA4 CDS that is upstream (exon 1 to 20) or of the ABCA4 CDS that is downstream (exon 21 to 50) the identified target site (Fig.

[0366] 6B). For the 5’ donor vector, constructs for integration via the NHEJ of ABCA4 donor DNAs were generated (SEQ ID NO: 1), together with constructs for integration via MMEJ of ABCA4 donor DNAs carrying either 20bp or 8bp-long microhomology arms (SEQ ID NO: 3 and 5, respectively), to test whether the size of the micro-homology arms, which preferably ranges from 5 to 25 bp, influenced the efficiency of integration. For the 3’ donor vector constructs for integration via the NHEJ were also generated (SEQ ID NO: 7).

[0367] Control vectors, including the expression cassette for a scramble gRNA, which do not cut in any site in the genome (5’ NHEJ scramble, SEQ ID NO: 2; 5’ MMEJ 20bp scramble, SEQ ID NO: 4; 5’ MMEJ 8bp scramble, SEQ ID NO: 6; and 3’ NHEJ scramble, SEQ ID NO: 8) were also generated to be used as negative control.

[0368] The generated donor vectors were tested side-by-side upon transfection in human embryonic kidney 293 (HEK293 cells) and found that they mediate efficient integration of the donor template in the target ABCA4 locus (Fig. 7A), resulting in ABCA4 expression (Fig. 7B). As already observed for the genome editing approach targeted to the murine Abca4 gene, the MMEJ-based donor DNA template results in more precise integration compared to NHEJ-based donor, independently of the length of homology arms used (Fig. 7A). EXAMPLE 4 Testing mutation-independent genome editing approaches for editing of the human ABCA4 gene in organoids.

[0369] In order to test the efficiency of the genome editing approaches developed to target the human ABCA4 gene in more relevant models towards clinical translation, induced pluripotent stem cells (iPSC) have been obtained from STGD1 patients, which carry either mutations in the 5’ (homozygous c.768G> T) or in the 3’ half (compound heterozygous: c.4539+2001G> A e c.4892T> C) of ABCA4. The iPSC lines were used for generation of both three-dimensional (3D) retinal organoids and retinal pigment epithelium (RPE) layers. Characterization by both light and fluorescence microscopy, confirmed proper maturation of organoids, as assessed by expression of relevant markers of mature photoreceptors (Fig. 7C).

[0370] Therefore, AAV2 vectors encoding either Cas9 from the photoreceptor-specific IRPE promoter or comprising each of the above-described donor vectors were prepared, and used to infect fully differentiated retinal organoids. Four weeks post-infection the organoids were harvested and, importantly, appropriate integration of the donor DNA in the ABCA4 locus was proved to occur (Fig. 7D).

[0371] EXAMPLE 5 Testing mutation-independent genome editing approaches for editing of the human CEP290 gene.

[0372] Efficiency of our genome editing approach is further confirmed by editing another large gene, CEP290, which is one of the most common causes of Leber Congenital Amaurosis.

[0373] Mutations of human CEP290 gene account for 15-20% of all cases of Leber Congenital Amaurosis. To this aim, similarly to the ABCA4 gene, the central region of CEP290 was screened in silico and the gRNA which presented the best on-target and off-target scores, cutting in intron 30 of the gene, was selected (gRNA 2 SEQ ID NO: 28). Accordingly, a donor DNA for correction of the 3’ half of CEP290 was generated (SEQ ID NO: 52), including exons from 31 to 54, relying on NHEJ for targeted integration. The generated donor vector was tested upon transfection in HEK293 cells.

[0374] Nested PCR analysis on the genomic DNA confirmed efficient integration of the donor template in the target locus (Fig. 8 A) and specific expression of CEP290 in transfected cells (Fig. 8B).

[0375] Sequences disclosed in conjunction with the present invention are enclosed and displayed hereafter. SEQUENCES

[0376] Seq ID NO: 1 HITI 5’ hABCA4

[0377] ITR

[0378] gRNA+PAM

[0379] CMV enhancer + promoter + chimeric intron

[0380] ABCA4 5' CDS

[0381] Splicing Donor

[0382] hU6 promoter

[0383] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCCCGCGGAT CC AT! GTTATAACTCAGCGGGGAAGCGGAGACTCGCGCGAGTCGAGGAGGGAAGATCTTCAATATTGGCCATTAGCCATA TTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATG TACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCA ATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGC TGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTC CATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGT CCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTT CCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGG CGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCAC CAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGG TGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGCGGTAGTTTATCAC AGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGC ACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGA AGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCAC TCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCCTCGAGAATTCACGC GTGGTACCTCTAGAGTCGACCCGGGCGGCCGCCATGGGCTTCGTGAGACAGATACAGCTTTTGCTCTGGAAGAAC TGGACCCTGCGGAAAAGGCAAAAGATTCGCTTTGTGGTGGAACTCGTGTGGCCTTTATCTTTATTTCTGGTCTTG ATCTGGTTAAGGAATGCCAACCCGCTCTACAGCCATCATGAATGCCATTTCCCCAACAAGGCGATGCCCTCAGCA GGAATGCTGCCGTGGCTCCAGGGGATCTTCTGCAATGTGAACAATCCCTGTTTTCAAAGCCCCACCCCAGGAGAA TCTCCTGGAATTGTGTCAAACTATAACAACTCCATCTTGGCAAGGGTATATCGAGATTTTCAAGAACTCCTCATG AATGCACCAGAGAGCCAGCACCTTGGCCGTATTTGGACAGAGCTACACATCTTGTCCCAATTCATGGACACCCTC CGGACTCACCCGGAGAGAATTGCAGGAAGAGGAATACGAATAAGGGATATCTTGAAAGATGAAGAAACACTGACA CTATTTCTCATTAAAAACATCGGCCTGTCTGACTCAGTGGTCTACCTTCTGATCAACTCTCAAGTCCGTCCAGAG CAGTTCGCTCATGGAGTCCCGGACCTGGCGCTGAAGGACATCGCCTGCAGCGAGGCCCTCCTGGAGCGCTTCATC ATCTTCAGCCAGAGACGCGGGGCAAAGACGGTGCGCTATGCCCTGTGCTCCCTCTCCCAGGGCACCCTACAGTGG ATAGAAGACACTCTGTATGCCAACGTGGACTTCTTCAAGCTCTTCCGTGTGCTTCCCACACTCCTAGACAGCCGT TCTCAAGGTATCAATCTGAGATCTTGGGGAGGAATATTATCTGATATGTCACCAAGAATTCAAGAGTTTATCCAT CGGCCGAGTATGCAGGACTTGCTGTGGGTGACCAGGCCCCTCATGCAGAATGGTGGTCCAGAGACCTTTACAAAG CTGATGGGCATCCTGTCTGACCTCCTGTGTGGCTACCCCGAGGGAGGTGGCTCTCGGGTGCTCTCCTTCAACTGG TATGAAGACAATAACTATAAGGCCTTTCTGGGGATTGACTCCACAAGGAAGGATCCTATCTATTCTTATGACAGA AGAACAACATCCTTTTGTAATGCATTGATCCAGAGCCTGGAGTCAAATCCTTTAACCAAAATCGCTTGGAGGGCG GCAAAGCCTTTGCTGATGGGAAAAATCCTGTACACTCCTGATTCACCTGCAGCACGAAGGATACTGAAGAATGCC AACTCAACTTTTGAAGAACTGGAACACGTTAGGAAGTTGGTCAAAGCCTGGGAAGAAGTAGGGCCCCAGATCTGG TACTTCTTTGACAACAGCACACAGATGAACATGATCAGAGATACCCTGGGGAACCCAACAGTAAAAGACTTTTTG AATAGGCAGCTTGGTGAAGAAGGTATTACTGCTGAAGCCATCCTAAACTTCCTCTACAAGGGCCCTCGGGAAAGC CAGGCTGACGACATGGCCAACTTCGACTGGAGGGACATATTTAACATCACTGATCGCACCCTCCGCCTTGTCAAT CAATACCTGGAGTGCTTGGTCCTGGATAAGTTTGAAAGCTACAATGATGAAACTCAGCTCACCCAACGTGCCCTC TCTCTACTGGAGGAAAACATGTTCTGGGCCGGAGTGGTATTCCCTGACATGTATCCCTGGACCAGCTCTCTACCA CCCCACGTGAAGTATAAGATCCGAATGGACATAGACGTGGTGGAGAAAACCAATAAGATTAAAGACAGGTATTGG GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGGATTCTGGT CCCAGAGCTGATCCCGTGGAAGATTTCCGGTACATCTGGGGCGGGTTTGCCTATCTGCAGGACATGGTTGAACAG GGGATCACAAGGAGCCAGGTGCAGGCGGAGGCTCCAGTTGGAATCTACCTCCAGCAGATGCCCTACCCCTGCTTC GTGGACGATTCTTTCATGATCATCCTGAACCGCTGTTTCCCTATCTTCATGGTGCTGGCATGGATCTACTCTGTC TCCATGACTGTGAAGAGCATCGTCTTGGAGAAGGAGTTGCGACTGAAGGAGACCTTGAAAAATCAGGGTGTCTCC AATGCAGTGATTTGGTGTACCTGGTTCCTGGACAGCTTCTCCATCATGTCGATGAGCATCTTCCTCCTGACGATA TTCATCATGCATGGAAGAATCCTACATTACAGCGACCCATTCATCCTCTTCCTGTTCTTGTTGGCTTTCTCCACT GCCACCATCATGCTGTGCTTTCTGCTCAGCACCTTCTTCTCCAAGGCCAGTCTGGCAGCAGCCTGTAGTGGTGTC ATCTATTTCACCCTCTACCTGCCACACATCCTGTGCTTCGCCTGGCAGGACCGCATGACCGCTGAGCTGAAGAAG GCTGTGAGCTTACTGTCTCCGGTGGCATTTGGATTTGGCACTGAGTACCTGGTTCGCTTTGAAGAGCAAGGCCTG GGGCTGCAGTGGAGCAACATCGGGAACAGTCCCACGGAAGGGGACGAATTCAGCTTCCTGCTGTCCATGCAGATG ATGCTCCTTGATGCTGCTGTCTATGGCTTACTCGCTTGGTACCTTGATCAGGTGTTTCCAGGAGACTATGGAACC CCACTTCCTTGGTACTTTCTTCTACAAGAGTCGTATTGGCTTGGCGGTGAAGGGTGTTCAACCAGAGAAGAAAGA GCCCTGGAAAAGACCGAGCCCCTAACAGAGGAAACGGAGGATCCAGAGCACCCAGAAGGAATACACGACTCCTTC TTTGAACGTGAGCATCCAGGGTGGGTTCCTGGGGTATGCGTGAAGAATCTGGTAAAGATTTTTGAGCCCTGTGGC CGGCCAGCTGTGGACCGTCTGAACATCACCTTCTACGAGAACCAGATCACCGCATTCCTGGGCCACAATGGAGCT GGGAAAACCACCACCTTGTCCATCCTGACGGGTCTGTTGCCACCAACCTCTGGGACTGTGCTCGTTGGGGGAAGG GACATTGAAACCAGCCTGGATGCAGTCCGGCAGAGCCTTGGCATGTGTCCACAGCACAACATCCTGTTCCACCAG TAAGTАТCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGTAAGCGAGACATCTATACTAACTAATGGAAGCGGACataccaatgttataactcagCGGACACACCGG TTGGATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGA CTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAA AATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCT TGTGGAAAGGACGAAACACCGATACCAATGTTATAACTCAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGCGGCCGCACCAATTGAGGAACCC CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGA CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0384] Seq ID NO: 2 HITI 5’ hABCA4 gRNA scramble

[0385] ITR

[0386] gRNA+PAM

[0387] CMV enhancer + promoter + chimeric intron

[0388] ABCA4 5' CDS

[0389] Splicing Donor

[0390] hU6 promoter

[0391] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCCCGCGGAT CC AT! GTTATAACTCAGCGGGGAAGCGGAGACTCGCGCGAGTCGAGGAGGGAAGATCTTCAATATTGGCCATTAGCCATA TTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATG TACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCA ATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGC TGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTC CATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGT CCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTT CCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGG CGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCAC CAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGG TGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGCGGTAGTTTATCAC AGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGC ACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGA AGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCAC TCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCCTCGAGAATTCACGC GTGGTACCTCTAGAGTCGACCCGGGCGGCCGCCATGGGCTTCGTGAGACAGATACAGCTTTTGCTCTGGAAGAAC TGGACCCTGCGGAAAAGGCAAAAGATTCGCTTTGTGGTGGAACTCGTGTGGCCTTTATCTTTATTTCTGGTCTTG ATCTGGTTAAGGAATGCCAACCCGCTCTACAGCCATCATGAATGCCATTTCCCCAACAAGGCGATGCCCTCAGCA GGAATGCTGCCGTGGCTCCAGGGGATCTTCTGCAATGTGAACAATCCCTGTTTTCAAAGCCCCACCCCAGGAGAA TCTCCTGGAATTGTGTCAAACTATAACAACTCCATCTTGGCAAGGGTATATCGAGATTTTCAAGAACTCCTCATG AATGCACCAGAGAGCCAGCACCTTGGCCGTATTTGGACAGAGCTACACATCTTGTCCCAATTCATGGACACCCTC CGGACTCACCCGGAGAGAATTGCAGGAAGAGGAATACGAATAAGGGATATCTTGAAAGATGAAGAAACACTGACA CTATTTCTCATTAAAAACATCGGCCTGTCTGACTCAGTGGTCTACCTTCTGATCAACTCTCAAGTCCGTCCAGAG CAGTTCGCTCATGGAGTCCCGGACCTGGCGCTGAAGGACATCGCCTGCAGCGAGGCCCTCCTGGAGCGCTTCATC ATCTTCAGCCAGAGACGCGGGGCAAAGACGGTGCGCTATGCCCTGTGCTCCCTCTCCCAGGGCACCCTACAGTGG ATAGAAGACACTCTGTATGCCAACGTGGACTTCTTCAAGCTCTTCCGTGTGCTTCCCACACTCCTAGACAGCCGT TCTCAAGGTATCAATCTGAGATCTTGGGGAGGAATATTATCTGATATGTCACCAAGAATTCAAGAGTTTATCCAT CGGCCGAGTATGCAGGACTTGCTGTGGGTGACCAGGCCCCTCATGCAGAATGGTGGTCCAGAGACCTTTACAAAG CTGATGGGCATCCTGTCTGACCTCCTGTGTGGCTACCCCGAGGGAGGTGGCTCTCGGGTGCTCTCCTTCAACTGG TATGAAGACAATAACTATAAGGCCTTTCTGGGGATTGACTCCACAAGGAAGGATCCTATCTATTCTTATGACAGA AGAACAACATCCTTTTGTAATGCATTGATCCAGAGCCTGGAGTCAAATCCTTTAACCAAAATCGCTTGGAGGGCG GCAAAGCCTTTGCTGATGGGAAAAATCCTGTACACTCCTGATTCACCTGCAGCACGAAGGATACTGAAGAATGCC AACTCAACTTTTGAAGAACTGGAACACGTTAGGAAGTTGGTCAAAGCCTGGGAAGAAGTAGGGCCCCAGATCTGG TACTTCTTTGACAACAGCACACAGATGAACATGATCAGAGATACCCTGGGGAACCCAACAGTAAAAGACTTTTTG AATAGGCAGCTTGGTGAAGAAGGTATTACTGCTGAAGCCATCCTAAACTTCCTCTACAAGGGCCCTCGGGAAAGC CAGGCTGACGACATGGCCAACTTCGACTGGAGGGACATATTTAACATCACTGATCGCACCCTCCGCCTTGTCAAT CAATACCTGGAGTGCTTGGTCCTGGATAAGTTTGAAAGCTACAATGATGAAACTCAGCTCACCCAACGTGCCCTC TCTCTACTGGAGGAAAACATGTTCTGGGCCGGAGTGGTATTCCCTGACATGTATCCCTGGACCAGCTCTCTACCA CCCCACGTGAAGTATAAGATCCGAATGGACATAGACGTGGTGGAGAAAACCAATAAGATTAAAGACAGGTATTGG GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGGATTCTGGT CCCAGAGCTGATCCCGTGGAAGATTTCCGGTACATCTGGGGCGGGTTTGCCTATCTGCAGGACATGGTTGAACAG GGGATCACAAGGAGCCAGGTGCAGGCGGAGGCTCCAGTTGGAATCTACCTCCAGCAGATGCCCTACCCCTGCTTC GTGGACGATTCTTTCATGATCATCCTGAACCGCTGTTTCCCTATCTTCATGGTGCTGGCATGGATCTACTCTGTC TCCATGACTGTGAAGAGCATCGTCTTGGAGAAGGAGTTGCGACTGAAGGAGACCTTGAAAAATCAGGGTGTCTCC AATGCAGTGATTTGGTGTACCTGGTTCCTGGACAGCTTCTCCATCATGTCGATGAGCATCTTCCTCCTGACGATA TTCATCATGCATGGAAGAATCCTACATTACAGCGACCCATTCATCCTCTTCCTGTTCTTGTTGGCTTTCTCCACT GCCACCATCATGCTGTGCTTTCTGCTCAGCACCTTCTTCTCCAAGGCCAGTCTGGCAGCAGCCTGTAGTGGTGTC ATCTATTTCACCCTCTACCTGCCACACATCCTGTGCTTCGCCTGGCAGGACCGCATGACCGCTGAGCTGAAGAAG GCTGTGAGCTTACTGTCTCCGGTGGCATTTGGATTTGGCACTGAGTACCTGGTTCGCTTTGAAGAGCAAGGCCTG GGGCTGCAGTGGAGCAACATCGGGAACAGTCCCACGGAAGGGGACGAATTCAGCTTCCTGCTGTCCATGCAGATG ATGCTCCTTGATGCTGCTGTCTATGGCTTACTCGCTTGGTACCTTGATCAGGTGTTTCCAGGAGACTATGGAACC CCACTTCCTTGGTACTTTCTTCTACAAGAGTCGTATTGGCTTGGCGGTGAAGGGTGTTCAACCAGAGAAGAAAGA GCCCTGGAAAAGACCGAGCCCCTAACAGAGGAAACGGAGGATCCAGAGCACCCAGAAGGAATACACGACTCCTTC TTTGAACGTGAGCATCCAGGGTGGGTTCCTGGGGTATGCGTGAAGAATCTGGTAAAGATTTTTGAGCCCTGTGGC CGGCCAGCTGTGGACCGTCTGAACATCACCTTCTACGAGAACCAGATCACCGCATTCCTGGGCCACAATGGAGCT GGGAAAACCACCACCTTGTCCATCCTGACGGGTCTGTTGCCACCAACCTCTGGGACTGTGCTCGTTGGGGGAAGG GACATTGAAACCAGCCTGGATGCAGTCCGGCAGAGCCTTGGCATGTGTCCACAGCACAACATCCTGTTCCACCAG TAAGTАТCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGTAAGCGAGACATCTATACTAACTAATGGAAGCGGACataccaatgttataactcagCGGACACACCGG TTTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGT AAACACAAAGAT AT T AGT ACAAAAT AC GT GAG GT AGAAAGT AAT AAT T T CT T GGGT AGT T T GCAGT T T T AAAAT T ATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTG GAAAGGACGAAACACCGGACTCGCGCGAGTCGAGGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGCGGCCGCACCAATTGATTGAGGAACCC CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGA CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0392] Seq ID NO: 3 MITI plasmid 20HAhABCA45’ gRNA

[0393] ITR

[0394] gRNA+PAM

[0395] microhomology arms

[0396] CMV enhancer + promoter + chimeric intron

[0397] ABCA4 5' CDS

[0398] Splicing Donor

[0399] hU6 promoter

[0400] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCATACCAATGTTATAACTCAGCGG ctactcagagataaccgctgGGAAGCGGAGACTCGCGCGAGTCGAGGAGGGAAGATCTTCAATATT GGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATC TATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTT ATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAA ATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGC CAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT ATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA CCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC AGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGA GTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCG GTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTG CGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAA GTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCT TGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCT CCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCC TCGAGAATTCACGCGTGGTACCTCTAGAGTCGACCCGGGCGGCCGCCATGGGCTTCGTGAGACAGATACAGCTTT TGCTCTGGAAGAACTGGACCCTGCGGAAAAGGCAAAAGATTCGCTTTGTGGTGGAACTCGTGTGGCCTTTATCTT TATTTCTGGTCTTGATCTGGTTAAGGAATGCCAACCCGCTCTACAGCCATCATGAATGCCATTTCCCCAACAAGG CGATGCCCTCAGCAGGAATGCTGCCGTGGCTCCAGGGGATCTTCTGCAATGTGAACAATCCCTGTTTTCAAAGCC CCACCCCAGGAGAATCTCCTGGAATTGTGTCAAACTATAACAACTCCATCTTGGCAAGGGTATATCGAGATTTTC AAGAACTCCTCATGAATGCACCAGAGAGCCAGCACCTTGGCCGTATTTGGACAGAGCTACACATCTTGTCCCAAT TCATGGACACCCTCCGGACTCACCCGGAGAGAATTGCAGGAAGAGGAATACGAATAAGGGATATCTTGAAAGATG AAGAAACACTGACACTATTTCTCATTAAAAACATCGGCCTGTCTGACTCAGTGGTCTACCTTCTGATCAACTCTC AAGTCCGTCCAGAGCAGTTCGCTCATGGAGTCCCGGACCTGGCGCTGAAGGACATCGCCTGCAGCGAGGCCCTCC TGGAGCGCTTCATCATCTTCAGCCAGAGACGCGGGGCAAAGACGGTGCGCTATGCCCTGTGCTCCCTCTCCCAGG GCACCCTACAGTGGATAGAAGACACTCTGTATGCCAACGTGGACTTCTTCAAGCTCTTCCGTGTGCTTCCCACAC TCCTAGACAGCCGTTCTCAAGGTATCAATCTGAGATCTTGGGGAGGAATATTATCTGATATGTCACCAAGAATTC AAGAGTTTATCCATCGGCCGAGTATGCAGGACTTGCTGTGGGTGACCAGGCCCCTCATGCAGAATGGTGGTCCAG AGACCTTTACAAAGCTGATGGGCATCCTGTCTGACCTCCTGTGTGGCTACCCCGAGGGAGGTGGCTCTCGGGTGC TCTCCTTCAACTGGTATGAAGACAATAACTATAAGGCCTTTCTGGGGATTGACTCCACAAGGAAGGATCCTATCT ATTCTTATGACAGAAGAACAACATCCTTTTGTAATGCATTGATCCAGAGCCTGGAGTCAAATCCTTTAACCAAAA TCGCTTGGAGGGCGGCAAAGCCTTTGCTGATGGGAAAAATCCTGTACACTCCTGATTCACCTGCAGCACGAAGGA TACTGAAGAATGCCAACTCAACTTTTGAAGAACTGGAACACGTTAGGAAGTTGGTCAAAGCCTGGGAAGAAGTAG GGCCCCAGATCTGGTACTTCTTTGACAACAGCACACAGATGAACATGATCAGAGATACCCTGGGGAACCCAACAG TAAAAGACTTTTTGAATAGGCAGCTTGGTGAAGAAGGTATTACTGCTGAAGCCATCCTAAACTTCCTCTACAAGG GCCCTCGGGAAAGCCAGGCTGACGACATGGCCAACTTCGACTGGAGGGACATATTTAACATCACTGATCGCACCC TCCGCCTTGTCAATCAATACCTGGAGTGCTTGGTCCTGGATAAGTTTGAAAGCTACAATGATGAAACTCAGCTCA CCCAACGTGCCCTCTCTCTACTGGAGGAAAACATGTTCTGGGCCGGAGTGGTATTCCCTGACATGTATCCCTGGA CCAGCTCTCTACCACCCCACGTGAAGTATAAGATCCGAATGGACATAGACGTGGTGGAGAAAACCAATAAGATTA AAGACAGGTATTGGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATG ACAAGGATTCTGGTCCCAGAGCTGATCCCGTGGAAGATTTCCGGTACATCTGGGGCGGGTTTGCCTATCTGCAGG ACATGGTTGAACAGGGGATCACAAGGAGCCAGGTGCAGGCGGAGGCTCCAGTTGGAATCTACCTCCAGCAGATGC CCTACCCCTGCTTCGTGGACGATTCTTTCATGATCATCCTGAACCGCTGTTTCCCTATCTTCATGGTGCTGGCAT GGATCTACTCTGTCTCCATGACTGTGAAGAGCATCGTCTTGGAGAAGGAGTTGCGACTGAAGGAGACCTTGAAAA ATCAGGGTGTCTCCAATGCAGTGATTTGGTGTACCTGGTTCCTGGACAGCTTCTCCATCATGTCGATGAGCATCT TCCTCCTGACGATATTCATCATGCATGGAAGAATCCTACATTACAGCGACCCATTCATCCTCTTCCTGTTCTTGT TGGCTTTCTCCACTGCCACCATCATGCTGTGCTTTCTGCTCAGCACCTTCTTCTCCAAGGCCAGTCTGGCAGCAG CCTGTAGTGGTGTCATCTATTTCACCCTCTACCTGCCACACATCCTGTGCTTCGCCTGGCAGGACCGCATGACCG CTGAGCTGAAGAAGGCTGTGAGCTTACTGTCTCCGGTGGCATTTGGATTTGGCACTGAGTACCTGGTTCGCTTTG AAGAGCAAGGCCTGGGGCTGCAGTGGAGCAACATCGGGAACAGTCCCACGGAAGGGGACGAATTCAGCTTCCTGC TGTCCATGCAGATGATGCTCCTTGATGCTGCTGTCTATGGCTTACTCGCTTGGTACCTTGATCAGGTGTTTCCAG GAGACTATGGAACCCCACTTCCTTGGTACTTTCTTCTACAAGAGTCGTATTGGCTTGGCGGTGAAGGGTGTTCAA CCAGAGAAGAAAGAGCCCTGGAAAAGACCGAGCCCCTAACAGAGGAAACGGAGGATCCAGAGCACCCAGAAGGAA TACACGACTCCTTCTTTGAACGTGAGCATCCAGGGTGGGTTCCTGGGGTATGCGTGAAGAATCTGGTAAAGATTT TTGAGCCCTGTGGCCGGCCAGCTGTGGACCGTCTGAACATCACCTTCTACGAGAACCAGATCACCGCATTCCTGG GCCACAATGGAGCTGGGAAAACCACCACCTTGTCCATCCTGACGGGTCTGTTGCCACCAACCTCTGGGACTGTGC TCGTTGGGGGAAGGGACATTGAAACCAGCCTGGATGCAGTCCGGCAGAGCCTTGGCATGTGTCCACAGCACAACA TCCTGTTCCACCAGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACA GAGAAGACTCTTGCGTTTCTGTAAGCGAGACATCTATACTAACTAATGGAAGCGGACagttataacattggtata taCCGctgagttataacattggtatACCGGTTGGATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGG CT GT T AGAGAGAT AAT T GGAAT T AAT T T GACT GT AAACACAAAGAT AT T AGT ACAAAAT AC GT GAG GT AGAAAGT AATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAA AGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGataccaatgttataactcagGTTT TAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT TTTTGCGCGGCCGCACCAATTGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AG

[0401] Seq ID NO: 4 MITI 20HA hABCA4 5’ gRNA scramble

[0402] ITR gRNA+PAM

[0403] microhomology arms

[0404] CMV enhancer + promoter + chimeric intron

[0405] ABCA4 5' CDS

[0406] Splicing Donor

[0407] hU6 promoter

[0408] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCATACCAATGTTATAACTCAGCGG ctactcagagataaccgctgGGAAGCGGAGACTCGCGCGAGTCGAGGAGGGAAGATCTTCAATATT GGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATC TATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTT ATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAA ATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGC CAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT ATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA CCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC AGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGA GTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCG GTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTG CGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAA GTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCT TGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCT CCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCC TCGAGAATTCACGCGTGGTACCTCTAGAGTCGACCCGGGCGGCCGCCATGGGCTTCGTGAGACAGATACAGCTTT TGCTCTGGAAGAACTGGACCCTGCGGAAAAGGCAAAAGATTCGCTTTGTGGTGGAACTCGTGTGGCCTTTATCTT TATTTCTGGTCTTGATCTGGTTAAGGAATGCCAACCCGCTCTACAGCCATCATGAATGCCATTTCCCCAACAAGG CGATGCCCTCAGCAGGAATGCTGCCGTGGCTCCAGGGGATCTTCTGCAATGTGAACAATCCCTGTTTTCAAAGCC CCACCCCAGGAGAATCTCCTGGAATTGTGTCAAACTATAACAACTCCATCTTGGCAAGGGTATATCGAGATTTTC AAGAACTCCTCATGAATGCACCAGAGAGCCAGCACCTTGGCCGTATTTGGACAGAGCTACACATCTTGTCCCAAT TCATGGACACCCTCCGGACTCACCCGGAGAGAATTGCAGGAAGAGGAATACGAATAAGGGATATCTTGAAAGATG AAGAAACACTGACACTATTTCTCATTAAAAACATCGGCCTGTCTGACTCAGTGGTCTACCTTCTGATCAACTCTC AAGTCCGTCCAGAGCAGTTCGCTCATGGAGTCCCGGACCTGGCGCTGAAGGACATCGCCTGCAGCGAGGCCCTCC TGGAGCGCTTCATCATCTTCAGCCAGAGACGCGGGGCAAAGACGGTGCGCTATGCCCTGTGCTCCCTCTCCCAGG GCACCCTACAGTGGATAGAAGACACTCTGTATGCCAACGTGGACTTCTTCAAGCTCTTCCGTGTGCTTCCCACAC TCCTAGACAGCCGTTCTCAAGGTATCAATCTGAGATCTTGGGGAGGAATATTATCTGATATGTCACCAAGAATTC AAGAGTTTATCCATCGGCCGAGTATGCAGGACTTGCTGTGGGTGACCAGGCCCCTCATGCAGAATGGTGGTCCAG AGACCTTTACAAAGCTGATGGGCATCCTGTCTGACCTCCTGTGTGGCTACCCCGAGGGAGGTGGCTCTCGGGTGC TCTCCTTCAACTGGTATGAAGACAATAACTATAAGGCCTTTCTGGGGATTGACTCCACAAGGAAGGATCCTATCT ATTCTTATGACAGAAGAACAACATCCTTTTGTAATGCATTGATCCAGAGCCTGGAGTCAAATCCTTTAACCAAAA TCGCTTGGAGGGCGGCAAAGCCTTTGCTGATGGGAAAAATCCTGTACACTCCTGATTCACCTGCAGCACGAAGGA TACTGAAGAATGCCAACTCAACTTTTGAAGAACTGGAACACGTTAGGAAGTTGGTCAAAGCCTGGGAAGAAGTAG GGCCCCAGATCTGGTACTTCTTTGACAACAGCACACAGATGAACATGATCAGAGATACCCTGGGGAACCCAACAG TAAAAGACTTTTTGAATAGGCAGCTTGGTGAAGAAGGTATTACTGCTGAAGCCATCCTAAACTTCCTCTACAAGG GCCCTCGGGAAAGCCAGGCTGACGACATGGCCAACTTCGACTGGAGGGACATATTTAACATCACTGATCGCACCC TCCGCCTTGTCAATCAATACCTGGAGTGCTTGGTCCTGGATAAGTTTGAAAGCTACAATGATGAAACTCAGCTCA CCCAACGTGCCCTCTCTCTACTGGAGGAAAACATGTTCTGGGCCGGAGTGGTATTCCCTGACATGTATCCCTGGA CCAGCTCTCTACCACCCCACGTGAAGTATAAGATCCGAATGGACATAGACGTGGTGGAGAAAACCAATAAGATTA AAGACAGGTATTGGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATG ACAAGGATTCTGGTCCCAGAGCTGATCCCGTGGAAGATTTCCGGTACATCTGGGGCGGGTTTGCCTATCTGCAGG ACATGGTTGAACAGGGGATCACAAGGAGCCAGGTGCAGGCGGAGGCTCCAGTTGGAATCTACCTCCAGCAGATGC CCTACCCCTGCTTCGTGGACGATTCTTTCATGATCATCCTGAACCGCTGTTTCCCTATCTTCATGGTGCTGGCAT GGATCTACTCTGTCTCCATGACTGTGAAGAGCATCGTCTTGGAGAAGGAGTTGCGACTGAAGGAGACCTTGAAAA ATCAGGGTGTCTCCAATGCAGTGATTTGGTGTACCTGGTTCCTGGACAGCTTCTCCATCATGTCGATGAGCATCT TCCTCCTGACGATATTCATCATGCATGGAAGAATCCTACATTACAGCGACCCATTCATCCTCTTCCTGTTCTTGT TGGCTTTCTCCACTGCCACCATCATGCTGTGCTTTCTGCTCAGCACCTTCTTCTCCAAGGCCAGTCTGGCAGCAG CCTGTAGTGGTGTCATCTATTTCACCCTCTACCTGCCACACATCCTGTGCTTCGCCTGGCAGGACCGCATGACCG CTGAGCTGAAGAAGGCTGTGAGCTTACTGTCTCCGGTGGCATTTGGATTTGGCACTGAGTACCTGGTTCGCTTTG AAGAGCAAGGCCTGGGGCTGCAGTGGAGCAACATCGGGAACAGTCCCACGGAAGGGGACGAATTCAGCTTCCTGC TGTCCATGCAGATGATGCTCCTTGATGCTGCTGTCTATGGCTTACTCGCTTGGTACCTTGATCAGGTGTTTCCAG GAGACTATGGAACCCCACTTCCTTGGTACTTTCTTCTACAAGAGTCGTATTGGCTTGGCGGTGAAGGGTGTTCAA CCAGAGAAGAAAGAGCCCTGGAAAAGACCGAGCCCCTAACAGAGGAAACGGAGGATCCAGAGCACCCAGAAGGAA TACACGACTCCTTCTTTGAACGTGAGCATCCAGGGTGGGTTCCTGGGGTATGCGTGAAGAATCTGGTAAAGATTT TTGAGCCCTGTGGCCGGCCAGCTGTGGACCGTCTGAACATCACCTTCTACGAGAACCAGATCACCGCATTCCTGG GCCACAATGGAGCTGGGAAAACCACCACCTTGTCCATCCTGACGGGTCTGTTGCCACCAACCTCTGGGACTGTGC TCGTTGGGGGAAGGGACATTGAAACCAGCCTGGATGCAGTCCGGCAGAGCCTTGGCATGTGTCCACAGCACAACA TCCTGTTCCACCAGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACA GAGAAGACTCTTGCGTTTCTGTAAGCGAGACATCTATACTAACTAATGGAAGCGGACagttataacattggtata taCCGctgagttataacattggtatACCGGTTTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAgATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGACTCGCGCGAGTCGAGGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGCGGCCGCACCAATTGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0409] Seq ID NO: 5 MITI 8HA hABCA45’ gRNA

[0410] ITR

[0411] gRNA+PAM

[0412] microhomology arms

[0413] CMV enhancer + promoter + chimeric intron

[0414] ABCA4 5' CDS

[0415] Splicing Donor

[0416] hU6 promoter

[0417] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAG AGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCATACCAATGTTATAACTCAGCGGAACCGCTGGGAAG CGGAGACTCGCGCGAGTCGAGGAGGGAAGATCTTCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGC ATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATG TCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCA TAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCG CCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGA GTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTA CGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTC ACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCC AAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG AGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGT CAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAA GGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATA GGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTC TTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCCTCGAGAATTCACGCGTGGTACCTCTAGAGTCGAC CCGGGCGGCCGCCATGGGCTTCGTGAGACAGATACAGCTTTTGCTCTGGAAGAACTGGACCCTGCGGAAAAGGCA AAAGATTCGCTTTGTGGTGGAACTCGTGTGGCCTTTATCTTTATTTCTGGTCTTGATCTGGTTAAGGAATGCCAA CCCGCTCTACAGCCATCATGAATGCCATTTCCCCAACAAGGCGATGCCCTCAGCAGGAATGCTGCCGTGGCTCCA GGGGATCTTCTGCAATGTGAACAATCCCTGTTTTCAAAGCCCCACCCCAGGAGAATCTCCTGGAATTGTGTCAAA CTATAACAACTCCATCTTGGCAAGGGTATATCGAGATTTTCAAGAACTCCTCATGAATGCACCAGAGAGCCAGCA CCTTGGCCGTATTTGGACAGAGCTACACATCTTGTCCCAATTCATGGACACCCTCCGGACTCACCCGGAGAGAAT TGCAGGAAGAGGAATACGAATAAGGGATATCTTGAAAGATGAAGAAACACTGACACTATTTCTCATTAAAAACAT CGGCCTGTCTGACTCAGTGGTCTACCTTCTGATCAACTCTCAAGTCCGTCCAGAGCAGTTCGCTCATGGAGTCCC GGACCTGGCGCTGAAGGACATCGCCTGCAGCGAGGCCCTCCTGGAGCGCTTCATCATCTTCAGCCAGAGACGCGG GGCAAAGACGGTGCGCTATGCCCTGTGCTCCCTCTCCCAGGGCACCCTACAGTGGATAGAAGACACTCTGTATGC CAACGTGGACTTCTTCAAGCTCTTCCGTGTGCTTCCCACACTCCTAGACAGCCGTTCTCAAGGTATCAATCTGAG ATCTTGGGGAGGAATATTATCTGATATGTCACCAAGAATTCAAGAGTTTATCCATCGGCCGAGTATGCAGGACTT GCTGTGGGTGACCAGGCCCCTCATGCAGAATGGTGGTCCAGAGACCTTTACAAAGCTGATGGGCATCCTGTCTGA CCTCCTGTGTGGCTACCCCGAGGGAGGTGGCTCTCGGGTGCTCTCCTTCAACTGGTATGAAGACAATAACTATAA GGCCTTTCTGGGGATTGACTCCACAAGGAAGGATCCTATCTATTCTTATGACAGAAGAACAACATCCTTTTGTAA TGCATTGATCCAGAGCCTGGAGTCAAATCCTTTAACCAAAATCGCTTGGAGGGCGGCAAAGCCTTTGCTGATGGG AAAAATCCTGTACACTCCTGATTCACCTGCAGCACGAAGGATACTGAAGAATGCCAACTCAACTTTTGAAGAACT GGAACACGTTAGGAAGTTGGTCAAAGCCTGGGAAGAAGTAGGGCCCCAGATCTGGTACTTCTTTGACAACAGCAC ACAGATGAACATGATCAGAGATACCCTGGGGAACCCAACAGTAAAAGACTTTTTGAATAGGCAGCTTGGTGAAGA AGGTATTACTGCTGAAGCCATCCTAAACTTCCTCTACAAGGGCCCTCGGGAAAGCCAGGCTGACGACATGGCCAA CTTCGACTGGAGGGACATATTTAACATCACTGATCGCACCCTCCGCCTTGTCAATCAATACCTGGAGTGCTTGGT CCTGGATAAGTTTGAAAGCTACAATGATGAAACTCAGCTCACCCAACGTGCCCTCTCTCTACTGGAGGAAAACAT GTTCTGGGCCGGAGTGGTATTCCCTGACATGTATCCCTGGACCAGCTCTCTACCACCCCACGTGAAGTATAAGAT CCGAATGGACATAGACGTGGTGGAGAAAACCAATAAGATTAAAGACAGGTATTGGGACTACAAAGACCATGACGG TGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGGATTCTGGTCCCAGAGCTGATCCCGTGGA AGATTTCCGGTACATCTGGGGCGGGTTTGCCTATCTGCAGGACATGGTTGAACAGGGGATCACAAGGAGCCAGGT GCAGGCGGAGGCTCCAGTTGGAATCTACCTCCAGCAGATGCCCTACCCCTGCTTCGTGGACGATTCTTTCATGAT CATCCTGAACCGCTGTTTCCCTATCTTCATGGTGCTGGCATGGATCTACTCTGTCTCCATGACTGTGAAGAGCAT CGTCTTGGAGAAGGAGTTGCGACTGAAGGAGACCTTGAAAAATCAGGGTGTCTCCAATGCAGTGATTTGGTGTAC CTGGTTCCTGGACAGCTTCTCCATCATGTCGATGAGCATCTTCCTCCTGACGATATTCATCATGCATGGAAGAAT CCTACATTACAGCGACCCATTCATCCTCTTCCTGTTCTTGTTGGCTTTCTCCACTGCCACCATCATGCTGTGCTT TCTGCTCAGCACCTTCTTCTCCAAGGCCAGTCTGGCAGCAGCCTGTAGTGGTGTCATCTATTTCACCCTCTACCT GCCACACATCCTGTGCTTCGCCTGGCAGGACCGCATGACCGCTGAGCTGAAGAAGGCTGTGAGCTTACTGTCTCC GGTGGCATTTGGATTTGGCACTGAGTACCTGGTTCGCTTTGAAGAGCAAGGCCTGGGGCTGCAGTGGAGCAACAT CGGGAACAGTCCCACGGAAGGGGACGAATTCAGCTTCCTGCTGTCCATGCAGATGATGCTCCTTGATGCTGCTGT CTATGGCTTACTCGCTTGGTACCTTGATCAGGTGTTTCCAGGAGACTATGGAACCCCACTTCCTTGGTACTTTCT TCTACAAGAGTCGTATTGGCTTGGCGGTGAAGGGTGTTCAACCAGAGAAGAAAGAGCCCTGGAAAAGACCGAGCC CCTAACAGAGGAAACGGAGGATCCAGAGCACCCAGAAGGAATACACGACTCCTTCTTTGAACGTGAGCATCCAGG GTGGGTTCCTGGGGTATGCGTGAAGAATCTGGTAAAGATTTTTGAGCCCTGTGGCCGGCCAGCTGTGGACCGTCT GAACATCACCTTCTACGAGAACCAGATCACCGCATTCCTGGGCCACAATGGAGCTGGGAAAACCACCACCTTGTC CATCCTGACGGGTCTGTTGCCACCAACCTCTGGGACTGTGCTCGTTGGGGGAAGGGACATTGAAACCAGCCTGGA TGCAGTCCGGCAGAGCCTTGGCATGTGTCCACAGCACAACATCCTGTTCCACCAGTAAGTATCAAGGTTACAAGA CAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGTAAGCGAGACATC TATACTAACTAATGGAAGCGGACagttataaCCGctgagttataacattggtatACCGGTTGGATTTCCCATGAT TCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGAT AT T AGT ACAAAAT AC GT GAG GT AGAAAGT AAT AAT T T CT T GGGT AGT T T GCAGT T T T AAAAT TAT GT T T T AAAAT GGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA CACCGataccaatgttataactcagGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGCGGCCGCACCAATTGATTGAGGAACCCCTAGTGATGGAG TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTT GCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0418] Seq ID NO: 6 MITI 8HA hABCA4 5’ gRNA scramble

[0419] ITR

[0420] gRNA+PAM

[0421] microhomology arms

[0422] CMV enhancer + promoter + chimeric intron

[0423] ABCA4 5' CDS

[0424] Splicing Donor

[0425] hU6 promoter

[0426] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCATACCAATGTTATA ACTCAGCGGAACCGCTGGGAAGCGGAGACTCGCGCGAGTCGAGGAGGGAAGATCTTCAATATTGGCCATTAGCCA TATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATA TGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAAT CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTG GCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTT TCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAA GTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACT TTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATG GGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGC ACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTAC GGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGCGGTAGTTTATC ACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAG GCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGA GAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCC ACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCCTCGAGAATTCAC GCGTGGTACCTCTAGAGTCGACCCGGGCGGCCGCCATGGGCTTCGTGAGACAGATACAGCTTTTGCTCTGGAAGA ACTGGACCCTGCGGAAAAGGCAAAAGATTCGCTTTGTGGTGGAACTCGTGTGGCCTTTATCTTTATTTCTGGTCT TGATCTGGTTAAGGAATGCCAACCCGCTCTACAGCCATCATGAATGCCATTTCCCCAACAAGGCGATGCCCTCAG CAGGAATGCTGCCGTGGCTCCAGGGGATCTTCTGCAATGTGAACAATCCCTGTTTTCAAAGCCCCACCCCAGGAG AATCTCCTGGAATTGTGTCAAACTATAACAACTCCATCTTGGCAAGGGTATATCGAGATTTTCAAGAACTCCTCA TGAATGCACCAGAGAGCCAGCACCTTGGCCGTATTTGGACAGAGCTACACATCTTGTCCCAATTCATGGACACCC TCCGGACTCACCCGGAGAGAATTGCAGGAAGAGGAATACGAATAAGGGATATCTTGAAAGATGAAGAAACACTGA CACTATTTCTCATTAAAAACATCGGCCTGTCTGACTCAGTGGTCTACCTTCTGATCAACTCTCAAGTCCGTCCAG AGCAGTTCGCTCATGGAGTCCCGGACCTGGCGCTGAAGGACATCGCCTGCAGCGAGGCCCTCCTGGAGCGCTTCA TCATCTTCAGCCAGAGACGCGGGGCAAAGACGGTGCGCTATGCCCTGTGCTCCCTCTCCCAGGGCACCCTACAGT GGATAGAAGACACTCTGTATGCCAACGTGGACTTCTTCAAGCTCTTCCGTGTGCTTCCCACACTCCTAGACAGCC GTTCTCAAGGTATCAATCTGAGATCTTGGGGAGGAATATTATCTGATATGTCACCAAGAATTCAAGAGTTTATCC ATCGGCCGAGTATGCAGGACTTGCTGTGGGTGACCAGGCCCCTCATGCAGAATGGTGGTCCAGAGACCTTTACAA AGCTGATGGGCATCCTGTCTGACCTCCTGTGTGGCTACCCCGAGGGAGGTGGCTCTCGGGTGCTCTCCTTCAACT GGTATGAAGACAATAACTATAAGGCCTTTCTGGGGATTGACTCCACAAGGAAGGATCCTATCTATTCTTATGACA GAAGAACAACATCCTTTTGTAATGCATTGATCCAGAGCCTGGAGTCAAATCCTTTAACCAAAATCGCTTGGAGGG CGGCAAAGCCTTTGCTGATGGGAAAAATCCTGTACACTCCTGATTCACCTGCAGCACGAAGGATACTGAAGAATG CCAACTCAACTTTTGAAGAACTGGAACACGTTAGGAAGTTGGTCAAAGCCTGGGAAGAAGTAGGGCCCCAGATCT GGTACTTCTTTGACAACAGCACACAGATGAACATGATCAGAGATACCCTGGGGAACCCAACAGTAAAAGACTTTT TGAATAGGCAGCTTGGTGAAGAAGGTATTACTGCTGAAGCCATCCTAAACTTCCTCTACAAGGGCCCTCGGGAAA GCCAGGCTGACGACATGGCCAACTTCGACTGGAGGGACATATTTAACATCACTGATCGCACCCTCCGCCTTGTCA ATCAATACCTGGAGTGCTTGGTCCTGGATAAGTTTGAAAGCTACAATGATGAAACTCAGCTCACCCAACGTGCCC TCTCTCTACTGGAGGAAAACATGTTCTGGGCCGGAGTGGTATTCCCTGACATGTATCCCTGGACCAGCTCTCTAC CACCCCACGTGAAGTATAAGATCCGAATGGACATAGACGTGGTGGAGAAAACCAATAAGATTAAAGACAGGTATTGGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGGATTCTG GTCCCAGAGCTGATCCCGTGGAAGATTTCCGGTACATCTGGGGCGGGTTTGCCTATCTGCAGGACATGGTTGAAC AGGGGATCACAAGGAGCCAGGTGCAGGCGGAGGCTCCAGTTGGAATCTACCTCCAGCAGATGCCCTACCCCTGCT TCGTGGACGATTCTTTCATGATCATCCTGAACCGCTGTTTCCCTATCTTCATGGTGCTGGCATGGATCTACTCTG TCTCCATGACTGTGAAGAGCATCGTCTTGGAGAAGGAGTTGCGACTGAAGGAGACCTTGAAAAATCAGGGTGTCT CCAATGCAGTGATTTGGTGTACCTGGTTCCTGGACAGCTTCTCCATCATGTCGATGAGCATCTTCCTCCTGACGA TATTCATCATGCATGGAAGAATCCTACATTACAGCGACCCATTCATCCTCTTCCTGTTCTTGTTGGCTTTCTCCA CTGCCACCATCATGCTGTGCTTTCTGCTCAGCACCTTCTTCTCCAAGGCCAGTCTGGCAGCAGCCTGTAGTGGTG TCATCTATTTCACCCTCTACCTGCCACACATCCTGTGCTTCGCCTGGCAGGACCGCATGACCGCTGAGCTGAAGA AGGCTGTGAGCTTACTGTCTCCGGTGGCATTTGGATTTGGCACTGAGTACCTGGTTCGCTTTGAAGAGCAAGGCC TGGGGCTGCAGTGGAGCAACATCGGGAACAGTCCCACGGAAGGGGACGAATTCAGCTTCCTGCTGTCCATGCAGA TGATGCTCCTTGATGCTGCTGTCTATGGCTTACTCGCTTGGTACCTTGATCAGGTGTTTCCAGGAGACTATGGAA CCCCACTTCCTTGGTACTTTCTTCTACAAGAGTCGTATTGGCTTGGCGGTGAAGGGTGTTCAACCAGAGAAGAAA GAGCCCTGGAAAAGACCGAGCCCCTAACAGAGGAAACGGAGGATCCAGAGCACCCAGAAGGAATACACGACTCCT TCTTTGAACGTGAGCATCCAGGGTGGGTTCCTGGGGTATGCGTGAAGAATCTGGTAAAGATTTTTGAGCCCTGTG GCCGGCCAGCTGTGGACCGTCTGAACATCACCTTCTACGAGAACCAGATCACCGCATTCCTGGGCCACAATGGAG CTGGGAAAACCACCACCTTGTCCATCCTGACGGGTCTGTTGCCACCAACCTCTGGGACTGTGCTCGTTGGGGGAA GGGACATTGAAACCAGCCTGGATGCAGTCCGGCAGAGCCTTGGCATGTGTCCACAGCACAACATCCTGTTCCACC GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTT GCGTTTCTGTAAGCGAGACATCTATACTAACTAATGGAAGCGGACagttataaCCGctgagttataacattggta tACCGGTTGGATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTA ATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAG TTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTAT ATATCTTGTGGAAAGGACGAAACACCGGACTCGCGCGAGTCGAGGAGGTTTTAGAGCTAGAAATAGCAAGTTAAA ATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGCGGCCGCACCAATTGAT TGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAA GGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0427] Seq ID NO: 7 HITI hABCA43’ gRNA

[0428] ITR

[0429] gRNA+PAM

[0430] Splicing acceptor

[0431] ABCA4 3 ' CDS

[0432] SV40 PolyA

[0433] hU6 promoter

[0434] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCCCGCGGATACCAATGTTATAACTCAGCGGGACTCGCGCGAGTCGAGGAGGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTT TCTCTCCACAGCCTCACGGTGGCTGAGCACATGCTGTTCTATGCCCAGCTGAAAGGAAAGTCCCAGGAGGAGGCC CAGCTGGAGATGGAAGCCATGTTGGAGGACACAGGCCTCCACCACAAGCGGAATGAAGAGGCTCAGGACCTATCA GGTGGCATGCAGAGAAAGCTGTCGGTTGCCATTGCCTTTGTGGGAGATGCCAAGGTGGTGATTCTGGACGAACCC ACCTCTGGGGTGGACCCTTACTCGAGACGCTCAATCTGGGATCTGCTCCTGAAGTATCGCTCAGGCAGAACCATC ATCATGTCCACTCACCACATGGACGAGGCCGACCTCCTTGGGGACCGCATTGCCATCATTGCCCAGGGAAGGCTC TACTGCTCAGGCACCCCACTCTTCCTGAAGAACTGCTTTGGCACAGGCTTGTACTTAACCTTGGTGCGCAAGATG AAAAACATCCAGAGCCAAAGGAAAGGCAGTGAGGGGACCTGCAGCTGCTCGTCTAAGGGTTTCTCCACCACGTGT CCAGCCCACGTCGATGACCTAACTCCAGAACAAGTCCTGGATGGGGATGTAAATGAGCTGATGGATGTAGTTCTC CACCATGTTCCAGAGGCAAAGCTGGTGGAGTGCATTGGTCAAGAACTTATCTTCCTTCTTCCAAATAAGAACTTC AAGCACAGAGCATATGCCAGCCTTTTCAGAGAGCTGGAGGAGACGCTGGCTGACCTTGGTCTCAGCAGTTTTGGA ATTTCTGACACTCCCCTGGAAGAGATTTTTCTGAAGGTCACGGAGGATTCTGATTCAGGACCTCTGTTTGCGGGT GGCGCTCAGCAGAAAAGAGAAAACGTCAACCCCCGACACCCCTGCTTGGGTCCCAGAGAGAAGGCTGGACAGACA CCCCAGGACTCCAATGTCTGCTCCCCAGGGGCGCCGGCTGCTCACCCAGAGGGCCAGCCTCCCCCAGAGCCAGAG TGCCCAGGCCCGCAGCTCAACACGGGGACACAGCTGGTCCTCCAGCATGTGCAGGCGCTGCTGGTCAAGAGATTC CAACACACCATCCGCAGCCACAAGGACTTCCTGGCGCAGATCGTGCTCCCGGCTACCTTTGTGTTTTTGGCTCTG ATGCTTTCTATTGTTATCCCTCCTTTTGGCGAATACCCCGCTTTGACCCTTCACCCCTGGATATATGGGCAGCAG TACACCTTCTTCAGCATGGATGAACCAGGCAGTGAGCAGTTCACGGTACTTGCAGACGTCCTCCTGAATAAGCCA GGCTTTGGCAACCGCTGCCTGAAGGAAGGGTGGCTTCCGGAGTACCCCTGTGGCAACTCAACACCCTGGAAGACT CCTTCTGTGTCCCCAAACATCACCCAGCTGTTCCAGAAGCAGAAATGGACACAGGTCAACCCTTCACCATCCTGC AGGTGCAGCACCAGGGAGAAGCTCACCATGCTGCCAGAGTGCCCCGAGGGTGCCGGGGGCCTCCCGCCCCCCCAG AGAACACAGCGCAGCACGGAAATTCTACAAGACCTGACGGACAGGAACATCTCCGACTTCTTGGTAAAAACGTAT CCTGCTCTTATAAGAAGCAGCTTAAAGAGCAAATTCTGGGTCAATGAACAGAGGTATGGAGGAATTTCCATTGGA GGAAAGCTCCCAGTCGTCCCCATCACGGGGGAAGCACTTGTTGGGTTTTTAAGCGACCTTGGCCGGATCATGAAT GTGAGCGGGGGCCCTATCACTAGAGAGGCCTCTAAAGAAATACCTGATTTCCTTAAACATCTAGAAACTGAAGAC AACATTAAGGTGTGGTTTAATAACAAAGGCTGGCATGCCCTGGTCAGCTTTCTCAATGTGGCCCACAACGCCATC TTACGGGCCAGCCTGCCTAAGGACAGGAGCCCCGAGGAGTATGGAATCACCGTCATTAGCCAACCCCTGAACCTG ACCAAGGAGCAGCTCTCAGAGATTACAGTGCTGACCACTTCAGTGGATGCTGTGGTTGCCATCTGCGTGATTTTC TCCATGTCCTTCGTCCCAGCCAGCTTTGTCCTTTATTTGATCCAGGAGCGGGTGAACAAATCCAAGCACCTCCAG TTTATCAGTGGAGTGAGCCCCACCACCTACTGGGTAACCAACTTCCTCTGGGACATCATGAATTATTCCGTGAGT GCTGGGCTGGTGGTGGGCATCTTCATCGGGTTTCAGAAGAAAGCCTACACTTCTCCAGAAAACCTTCCTGCCCTT GTGGCACTGCTCCTGCTGTATGGATGGGCGGTCATTCCCATGATGTACCCAGCATCCTTCCTGTTTGATGTCCCC AGCACAGCCTATGTGGCTTTATCTTGTGCTAATCTGTTCATCGGCATCAACAGCAGTGCTATTACCTTCATCTTG GAATTATTTGAGAATAACCGGACGCTGCTCAGGTTCAACGCCGTGCTGAGGAAGCTGCTCATTGTCTTCCCCCAC TTCTGCCTGGGCCGGGGCCTCATTGACCTTGCACTGAGCCAGGCTGTGACAGATGTCTATGCCCGGTTTGGTGAG GAGCACTCTGCAAATCCGTTCCACTGGGACCTGATTGGGAAGAACCTGTTTGCCATGGTGGTGGAAGGGGTGGTG TACTTCCTCCTGACCCTGCTGGTCCAGCGCCACTTCTTCCTCTCCCAATGGATTGCCGAGCCCACTAAGGAGCCC ATTGTTGATGAAGATGATGATGTGGCTGAAGAAAGACAAAGAATTATTACTGGTGGAAATAAAACTGACATCTTA AGGCTACATGAACTAACCAAGATTTATCCAGGCACCTCCAGCCCAGCAGTGGACAGGCTGTGTGTCGGAGTTCGC CCTGGAGAGTGCTTTGGCCTCCTGGGAGTGAATGGTGCCGGCAAAACAACCACATTCAAGATGCTCACTGGGGAC ACCACAGTGACCTCAGGGGATGCCACCGTAGCAGGCAAGAGTATTTTAACCAATATTTCTGAAGTCCATCAAAAT ATGGGCTACTGTCCTCAGTTTGATGCAATCGATGAGCTGCTCACAGGACGAGAACATCTTTACCTTTATGCCCGG CTTCGAGGTGTACCAGCAGAAGAAATCGAAAAGGTTGCAAACTGGAGTATTAAGAGCCTGGGCCTGACTGTCTAC GCCGACTGCCTGGCTGGCACGTACAGTGGGGGCAACAAGCGGAAACTCTCCACAGCCATCGCACTCATTGGCTGC CCACCGCTGGTGCTGCTGGATGAGCCCACCACAGGGATGGACCCCCAGGCACGCCGCATGCTGTGGAACGTCATC GTGAGCATCATCAGAGAAGGGAGGGCTGTGGTCCTCACATCCCACAGCATGGAAGAATGTGAGGCACTGTGTACC CGGCTGGCCATCATGGTAAAGGGCGCCTTTCGATGTATGGGCACCATTCAGCATCTCAAGTCCAAATTTGGAGAT GGCTATATCGTCACAATGAAGATCAAATCCCCGAAGGACGACCTGCTTCCTGACCTGAACCCTGTGGAGCAGTTC TTCCAGGGGAACTTCCCAGGCAGTGTGCAGAGGGAGAGGCACTACAACATGCTCCAGTTCCAGGTCTCCTCCTCC TCCCTGGCGAGGATCTTCCAGCTCCTCCTCTCCCACAAGGACAGCCTGCTCATCGAGGAGTACTCAGTCACACAG ACCACACTGGACCAGGTGTTTGTAAATTTTGCTAAACAGCAGACTGAAAGTCATGACCTCCCTCTGCACCCTCGA GCTGCTGGAGCCAGTCGACAAGCCCAGGACGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGAC TACAAGGATGACGATGACAAGTGAGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAAC CACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTAT AAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGT TTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCAAGCGACACAGGACATCTATACTAA CTAATGAGGAAGCGGACATACCAATGTTATAACTCAGCGGTACCGGTTTTCCCATGATTCCTTCATATTTGCATA TACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGT GACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTA CCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGATACCAATGTTA TAACTCAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG AGTCGGTGCTTTTTTTGCGCGGCCGCACCAATTGATTGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAG

[0435] Seq ID NO: 8 HITI hABCA43’ gRNA scramble

[0436] ITR

[0437] gRNA+PAM

[0438] Splicing acceptor

[0439] ABCA4 3 ' CDS

[0440] SV40 PolyA

[0441] hU6 promoter

[0442] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAG AGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCCCGCGGATACCAATGTTATAACTCAGCGGGACTCGC GCGAGTCGAGGAGGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGCCTCACGGTGG CTGAGCACATGCTGTTCTATGCCCAGCTGAAAGGAAAGTCCCAGGAGGAGGCCCAGCTGGAGATGGAAGCCATGT TGGAGGACACAGGCCTCCACCACAAGCGGAATGAAGAGGCTCAGGACCTATCAGGTGGCATGCAGAGAAAGCTGT CGGTTGCCATTGCCTTTGTGGGAGATGCCAAGGTGGTGATTCTGGACGAACCCACCTCTGGGGTGGACCCTTACT CGAGACGCTCAATCTGGGATCTGCTCCTGAAGTATCGCTCAGGCAGAACCATCATCATGTCCACTCACCACATGG ACGAGGCCGACCTCCTTGGGGACCGCATTGCCATCATTGCCCAGGGAAGGCTCTACTGCTCAGGCACCCCACTCT TCCTGAAGAACTGCTTTGGCACAGGCTTGTACTTAACCTTGGTGCGCAAGATGAAAAACATCCAGAGCCAAAGGA AAGGCAGTGAGGGGACCTGCAGCTGCTCGTCTAAGGGTTTCTCCACCACGTGTCCAGCCCACGTCGATGACCTAA CTCCAGAACAAGTCCTGGATGGGGATGTAAATGAGCTGATGGATGTAGTTCTCCACCATGTTCCAGAGGCAAAGC TGGTGGAGTGCATTGGTCAAGAACTTATCTTCCTTCTTCCAAATAAGAACTTCAAGCACAGAGCATATGCCAGCC TTTTCAGAGAGCTGGAGGAGACGCTGGCTGACCTTGGTCTCAGCAGTTTTGGAATTTCTGACACTCCCCTGGAAG AGATTTTTCTGAAGGTCACGGAGGATTCTGATTCAGGACCTCTGTTTGCGGGTGGCGCTCAGCAGAAAAGAGAAA ACGTCAACCCCCGACACCCCTGCTTGGGTCCCAGAGAGAAGGCTGGACAGACACCCCAGGACTCCAATGTCTGCT CCCCAGGGGCGCCGGCTGCTCACCCAGAGGGCCAGCCTCCCCCAGAGCCAGAGTGCCCAGGCCCGCAGCTCAACA CGGGGACACAGCTGGTCCTCCAGCATGTGCAGGCGCTGCTGGTCAAGAGATTCCAACACACCATCCGCAGCCACA AGGACTTCCTGGCGCAGATCGTGCTCCCGGCTACCTTTGTGTTTTTGGCTCTGATGCTTTCTATTGTTATCCCTC CTTTTGGCGAATACCCCGCTTTGACCCTTCACCCCTGGATATATGGGCAGCAGTACACCTTCTTCAGCATGGATG AACCAGGCAGTGAGCAGTTCACGGTACTTGCAGACGTCCTCCTGAATAAGCCAGGCTTTGGCAACCGCTGCCTGA AGGAAGGGTGGCTTCCGGAGTACCCCTGTGGCAACTCAACACCCTGGAAGACTCCTTCTGTGTCCCCAAACATCA CCCAGCTGTTCCAGAAGCAGAAATGGACACAGGTCAACCCTTCACCATCCTGCAGGTGCAGCACCAGGGAGAAGC TCACCATGCTGCCAGAGTGCCCCGAGGGTGCCGGGGGCCTCCCGCCCCCCCAGAGAACACAGCGCAGCACGGAAA TTCTACAAGACCTGACGGACAGGAACATCTCCGACTTCTTGGTAAAAACGTATCCTGCTCTTATAAGAAGCAGCT TAAAGAGCAAATTCTGGGTCAATGAACAGAGGTATGGAGGAATTTCCATTGGAGGAAAGCTCCCAGTCGTCCCCA TCACGGGGGAAGCACTTGTTGGGTTTTTAAGCGACCTTGGCCGGATCATGAATGTGAGCGGGGGCCCTATCACTA GAGAGGCCTCTAAAGAAATACCTGATTTCCTTAAACATCTAGAAACTGAAGACAACATTAAGGTGTGGTTTAATA ACAAAGGCTGGCATGCCCTGGTCAGCTTTCTCAATGTGGCCCACAACGCCATCTTACGGGCCAGCCTGCCTAAGG ACAGGAGCCCCGAGGAGTATGGAATCACCGTCATTAGCCAACCCCTGAACCTGACCAAGGAGCAGCTCTCAGAGA TTACAGTGCTGACCACTTCAGTGGATGCTGTGGTTGCCATCTGCGTGATTTTCTCCATGTCCTTCGTCCCAGCCA GCTTTGTCCTTTATTTGATCCAGGAGCGGGTGAACAAATCCAAGCACCTCCAGTTTATCAGTGGAGTGAGCCCCA CCACCTACTGGGTAACCAACTTCCTCTGGGACATCATGAATTATTCCGTGAGTGCTGGGCTGGTGGTGGGCATCT TCATCGGGTTTCAGAAGAAAGCCTACACTTCTCCAGAAAACCTTCCTGCCCTTGTGGCACTGCTCCTGCTGTATG GATGGGCGGTCATTCCCATGATGTACCCAGCATCCTTCCTGTTTGATGTCCCCAGCACAGCCTATGTGGCTTTAT CTTGTGCTAATCTGTTCATCGGCATCAACAGCAGTGCTATTACCTTCATCTTGGAATTATTTGAGAATAACCGGA CGCTGCTCAGGTTCAACGCCGTGCTGAGGAAGCTGCTCATTGTCTTCCCCCACTTCTGCCTGGGCCGGGGCCTCA TTGACCTTGCACTGAGCCAGGCTGTGACAGATGTCTATGCCCGGTTTGGTGAGGAGCACTCTGCAAATCCGTTCC ACTGGGACCTGATTGGGAAGAACCTGTTTGCCATGGTGGTGGAAGGGGTGGTGTACTTCCTCCTGACCCTGCTGG TCCAGCGCCACTTCTTCCTCTCCCAATGGATTGCCGAGCCCACTAAGGAGCCCATTGTTGATGAAGATGATGATG TGGCTGAAGAAAGACAAAGAATTATTACTGGTGGAAATAAAACTGACATCTTAAGGCTACATGAACTAACCAAGATTTATCCAGGCACCTCCAGCCCAGCAGTGGACAGGCTGTGTGTCGGAGTTCGCCCTGGAGAGTGCTTTGGCCTCC TGGGAGTGAATGGTGCCGGCAAAACAACCACATTCAAGATGCTCACTGGGGACACCACAGTGACCTCAGGGGATG CCACCGTAGCAGGCAAGAGTATTTTAACCAATATTTCTGAAGTCCATCAAAATATGGGCTACTGTCCTCAGTTTG ATGCAATCGATGAGCTGCTCACAGGACGAGAACATCTTTACCTTTATGCCCGGCTTCGAGGTGTACCAGCAGAAG AAATCGAAAAGGTTGCAAACTGGAGTATTAAGAGCCTGGGCCTGACTGTCTACGCCGACTGCCTGGCTGGCACGT ACAGTGGGGGCAACAAGCGGAAACTCTCCACAGCCATCGCACTCATTGGCTGCCCACCGCTGGTGCTGCTGGATG AGCCCACCACAGGGATGGACCCCCAGGCACGCCGCATGCTGTGGAACGTCATCGTGAGCATCATCAGAGAAGGGA GGGCTGTGGTCCTCACATCCCACAGCATGGAAGAATGTGAGGCACTGTGTACCCGGCTGGCCATCATGGTAAAGG GCGCCTTTCGATGTATGGGCACCATTCAGCATCTCAAGTCCAAATTTGGAGATGGCTATATCGTCACAATGAAGA TCAAATCCCCGAAGGACGACCTGCTTCCTGACCTGAACCCTGTGGAGCAGTTCTTCCAGGGGAACTTCCCAGGCA GTGTGCAGAGGGAGAGGCACTACAACATGCTCCAGTTCCAGGTCTCCTCCTCCTCCCTGGCGAGGATCTTCCAGC TCCTCCTCTCCCACAAGGACAGCCTGCTCATCGAGGAGTACTCAGTCACACAGACCACACTGGACCAGGTGTTTG TAAATTTTGCTAAACAGCAGACTGAAAGTCATGACCTCCCTCTGCACCCTCGAGCTGCTGGAGCCAGTCGACAAG CCCAGGACGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGT GAGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAA AAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAAC AACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTC TACAAATGTGGTAAAATCGATAAGGATCAAGCGACACAGGACATCTATACTAACTAATGAGGAAGCGGACATACC AATGTTATAACTCAGCGGTACCGGTTTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGA GAT AAT T GGAAT T AAT T T GACT GT AAACACAAAGAT AT T AGT ACAAAAT AC GT GAG GT AGAAAGT AAT AAT T T CT TGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGA TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGACTCGCGCGAGTCGAGGAGGTTTTAGAGCTAGA AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGCGG CCGCACCAATTGATTGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAG GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0443] SeqIDNO: 9-14 skip

[0444] SEQ ID NO: 15 HITI 5’ mABCA4 gRNA

[0445] ITR

[0446] gRNA+PAM

[0447] enhancer + GRK 1 promoter

[0448] ABCA4 5' cDNA

[0449] ABCA4 intron 21 (acting as splicing’ donor signal)

[0450] hU6 promoter

[0451] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGC CTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCCCCCAG TGGTTCTCAACGTCCTATCTACGTAGCCATGCTCTGGGAAGATCTTCAATATTGGCCATTAGCCATATTAT TCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATG TACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTGGGCCCCAGAA GCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGGAGGAAGGGGCCGGGCAGAATG ATCTAATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAAGCGTCCT CCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCCGGGCTCCCAGGGGCTTCCCAGTGGTCCC CAGGAACCCTCGACAGGGCCAGGGCGTCTCTCTCGTCCAGCAAGGGCAGGGACGGGCCACAGGCAAGGGCT C G AGAAT T CGGCACGAGGTCCTAGCGGCCTTTGTGTCCGGTGCTTGCCTGAGCCCCAGCTCGGGGTGATTC TCCCGGTGCTGGGGTGGTCCGGCATGGGCTTCCTCAGACAGATACAGCTTTTGCTTTGGAAGAACTGGACT CTGAGGAAAAGGCAGAAGATTCGCTTTGTAGTGGAACTCGTGTGGCCTTTGTCTTTGTTTTTGGTGTTAAT CTGGCTGAGGAATGCCAACCCACTCTATAGTCAGCATGAATGCCATTTTCCCAACAAGGCGATGCCTTCAG CAGGACTGTTACCGTGGCTCCAGGGGATTTTCTGCAATATGAACAACCCTTGTTTTCAAAACCCCACCCCT GGAGAGTCTCCTGGAACTGTCTCAAACTATAACAACTCCATCTTGGCAAGAGTATATCGAGATTTTCAAGA ACTCTTCATGGACACCCCGGAGGTCCAGCACCTTGGCCAGGTTTGGGCCGAGCTCCGCACCTTGTCGCAGT TCATGGACACCCTGAGGACTCACCCTGAGAGATTTGCAGGAAGAGGATTACAAATCCGAGACATCCTAAAA GATGAAGAGGCGCTGACCCTATTTCTCATGAGAAACATTGGCCTGTCTGACTCGGTTGCCCATCTTCTGGT CAACTCCCAAGTTCGTGTGGAGCAGTTTGCTTATGGAGTCCCAGACTTGGAACTGACAGACATTGCCTGCA GCGAGGCCCTCCTGCAGCGCTTCATCATCTTCAGCCAGCGTCGGGGGGCACAGACGGTACGCGATGCCCTG TGTCCCCTCTCCCAGGTCACCCTACAGTGGATAGAAGACACTCTGTATGCCGATGTGGACTTCTTCAAACT CTTCCATGTGCTCCCCACACTCCTGGACAGCAGTTCTCAAGGAATCAACTTGAGATTTTGGGGAGGAATAT TATCTGATCTGTCACCAAGAATGCAAAAGTTTATCCATCGGCCAAGTGTTCAAGACTTGCTATGGGTGAGC AGACCTCTCCTGCAGAACGGTGGTCCTGAGACCTTCACACAGCTGATGAGCATTCTGTCTGACCTCCTGTG TGGGTACCCAGAGGGAGGAGGCTCCCGAGTGTTCTCCTTTAACTGGTATGAAGACAATAACTATAAAGCCT TCCTGGGGATTGATTCCACAAGGAAAGACCCCGCCTATTCTTACGACAAAAGAACAACATCCTTTTGTAAT TCATTGATCCAGAGCCTGGAGTCAAACCCTTTAACCAAAATAGCCTGGAGGGCGGCAAAGCCATTGCTGAT GGGAAAAATCCTCTTTACTCCAGATTCCCCTGCTGCTCGAAGGATAATGAAGAATGCCAACTCAACTTTTG AAGAACTGGATCGAGTTAGGAAGTTGGTAAAAGCCTGGGAGGAAGTGGGGCCCCAGATCTGGTACTTCTTT GAGAAGAGCACACAGATGACCGTGATCCGAGACACCCTGCAGCACCCAACCGTCAAAGACTTCATAAATAG GCAGCTCGGAGAAGAAGGCATTACCACCGAAGCCGTATTAAACTTCTTCTCTAACGGTCCCCAAGAGAAGC AGGCTGATGATATGACCAGCTTTGACTGGAGGGACATATTCAACATCACTGACCGATTCCTACGCTTGGCT AATCAATACCTGGAGTGTCTGGTCCTGGATAAGTTTGAAAGTTATGATGATGAAGTGCAGCTCACCCAACG AGCCCTGTCTCTCCTGGAGGAGAACAGGTTCTGGGCCGGAGTGGTGTTCCCTGGCATGTATCCCTGGGCCA GCTCCTTACCTCCTCATGTGAAGTACAAGATTCGGATGGACATAGATGTGGTGGAGAAGACCAATAAGATC AAAGACAGGTACTGGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGA CGATGACAAGGATTCTGGTCCCAGGGCGGATCCTGTCGAAGATTTCCGGTACATCTGGGGAGGTTTTGCCT ATCTACAGGACATGGTGGAGCAAGGAATCGTGAAGAGTCAGATGCAGGCAGAGCCTCCAATTGGAGTCTAT CTCCAACAGATGCCTTATCCCTGCTTTGTGGATGACTCCTTCATGATCATCCTGAATCGCTGTTTCCCTAT CTTCATGGTGCTGGCGTGGATCTACTCTGTCTCCATGACCGTTAAGGGCATTGTTTTGGAGAAGGAGCTGA GGCTGAAGGAGACCTTGAAAAACCAAGGCGTCTCTAATGCTGTCATCTGGTGTACCTGGTTCCTGGATAGC TTCTCCATCATGGCGCTGAGTATCTTCCTCCTGACGCTGTTCATCATGCATGGAAGGATCCTCCATTACAG CGATCCCTTCATTCTCTTCCTGTTCTTGTTGGCCTTTGCCACTGCGACCATCATGCAGAGCTTCCTGCTCA GTACTTTGTTTTCCAAGGCCAGCCTTGCAGCAGCCTGCAGTGGGGTCATCTACTTCACCCTCTACCTACCA CACGTTCTGTGCTTTGCCTGGCAGGACCGGATGACGGCCGACCTGAAGACGACTGTGAGCCTACTTTCTTC CGTGGCATTTGGGTTTGGCACCGAGTACCTGGTCCGCTTTGAGGAGCAAGGCCTGGGGCTGCAATGGAGCA ACATTGGGAAGAGTCCCCTGGAAGGGGATGAGTTCAGTTTCCTGCTGTCTATGAAGATGATGCTTCTCGAT GCTGCTCTCTACGGCTTGCTTGCTTGGTATCTTGACCAGGTTTTCCCAGGAGACTATGGGACCCCACTTCC CTGGTACTTCCTTCTGCAGGAGTCCTACTGGCTTGGTGGTGAAGGTTGTTCGACCAGAGAAGAAAGGGCTC TGGAAAAGACTGAACCCTTAACAGAGGAGATGGAGGATCCAGAGCACCCAGAAGGAATGAATGACTCCTTT TTTGAACGCGAGCTTCCAGGGCTGGTGCCTGGTGTGTGTGTGAAGAACCTGGTGAAGGTTTTTGAGCCCAG TGGCCGGCCAGCTGTGGACCGTCTGAACATCACTTTCTATGAGAACCAAATCACGGCGTTCCTGGGTCACA ACGGAGCGGGAAAGACCACCACCTTGTCCATCCTGACAGGACTGTTGCCACCGACGTCAGGAACTGTGCTC ATTGGGGGAAAAGACATTGAAACCAACCTGGATGTAGTACGGCAGAGCCTGGGCATGTGTCCACAACACAA CATCCTGTTTCATCACCTCACGGTGGCTGAGCACATCTTGTTCTATGCCCAGCTGAAAGGGAGATCCTGGG AGGAGGCCCAGCTTGAGATGGAAGCCATGCTAGAAGACACGGGCCTCCACCATAAGAGGAATGAAGAAGCT CAGGACCTTTCAGGTGCTTGGTATTGGGAGGAGACGGGGTCACTTACGGAGCATCCGTTGCCTCCCCAGTG GTTCTCAACGTCCTAATCGATCTTTCGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTAGGACGTTGAGAACCACTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCAATTGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0452] Seq ID NO: 16 HITI 5’ mABCA4 gRNA scramble

[0453] ITR

[0454] gRNA+PAM

[0455] enhancer + GRK 1 promoter

[0456] ABCA4 5' cDNA

[0457] ABCA4 intron 21 (acting as splicing’ donor signal)

[0458] hU6 promoter

[0459] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGC CTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCCCCCAG TGGTTCTCAACGTCCTATCTACGTAGCCATGCTCTGGGAAGATCTTCAATATTGGCCATTAGCCATATTAT TCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATG TACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTGGGCCCCAGAA GCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGGAGGAAGGGGCCGGGCAGAATG ATCTAATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAAGCGTCCT CCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCCGGGCTCCCAGGGGCTTCCCAGTGGTCCC CAGGAACCCTCGACAGGGCCAGGGCGTCTCTCTCGTCCAGCAAGGGCAGGGACGGGCCACAGGCAAGGGCT C G AGAAT T CGGCACGAGGTCCTAGCGGCCTTTGTGTCCGGTGCTTGCCTGAGCCCCAGCTCGGGGTGATTC TCCCGGTGCTGGGGTGGTCCGGCATGGGCTTCCTCAGACAGATACAGCTTTTGCTTTGGAAGAACTGGACT CTGAGGAAAAGGCAGAAGATTCGCTTTGTAGTGGAACTCGTGTGGCCTTTGTCTTTGTTTTTGGTGTTAAT CTGGCTGAGGAATGCCAACCCACTCTATAGTCAGCATGAATGCCATTTTCCCAACAAGGCGATGCCTTCAG CAGGACTGTTACCGTGGCTCCAGGGGATTTTCTGCAATATGAACAACCCTTGTTTTCAAAACCCCACCCCT GGAGAGTCTCCTGGAACTGTCTCAAACTATAACAACTCCATCTTGGCAAGAGTATATCGAGATTTTCAAGA ACTCTTCATGGACACCCCGGAGGTCCAGCACCTTGGCCAGGTTTGGGCCGAGCTCCGCACCTTGTCGCAGT TCATGGACACCCTGAGGACTCACCCTGAGAGATTTGCAGGAAGAGGATTACAAATCCGAGACATCCTAAAA GATGAAGAGGCGCTGACCCTATTTCTCATGAGAAACATTGGCCTGTCTGACTCGGTTGCCCATCTTCTGGT CAACTCCCAAGTTCGTGTGGAGCAGTTTGCTTATGGAGTCCCAGACTTGGAACTGACAGACATTGCCTGCA GCGAGGCCCTCCTGCAGCGCTTCATCATCTTCAGCCAGCGTCGGGGGGCACAGACGGTACGCGATGCCCTG TGTCCCCTCTCCCAGGTCACCCTACAGTGGATAGAAGACACTCTGTATGCCGATGTGGACTTCTTCAAACT CTTCCATGTGCTCCCCACACTCCTGGACAGCAGTTCTCAAGGAATCAACTTGAGATTTTGGGGAGGAATAT TATCTGATCTGTCACCAAGAATGCAAAAGTTTATCCATCGGCCAAGTGTTCAAGACTTGCTATGGGTGAGC AGACCTCTCCTGCAGAACGGTGGTCCTGAGACCTTCACACAGCTGATGAGCATTCTGTCTGACCTCCTGTG TGGGTACCCAGAGGGAGGAGGCTCCCGAGTGTTCTCCTTTAACTGGTATGAAGACAATAACTATAAAGCCT TCCTGGGGATTGATTCCACAAGGAAAGACCCCGCCTATTCTTACGACAAAAGAACAACATCCTTTTGTAAT TCATTGATCCAGAGCCTGGAGTCAAACCCTTTAACCAAAATAGCCTGGAGGGCGGCAAAGCCATTGCTGAT GGGAAAAATCCTCTTTACTCCAGATTCCCCTGCTGCTCGAAGGATAATGAAGAATGCCAACTCAACTTTTG AAGAACTGGATCGAGTTAGGAAGTTGGTAAAAGCCTGGGAGGAAGTGGGGCCCCAGATCTGGTACTTCTTT GAGAAGAGCACACAGATGACCGTGATCCGAGACACCCTGCAGCACCCAACCGTCAAAGACTTCATAAATAG GCAGCTCGGAGAAGAAGGCATTACCACCGAAGCCGTATTAAACTTCTTCTCTAACGGTCCCCAAGAGAAGC AGGCTGATGATATGACCAGCTTTGACTGGAGGGACATATTCAACATCACTGACCGATTCCTACGCTTGGCT AATCAATACCTGGAGTGTCTGGTCCTGGATAAGTTTGAAAGTTATGATGATGAAGTGCAGCTCACCCAACG AGCCCTGTCTCTCCTGGAGGAGAACAGGTTCTGGGCCGGAGTGGTGTTCCCTGGCATGTATCCCTGGGCCA GCTCCTTACCTCCTCATGTGAAGTACAAGATTCGGATGGACATAGATGTGGTGGAGAAGACCAATAAGATC AAAGACAGGTACTGGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGA CGATGACAAGGATTCTGGTCCCAGGGCGGATCCTGTCGAAGATTTCCGGTACATCTGGGGAGGTTTTGCCT ATCTACAGGACATGGTGGAGCAAGGAATCGTGAAGAGTCAGATGCAGGCAGAGCCTCCAATTGGAGTCTAT CTCCAACAGATGCCTTATCCCTGCTTTGTGGATGACTCCTTCATGATCATCCTGAATCGCTGTTTCCCTAT CTTCATGGTGCTGGCGTGGATCTACTCTGTCTCCATGACCGTTAAGGGCATTGTTTTGGAGAAGGAGCTGA GGCTGAAGGAGACCTTGAAAAACCAAGGCGTCTCTAATGCTGTCATCTGGTGTACCTGGTTCCTGGATAGC TTCTCCATCATGGCGCTGAGTATCTTCCTCCTGACGCTGTTCATCATGCATGGAAGGATCCTCCATTACAG CGATCCCTTCATTCTCTTCCTGTTCTTGTTGGCCTTTGCCACTGCGACCATCATGCAGAGCTTCCTGCTCA GTACTTTGTTTTCCAAGGCCAGCCTTGCAGCAGCCTGCAGTGGGGTCATCTACTTCACCCTCTACCTACCA CACGTTCTGTGCTTTGCCTGGCAGGACCGGATGACGGCCGACCTGAAGACGACTGTGAGCCTACTTTCTTC CGTGGCATTTGGGTTTGGCACCGAGTACCTGGTCCGCTTTGAGGAGCAAGGCCTGGGGCTGCAATGGAGCA ACATTGGGAAGAGTCCCCTGGAAGGGGATGAGTTCAGTTTCCTGCTGTCTATGAAGATGATGCTTCTCGAT GCTGCTCTCTACGGCTTGCTTGCTTGGTATCTTGACCAGGTTTTCCCAGGAGACTATGGGACCCCACTTCC CTGGTACTTCCTTCTGCAGGAGTCCTACTGGCTTGGTGGTGAAGGTTGTTCGACCAGAGAAGAAAGGGCTC TGGAAAAGACTGAACCCTTAACAGAGGAGATGGAGGATCCAGAGCACCCAGAAGGAATGAATGACTCCTTT TTTGAACGCGAGCTTCCAGGGCTGGTGCCTGGTGTGTGTGTGAAGAACCTGGTGAAGGTTTTTGAGCCCAG TGGCCGGCCAGCTGTGGACCGTCTGAACATCACTTTCTATGAGAACCAAATCACGGCGTTCCTGGGTCACA ACGGAGCGGGAAAGACCACCACCTTGTCCATCCTGACAGGACTGTTGCCACCGACGTCAGGAACTGTGCTC ATTGGGGGAAAAGACATTGAAACCAACCTGGATGTAGTACGGCAGAGCCTGGGCATGTGTCCACAACACAA CATCCTGTTTCATCACCTCACGGTGGCTGAGCACATCTTGTTCTATGCCCAGCTGAAAGGGAGATCCTGGG AGGAGGCCCAGCTTGAGATGGAAGCCATGCTAGAAGACACGGGCCTCCACCATAAGAGGAATGAAGAAGCT CAGGACCTTTCAGGTGCTTGGTATTGGGAGGAGACGGGGTCACTTACGGAGCATCCGTTGCCTCCCCAGTG GTTCTCAACGTCCTAATCGATACATGTGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGAGGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGACTCGCGCGAGTCGAGGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCAATTGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0460] Seq ID NO: 17 MITI 5’ mABCA4 gRNA

[0461] ITR

[0462] gRNA+PAM

[0463] Homology arms

[0464] enhancer + GRK 1 promoter

[0465] ABCA4 5' cDNA ABCA4 intron 21 (acting' as splicing' donor signal)

[0466] hU6 promoter

[0467] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCTAGGACGTTGAGAA CCACTGGGGCATTAGGACGTTGAGAACCATCTACGTAGCCATGCTCTGGGAAGATCTTCAATATTGGCCATTAGC CATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAA TATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTGGGCCCCAGAA GCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGGAGGAAGGGGCCGGGCAGAATGATCT AATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAAGCGTCCTCCGTGACC CCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCCGGGCTCCCAGGGGCTTCCCAGTGGTCCCCAGGAACCCTCG ACAGGGCCAGGGCGTCTCTCTCGTCCAGCAAGGGCAGGGACGGGCCACAGGCAAGGGCTCGAGAATTCGGCACGA GGTCCTAGCGGCCTTTGTGTCCGGTGCTTGCCTGAGCCCCAGCTCGGGGTGATTCTCCCGGTGCTGGGGTGGTCC GGCATGGGCTTCCTCAGACAGATACAGCTTTTGCTTTGGAAGAACTGGACTCTGAGGAAAAGGCAGAAGATTCGC TTTGTAGTGGAACTCGTGTGGCCTTTGTCTTTGTTTTTGGTGTTAATCTGGCTGAGGAATGCCAACCCACTCTAT AGTCAGCATGAATGCCATTTTCCCAACAAGGCGATGCCTTCAGCAGGACTGTTACCGTGGCTCCAGGGGATTTTC TGCAATATGAACAACCCTTGTTTTCAAAACCCCACCCCTGGAGAGTCTCCTGGAACTGTCTCAAACTATAACAAC TCCATCTTGGCAAGAGTATATCGAGATTTTCAAGAACTCTTCATGGACACCCCGGAGGTCCAGCACCTTGGCCAG GTTTGGGCCGAGCTCCGCACCTTGTCGCAGTTCATGGACACCCTGAGGACTCACCCTGAGAGATTTGCAGGAAGA GGATTACAAATCCGAGACATCCTAAAAGATGAAGAGGCGCTGACCCTATTTCTCATGAGAAACATTGGCCTGTCT GACTCGGTTGCCCATCTTCTGGTCAACTCCCAAGTTCGTGTGGAGCAGTTTGCTTATGGAGTCCCAGACTTGGAA CTGACAGACATTGCCTGCAGCGAGGCCCTCCTGCAGCGCTTCATCATCTTCAGCCAGCGTCGGGGGGCACAGACG GTACGCGATGCCCTGTGTCCCCTCTCCCAGGTCACCCTACAGTGGATAGAAGACACTCTGTATGCCGATGTGGAC TTCTTCAAACTCTTCCATGTGCTCCCCACACTCCTGGACAGCAGTTCTCAAGGAATCAACTTGAGATTTTGGGGA GGAATATTATCTGATCTGTCACCAAGAATGCAAAAGTTTATCCATCGGCCAAGTGTTCAAGACTTGCTATGGGTG AGCAGACCTCTCCTGCAGAACGGTGGTCCTGAGACCTTCACACAGCTGATGAGCATTCTGTCTGACCTCCTGTGT GGGTACCCAGAGGGAGGAGGCTCCCGAGTGTTCTCCTTTAACTGGTATGAAGACAATAACTATAAAGCCTTCCTG GGGATTGATTCCACAAGGAAAGACCCCGCCTATTCTTACGACAAAAGAACAACATCCTTTTGTAATTCATTGATC CAGAGCCTGGAGTCAAACCCTTTAACCAAAATAGCCTGGAGGGCGGCAAAGCCATTGCTGATGGGAAAAATCCTC TTTACTCCAGATTCCCCTGCTGCTCGAAGGATAATGAAGAATGCCAACTCAACTTTTGAAGAACTGGATCGAGTT AGGAAGTTGGTAAAAGCCTGGGAGGAAGTGGGGCCCCAGATCTGGTACTTCTTTGAGAAGAGCACACAGATGACC GTGATCCGAGACACCCTGCAGCACCCAACCGTCAAAGACTTCATAAATAGGCAGCTCGGAGAAGAAGGCATTACC ACCGAAGCCGTATTAAACTTCTTCTCTAACGGTCCCCAAGAGAAGCAGGCTGATGATATGACCAGCTTTGACTGG AGGGACATATTCAACATCACTGACCGATTCCTACGCTTGGCTAATCAATACCTGGAGTGTCTGGTCCTGGATAAG TTTGAAAGTTATGATGATGAAGTGCAGCTCACCCAACGAGCCCTGTCTCTCCTGGAGGAGAACAGGTTCTGGGCC GGAGTGGTGTTCCCTGGCATGTATCCCTGGGCCAGCTCCTTACCTCCTCATGTGAAGTACAAGATTCGGATGGAC ATAGAT GT GGT GGAGAAGACCAATAAGAT CAAAGACAGGTACT GGGACTACAAAGACCAT GACGGT GATTATAAA GATCATGACATCGACTACAAGGATGACGATGACAAGGATTCTGGTCCCAGGGCGGATCCTGTCGAAGATTTCCGG TACATCTGGGGAGGTTTTGCCTATCTACAGGACATGGTGGAGCAAGGAATCGTGAAGAGTCAGATGCAGGCAGAG CCTCCAATTGGAGTCTATCTCCAACAGATGCCTTATCCCTGCTTTGTGGATGACTCCTTCATGATCATCCTGAAT CGCTGTTTCCCTATCTTCATGGTGCTGGCGTGGATCTACTCTGTCTCCATGACCGTTAAGGGCATTGTTTTGGAG AAGGAGCTGAGGCTGAAGGAGACCTTGAAAAACCAAGGCGTCTCTAATGCTGTCATCTGGTGTACCTGGTTCCTG GATAGCTTCTCCATCATGGCGCTGAGTATCTTCCTCCTGACGCTGTTCATCATGCATGGAAGGATCCTCCATTAC AGCGATCCCTTCATTCTCTTCCTGTTCTTGTTGGCCTTTGCCACTGCGACCATCATGCAGAGCTTCCTGCTCAGT ACTTTGTTTTCCAAGGCCAGCCTTGCAGCAGCCTGCAGTGGGGTCATCTACTTCACCCTCTACCTACCACACGTT CTGTGCTTTGCCTGGCAGGACCGGATGACGGCCGACCTGAAGACGACTGTGAGCCTACTTTCTTCCGTGGCATTT GGGTTTGGCACCGAGTACCTGGTCCGCTTTGAGGAGCAAGGCCTGGGGCTGCAATGGAGCAACATTGGGAAGAGT CCCCTGGAAGGGGATGAGTTCAGTTTCCTGCTGTCTATGAAGATGATGCTTCTCGATGCTGCTCTCTACGGCTTG CTTGCTTGGTATCTTGACCAGGTTTTCCCAGGAGACTATGGGACCCCACTTCCCTGGTACTTCCTTCTGCAGGAG TCCTACTGGCTTGGTGGTGAAGGTTGTTCGACCAGAGAAGAAAGGGCTCTGGAAAAGACTGAACCCTTAACAGAG GAGATGGAGGATCCAGAGCACCCAGAAGGAATGAATGACTCCTTTTTTGAACGCGAGCTTCCAGGGCTGGTGCCT GGTGTGTGTGTGAAGAACCTGGTGAAGGTTTTTGAGCCCAGTGGCCGGCCAGCTGTGGACCGTCTGAACATCACT TTCTATGAGAACCAAATCACGGCGTTCCTGGGTCACAACGGAGCGGGAAAGACCACCACCTTGTCCATCCTGACA GGACTGTTGCCACCGACGTCAGGAACTGTGCTCATTGGGGGAAAAGACATTGAAACCAACCTGGATGTAGTACGG CAGAGCCTGGGCATGTGTCCACAACACAACATCCTGTTTCATCACCTCACGGTGGCTGAGCACATCTTGTTCTAT GCCCAGCTGAAAGGGAGATCCTGGGAGGAGGCCCAGCTTGAGATGGAAGCCATGCTAGAAGACACGGGCCTCCAC CATAAGAGGAATGAAGAAGCTCAGGACCTTTCAGGTGCTTGGTATTGGGAGGAGACGGGGTCACTTACGGAGCAT CCGTTGCCTCTGGGGTAGGGCCTTATAA CCCCCAGTGGTTCTCAACGTCCTAAT C GAT AC AT GTGAGGGCCTAT TTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAA ACACAAAGAT AT T AGT ACAAAAT AC GT GAG GT AGAAAGT AAT AAT T T CT T GGGT AGT T T GCAGT T T T AAAAT TAT GTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGA AAGGACGAAACACCGTAGGACGTTGAGAACCACTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCAATTGAGGAACCCCTAGTGATGGA GTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTT TGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0468] Seq ID NO: 18 MITI 5’ mABCA4 gRNA scramble

[0469] ITR

[0470] gRNA+PAM

[0471] Homology arms

[0472] enhancer + GRK 1 promoter

[0473] ABCA4 5' cDNA

[0474] ABCA4 intron 21 (acting as splicing donor signal)

[0475] hU6 promoter

[0476] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCTAGGACGTTGAGAA CCACTGGGGCATTAGGACGTTGAGaACCATCTACGTAGCCATGCTCTGGGAAGATCTTCAATATTGGCCATTAGC CATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAA TATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTGGGCCCCAGAA GCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGGAGGAAGGGGCCGGGCAGAATGATCT AATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAAGCGTCCTCCGTGACC CCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCCGGGCTCCCAGGGGCTTCCCAGTGGTCCCCAGGAACCCTCG ACAGGGCCAGGGCGTCTCTCTCGTCCAGCAAGGGCAGGGACGGGCCACAGGCAAGGGCTCGAGAATTCGGCACGA GGTCCTAGCGGCCTTTGTGTCCGGTGCTTGCCTGAGCCCCAGCTCGGGGTGATTCTCCCGGTGCTGGGGTGGTCC GGCATGGGCTTCCTCAGACAGATACAGCTTTTGCTTTGGAAGAACTGGACTCTGAGGAAAAGGCAGAAGATTCGC TTTGTAGTGGAACTCGTGTGGCCTTTGTCTTTGTTTTTGGTGTTAATCTGGCTGAGGAATGCCAACCCACTCTAT AGTCAGCATGAATGCCATTTTCCCAACAAGGCGATGCCTTCAGCAGGACTGTTACCGTGGCTCCAGGGGATTTTC TGCAATATGAACAACCCTTGTTTTCAAAACCCCACCCCTGGAGAGTCTCCTGGAACTGTCTCAAACTATAACAAC TCCATCTTGGCAAGAGTATATCGAGATTTTCAAGAACTCTTCATGGACACCCCGGAGGTCCAGCACCTTGGCCAG GTTTGGGCCGAGCTCCGCACCTTGTCGCAGTTCATGGACACCCTGAGGACTCACCCTGAGAGATTTGCAGGAAGA GGATTACAAATCCGAGACATCCTAAAAGATGAAGAGGCGCTGACCCTATTTCTCATGAGAAACATTGGCCTGTCT GACTCGGTTGCCCATCTTCTGGTCAACTCCCAAGTTCGTGTGGAGCAGTTTGCTTATGGAGTCCCAGACTTGGAA CTGACAGACATTGCCTGCAGCGAGGCCCTCCTGCAGCGCTTCATCATCTTCAGCCAGCGTCGGGGGGCACAGACG GTACGCGATGCCCTGTGTCCCCTCTCCCAGGTCACCCTACAGTGGATAGAAGACACTCTGTATGCCGATGTGGAC TTCTTCAAACTCTTCCATGTGCTCCCCACACTCCTGGACAGCAGTTCTCAAGGAATCAACTTGAGATTTTGGGGA GGAATATTATCTGATCTGTCACCAAGAATGCAAAAGTTTATCCATCGGCCAAGTGTTCAAGACTTGCTATGGGTG AGCAGACCTCTCCTGCAGAACGGTGGTCCTGAGACCTTCACACAGCTGATGAGCATTCTGTCTGACCTCCTGTGT GGGTACCCAGAGGGAGGAGGCTCCCGAGTGTTCTCCTTTAACTGGTATGAAGACAATAACTATAAAGCCTTCCTG GGGATTGATTCCACAAGGAAAGACCCCGCCTATTCTTACGACAAAAGAACAACATCCTTTTGTAATTCATTGATC CAGAGCCTGGAGTCAAACCCTTTAACCAAAATAGCCTGGAGGGCGGCAAAGCCATTGCTGATGGGAAAAATCCTC TTTACTCCAGATTCCCCTGCTGCTCGAAGGATAATGAAGAATGCCAACTCAACTTTTGAAGAACTGGATCGAGTT AGGAAGTTGGTAAAAGCCTGGGAGGAAGTGGGGCCCCAGATCTGGTACTTCTTTGAGAAGAGCACACAGATGACC GTGATCCGAGACACCCTGCAGCACCCAACCGTCAAAGACTTCATAAATAGGCAGCTCGGAGAAGAAGGCATTACC ACCGAAGCCGTATTAAACTTCTTCTCTAACGGTCCCCAAGAGAAGCAGGCTGATGATATGACCAGCTTTGACTGG AGGGACATATTCAACATCACTGACCGATTCCTACGCTTGGCTAATCAATACCTGGAGTGTCTGGTCCTGGATAAG TTTGAAAGTTATGATGATGAAGTGCAGCTCACCCAACGAGCCCTGTCTCTCCTGGAGGAGAACAGGTTCTGGGCC GGAGTGGTGTTCCCTGGCATGTATCCCTGGGCCAGCTCCTTACCTCCTCATGTGAAGTACAAGATTCGGATGGAC ATAGAT GT GGT GGAGAAGACCAATAAGAT CAAAGACAGGTACT GGGACTACAAAGACCAT GACGGT GATTATAAA GATCATGACATCGACTACAAGGATGACGATGACAAGGATTCTGGTCCCAGGGCGGATCCTGTCGAAGATTTCCGG TACATCTGGGGAGGTTTTGCCTATCTACAGGACATGGTGGAGCAAGGAATCGTGAAGAGTCAGATGCAGGCAGAG CCTCCAATTGGAGTCTATCTCCAACAGATGCCTTATCCCTGCTTTGTGGATGACTCCTTCATGATCATCCTGAAT CGCTGTTTCCCTATCTTCATGGTGCTGGCGTGGATCTACTCTGTCTCCATGACCGTTAAGGGCATTGTTTTGGAG AAGGAGCTGAGGCTGAAGGAGACCTTGAAAAACCAAGGCGTCTCTAATGCTGTCATCTGGTGTACCTGGTTCCTG GATAGCTTCTCCATCATGGCGCTGAGTATCTTCCTCCTGACGCTGTTCATCATGCATGGAAGGATCCTCCATTAC AGCGATCCCTTCATTCTCTTCCTGTTCTTGTTGGCCTTTGCCACTGCGACCATCATGCAGAGCTTCCTGCTCAGT ACTTTGTTTTCCAAGGCCAGCCTTGCAGCAGCCTGCAGTGGGGTCATCTACTTCACCCTCTACCTACCACACGTT CTGTGCTTTGCCTGGCAGGACCGGATGACGGCCGACCTGAAGACGACTGTGAGCCTACTTTCTTCCGTGGCATTT GGGTTTGGCACCGAGTACCTGGTCCGCTTTGAGGAGCAAGGCCTGGGGCTGCAATGGAGCAACATTGGGAAGAGT CCCCTGGAAGGGGATGAGTTCAGTTTCCTGCTGTCTATGAAGATGATGCTTCTCGATGCTGCTCTCTACGGCTTG CTTGCTTGGTATCTTGACCAGGTTTTCCCAGGAGACTATGGGACCCCACTTCCCTGGTACTTCCTTCTGCAGGAG TCCTACTGGCTTGGTGGTGAAGGTTGTTCGACCAGAGAAGAAAGGGCTCTGGAAAAGACTGAACCCTTAACAGAG GAGATGGAGGATCCAGAGCACCCAGAAGGAATGAATGACTCCTTTTTTGAACGCGAGCTTCCAGGGCTGGTGCCT GGTGTGTGTGTGAAGAACCTGGTGAAGGTTTTTGAGCCCAGTGGCCGGCCAGCTGTGGACCGTCTGAACATCACT TTCTATGAGAACCAAATCACGGCGTTCCTGGGTCACAACGGAGCGGGAAAGACCACCACCTTGTCCATCCTGACA GGACTGTTGCCACCGACGTCAGGAACTGTGCTCATTGGGGGAAAAGACATTGAAACCAACCTGGATGTAGTACGG CAGAGCCTGGGCATGTGTCCACAACACAACATCCTGTTTCATCACCTCACGGTGGCTGAGCACATCTTGTTCTAT GCCCAGCTGAAAGGGAGATCCTGGGAGGAGGCCCAGCTTGAGATGGAAGCCATGCTAGAAGACACGGGCCTCCAC CATAAGAGGAATGAAGAAGCTCAGGACCTTTCAGGTGCTTGGTATTGGGAGGAGACGGGGTCACTTACGGAGCAT CCGTTGCCTCTGGGGTAGGGCCTTATAACCCCCAGTGGTTCTCAACGTCCTAATCGATCTTTCGAGGGCCTATTT CCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAAC ACAAAGAT AT T AGT ACAAAAT AC GT GAG GT AGAAAGT AAT AAT T T CT T GGGT AGT T T GCAGT T T T AAAAT TAT GT TTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAA GGACGAAACACCGACTCGCGCGAGTCGAGGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGT TATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGCGGCCGCAATTGAGGAACCCCTAGTGATGGAGTT GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGC CCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0477] Seq ID NO: 19 AAV-IRBP-Cas9 TGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACT CATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTT CACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCTGCGCGCTCGCTCGCTCACTGAGGCCG CCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAG TGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCT CTAGGAAGATCGGAATTCGCCCTTAAGCTAGTAGCACAGTGTCTGGCATGTAGCAGGAACTAAAATAATGGCAGT GATTAATGTTATGATATGCAGACACAACACAGCAAGATAAGATGCAATGTACCTTCTGGGTCAAACCACCCTGGC CACTCCTCCCCGATACCCAGGGTTGATGTGCTTGAATTAGACAGGATTAAAGGCTTACTGGAGCTGGAAGCCTTG CCCCAACTCAGGAGTTTAGCCCCAGACCTTCTGTCCACCAGCGCGGCCGACCGGCCAAGGGCGAATTCTGCAGAT ATCCATCACACTGGCGGCCGATCCCCGGGTACCGGTGCCACCATGTACCCATACGATGTTCCAGATTACGCTTCG CCGAAGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTG GGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCAC AGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGA ACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCC AAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCAC CCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAA CTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGC CACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACC TACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTG AGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTG ATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTG AGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTG GCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCC CTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAG CAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGA GCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTG AAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTG GGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAG AAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACC AGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTC ATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAG TACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGC GGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAG GACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTG GGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTG GAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCAC CTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTG ATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGA AACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAG GGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTG AAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAG AACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTG GGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTG CAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATC GTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAG AGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTG ATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTC ATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACT AAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTC CGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCC GTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTAC GACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAAC ATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAAC GGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAA GTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGC GATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTAT TCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATC ACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAA AAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCT GCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCAC TATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTG GACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTG TCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACC AATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAG GTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGA GGCGACAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCTAAGAATTCAATAAAAGATCTTTATTTTCATTAGAT CTGTGTGTTGGTTTTTTGTGTGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG CTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCG AGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAT ACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTG ACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGC TTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAA AAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAG TCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGAT TTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTT AACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAG CCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCT GTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTC GTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGA AATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCC TGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTT TTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTG GGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGT TTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAA CTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGAT GGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACA ACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGG GAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTG CGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAA GTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGT GGAAGCCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGG AGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTG TCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAG ATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTA CCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAG ATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATAC CTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGA CGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACG ACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGAC AGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTT TATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCT GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACG ACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGG C C GATT CAT TAA

[0478] Seq ID NO: 20 mAbca4_gRNA2 mAbca4

[0479] G T AG GAG G T T GAGAAC GAG T G

[0480] Seq ID NO: 21 SpCas9_gRNAl hABCA4

[0481] GAT AC C AAT GTTATAACTCAG

[0482] Seq ID NO: 22 SpCas9_gRNA2 hABCA4

[0483] GATGGACATAGGAAACCCCAG

[0484] Seq ID NO: 23 SpCas9_gRNA3 hABCA4

[0485] GTCTGAGATGACTTCATACGG

[0486] Seq ID NO: 24 skip

[0487] Seq ID NO: 25 skip

[0488] Seq ID NO: 26 skip

[0489] Seq ID NO: 27 SpCas9_gRNAl hCEP290

[0490] GTGTAATCCCAGCTACCCAGG

[0491] Seq ID NO: 28 SpCas9_gRNA2 hCEP290

[0492] GT CAAAAGAT AT AC CAAC CAG

[0493] Seq ID NO: 29 SpCas9_gRNA3 hCEP290

[0494] GGTTTCCTCATACTGATGAG

[0495] Seq ID NO: 30 CL1 degradation signal GCCTGCAAGAACTGGTTCAGCAGCCTGAGCCACTTCGTGATCCACCTG SEQ ID NO: 31 pSpCas9 (BB)-2A-GFP plasmid gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaat ttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatat atcttGTGGAAAGGACGAAACACCggGTCTTCgaGAAGACctgttttagagctaGAAAtagcaagttaaaataag gctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTTgttttagagctagaaatagcaagtta aaataaggctagtccgtTTTTagcgcgtgcgccaattctgcagacaaatggctctagaggtacccgttacataac ttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatagtaacgccaataggga ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgc caagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattGtgcccagtacatgaccttatggg actttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgct tcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcga tgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcgga gaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggc cctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgcc gcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcct ccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattac ctggagcacctgcctgaaatcactttttttcaggttGGaccggtgccaccATGGACTATAAGGACCACGACGGAG ACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTA TCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCG TGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGA AGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAA GAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACG ACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCG GCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACA GCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGA TCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGC TGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCA GACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGA GCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACA CCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGA ACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCT CTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTG AGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGG AAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACA GAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGC ACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGA CCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCG AGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGA TGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCG TGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGA AAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCA AGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACC ACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCG TGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACG ACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCA TCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGC AGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCC TGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGG ACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCA CCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGA TCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGC GGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGA GCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACG TGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGA GAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGAC AGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACG AGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATT TCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAA CCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGA AGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACT TTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCG GGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCG TGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGA TCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGG T GGT GGCCAAAGT GGAAAAGGGCAAGT CCAAGAAACT GAAGAGT GT GAAAGAGCT GCT GGGGAT CACCAT CAT GG AAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGA TCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAAC TGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGC TGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCA TCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACA ACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAG CCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACG CCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAA GGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGgaattcGGCAGTGGAGAGGGCAGAGGAAGTC TGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGC CCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCA CCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCA CCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCA TGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGG GGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGG TGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCA TCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACG AGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACA AGgaattctaaCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCA TTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAgAA TAGCAGGCATGCTGGGGAgcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctc gctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagc gcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacg tcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgacc gctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggcttt ccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaa cttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtcc acgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgattta taagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaac aaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccc cgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtg accgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtg atacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaat gtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctga taaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattccctttttt gcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggt gcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgtttt ccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactc ggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggc atgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacg atcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaa ccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgc aaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagtt gcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgga agccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagt caggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtca gaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatc ctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaag atcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctacca gcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagata ccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctc gctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacga tagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacc tacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacagg tatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttat agtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatgg aaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

[0496] SEQ ID NO: 32 left HA 5’ MITI mABCA4

[0497] CAT TAGGACGTT GAGAAC C A SEQ ID NO: 33 right HA 5’ MITI mABCA4

[0498] CTGGGGTAGGGCCTTATAAC SEQ ID NO: 34 Splicing Donor signal GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTG CGTTTCT SEQ ID NO: 35 Splicing Acceptor signal GATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG SEQ ID NO: 368bp left HA 5’ MITI hABCA4

[0499] AACCGCTG SEQ ID NO: 378bp right HA 5’ MITI hABCA4

[0500] AGTTATAA SEQ ID NO: 3820bp left HA 5’ MITI hABCA4

[0501] CTACTCAGAGATAACCGCTG SEQ ID NO: 3920bp right HA 5’ MITI hABCA4

[0502] AGT T AT AACAT T GGT AT AT A SEQ ID NO: 408bp left HA 3’ MITI hABCA4

[0503] AACCGCTG SEQ ID NO: 41 8bp right HA 3’ MITI hABCA4

[0504] AGTTATAA SEQ ID NO: 4220bp left HA 3’ MITI hABCA4

[0505] CTACTCAGAGATAACCGCTG SEQ ID NO: 4320bp right HA 3’ MITI hABCA4

[0506] AGT TAT AACAT T GGT AT AT A SEQ ID NO: 44 skip

[0507] SEQ ID NO: 45 hU6 promoter TTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTA AACACAAAGAT AT T AGT ACAAAAT AC GT GAC GT AGAAAGT AAT AAT T T CT T GGGT AGT T T GCAGT T T T AAAAT TA TGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGG AAAGGACG SEQ ID NO: 46 SV40 poly A TTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTA TTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATT GCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTG GTAAAAT CGATAAGGAT C SEQ ID NO: 47 BGH polyA CCGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAG GTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTC TGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGG SEQ ID NO: 48 CMV promoter TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTAC GGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCA AGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA CATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTT TTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAAT GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGT SEQ ID NO: 49 skip

[0508] SEQ ID NO: 50 GRK1 promoter GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGGAGGAAGGGGCCGGG CAGAATGATCTAATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAAGCGT CCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCCGGGCTCCCAGGGGCTTCCCAGTGGTCCCC AGGAACCCTCGACAGGGCCAGGGCGTCTCTCTCGTCCAGCAAGGGCAGGGACGGGCCACAGGCAAGGGC SEQ IDNO: 51 skip

[0509] SEQ ID NO: 52 HITI 3' hCEP290

[0510] ITR

[0511] gRNA+PAM

[0512] Splicing acceptor

[0513] CEP 3 ' CDS

[0514] BGH PolyA

[0515] hU6 promoter

[0516] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCCCTCTGGTTGGTAT ATCTTTTGAGACTCGCGCGAGTCGAGGAGGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTC CACAGGCACAGTCTGATGAAAAGTCGCTCATTGCCAAGTTGCACCAACATAATGTCTCTCTTCAACTGAGTGAGG CTACTGCTCTTGGTAAGTTGGAGTCAATTACATCTAAACTGCAGAAGATGGAGGCCTACAACTTGCGCTTAGAGC AGAAACTTGATGAAAAAGAACAGGCTCTCTATTATGCTCGTTTGGAGGGAAGAAACAGAGCAAAACATCTGCGCC AAACAATTCAGTCTCTACGACGACAGTTTAGTGGAGCTTTACCCTTGGCACAACAGGAAAAGTTCTCCAAAACAA T GATT CAACTACAAAAT GACAAACTTAAGATAAT GCAAGAAAT GAAAAATT CT CAACAAGAACATAGAAATAT GG AGAACAAAACATTGGAGATGGAATTAAAATTAAAGGGCCTGGAAGAGTTAATAAGCACTTTAAAGGATACCAAAG GAGCCCAAAAGGTAATCAACTGGCATATGAAAATAGAAGAACTTCGTCTTCAAGAACTTAAACTAAATCGGGAAT T AGT C AAG GAT AAAGAAGAAAT AAAAT AT T T GAAT AAC AT AAT T T C T GAAT AT GAAC GT AC AAT CAGCAGTCTTG AAGAAGAAATTGTGCAACAGAACAAGTTTCATGAAGAAAGACAAATGGCCTGGGATCAAAGAGAAGTTGACCTGG AACGCCAACTAGACATTTTTGACCGTCAGCAAAATGAAATACTAAATGCGGCACAAAAGTTTGAAGAAGCTACAG GATCAATCCCTGACCCTAGTTTGCCCCTTCCAAATCAACTTGAGATCGCTCTAAGGAAAATTAAGGAGAACATTC GAATAATTCTAGAAACACGGGCAACTTGCAAATCACTAGAAGAGAAACTAAAAGAGAAAGAATCTGCTTTAAGGT TAGCAGAACAAAATATACTGTCAAGAGACAAAGTAATCAATGAACTGAGGCTTCGATTGCCTGCCACTGCAGAAA GAGAAAAGCT CATAGCT GAGCTAGGCAGAAAAGAGAT GGAACCAAAAT CT CACCACACATT GAAAATT GCT CAT C AAACCATTGCAAACATGCAAGCAAGGTTAAATCAAAAAGAAGAAGTATTAAAGAAGTATCAACGTCTTCTAGAAA AAGCCAGAGAGGAGCAAAGAGAAATTGTGAAGAAACATGAGGAAGACCTTCATATTCTTCATCACAGATTAGAAC TACAGGCTGATAGTTCACTAAATAAATTCAAACAAACGGCTTGGGATTTAATGAAACAGTCTCCCACTCCAGTTC CTACCAACAAGCATTTTATTCGTCTGGCTGAGATGGAACAGACAGTAGCAGAACAAGATGACTCTCTTTCCTCAC T CTT GGT CAAACTAAAGAAAGTAT CACAAGATTT GGAGAGACAAAGAGAAAT CACT GAATTAAAAGTAAAAGAAT TTGAAAATATCAAATTACAGCTTCAAGAAAACCATGAAGATGAAGTGAAAAAAGTAAAAGCGGAAGTAGAGGATT TAAAGTATCTTCTGGACCAGTCACAAAAGGAGTCACAGTGTTTAAAATCTGAACTTCAGGCTCAAAAAGAAGCAA ATTCAAGAGCTCCAACAACTACAATGAGAAATCTAGTAGAACGGCTAAAGAGCCAATTAGCCTTGAAGGAGAAAC AACAGAAAGCACTTAGTCGGGCACTTTTAGAACTCCGGGCAGAAATGACAGCAGCTGCTGAAGAACGTATTATTT CTGCAACTTCTCAAAAAGAGGCCCATCTCAATGTTCAACAAATCGTTGATCGACATACTAGAGAGCTAAAGACAC AAGTTGAAGATTTAAATGAAAATCTTTTAAAATTGAAAGAAGCACTTAAAACTAGTAAAAACAGAGAAAACTCAC TAACTGATAATTTGAATGACTTAAATAATGAACTGCAAAAGAAACAAAAAGCCTATAATAAAATACTTAGAGAGA AAGAGGAAATT GAT CAAGAGAAT GAT GAACT GAAAAGGCAAATTAAAAGACTAACCAGT GGATTACAGGGCAAAC CCCTGACAGATAATAAACAAAGTCTAATTGAAGAACTCCAAAGGAAAGTTAAAAAACTAGAGAACCAATTAGAGG GAAAGGTGGAGGAAGTAGACCTAAAACCTATGAAAGAAAAGAATGCTAAAGAAGAATTAATTAGGTGGGAAGAAG GTAAAAAGTGGCAAGCCAAAATAGAAGGAATTCGAAACAAGTTAAAAGAGAAAGAGGGGGAAGTCTTTACTTTAA CAAAGCAGTTGAATACTTTGAAGGATCTTTTTGCCAAAGCCGATAAAGAGAAACTTACTTTGCAGAGGAAACTAA AAACAACTGGCATGACTGTTGATCAGGTTTTGGGAATACGAGCTTTGGAGTCAGAAAAAGAATTGGAAGAATTAA AAAAGAGAAAT C T T GAG T T AGAAAAT GAT AT AT T GT A ta tgagggcccaccaagc tcttcctcgagattctgttg tagaagatttacatttacaaaatagatacc tccaagAAAAACTTCATGCTTTAGAAAAACAGTTTTCAAAGGATA CATATTCTAAGCCTTCAATTTCAGGAATAGAGTCAGATGATCATTGTCAGAGAGAACAGGAGCTTCAGAAGGAAA ACTTGAAGTTGTCATCTGAAAATATTGAACTGAAATTTCAGCTTGAACAAGCAAATAAAGATTTGCCAAGATTAA AGAATCAAGTCAGAGATTTGAAGGAAATGTGTGAATTTCTTAAGAAAGAAAAAGCAGAAGTTCAGCGGAAACTTG GCCATGTTAGAGGGTCTGGTAGAAGTGGAAAGACAATCCCAGAACTGGAAAAAACCATTGGTTTAATGAAAAAAG TAGTT GAAAAAGT CCAGAGAGAAAAT GAACAGTT GAAAAAAGCAT CAGGAATATT GACTAGT GAAAAAAT GGCTA ATATTGAGCAGGAAAATGAAAAATTGAAGGCTGAATTAGAAAAACTTAAAGCTCATCTTGGGCATCAGTTGAGCA TGCACTATGAATCCAAGACCAAAGGCACAGAAAAAATTATTGCTGAAAATGAAAGGCTTCGTAAAGAACTTAAAA AAGAAACTGATGCTGCAGAGAAATTACGGATAGCAAAGAATAATTTAGAGATATTAAATGAGAAGATGACAGTTC AACTAGAAGAGACTGGTAAGAGATTGCAGTTTGCAGAAAGCAGAGGTCCACAGCTTGAAGGTGCTGACAGTAAGA GCTGGAAATCCATTGTGGTTACAAGAATGTATGAAACCAAGTTAAAAGAATTGGAAACTGATATTGCCAAAAAAA ATCAAAGCATTACTGACCTTAAACAGCTTGTAAAAGAAGCAACAGAGAGAGAACAAAAAGTTAACAAATACAATG AAGACCTTGAACAACAGATTAAGATTCTTAAACATGTTCCTGAAGGTGCTGAGACAGAGCAAGGCCTTAAACGGG AGCTTCAAGTTCTTAGATTAGCTAATCATCAGCTGGATAAAGAGAAAGCAGAATTAATCCATCAGATAGAAGCTA ACAAGGACCAAAGT GGAGCT GAAAGCAC CAT ACCT GAT GCT GAT CAACTAAAGGAAAAAATAAAAGAT CTAGAGA CACAGCT CAAAAT GT CAGAT CTAGAAAAGCAGCATTT GAAGGAGGAAATAAAGAAGCT GAAAAAAGAACT GGAAA AT T T T GAT C C T T CAT T T T T T GAAGAAAT T GAAGAT C T T AAGT AT AAT T AC AAG GAAGAAGT GAAGAAGAAT AT T C TCTTAGAAGAGAAGGTAAAAAAACTTTCAGAACAATTGGGAGTTGAATTAACTAGCCCTGTTGCTGCTTCTGAAG AGT T T GAAGAT GAAGAAGAAAGT C CT GT T AAT T T C C C CAT T TAG GACT ACAAAGAC CAT GAC GGT GAT T AT AAAG ATCATGACATCGACTACAAGGATGACGATGACAAGTGAAAGCTTGATATCATCGATGGGCCCGGATCCGCCTCGA CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTC CCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGa ctcgagttaagggcgaa ttcccgataaggatcttcctagagCCTCTGGTTGGTATATCTTTTGAggtaccTTTCCCATGATTCCTTCATATT TGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAA ATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATA TGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCAAAAG ATATACCAACCAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG CACCGAGTCGGTGCTTTTTTTGCGCGGCCGCACCAATTGATTGAGGAACCCCTAGTGATGGAGTTGGCCACTCCC TCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCC TCAGTGAGCGAGCGAGCGCGCAG SEQ ID NO: 53 HITI 3' hCEP290 scramble

[0517] ITR

[0518] gRNA+PAM

[0519] Splicing acceptor

[0520] CEP 3 ' CDS

[0521] Bgh PolyA

[0522] hU6 promoter

[0523] gRNA + gRNA scaffold CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCTAGCCCTCTGGTTGGTAT ATCTTTTGAGACTCGCGCGAGTCGAGGAGGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTC CACAGGCACAGTCTGATGAAAAGTCGCTCATTGCCAAGTTGCACCAACATAATGTCTCTCTTCAACTGAGTGAGG CTACTGCTCTTGGTAAGTTGGAGTCAATTACATCTAAACTGCAGAAGATGGAGGCCTACAACTTGCGCTTAGAGC AGAAACTTGATGAAAAAGAACAGGCTCTCTATTATGCTCGTTTGGAGGGAAGAAACAGAGCAAAACATCTGCGCC AAACAATTCAGTCTCTACGACGACAGTTTAGTGGAGCTTTACCCTTGGCACAACAGGAAAAGTTCTCCAAAACAA T GATT CAACTACAAAAT GACAAACTTAAGATAAT G GAAGAAAT GAAAAATT CT CAACAAGAACATAGAAATAT GG AGAACAAAACATTGGAGATGGAATTAAAATTAAAGGGCCTGGAAGAGTTAATAAGCACTTTAAAGGATACCAAAG GAGCCCAAAAGGTAATCAACTGGCATATGAAAATAGAAGAACTTCGTCTTCAAGAACTTAAACTAAATCGGGAAT T AGT C AAG GAT AAAGAAGAAAT AAAAT AT T T GAAT AAC AT AAT T T C T GAAT AT GAAC GT AC AAT CAGCAGTCTTG AAGAAGAAATTGTGCAACAGAACAAGTTTCATGAAGAAAGACAAATGGCCTGGGATCAAAGAGAAGTTGACCTGG AACGCCAACTAGACATTTTTGACCGTCAGCAAAATGAAATACTAAATGCGGCACAAAAGTTTGAAGAAGCTACAG GATCAATCCCTGACCCTAGTTTGCCCCTTCCAAATCAACTTGAGATCGCTCTAAGGAAAATTAAGGAGAACATTC GAATAATTCTAGAAACACGGGCAACTTGCAAATCACTAGAAGAGAAACTAAAAGAGAAAGAATCTGCTTTAAGGT TAGCAGAACAAAATATACTGTCAAGAGACAAAGTAATCAATGAACTGAGGCTTCGATTGCCTGCCACTGCAGAAA GAGAAAAGCT CAT AGCT GAGCTAGGCAGAAAAGAGAT GGAAC CAAAAT CT GAG CACACATT GAAAATT GCT CAT C AAACCATTGCAAACATGCAAGCAAGGTTAAATCAAAAAGAAGAAGTATTAAAGAAGTATCAACGTCTTCTAGAAA AAGCCAGAGAGGAGCAAAGAGAAATTGTGAAGAAACATGAGGAAGACCTTCATATTCTTCATCACAGATTAGAAC TACAGGCTGATAGTTCACTAAATAAATTCAAACAAACGGCTTGGGATTTAATGAAACAGTCTCCCACTCCAGTTC CTACCAACAAGCATTTTATTCGTCTGGCTGAGATGGAACAGACAGTAGCAGAACAAGATGACTCTCTTTCCTCAC T CTT GGT CAAACTAAAGAAAGTAT CACAAGATTT GGAGAGACAAAGAGAAAT CACT GAATTAAAAGTAAAAGAAT TTGAAAATATCAAATTACAGCTTCAAGAAAACCATGAAGATGAAGTGAAAAAAGTAAAAGCGGAAGTAGAGGATT TAAAGTATCTTCTGGACCAGTCACAAAAGGAGTCACAGTGTTTAAAATCTGAACTTCAGGCTCAAAAAGAAGCAA ATTCAAGAGCTCCAACAACTACAATGAGAAATCTAGTAGAACGGCTAAAGAGCCAATTAGCCTTGAAGGAGAAAC AACAGAAAGCACTTAGTCGGGCACTTTTAGAACTCCGGGCAGAAATGACAGCAGCTGCTGAAGAACGTATTATTT CTGCAACTTCTCAAAAAGAGGCCCATCTCAATGTTCAACAAATCGTTGATCGACATACTAGAGAGCTAAAGACAC AAGTTGAAGATTTAAATGAAAATCTTTTAAAATTGAAAGAAGCACTTAAAACTAGTAAAAACAGAGAAAACTCAC TAACTGATAATTTGAATGACTTAAATAATGAACTGCAAAAGAAACAAAAAGCCTATAATAAAATACTTAGAGAGA AAGAGGAAATT GAT CAAGAGAAT GAT GAACT GAAAAGGCAAATTAAAAGACTAACCAGT GGATTACAGGGCAAAC CCCTGACAGATAATAAACAAAGTCTAATTGAAGAACTCCAAAGGAAAGTTAAAAAACTAGAGAACCAATTAGAGG GAAAGGTGGAGGAAGTAGACCTAAAACCTATGAAAGAAAAGAATGCTAAAGAAGAATTAATTAGGTGGGAAGAAG GTAAAAAGTGGCAAGCCAAAATAGAAGGAATTCGAAACAAGTTAAAAGAGAAAGAGGGGGAAGTCTTTACTTTAA CAAAGCAGTTGAATACTTTGAAGGATCTTTTTGCCAAAGCCGATAAAGAGAAACTTACTTTGCAGAGGAAACTAA AAACAACTGGCATGACTGTTGATCAGGTTTTGGGAATACGAGCTTTGGAGTCAGAAAAAGAATTGGAAGAATTAA AAAAGAGAAAT C T T GAG T T AGAAAAT GAT AT AT T GT A ta tgagggcccaccaagc tcttcctcgagattctgttg tagaagatttacatttacaaaatagatacc tccaagAAAAACTTCATGCTTTAGAAAAACAGTTTTCAAAGGATA CATATTCTAAGCCTTCAATTTCAGGAATAGAGTCAGATGATCATTGTCAGAGAGAACAGGAGCTTCAGAAGGAAA ACTTGAAGTTGTCATCTGAAAATATTGAACTGAAATTTCAGCTTGAACAAGCAAATAAAGATTTGCCAAGATTAA AGAATCAAGTCAGAGATTTGAAGGAAATGTGTGAATTTCTTAAGAAAGAAAAAGCAGAAGTTCAGCGGAAACTTG GCCATGTTAGAGGGTCTGGTAGAAGTGGAAAGACAATCCCAGAACTGGAAAAAACCATTGGTTTAATGAAAAAAG TAGTT GAAAAAGT CCAGAGAGAAAAT GAACAGTT GAAAAAAGCAT CAGGAATATT GACTAGT GAAAAAAT GGCTA ATATTGAGCAGGAAAATGAAAAATTGAAGGCTGAATTAGAAAAACTTAAAGCTCATCTTGGGCATCAGTTGAGCA TGCACTATGAATCCAAGACCAAAGGCACAGAAAAAATTATTGCTGAAAATGAAAGGCTTCGTAAAGAACTTAAAA AAGAAACTGATGCTGCAGAGAAATTACGGATAGCAAAGAATAATTTAGAGATATTAAATGAGAAGATGACAGTTC AACTAGAAGAGACTGGTAAGAGATTGCAGTTTGCAGAAAGCAGAGGTCCACAGCTTGAAGGTGCTGACAGTAAGA GCTGGAAATCCATTGTGGTTACAAGAATGTATGAAACCAAGTTAAAAGAATTGGAAACTGATATTGCCAAAAAAA ATCAAAGCATTACTGACCTTAAACAGCTTGTAAAAGAAGCAACAGAGAGAGAACAAAAAGTTAACAAATACAATG AAGACCTTGAACAACAGATTAAGATTCTTAAACATGTTCCTGAAGGTGCTGAGACAGAGCAAGGCCTTAAACGGG AGCTTCAAGTTCTTAGATTAGCTAATCATCAGCTGGATAAAGAGAAAGCAGAATTAATCCATCAGATAGAAGCTA ACAAGGACCAAAGT GGAGCT GAAAGCAC CAT ACCT GAT GCT GAT C AAC T AAAG GAAAAAAT AAAAGAT CT AGAGA CACAGCT CAAAAT GT CAGAT CTAGAAAAGCAGCATTT GAAGGAGGAAATAAAGAAGCT GAAAAAAGAACT GGAAA AT T T T GAT C C T T CAT T T T T T GAAGAAAT T GAAGAT C T T AAGT AT AAT T AC AAG GAAGAAGT GAAGAAGAAT AT T C TCTTAGAAGAGAAGGTAAAAAAACTTTCAGAACAATTGGGAGTTGAATTAACTAGCCCTGTTGCTGCTTCTGAAG AGT T T GAAGAT GAAGAAGAAAGT C CT GT T AAT T T C C C CAT T TAG GACT ACAAAGAC CAT GAC GGT GAT TAT AAAG ATCATGACATCGACTACAAGGATGACGATGACAAGTGAAAGCTTGATATCATCGATGGGCCCGGATCCGCCTCGA CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTC CCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGa ctcgagttaagggcgaa ttcccgataaggatcttcctagagCCTCTGGTTGGTATATCTTTTGAggtaccTTTCCCATGATTCCTTCATATT TGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAA ATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATA TGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGACTCGC GCGAGTCGAGGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG CACCGAGTCGGTGCTTTTTTTGCGCGGCCGCACCAATTGATTGAGGAACCCCTAGTGATGGAGTTGGCCACTCCC TCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCC TCAGTGAGCGAGCGAGCGCGCAG

Claims

CLAIMS1. A kit for gene editing of a target gene by non-homologous end-joining (NHEJ) or microhomology-mediated end joining (MMEJ) targeted integration, comprising:a) a nuclease capable of introducing a double strand break (DSB) in a target site of a target gene, or a polynucleotide encoding thereof, said target site being an intron of the target gene;b) a complementary strand oligonucleotide having sequence homologous to the target site sequence;c.l) a first donor nucleic acid comprising a first knock-in cassette for reconstitution of the target gene, said first knock-in cassette comprising from 5’ to 3’:- a promoter,- an exogenous polynucleotide (5’ exogenous polynucleotide) operably linked to said promoter and having sequence homologous to the coding sequence of the portion of the target gene upstream the DSB, and- a splicing donor signal;andc.2) a second donor nucleic acid comprising a second knock-in cassette for reconstitution of the target gene, said second knock-in cassette comprising:- a splicing acceptor signal,- an exogenous polynucleotide (3’ exogenous polynucleotide) having sequence homologous to the coding sequence of portion of the target gene downstream the DSB, and- a poly adenylation signal;wherein each of said first and second knock-in cassettes is flanked, at both the 5’ and 3’, by a target integration polynucleotide for NHEJ, having sequence homologous to the target site sequence and inverted (inverted targeting sequence), or by homology arms polynucleotides for MMEJ, having sequence of 5 to 25 base pairs (bp) in length (microhomology arms); and wherein the target gene has a coding sequence of at least 4 kb in length.

2. The kit of claim 1, wherein the target gene has a coding sequence not greater than 10 kb in length, preferably of from 4.5 kb to 9.5 kb in length.

3. The kit of claims 1 or 2, wherein each of the first and second donor nucleic acid is flanked by a 5 '-inverted terminal repeat (5'-ITR) sequence and 3 '-inverted terminal repeat (3'-ITR), said ITRs being derived from an AAV virus, preferably from an AAV of serotype selected from one or more of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 AAV9 and AAV 10 AAVSH19, AAVPHP. B, or a derivative thereof serotypes, more preferably of an AAV2 or AAV8 serotype.

4. The kit of any one of claims 1-3, wherein the target gene is a human gene selected from the group consisting of: MY07A, ABCA4, PCDH15, CACNA1F, GPR179, NBAS, CEP290, EYS, CFTR, COL4A3, COL4A4, SPG11, SPG15, FBN1, TSC2, NF1, LAMA2, FANCA, and ATM.

5. The kit of any one of claims 1-4, wherein the splicing donor signal has sequence comprising, or consisting of, SEQ ID NO: 34, or a variant thereof, and / or wherein the splicing acceptor signal has sequence comprising, or consisting of, SEQ ID NO: 35, or a variant thereof.

6. The kit of any one of claims 1-5, wherein the first donor nucleic acid comprises a degradation signal upstream the promoter, preferably a CL1 degradation signal or a variant thereof having sequence comprising or consisting of a sequence having at least 80% of identity to SEQ ID NO: 30.

7. The kit of anyone of claims 1-6, wherein the first donor nucleic acid and / or the second donor nucleic acid comprises a cassette for expressing the complementary strand oligonucleotide, preferably downstream the knock-in cassette, more preferably wherein expression of the complementary strand oligonucleotide is under the control of a human U6 promoter having sequence comprising, or consisting of, SEQ ID NO: 45, or a variant thereof.

8. The kit of any one of claims 1-7, wherein:b) the complementary strand oligonucleotide is a guide RNA (gRNA) capable of guiding the nuclease to the target site, preferably wherein the target gene and the relative target site is selected from anyone of those listed in Table 1::more preferably wherein:the target gene is human ABCA4 gene and said gRNA has sequence comprising, or consisting of, any one of sequences SEQ ID NO: 21-23, most preferably having sequence comprising, or consisting of SEQ ID NO: 22; and / or each of said first and second knock-in cassettes is flanked by a left microhomology arms having sequence selected from SEQ ID NO: 36, 38, 40, 42 and by a right microhomology arm having sequence selected from SEQ ID NO: 37, 39, 41, 43; or wherein the target gene is human CEP290 gene and said gRNA has sequence comprising, or consisting of, any one of sequences SEQ ID NO: 27-29, most preferably has sequence comprising, or consisting of SEQ ID NO: 28.

9. A vector comprising a polynucleotide encoding the first donor nucleic acid or encoding the second donor nucleic acid of claim 8.

10. The vector of claim 9 having sequence comprising any one of SEQ ID NO: 1, 3, 5, 7 or 52.

11. A viral particle comprising the vector of any of claims 9-10, preferably an AAV viral particle.

12. A pharmaceutical composition comprising the vector of anyone of claims 9-10 or the viral particle of claim 11, and suitable pharmaceutically acceptable excipients.

13. The kit of anyone of claims 1-8 or the vector of anyone of claims 9-10 or the viral particle of claim 11, or the pharmaceutical formulation of claim 12, for use as medicament, preferably for use as medicament in the treatment of a disease caused by mutation in the target gene.

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