Efficient and Safe Therapeutic Use of Paired Cas9-Nickases for Primary Hyperoxaluria Type 1
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ABSTRACT:
PROVIDER: PRJNA1029956 | ENA |
REPOSITORIES: ENA
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Project description:The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. Additionally, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.
2023-11-10 | GSE245791 | GEO
Project description:The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. Additionally, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.
| S-SCDT-10_1038-S44321-023-00008-8 | biostudies-other
Project description:The dysregulation of gene dosage due to duplication or haploinsufficiency is a major cause of autosomal dominant diseases such as Alzheimer’s disease. However, there is no rapid and efficient method for manipulating gene dosage in a human model system such as human induced pluripotent stem cells (iPSCs). Here, we describe a simple and precise method to simultaneously generate iPSC lines with different gene dosages using paired Cas9 nickases. We first generate a Cas9 nickase variant with broader protospacer-adjacent motif specificity to expand the targetability of double-nicking–mediated genome editing. As a proof-of-concept study, we examine the gene dosage effects on Alzheimer’s disease pathogenesis patient-derived iPSC line that carries three copies of APP (amyloid precursor protein). This method enables the rapid and simultaneous generation of iPSC lines with monoallelic, biallelic, or triallelic knockout of APP. The cortical neurons generated from isogenically corrected iPSCs exhibit gene dosage-dependent correction of disease-associated phenotypes of amyloid-beta secretion and Tau hyperphosphorylation. Thus, the rapid generation of iPSCs with different gene dosages using the method described herein can be a useful model system for investigating disease mechanisms and therapeutic development.
2021-02-04 | GSE160224 | GEO
Project description:Efficient manipulation of gene dosage in human iPSCs using CRISPR/Cas9 nickases
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Project description:Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in therapeutic genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal rearrangements induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following single or paired Cas9-based nickase editing. Conversely, whereas single nickase applications did not result in gross genomic aberrations, the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal rearrangements at on-target sites.
2024-03-15 | GSE241778 | GEO
Project description:Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired nickase editing. While the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal aberrations at on-target sites.
2024-03-15 | GSE255081 | GEO
Project description:Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in therapeutic genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal rearrangements induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following single or paired Cas9-based nickase editing. Conversely, whereas single nickase applications did not result in gross genomic aberrations, the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal rearrangements at on-target sites.
2024-03-15 | GSE241779 | GEO
Project description:Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in therapeutic genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal rearrangements induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following single or paired Cas9-based nickase editing. Conversely, whereas single nickase applications did not result in gross genomic aberrations, the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal rearrangements at on-target sites.
2024-03-15 | GSE254922 | GEO
Project description:Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in therapeutic genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal rearrangements induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following single or paired Cas9-based nickase editing. Conversely, whereas single nickase applications did not result in gross genomic aberrations, the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal rearrangements at on-target sites.
2024-03-15 | GSE242954 | GEO
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