A Mouse Model of X-Linked Chronic Granulomatous Disease for the Development of CRISPR/Cas9 Gene Therapy (2024)

2.1. Generation of CybbC517del Mice

CybbC517del mutant mice were generated by CRISPR/Cas9-mediated modification in zygotes. Two sgRNAs flanking codon 173 in exon 6 of the CYBB gene were designed using the CRISPOR tool and tested for in vitro cleavage efficiency using a PCR product spanning the target sequence. sgCybb#1 and sgCybb#2 exhibiting high cleavage efficiency were used for further experiments. Zygotes obtained from superovulated C57BL/6N females (Charles River, Sulzbach, Germany) were electroporated as described [12] with sgRNA/Cas9 RNPs complexes and a 125 nt single-stranded DNA oligonucleotide (IDT) that included the C517 deletion as a donor template. The electroporated zygotes were transferred into pseudo-pregnant NMRI females to obtain mutant founder mice. Animals were handled according to institutional guidelines under experimental procedures approved by the Landesamt für Gesundheit and Soziales (Berlin, Germany).

2.2. Genotyping of Animals

To genotype ROSA26-Cas9-CybbC517del mice for the specific point mutation in exon 6 of the CYBB gene, a 314-bp region was amplified using Cybb-for1 and Cybb-rev1 primers (Table 1) using DreamTaq Polymerase (Thermo Scientific, Darmstadt, Germany). The PCR program included an initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation, annealing (62 °C), and extension, with a final extension at 72 °C for 5 min. PCR products were verified on a 2% agarose gel before purification using the GeneJET PCR Purification Kit (Thermo Scientific). Purified PCR products were sent for Sanger sequencing with the Cybb-for1 primer.

Additionally, to confirm constitutive Cas9 expression, genomic DNA was amplified using Cas9-fw and Cas9-rev primers (Table 1), producing a 386 bp product indicative of the Cas9 expression cassette in the Rosa26 locus. This PCR followed a similar thermal-cycling protocol, with annealing at 61 °C. PCR products were analyzed on a 2% agarose gel.

2.3. Cybb Repair Template Cloning and rAAV6-Cybb Production

The repair template for the C517 deletion was generated by ligation of a 2000 bp synthetic gBlock DNA fragment (IDT) that included the exon 6 wildtype sequence and two silent nucleotide replacements into an AAV plasmid between the ITR regions of AAV2 (CellBiolabs). Recombinant AAV6-Cybb production with the coat of serotype 6 was performed as previously described [13,14,15]. Briefly, 5 million Hek293T cells were seeded per 150-mm dish in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 25 mM HEPES and grown to 80–90% confluency. The cells were co-transfected with pAAV-Helper (10 µg), pAAV6-Rep/Cap (5 µg), and AAV6-Cybb (5 µg) plasmids using polyethyleneimine (PEI). After four days, the cell pellet and supernatant were harvested and processed separately before they were pooled together. The supernatant was filtered through a 0.22 µm PES membrane and centrifuged at >3000× g for 30 min. The cleared supernatant was transferred into a new Falcon tube and incubated in a 5× solution of 40% Polyethylene Glycol 8000 (PEG)/2.5 M NaCl (final concentration 8% PEG/500 mM NaCl) overnight at 4 °C. The next day, the virus was precipitated by centrifugation at >3000× g for 30 min. The supernatant was discarded, and the virus precipitate was resuspended in 2 mL PBS/0.001% pluronic F68/200 mM NaCl and kept on ice. The cell pellet was resuspended in 2 mL PBS/0.001% pluronic F68/200 mM NaCl and lysed by three cycles of thaw-freeze on dry ice and 37 °C (10 min per cycle). After centrifugation at >3000× g for 15 min, the cleared lysate was combined with the virus resuspension from the supernatant and treated with DNA endonuclease Benzonase (50 units per mL of virus suspension) for 1.5 h at 37 °C. After cleaning by centrifugation, the virus-containing supernatant was prepared for iodixanol gradient ultracentrifugation. In an iodixanol gradient tube (Beckmann, Krefeld, Germany), the layers of gradients were overlayed by a 10 mL syringe with a long 18-gauge needle, as follows: 8 mL of 15% iodixanol, 6 mL of 25% iodixanol, 5 mL of 40% iodixanol, and 5 mL of 60% iodixanol, respectively. The translucent supernatant containing the virus was dripped slowly onto the top layer of the gradient tube using a 20-gauge needle. The tubes were centrifuged at 58,000 rpm for 2 h 10 min at 18 °C in a Beckman Type 70Ti rotor. After ultracentrifugation, the layer from the 40–60% iodixanol interface was extracted carefully via an 18-gauge needle and filtered through a 0.22 µm PES membrane. The virus solution was dialyzed overnight twice with PBS containing 0.001% F68 at 4 °C in dialysis cassettes (Thermo Scientific). Finally, the virus solution was concentrated using Amicon Ultra-15 100-kDa MWCO columns (Millipore, Darmstadt, Germany) until 500 µL⁻¹ mL of concentrated virus solution remained. The concentration of the virus was measured by real-time PCR using TaqMan probes (Table 1) specific for the left and right AAV inverted terminal repeats (ITRs).

2.4. Isolation and Culture of Mouse Sca-1⁺ Hematopoietic Stem and Progenitor Cells

The isolation of Sca-1⁺ cells was performed as described by Tran et al. [15]. Cells were isolated from the bone marrow of both ROSA26-Cas9-CybbC517del mice and wild-type littermates using the Sca1 enrichment kit according to the manufacturer’s manual (Miltenyi Biotec, Bergisch Gladbach, Germany). After isolation, the Sca-1⁺ cells were resuspended in 2 mL of serum-free HPSC medium (StemSpanTM SFEM II medium supplemented with mouse SCF (50 ng/mL), mouse TPO (50 ng/mL), mouse Flt3-L (50 ng/mL) and human IL-11 (50 ng/mL)) and seeded at a density of 2 × 10⁵ cells/mL in a well of a six-well plate.

2.5. sgRNA Electroporation and AAV Transduction

For sgRNA complex formation, crRNA and tracrRNA oligos were mixed in equimolar concentrations (5 µL of 200 µM each) in a sterile PCR tube, incubated at 95 °C for 5 min in a thermal cycler, and cooled to room temperature. Then, 48 h after isolation, Sca-1⁺ cells were collected in a 15 mL falcon tube and the total cell count was determined. The cells were washed once with PBS at room temperature, and 3 × 10⁵ cells were resuspended in 20 µL of AMAXA electroporation buffer. To this suspension, 1.2 µL (120 pmol) of the hybridized cr:tracrRNA complex was added. The final electroporation mixture was then carefully transferred to a well of a 16-well Nucleocuvette strip. Electroporation was conducted using the AMAXA 4D-NucleofectorTM (Lonza), following the program specified for ‘mouse B cells’ [15]. Immediately afterwards, 80 µL of pre-warmed complete HSPCs medium was added to each well. The cells were then transferred to a well in a six-well plate containing 1.5 mL of the same medium. Thirty minutes later, the cells were transduced with AAV6-Cybb at a multiplicity of infection (MOI) of 1 × 10⁶ genome copies per cell. Two days after electroporation, the cells were initiated into macrophage differentiation.

2.6. Differentiation of HSPCs into Mature Macrophages

Mouse HSPCs were cultured in serum-free HSPC medium (StemSpanTM SFEM II medium supplemented with mouse SCF (50 ng/mL), mouse TPO (50 ng/mL), mouse Flt3-L (50 ng/mL) and human IL-11 (50 ng/mL)) for four days after isolation. At day four, the medium was replaced with a macrophage-differentiation medium supplemented with GM-CSF (20 ng/mL). The cells were then cultured for an additional 14 days in this complete macrophage medium. Mature macrophages were identified by flow cytometry using APC anti-mouse F4/80 (Rat IgG2a, κ, Clone: BM8).

2.7. Off-Target Analysis by T7 Endonuclease I (T7EI) Cleavage Assay

Off-target sites for sgRNA-Cybb were selected using CRISPOR (http://crispor.tefor.net/ (accessed on 14 May 2021)). Wizard Genomic DNA Purification Kit (Promega, Walldorf, Germany) following the manufacturer’s instructions. For the on-target site, PCR was done using PrimeStar GXL (Takara, Mountain View, CA, USA) with Cybb-geno-for and Cybb-geno-rev primers (Table 1) using the following conditions: 98 °C for 2 min, 30 cycles (98 °C for 10 s, 55.1 °C for 10 s, 68 °C for 50 s), and 68 °C for 10 min. For the off-target sites, PCR was done using Q5^® High-Fidelity DNA Polymerase (NEB) with site-specific primers (Table 1) using the following conditions: 98 °C for 30 s, 35 cycles (98 °C for 10 s, 56.2 °C (for Actn1 site)/62 °C (for Chd5, Tmem82 and K23Rik sites)/60 °C (for Chchd7, Tceb3 and Qdpr sites) for 30 s, 72 °C for 30 s), and 72 °C for 2 min. PCR amplicons were purified using GeneJET PCR Purification Kit (ThermoFischer Scientific).

The purified PCR products were denatured and annealed in NEBuffer 2 (NEB) in a thermocycler using the following conditions: denaturation at 95 °C for 5 min, re-annealing at 95–85 °C (−2 °C/s) and 85–25 °C (−0.1 °C/s). For the T7EI assay, 1000 ng of the heterocomplex PCR product were digested with T7EI (NEB) according to the manufacturer’s instructions. Cleaved DNA fragments were separated on 2% agarose gels, and the DNA concentration of each band was quantified by the ImageJ version 1.53t software. The percentages of HDR and NEHJ rates was calculated as previously described.

2.8. On- and Off-Target Analysis by Amplicon Sequencing

The quantification of on- and off-target editing frequency was performed by Illumina amplicon sequencing via Genewiz (Amplicon EZ; GENEWIZ Germany GmbH, Leipzig, Germany). For this purpose, genomic DNA (gDNA) was extracted from targeted and control cells using the Wizard Genomic DNA Purification Kit (Promega) following the manufacturer’s protocol.

For the on-target analysis, an outer PCR reaction (2.2 kb) was performed using primers (Table 1) located outside the hom*ology arms, while a second PCR reaction was performed using primers amplifying a shorter PCR product of 274 bp. For the first PCR reaction, primers Cybb-geno-for and Cybb-geno-rev (Table 1) were used along with PrimeStar GXL DNA polymerase (Takara) and 200 ng DNA per reaction under the following conditions: initial denaturation at 98 °C for 2 min, followed by 30 cycles (98 °C for 10 s, 55.1 °C for 10 s, 68 °C for 50 s), and final extension at 68 °C for 10 min. The second PCR amplification was conducted with Cybb-Amplicon-fow and Cybb-Amplicon-rev (Table 1), Q5 High Fidelity DNA-Polymerase (NEB), and 20 ng of purified 1^st PCR product under the following conditions: initial denaturation at 98 °C for 30 s, 30 cycles (98 °C for 10 s, 61 °C for 30 s, 72 °C for 20 s), and final extension 72 °C for 2 min.

For the off-target sites, PCR was done using Q5^® High-Fidelity DNA Polymerase (NEB) with site-specific primers (Table 1) using the following conditions: 98 °C for 30 s, 35 cycles (98 °C for 10 s, 56.2 °C (for Actn1 site)/62 °C (for Chd5, Tmem82 and K23Rik sites)/60 °C (for Chchd7, Tceb3 and Qdpr sites) for 30 s, 72 °C for 30 s), and 72 °C for 2 min. The PCR products were purified on columns using the GeneJET PCR Purification Kit (Thermo Scientific, #K0702). Next, 2 µg of each purified PCR reaction was submitted for deep sequencing.

A custom amplicon-sequencing analysis pipeline licensed from Bioinformatics Expert UG (Berlin, Germany) was employed for data analysis. This pipeline requires the raw data fastq files for paired reads and the reference sequence provided as a fasta file. The core functionalities of the pipeline were implemented using R (version 3.6.0). The alignment algorithm utilized the “pairwiseAlignment()” function from the R package Biostrings (version 2.52.0). Initially, paired reads were aggregated and cleaned to minimize potential sequencing errors. The process for each read pair included the following steps. First, the R2 read was converted to its reverse complement and the quality score was reversed. Second, the R1 and R2 reads were locally aligned to identify the overlapping region, using high penalty values for gap opening and extension to prevent gaps that could misalign bases during aggregation. Third, the R1 read and the reverse complement of the R2 read (rcR2) were stitched together at the overlap, with the base with the highest quality score selected at positions with conflicts and missing bases filled with “N” at the ends. Fourth, the initial 15 bases from the 5′ and 3′ ends of the reference sequence were aligned locally to the aggregated sequence to determine the sequence boundaries, allowing for two mismatches or indels. Bases outside the aligned boundaries were trimmed, resulting in a final aggregated sequence for each read that begins and ends with the reference sequence boundaries. In the second phase of the pipeline, all aggregated reads were globally aligned to the reference sequence using alignment reward/penalty scores (match = 1, mismatch = 0, gapOpen = −2, gapExtension = 0). These scores favored long gaps over small gaps and mismatches. Finally, all unique aggregated sequences were sorted and counted.

2.9. Western Blot Analysis

Western blot analysis was performed as described previously [16]. Cell lysis was performed in RIPA buffer supplemented with a protease-inhibitor co*cktail (Roche, Basel, Switzerland). Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Life Technologies, Darmstadt, Germany) following the manufacturer’s protocol. Proteins were separated in Mini-PROTEAN TGX Stain-Free Protein Gels, transferred to nitrocellulose membrane, and blocked in 5% milk/TBS-T for 1 h at room temperature. Membranes were incubated with appropriate antibodies (mouse anti-gp91^phox monoclonal antibody 1:2000, anti-ß-actin antibody 1:5000) overnight at 4 °C. Following incubation with the primary antibody, membranes were incubated with corresponding secondary antibodies for 1 h at room temperature and developed using the Pierce ECL Western blotting Substrate Kit.

2.10. DHR123 Functional Assay

The DHR123 functional assay, adapted from Bustamante et al. [2], was employed to measure ROS production in mature macrophages and neutrophils. Initially, cells were stimulated with phorbol 12-myristate 13-acetate (PMA) at a final concentration of 0.065 µM and incubated at 37 °C for 2 h. Following this, cells were incubated with dihydrororhodamine 123 (20 µM, Sigma, Setagaya City, Tokyo) in the dark at 37 °C for 5 min, with catalase (1300 IU/mL) included to regulate the reaction. After dihydrororhodamine 123 (DHR123) incubation, the cells were washed thrice with PBS to ensure the removal of excess dye. Accutase (500 µL) was then added to detach the cells, and after a 5-min incubation at 37 °C, the reaction was stopped by adding 500 µL of FACS buffer (PBS &1% BSA). The cell suspension was transferred to a microcentrifuge tube and centrifuged at 1000× g for 9 min to pellet the cells. The supernatant was discarded, and the cells were resuspended in 250 µL of FACS buffer for cell counting. For the identification of macrophages, cells were incubated with 0.25 µg of APC-conjugated anti-mouse F4/80 antibody per 10⁶ cells for 30 min on ice. After the incubation, cells were washed thrice with PBS and fixed in 4% paraformaldehyde (PFA) for 20 min on ice. Following fixation, cells were washed three times with PBS and finally resuspended in 400 µL of FACS buffer. The prepared samples were stored on ice in the dark until they were subjected to immediate analysis by flow cytometry.

2.11. Statistical Analysis

For all experiments, data are shown as mean ± SD if not stated otherwise. Statistical analysis was performed using GraphPad Prism 9.4 (GraphPad software, Boston, MA, USA). Sample sizes and the statistical tests used are described in the figure legends. A p value of less than 0.05 was considered statistically significant.

3.1. Generation of a Mouse Model Carrying a Patient-Specific Cybb Mutation

In order to create a mouse model for X-linked chronic granulomatous disease (X-CGD), we generated an edited mouse line that carries a patient-specific Cybb mutation. The specific mutation is a deletion of a single nucleotide (C517) in exon 6 of the CYBB gene, which results in a frameshift beyond codon 173 and Cybb deficiency (Figure 1A). To delete this single nucleotide using CRISPR/Cas9, we selected two guide RNAs (sgCybb#1 and sgCybb#2) targeting the exon 6 region (Figure 1A). The activity of both guide RNAs was assessed using a Cas9 in vitro cleavage assay (Figure 1B). Recombinant Cas9 nuclease was incubated with the candidate crRNAs (crCybb#1 and crCybb#2), tracrRNA, and a PCR product (272 bp) amplified from the CYBB target DNA sequence. The resulting digestion products were analyzed by agarose gel electrophoresis to confirm the cleavage of the PCR product. The results demonstrated the activity of crCybb#1 and crCybb#2, as shown by the appearance of the predicted cleavage products (~130, ~141 bp) upon incubation with tracrRNA and Cas9 protein (Figure 1B).

For production of the mutant CYBB allele, zygotes isolated from C57BL/6 mice were electroporated with Cas9 protein, one of the validated guide RNAs (sgCybb#1 or sgCybb#2), and a modified single-stranded DNA oligonucleotide (ODN-Cybb#1 with sgCybb#1 or ODN-Cybb#2 with sgCybb#2) (Figure 1C). The DNA oligonucleotide had hom*ology sequences flanking the targeted codon but lacked nucleotide C517 (Figure 1A,C). The induction of a double-strand break (DSB) near C517 allowed editing by hom*ologous recombination using ODN-Cybb as a donor template, leading to the introduction of the single-nucleotide deletion into the coding region of CYBB. From the electroporation of C57BL/6 mouse zygotes, we obtained nine pups, which were genotyped by PCR amplification using the primer pair Cybb_F1/Cybb_R1 and sequencing of the CYBB exon 6 region. Among these offspring, we identified seven founder mutants carrying random indels, while two mice exhibited only the desired deletion of C517 (Figure 1C, #8326 and #8327).

Since the CYBB gene is located on the X chromosome, Cybb-deficient males are hemizygous for the CybbC517del allele. Since both of the founders were males, the sequence reads of the PCR product could be easily compared to the wildtype reference, confirming the deletion of C517 (Figure 1C, #8327). In the mutant allele, the single-nucleotide deletion caused a reading frameshift, leading to a stretch of 15 missense codons followed by a stop codon.

Founder male #8327 was subsequently mated to C57BL/6 wildtype females to establish the CybbC517del mouse line. Approximately half of the offspring from this founder carried the C517del allele, therefore confirming germline transmission (Figure 1D). The CybbC517del mouse line is maintained in a heterozygous breeding scheme and housed in specified pathogen-free environment. The mutant mice are healthy and do not display a disease phenotype due to their selective immunodeficiency.

3.2. C517 Deletion Leads to Impaired gp91^phox Expression in Mature Macrophages

CYBB encodes the β-chain of flavocytochrome b558, also known as gp91^phox or NOX2, which is a crucial component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex found in phagocytes such as granulocytes, monocytes, and macrophages. While its expression is lower in other cell types like dendritic cells and B lymphocytes [2], disruptions in the CYBB gene commonly result in the loss of gp91^phox expression due to mRNA or protein instability. This defect is often associated with frameshift and early termination of protein synthesis [9]. The CybbC517del mouse line carries a patient-specific mutation that causes a frameshift in codon 173 and leads to premature termination following 15 missense codons.

To validate the expected phenotype of the CybbC517del mouse line, we investigated gp91^phox expression in in vitro-derived macrophages obtained from Sca1⁺ bone-marrow cells isolated from CybbC517del mutant mice (Figure 2A). In the wild-type control littermates, a specific gp91^phox band at approximately 58 kDa was observed, while this band was absent in the mutant sample Figure 2C,D). The absence of the signal in the mutant sample indicates that the C517 deletion leads to impaired expression of gp91^phox.

Furthermore, we investigated potential defects in the differentiation of hematopoietic stem and progenitor cells (HSPCs) resulting from the C517 deletion. To assess this outcome, we quantified and compared the population of F4/80-expressing macrophages in the mature cell pool derived from wild-type littermates and mutant HSPCs using flow cytometry. FACS analysis revealed similar quantities of mature macrophages in mutant and control (wild-type) samples (Figure 2B). As expected, these findings suggest that NADPH oxidase does not play a significant role in the development of macrophages.

3.3. C517 Deletion Leads to Impaired NADPH Oxidase Activity in Mature Macrophages

The phagocytic respiratory burst is a critical defense mechanism employed by the host to eliminate ingested microorganisms. It involves a series of reactions in which activated NADPH oxidase transfers electrons from cytosolic NADPH to external O₂, resulting in the generation of superoxide anions (O₂⁻) [8]. These superoxide anions are further metabolized into potent antimicrobial oxidants, including hydrogen peroxide (H₂O₂), hydroxyl anions (OH⁻), hypochlorous acid (HOCl), and nitryl chloride (NO₂Cl) [8]. In X-CGD patients, phagocytes are unable to generate the respiratory burst due to impaired NADPH oxidase activity.

In this study, we examined the respiratory-burst activity of mature macrophages derived from HSPCs of CybbC517del mice using dihydrorhodamine 123 (DHR123) and flow cytometry. DHR123 is a non-fluorescent dye that can be oxidized to fluorescent rhodamine 123 (R123) by hydrogen peroxide, a byproduct of the respiratory burst [17].

Mature macrophages were obtained from HSPCs of CybbC517del mice, as previously described (Figure 2A). These cells were stimulated with phorbol 12-myristate 13-acetate (PMA) in the presence of catalase, and the production of H₂O₂ was assessed by measuring the levels of rhodamine 123 (R123).

Control macrophages derived from wild-type littermates displayed robust respiratory-burst activity, with over 30% of stimulated macrophages showing normal H₂O₂ production, as indicated by detectable signals of rhodamine 123 (Figure 2E). In contrast, macrophages derived from CybbC517del mutant mice failed to generate a respiratory burst, with nearly no macrophages displaying any detectable rhodamine 123 signals (Figure 2E). The absence of H₂O₂ production in mutant macrophages indicates impaired NADPH oxidase activity resulting from the C517 deletion. These findings confirm that the expected outcome of the patient’s mutation is consistent with our observations of gp91^phox protein expression in mutant macrophages and validate the new mouse line as a model recapitulating the disease phenotype.

3.4. CRISPR/Cas9-Mediated Gene Correction of C517 Deletion in Mouse HSPCs

To correct the C517del mutation in mouse HSPCs, we employed CRISPR-mediated hom*ology-directed repair (HDR). For this purpose, we designed a repair template consisting of the wild-type coding sequence and hom*ology arms of at least 650 bp on both sides of the targeted codon. In addition to the reintroduced missing nucleotide at position 517, the repair template also contained two silent replacements in the guide RNA and PAM sequence to prevent recurrent double-strand breaks (DSBs) in the corrected allele. To target the C517del locus, we modified the guide sequence of sgCybb#2 by extending it by one nucleotide in the 5′ direction, resulting in a gRNA termed sgCybb#3. This modification ensured that sgCybb#3 would bind with 100% complementarity to the CYBB mutant allele. To test our strategy, we conducted the gene-editing experiment using HSPCs from CybbC517del mice with constitutive Cas9 expression. To achieve this, we crossed CybbC517del mice with ROSA26-Cas9 transgenic mice, which express Cas9 ubiquitously from the ROSA26 locus (Figure 3A).

To assess HDR efficiency, we isolated Sca1⁺ HSPCs from ROSA26-Cas9- CybbC517del mice, activated them with mouse SCF, TPO, Flt3L, and human IL-11 for 2 days, and then performed electroporation with Cas9 and sgCybb#3. The pretreated HSPCs were subsequently infected with recombinant AAV-6 viral vectors carrying the repair templates (AAV-Cybb) at a multiplicity of infection (MOI) of 1,000,000 viral genome copies (GCs) per cell. The targeted HSPCs were then differentiated into macrophages in the presence of GM-CSF for the next 20 days (Figure 3B). After targeting and differentiation, we quantitatively evaluated the HDR efficiency of the edited mature macrophages by analyzing the recovery of gp91^phox using western blot analysis (Figure 3C). In the experimental groups receiving both sgRNA and the AAV-6 repair template (HDR), we observed a 16% recovery of gp91^phox expression. In contrast, untreated cells and cells receiving only the AAV-6 repair template (donor control) did not exhibit expression of the gp91^phox protein (Figure 3C,D). Furthermore, cells treated with both sgRNA and the AAV-6 repair template (HDR) showed improved respiratory-burst activity, with 7% of stimulated macrophages displaying normal H₂O₂ production, compared to 2% in untreated cells and donor control-treated cells (Figure 3E,F).

To genetically confirm the correction events, we amplified the target region from genomic DNA of wild-type, untreated, and treated cells and quantified the editing events through amplicon sequencing (Figure 4D). In cells treated with both sgRNA and the AAV-6 repair template, we observed a 19% HDR correction of the pathogenic mutation (Figure 4E). In comparison, untreated cells and cells treated with sgRNA alone did not show any correction (Figure 4E). The frequencies of indels were high, reaching 77% in cells treated with sgRNA alone and 94% in cells treated with sgRNA and the repair template (Figure 4E). These results collectively demonstrate that our HDR strategy successfully corrects the patient-derived C517 deletion in primary HSPCs and partially rescues the molecular and phenotypic consequences of the mutation.

3.5. Off-Target Analysis in Edited HSPCs

To assess the safety and specificity of our gene-editing approach, we performed an off-target analysis in edited HSPCs. Off-target effects refer to unintended modifications occurring at genomic sites other than the intended target locus. Using the online tool CRISPOR (https://crispor.tefor.net/ (accessed on 14 May 2021)), we identified a list of 7 potential off-target sites that exhibit moderate sequence similarity to the target site, with 3–4 mismatches (Figure 4A). Notably, all of these off-target sites were located within exonic regions of genes. The presence of off-target sites in exonic regions raises specific concerns, as modifications within these regions have a higher likelihood of affecting gene function and potentially leading to unintended consequences. Therefore, our primary focus was directed towards evaluating the potential off-target effects within exonic sequences.

To assess the presence of off-target effects, we amplified all seven off-target sites in sgCybb#3 in the targeted HSPCs. Initially, we estimated the indel frequencies at both the on-target and off-target sites using T7 endonuclease I (T7EI) assay (Figure 4B). The results of this assay revealed an indel frequency of 31% at the on-target site, while none of the off-target sites led to a detectable cleavage product, suggesting that non-hom*ologous end joining (NHEJ) was not evident at these off-target sites (Figure 4C). To validate these data and obtain a more comprehensive analysis, we performed amplicon sequencing of three selected off-target and on-target sites in wild type, untreated, and treated cells (Figure 4D). Consistent with the results of the T7EI assay, the amplicon-sequencing data demonstrated no detectable off-target mutations or indels at the predicted off-target sites in the edited HSPCs (Figure 4E). These data suggest that our gene-editing approach exhibited high specificity and demonstrated low off-target activity. Notably, the indel frequency at the on-target site observed in the amplicon sequencing data was approximately three-fold higher in the edited samples compared to the indel frequency obtained from the T7EI assay (Figure 4E). This difference in indel frequencies can be attributed to the different sensitivities and methodologies employed by the two assays. This highlights the importance of employing multiple complementary approaches to thoroughly evaluate potential off-target effects and ensure the accuracy of the findings.