Precise CRISPR/Cas9 and Cas12 Correction Using Lipoplexes in Retinal Models Derived from Patients with Inherited Retinal Dystrophies

Most therapeutic strategies currently under investigation for inherited retinal dystrophies are gene- or mutation-specific, which limits their broad applicability.

“Although several therapeutic strategies are currently under investigation, most are gene- or mutation-specific, which limits their broad applicability [5,6].”

• cites [5] Georgiadis, 2023, Genetic treatment for autosomal dominant inherited retinal dystrophies: Approaches, challenges and targeted genotypes, Br. J. Ophthalmol. sourcePartly supported

  The Gene-independent approaches section says mutation-specific therapies may be time- or economically inefficient, and contrasts them with approaches that could apply to many genes. This supports the concern about limited applicability, but the paper focuses on autosomal dominant inherited retinal dystrophies and does not establish that most strategies under investigation across all inherited retinal dystrophies are gene- or mutation-specific. No page number is available in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [6] Hu, 2021, Gene therapy for inherited retinal diseases: Progress and possibilities, Clin. Exp. Optom. sourceNot checked — no full text of the source

In this study, several gene-edited clones exhibited cleavage of the wild-type allele followed by repair using the sister chromatid rather than the exogenous ssODN.

“In this study, several gene-edited clones exhibited cleavage the wild-type allele followed by repair using the sister chromatid rather than the exogenous ssODN, as reported previously [36].”

• cites [36] Siles, 2025, Rescue of the disease-associated phenotype in CRISPR-corrected hiPSCs as a therapeutic approach for inherited retinal dystrophies, Mol. Ther. Nucleic Acids sourceSupported

On-target defects were significantly lower than those obtained in hiPSCs, ranging from 2% to 14.29%, compared with nearly 40% reported in hiPSCs.

“Importantly, on-target defects were significantly lower than those obtained in hiPSCs, ranging from 2% to 14.29%, compared with nearly 40% reported in hiPSCs [36].”

• cites [36] Siles, 2025, Rescue of the disease-associated phenotype in CRISPR-corrected hiPSCs as a therapeutic approach for inherited retinal dystrophies, Mol. Ther. Nucleic Acids sourcePartly supported

  The Results section (page unavailable) reports on-target aberrations below 40% in all assays, which supports the claim's approximate hiPSC benchmark. It does not report a 2%–14.29% range or a statistically significant comparison with another group; the assays described were themselves conducted in hiPSCs. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

An HDR enhancer and NHEJ inhibitor were used to improve homology-directed repair and efficient genome editing, particularly for precise single-nucleotide modifications.

“An HDR enhancer and NHEJ inhibitor were used to improve homology-directed repair and efficient genome editing, particularly for precise single-nucleotide modifications (Figure 4A) [45,46].”

• cites [45] Azhagiri, 2021, Homology-directed gene-editing approaches for hematopoietic stem and progenitor cell gene therapy, Stem Cell Res. Ther. sourcePartly supported

  The ‘Inhibition of competitive NHEJ pathway’ section reports that NHEJ inhibitors improve HDR and that a mixture including HDR-promoting molecules increased HDR in iPSCs. The ‘HDR gene editing’ section says HDR templates are tested for single-nucleotide changes. The paper does not establish that this enhancer–inhibitor approach was used particularly to improve precise single-nucleotide modifications. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [46] Yu, 2015, Small molecules enhance crispr genome editing in pluripotent stem cells, Cell Stem Cell sourcePartly supported

  In the passage on SNP editing (indexed under Abstract; page unavailable), L755507 increased precise A4V single-nucleotide editing in human iPS cells almost ninefold and reduced indel mutations. The paper does not report using a separate NHEJ inhibitor alongside an HDR enhancer; it describes L755507 as an HDR enhancer that also reduced indels. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

The gene editing outcomes in Experiment A are consistent with previous reports in hiPSCs using the same sgRNA, which showed cleavage of both mutant and wild-type alleles.

“These results are consistent with previous reports in hiPSCs using the same sgRNA, which showed cleavage of both mutant and wild-type alleles [36].”

• cites [36] Siles, 2025, Rescue of the disease-associated phenotype in CRISPR-corrected hiPSCs as a therapeutic approach for inherited retinal dystrophies, Mol. Ther. Nucleic Acids sourcePartly supported

  The Results section (‘Precise HDR correction of pathogenic variants by using the endogenous sister chromatid’; page unavailable) reports editing consistent with cleavage of both mutant and wild-type alleles in hiPSCs, particularly with sgRNAs having one mismatch to the wild-type allele. The claim that the prior report used the same sgRNA as Experiment A cannot be verified from the supplied claim and our copy, which do not identify Experiment A’s guide. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Edited RPE cells preserved normal morphology.

“Edited RPE cells preserved normal morphology and expressed specific maturity markers.”

Edited RPE cells expressed specific maturity markers.

“Edited RPE cells preserved normal morphology and expressed specific maturity markers.”

Gene editing levels remained low in retinal organoids.

“Remarkably, we observed efficient Lipofectamine-mediated transfection and sgRNA-induced DNA cleavage in hiPSC-RPE cells, whereas gene editing levels remained low in RO, highlighting the need for improved delivery strategies.”

Retinal organoids showed no detectable CRISPR/Cas-induced DNA cleavage.

“In contrast, retinal organoids exhibited moderate transfection efficiency but showed no detectable CRISPR/Cas-induced DNA cleavage, highlighting the need for further optimization of gene editing in more complex cellular tissues.”

Cas12 yielded the highest HDR correction efficiency, exceeding 10% of HDR correction, for the pathogenic BEST1 variant in hiPS-derived RPE.

“Precise repair of the pathogenic BEST1 variant was successfully achieved in hiPS-derived RPE cells using both nucleases, with Cas12 yielding the highest efficiency, exceeding 10% of HDR correction.”

Precise correction of a heterozygous single-nucleotide mutation in hiPS-derived RPE from a patient with Best disease was achieved using both CRISPR/Cas9 and Cas12.

“Finally, we achieved precise correction of a heterozygous single-nucleotide mutation in hiPS-derived RPE from a patient with Best disease using both CRISPR/Cas9 and Cas12 systems.”

Retinal organoids exhibited moderate transfection efficiency.

“In contrast, retinal organoids exhibited moderate transfection efficiency but showed no detectable CRISPR/Cas-induced DNA cleavage, highlighting the need for further optimization of gene editing in more complex cellular tissues.”

Improved HDR correction was observed when using Cas12 nuclease compared to Cas9.

“Consistent with these advantages, we observed increased DNA cleavage efficiency, improved HDR correction, and reduced on- and off-target effects when using Cas12 nuclease compared to Cas9.”

hiPS-RO exhibited pronounced disorganization of cellular layers and signs of apoptosis after electroporation.

“In contrast, hiPS-RO, which are highly structured three-dimensional models, exhibited pronounced disorganization of cellular layers and signs of apoptosis after electroporation (Figure 2A).”

In the CRISPRMAX assay, only 2.63% harbored the pathogenic variant in homozygosis.

“whereas only 2.63% harbored the pathogenic variant in homozygosis (Figure 4F).”

In Experiment B, 5.88% of clones were repaired via the sister chromatid.

“with 5.88% repaired via the sister chromatid”

Experiment B yielded 15.68% (8/51) gene-edited clones.

“Surprisingly, this approach yielded 15.68% gene-edited clones (8/51).”

hiPS-RPE and immature RO showed approximately 30% of GFP+ cells after transfection with Lipofectamine 3000.

“In turn, hiPS-RPE and immature RO showed approximately 30% of GFP+ cells after transfection with Lipofectamine 3000 (Figure 1C,D).”

Conservation of OS integrity was observed four days after treatment using Lipofectamine Stem and CRISPRMAX.

“Remarkably, we observed conservation of OS integrity four days after treatment using Lipofectamine Stem and CRISPRMAX (Figure 2B).”

hiPS-RPE cells exhibited considerable DNA cleavage, reaching the highest levels using Cas12.

“We showed that hiPS-RPE cells exhibited considerable DNA cleavage, reaching the highest levels using Cas12.”

Lipoplex-mediated delivery achieved a superior balance between transfection efficiency, cellular integrity, and DNA cleavage compared to electroporation.

“Moreover, while electroporation produced high overall transfection rates, lipoplex-mediated delivery achieved a superior balance between transfection efficiency, cellular integrity, and DNA cleavage.”

Of the 24% gene editing with Lipofectamine Stem, 10% were clones with the pathogenic variant properly corrected.

“from which the 10% were clones with the pathogenic variant properly corrected”

In Experiment B, the other half of gene-edited clones displayed the BEST1 mutation and PAM modification in heterozygosis.

“The other half displayed the BEST1 mutation and PAM modification in heterozygosis”

The 7.84% of corrected clones in Experiment B was comparable to the proportion of clones carrying the homozygous mutation in Experiment A, which were repaired using the sister chromatid.

“Notably, the 7.84% of corrected clones in Experiment B was comparable to the proportion of clones carrying the homozygous mutation in Experiment A, which were repaired using the sister chromatid (Figure 4D).”

In all gene editing assays, the on-target abnormalities were below 15% of the screened clones.

“Notably, in all gene editing assays, the on-target abnormalities were below 15% of the screened clones.”

Homologous regions prediction identified only two regions with less than three mismatches for sgBEST1_4 and one for sgBEST1-Cas12_3 besides the target sequence itself.

“Regarding off-target analysis, homologous regions prediction identified only two regions with less than three mismatches for sgBEST1_4 and one for sgBEST1-Cas12_3 besides to the target sequence itself (Table S2).”

For ABCA4, Cas9 and Cas12 exhibited comparable efficiencies, ranging between 10% and 20%.

“In this case, Cas9 and Cas12 exhibited comparable efficiencies, ranging between 10% and 20% (Figure 3E,F).”

Analysis of sgRNA-mediated DNA cleavage rendered similar results (10-15%) for Cas9 targeting both the BEST1 and ABCA4 pathogenic variants, independent of the delivery method.

“The analysis of sgRNA-mediated DNA cleavage rendered similar results (10–15%) for Cas9 targeting both the BEST1 and ABCA4 pathogenic variants, independent of the delivery method (Neon electroporation or Lipofectamine) (Figure 3C,E).”

A pathogenic BEST1 variant was successfully corrected.

“and successfully corrected a pathogenic BEST1 variant”

Differentiated retinal cells exhibited moderate levels of GFP+ cells compared with undifferentiated hiPSCs.

“Differentiated retinal cells exhibited moderate levels of GFP+ cells compared with undifferentiated hiPSCs, which reached nearly 90% fluorescence following electroporation and more than 50% after lipofection (Figure 1C,D).”

Control transfections using either sgRNA or Cas nuclease alone did not generate detectable DNA DSBs.

“As expected, control transfections using either sgRNA or Cas nuclease alone did not generate detectable DNA DSBs (Figure S1C).”

Precise correction of the BEST1 c.229C>T mutation was achieved using CRISPR/Cas9 and Cas12, achieving approximately 10% gene correction in hiPS-RPE via lipoplex-mediated delivery.

“Here we show the precise correction of the BEST1 c.229C>T mutation using CRISPR/Cas9 and Cas12, achieving approximately 10% gene correction in hiPS-RPE via lipoplex-mediated delivery.”

Substantial DNA cleavage was achieved with both Cas9 and Cas12 nucleases in hiPS-RPE.

“Moreover, we achieved substantial DNA cleavage with both Cas9 and Cas12 nucleases in hiPS-RPE”

Cas12 yielded the highest efficiency, above 10% of properly repaired clones.

“with Cas12 yielding the highest efficiency, above 10% of properly repaired clones.”

DNA cut in BEST1 was higher using Cas12 than using Cas9 with the guides used.

“Overall, DNA cut in BEST1 was higher using Cas12 than using Cas9 with the guides used (Figure 3C,D).”

In Experiment A, 7.14% of clones carried the heterozygous BEST1 mutation in homozygosis instead of corrected.

“with 7.14% carrying the heterozygous BEST1 mutation in homozygosis instead of corrected”

Electroporation did not improve transfection efficiency compared with Lipofectamine-based transfection in hiPS-RO.

“Notably, electroporation did not improve transfection efficiency compared with Lipofectamine-based transfection in hiPS-RO (Figure 1D).”

Transfection efficiency in mature retinal organoids was markedly lower, with GFP expression detected only in limited areas following Lipofectamine Stem treatment.

“Unfortunately, transfection efficiency in mature retinal organoids was markedly lower, with GFP expression detected only in limited areas following Lipofectamine Stem treatment (Figure 1C,D).”

Comparable sgRNA-mediated cleavage efficiencies were observed using CRISPR/Cas12 across all delivery methods tested.

“Similarly, comparable sgRNA-mediated cleavage efficiencies were observed using CRISPR/Cas12 across all delivery methods tested (Figure 2D and Figure S1B).”

In Experiment B, 7.84% of clones exhibited pathogenic variant correction.

“Among these, 7.84% exhibited pathogenic variant correction”

hiPS-RO preserved their morphology after treatment with Lipofectamine, particularly following CRISPRMAX delivery, maintaining a conserved structure comparable to untreated RO.

“Conversely, they preserved their morphology after treatment with Lipofectamine, particularly following CRISPRMAX delivery, maintaining a conserved structure comparable to untreated RO (Figure 2A).”

hiPS-RPE did not display significant alterations in their appearance or behavior following any of the transfection methodologies tested, compared with untreated controls.

“hiPS-RPE did not display significant alterations in their appearance or behavior following any of the transfection methodologies tested, compared with untreated controls (Figure 2A).”

The highest gene-editing efficiencies were observed with Lipofectamine Stem, in line with the highest percentages of DNA cleavage, compared to CRISPRMAX.

“The highest gene-editing efficiencies were observed with Lipofectamine Stem, in line with the highest percentages of DNA cleavage, compared to CRISPRMAX (Figure 4F).”

No off-target effects were found in edited clones, except in one clone in which a single-nucleotide change was observed in the experiment performed using Cas9.

“Notably, we found no off-target effects in edited clones, except in one clone in which we observed a single-nucleotide change in the experiment performed using Cas9 (Table 2 and Figure S2B,C).”

In Experiment B, 1.96% of clones incorporated the ssODN-mediated PAM modification.

“and 1.96% incorporating the ssODN-mediated PAM modification”

Cas12-mediated cleavage exhibited more variable efficiencies, depending on both the delivery system and the sgRNA used, ranging from 5% to 30%.

“In contrast, Cas12-mediated cleavage exhibited more variable efficiencies, depending on both the delivery system and the sgRNA used, ranging from 5% to 30%.”

In Experiment A, 10.71% (3/28) of clones were gene-edited.

“Experiment A yielded a total of 10.71% gene-edited clones (3/28)”

In Experiment A, no indels in the mutant allele were observed.

“However, no indels in the mutant allele were observed.”

In Experiment A, 3.57% of clones harbored both the mutation and PAM modification in heterozygosis.

“and a 3.57% harboring both the mutation and PAM modification in heterozygosis”

The best results regarding on-target effects were obtained with CRISPR/Cas12 using Lipofectamine Stem similarly to Experiment B performed with Cas9.

“Additionally, the best results regarding on-target effects were obtained with CRISPR/Cas12 using Lipofectamine Stem similarly to Experiment B performed with Cas9 (Figure 4H).”

For Cas9 nuclease, the highest HPRT cleavage was obtained using Lipofectamine Stem, reaching almost 30%, slightly lower than that obtained in undifferentiated hiPSCs.

“For Cas9 nuclease, the highest HPRT cleavage was obtained using Lipofectamine Stem reaching almost 30%, slightly lower than that obtained in undifferentiated hiPSCs (Figure 2C and Figure S1A).”

The two Cas12 guides that yielded the highest cleavage results for each pathogenic variant shared the same TTG PAM sequence.

“Interestingly, these two Cas12 guides that yielded the highest cleavage results for each pathogenic variant (sgBEST1-Cas12_3 and sgABCA4-Cas12_1) shared the same TTG PAM sequence, suggesting a better affinity of Cas12 to this motif in our assay (Table 1).”

A maximum of 24% gene editing was reached using Lipofectamine Stem.

“Specifically, we reached a maximum of 24% of gene editing using Lipofectamine Stem”

Very low or undetectable levels of cleavage were observed in hiPS-RO using either Cas9 or Cas12.

“Unfortunately, very low or undetectable levels of cleavage were observed in hiPS-RO using either Cas9 or Cas12 (Figure 2C–E).”

Increased DNA cleavage efficiency was observed when using Cas12 nuclease compared to Cas9.

“Consistent with these advantages, we observed increased DNA cleavage efficiency, improved HDR correction, and reduced on- and off-target effects when using Cas12 nuclease compared to Cas9.”

In hiPSC-derived RPE cells, both Cas9 and Cas12 nucleases induced significant levels of DNA DSBs, with cleavage efficiencies ranging from approximately 15% to 30% following Lipofectamine-based delivery, comparable to those achieved by electroporation.

“In hiPSC-derived RPE cells, both nucleases induced significant levels of DNA DSBs, with cleavage efficiencies ranging from approximately 15% to 30% following Lipofectamine-based delivery, comparable to those achieved by electroporation (Figure 2C,D).”

Robust DNA cleavage was demonstrated using both Cas9 and Cas12 nucleases.

“We demonstrate robust DNA cleavage using both Cas9 and Cas12 nucleases”

Reduced on- and off-target effects were observed when using Cas12 nuclease compared to Cas9.

“Consistent with these advantages, we observed increased DNA cleavage efficiency, improved HDR correction, and reduced on- and off-target effects when using Cas12 nuclease compared to Cas9.”

Successful HDR-mediated correction of an autosomal dominant pathogenic variant in RPE cells causing Best disease was reported.

“and report successful HDR-mediated correction of an autosomal dominant pathogenic variant in RPE cells causing Best disease.”

Lipid-based transfection is a suitable approach for transient gene editing delivery in human retinal tissues.

“Collectively, these findings contribute to the development of CRISPR-based therapeutic strategies and support lipid-based transfection as a suitable approach for transient gene editing delivery in human retinal tissues.”

• AssumptionRests on an assumption: Lipofection is a more suitable method for a potential therapeutic in vivo application.

This study demonstrates, for the first time, precise correction of a single-nucleotide mutation in patient-derived RPE using CRISPR/Cas9 and Cas12 delivered using lipoplexes.

“This study demonstrates, for the first time, precise correction of a single-nucleotide mutation in patient-derived RPE using CRISPR/Cas9 and Cas12 delivered using lipoplexes.”

The concentration of ssODN can influence gene-editing outcomes in cases in which the sgRNA can recognize and cut the wild-type allele.

“and that the concentration of ssODN could influence gene-editing outcomes in cases in which the sgRNA could recognize and cut the wild-type allele.”

There is a correlation between cellular maturity and transfection efficacy.

“suggesting a correlation between cellular maturity and transfection efficacy.”

The presence of a rare variant carried by the patient was checked and confirmed to discard potential contamination with another cell line.

“Finally, to discard potential contamination with another cell line, and considering that the strategy followed in the screening was single-cell cloning, we checked and confirmed the presence of a rare variant carried by the patient (Figure S2D).”

The other half in Experiment B indicates the cut of the wild-type allele and ssODN-mediated repair.

“indicating the cut of the wild-type allele and ssODN-mediated repair.”

Cas12 has better affinity to the TTG PAM motif in our assay.

“Interestingly, these two Cas12 guides that yielded the highest cleavage results for each pathogenic variant (sgBEST1-Cas12_3 and sgABCA4-Cas12_1) shared the same TTG PAM sequence, suggesting a better affinity of Cas12 to this motif in our assay (Table 1).”

Increasing Cas9 can trigger higher percentages of DNA DSB.

“Altogether indicated that the increase in Cas9 could trigger higher percentages of DNA DSB”

The results indicate a potential use of CRISPR without using exogenous DNA templates for correcting heterozygous pathogenic variants.

“In addition, our results indicate a potential use of CRISPR without using exogenous DNA templates for correcting heterozygous pathogenic variants.”