1313 paav u6 bbsi mlui grna sa hlp sacas9 ha ollas spa  (Addgene inc)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-invasive detection of allele-specific CRISPR-SaCas9-KKH disruption of TOR1A DYT1 allele in a xenograft mouse model

doi: 10.1016/j.omtn.2025.102466

Figure Lengend Snippet: TOR1A DYT1 allele disruption by SaCas9-KKH nuclease activity in DYT1 hNPCs (A) gRNA design to target the GAG deletion (ΔGAG) signature in DYT1 dystonia. The genomic sequence of the TOR1A WT and TOR1A DYT1 alleles in DYT1 dystonia patients. The 3-nucleotide (nt) difference between both alleles is indicated with a dotted line. The TOR1A DYT1 allele encodes a target site for gRNA1 to gRNA4 (highlighted in red) and a protospacer-adjacent motif (PAM; highlighted in green) for SaCas9-KKH. The gRNAs differ in length by the nucleotides upstream of the ΔGAG region, which are indicated in purple. (B) Screening of gRNAs in DYT1 hNPCs. This cartoon illustrates our screening process, where gRNA and SaCas9-KKH expression plasmids were electroporated into DYT1 hNPCs. Based on eGFP fluorescence encoded in the SaCas9-KKH plasmid, potentially gene-edited cells were isolated through FACS. The non-eGFP-expressing hNPCs were used as control. ΔGAG disruption was confirmed by analysis of next-generation sequencing. (C) Isolation of SaCas9-KKH-expressing DYT1 hNPCs. FACS gating strategy for selecting single live hNPC events (DAPI NEG eGFP POS ). Post electroporation, 9%–15% of the single live hNPC events are eGFP POS . Images of SaCas9-KKH and eGFP-expressing hNPCs were taken using a confocal microscope. Scale bar represents 200 μM. (D) Gene editing of DYT1 allele in hNPCs with gRNA1–4 and SaCas9-KKH. Bar graph illustrating the percentage of editing of the TOR1A DYT1 allele based on next-generation sequencing analysis. gRNAs 1–4 targeted ΔGAG compared to hNPCs that did not express SaCas9-KKH (control). Statistical analysis was performed using a one-way ANOVA and GraphPad Prism 10.2.1 software. ∗ p < 0.05. (E) Types of genetic modifications introduced into the target DNA after gene editing of the TOR1A DYT1 allele in hNPCs. A pie chart generated by CRISPResso2 analysis illustrates the percentage of TOR1A DYT1 allele disruptions (i.e., premature stop codon), other mutations (i.e., insertions or deletions that did not result in a TOR1A DYT1 allele disruption or premature stop codon), and unedited alleles for gRNAs 1–4, with gRNA3 showing the highest level (37.3%) of premature stop codons.

Article Snippet: Dyt1 SaCas9-KKH gRNA oligos , .

Techniques: Disruption, Activity Assay, Sequencing, Expressing, Fluorescence, Plasmid Preparation, Isolation, Control, Next-Generation Sequencing, Electroporation, Microscopy, Software, Generated

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-invasive detection of allele-specific CRISPR-SaCas9-KKH disruption of TOR1A DYT1 allele in a xenograft mouse model

doi: 10.1016/j.omtn.2025.102466

Figure Lengend Snippet: Primers and oligos used in Maalouf and Frederick et al.

Article Snippet: Dyt1 SaCas9-KKH gRNA oligos , .

Techniques: Next-Generation Sequencing, Plasmid Preparation, CRISPR, Cloning

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-invasive detection of allele-specific CRISPR-SaCas9-KKH disruption of TOR1A DYT1 allele in a xenograft mouse model

doi: 10.1016/j.omtn.2025.102466

Figure Lengend Snippet: TaqMan probe assay for rapid detection of the ΔGAG signature in DYT1 dystonia (A) ΔGAG-specific-TaqMan probe assay. Outline of genomic and transcript analysis of DYT1 and WT samples using GAG- or ΔGAG-targeting TaqMan probes labeled with either VIC or ΔGAG-FAM fluorophores, respectively. (B) hNPC analysis using a ΔGAG-specific TaqMan probe assay. The assay was evaluated with both gDNA (right) and cDNA (left) extracted from three DYT1 hNPCs (blue) and three WT control hNPCs (red). To validate the assay, gBlocks encoding exon 5 of the TOR1A allele with (green) and without (yellow) the ΔGAG mutation were used. A no-sample control was included (black dot).

Article Snippet: Dyt1 SaCas9-KKH gRNA oligos , .

Techniques: TaqMan Probe Assay, Labeling, Control, Mutagenesis

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-invasive detection of allele-specific CRISPR-SaCas9-KKH disruption of TOR1A DYT1 allele in a xenograft mouse model

doi: 10.1016/j.omtn.2025.102466

Figure Lengend Snippet: DYT1 and WT synthetic DNA sequences used in Maalouf and Frederick et al.

Article Snippet: Dyt1 SaCas9-KKH gRNA oligos , .

Techniques: Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-invasive detection of allele-specific CRISPR-SaCas9-KKH disruption of TOR1A DYT1 allele in a xenograft mouse model

doi: 10.1016/j.omtn.2025.102466

Figure Lengend Snippet: EVs carry the ΔGAG signature when secreted from DYT1 hNPCs and are present in DYT1 patient plasma (A) Screening for ΔGAG in EVs derived from DYT1 patients. Cartoon illustrating our setup for isolating exRNA from hNPC-derived EVs in conditioned culture medium as well as EVs from patient plasma. Following exRNA isolation and cDNA conversion, we performed preamplification of the ΔGAG region, which was subsequently analyzed using a ΔGAG-specific TaqMan probe assay. (B) The ΔGAG region pre-amplicon is exclusively found in EV-derived exRNA. We evaluated the presence of the ΔGAG region in fractions containing EVs and ribonucleoproteins (RNPs) following SEC of conditioned hNPCs culture medium. Our data, represented by preamplification PCR results loaded on agarose gel, showed bands in the EV fractions for both WT and DYT1 samples, while no bands were observed in the RNP fractions. (C) ΔGAG-specific TaqMan probe assay as a screening tool to detect ΔGAG in EVs derived from DYT1 hNPCs. A 2D plot distinguishes exRNA from three DYT1 hNPCs (blue) and three WT control hNPCs (red). The assay involved performing preamplification PCR on cDNA-transformed exRNA to ensure the detection of the GAG-VIC and ΔGAG-FAM probes, specifically within hNPC-derived EVs. gBlocks controls with (green) and without (yellow) the ΔGAG mutation were used. A no-sample control was included (black dot). (D) ΔGAG in plasma from DYT1 patients. A 2D plot distinguishes exRNA from plasma samples collected from eight patients using a ΔGAG-specific TaqMan probe assay. The colors and controls correspond to those in (C). P and NP denote DYT1 patients presenting with or without symptoms, respectively.

Article Snippet: Dyt1 SaCas9-KKH gRNA oligos , .

Techniques: Clinical Proteomics, Derivative Assay, Isolation, TaqMan Probe Assay, Amplification, Agarose Gel Electrophoresis, Control, Transformation Assay, Mutagenesis

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-invasive detection of allele-specific CRISPR-SaCas9-KKH disruption of TOR1A DYT1 allele in a xenograft mouse model

doi: 10.1016/j.omtn.2025.102466

Figure Lengend Snippet: EVs as non-invasive biomarkers for mutant allele disruption by SaCas9-KKH nuclease activity in DYT1 hNPCs (A) ΔGAG signature in DYT1 hNPCs. Next-generation sequencing analysis of gDNA and cDNA-converted transcript isolates. (B) ΔGAG signature in EVs derived from DYT1 hNPCs. Next-generation sequencing analysis of cDNA-converted exRNA. (C) exRNA as a biomarker for gene editing of the ΔGAG region in the DYT1 allele. Representative next-generation sequencing result revealing indels in exRNA-converted cDNA from DYT1 hNPCs expressing gRNA 3 and SaCas9-KKH. (D) Underreporting of DYT1 allele editing by EVs compared to donor cells. A comparison of indels from next-generation sequencing analysis of DYT1 hNPCs electroporated with SaCas9-KKH and gRNA 3 and their corresponding EVs. (E) Gene-editing reporting in EVs vs. cells. Schematic of data (F). DYT1 hNPCs were electroporated with SaCas9-KKH and gRNAs 1–3, followed by FACS of eGFP-positive (DAPI NEG eGFP POS ) and eGFP-negative (DAPI NEG eGFP NEG ) cells. These sorted hNPCs were cultured and analyzed using the ΔGAG-specific TaqMan probe assay as well as their exRNA in the conditioned medium. (F) Changes in ΔGAG levels in DYT1 hNPCs following CRISPR-Cas activity are mirrored in their EVs. A 2D plot displays ΔGAG-FAM levels in cDNA from cell transcripts and EV exRNA from edited and non-edited cells. These levels were normalized to the GAG-VIC probe to establish a correlation between hNPCs and their EVs. The drop along the dotted line in the cluster after gene editing indicates that DYT1 allele targeting can be detected in both EVs and cells. Label colors represent the different gRNAs used.

Article Snippet: Dyt1 SaCas9-KKH gRNA oligos , .

Techniques: Mutagenesis, Disruption, Activity Assay, Next-Generation Sequencing, Derivative Assay, Biomarker Discovery, Expressing, Comparison, Cell Culture, TaqMan Probe Assay, CRISPR

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-invasive detection of allele-specific CRISPR-SaCas9-KKH disruption of TOR1A DYT1 allele in a xenograft mouse model

doi: 10.1016/j.omtn.2025.102466

Figure Lengend Snippet: In vivo targeting of ΔGAG with AAV9-CRISPR (A) hNPC-implantation mouse model to test gene therapy for treating ΔGAG in the brain. A schematic illustrates the i.c. injection of DYT1 hNPCs treated with either AAV9-CRISPR or AAV9-null. After 21 days, cardiac blood and brain tissues were collected, followed by the isolation of exRNA from the plasma and gDNA from brain sections with hNPCs, and subsequent next-generation sequencing analysis. The AAV9-CRISPR construct is illustrated at the top. (B) Detection of ΔGAG edits in the brain. CRISPResso2 analysis reveals a higher percentage of DYT1 allele edits in injected hNPCs from AAV9-CRISPR samples (pink) compared to AAV9-null samples (gray). Statistical analysis was conducted using Student’s t test; ∗ p < 0.05. (C) Detection of ΔGAG edits in plasma. CRISPResso2 analysis of exRNA-converted cDNA isolated from the plasma of injected mice shows edits in the mutant allele of the released TOR1A transcript in AAV9-CRISPR samples (pink), but not in AAV9-null samples (gray). Statistical analysis was conducted using Student’s t test; ∗ p < 0.05.

Article Snippet: Dyt1 SaCas9-KKH gRNA oligos , .

Techniques: In Vivo, CRISPR, Injection, Isolation, Clinical Proteomics, Next-Generation Sequencing, Construct, Mutagenesis