Journal: bioRxiv

Article Title: Allele-specific gene editing approach for vision loss restoration in RHO -associated Retinitis Pigmentosa

doi: 10.1101/2022.11.16.516784

Figure Lengend Snippet: In v i tro knock-down of human RHO- T17M expression. (A) Schematic view of construction of 293T stably expressing human RHO protein and transfection of pX601-EFS-SaCas9-U6-sgRNA (SaCas9/SgRNA) plasmid. (B) T7E1 assay indicated that SaCas9/17-Sg1 and SaCas9/17-Sg2 were appeared to cut the mutant sequence specifically, the full-length amplicon was 760bp, the two truncated amplicons were 510 bp and 250 bp, respectively. (C) The cutting efficacy of two sgRNAs with SaCas9 determined by TA & Sanger sequencing in 293T cells. (D) Rhodopsin expression reduction was determined by western blotting in 293T cells transfected with RHO -T17M-pEGFPN1(RHO17 cells) transfected with 17-Sg1 and Sg2 plasmid, comparing to the RHO wt cells with 17-Sg1 and Sg2 plasmid.

Article Snippet: The selected sgRNA was cloned into the plasmid pX601-AAVCMV: NLS-SaCas9-NLS-3xHA-bGHpA; U6: BsaI-sgRNA (pX601, #61591, Addgene).

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Sequencing, Amplification, Western Blot

Journal: bioRxiv

Article Title: Allele-specific gene editing approach for vision loss restoration in RHO -associated Retinitis Pigmentosa

doi: 10.1101/2022.11.16.516784

Figure Lengend Snippet: Determination of specificity and cutting efficiency of SaCas9 and sgRNA in induced pluripotent stem cells (iPSCs) from patients’ urine cells (UCs). (A)Photograph of UCs. Scale bar=100 μm. (B) Photograph of iPSCs. Scale bar = 200 μm. (C-D) Sanger sequencing results showed that P1 iPS had the heterozygous c.50C>T mutation. IF staining showed the iPSCs expressed human embryonic stem cell-specific surface antigens including NANOG (E), OCT4 (F), TRA-1-60 (G) and SSEA-4 (H) protein. (I) Chromosomal content analysis revealed a normal 46, XY karyotype of patient P1. (J) Schematic view of AAV-EFS-SaCas9-U6-sgRNA-p2a-Puro plasmid. (K) T7E1 assay indicated that SaCas9/17-Sg2 was appeared to cut the mutant sequence specifically. (L) The cutting efficacy of SaCas9/17-Sg2 is determined by TA & Sanger sequencing in iPSCs.

Article Snippet: The selected sgRNA was cloned into the plasmid pX601-AAVCMV: NLS-SaCas9-NLS-3xHA-bGHpA; U6: BsaI-sgRNA (pX601, #61591, Addgene).

Techniques: Sequencing, Mutagenesis, Staining, Plasmid Preparation

Journal: bioRxiv

Article Title: Allele-specific gene editing approach for vision loss restoration in RHO -associated Retinitis Pigmentosa

doi: 10.1101/2022.11.16.516784

Figure Lengend Snippet: (A) (Top) AAV2/8 vectors expressing EGFP under the control of EFS promoter; (Bottom) AAV2/8 vectors expressing SaCas9 under the control of EFS promoter and sgRNA under the control of U6 promoter were schematized. (B) Fluorescein shows AAV vectors distribution following subretinal injection immediately. (C) Infected areas treated with AAV2/8-EFS-EGFP vector at dose of 1×10 9 vg per eye (1×10 9 dose) were labelled by GFP expression (GFP+ area). ONL = outer nuclear layer; OS = outer segment; IS = inner segment. Scale bar = 50 μm. (D) IF staining indicates GFP expresses in mouse retinas at 1m and 11 mpi treated with AAV2/8-EFS-EGFP vector at 3×10 9 dose. (E) The quantitative PCR analysis indicated the expression of SaCas9, 17-Sg2 and GFP in Rho wt/hum retinas at 3 mpi treated with AAV2/8-EFS-SaCas9-U6-17-Sg2 and AAV2/8-EFS-EGFP vector (1:1 mixture) at different doses. (F) Determination of SaCas9 expression by western blotting at 3 mpi at 1×10 9 and 3×10 9 dose. (Left) Mouse #h, i and j ( Rho wt/hum ) were injected with AAV2/8-EFS-SaCas9-U6-17-Sg2 and AAV2/8-EFS-EGFP vector (1:1 mixture) at 1×10 9 dose; (Right) Mouse #k, l, m and n ( Rho wt/hum ) were injected with AAV2/8-EFS-SaCas9-U6-17-Sg2 and AAV2/8-EFS-EGFP vector (1:1 mixture) at 3×10 9 dose.

Article Snippet: The selected sgRNA was cloned into the plasmid pX601-AAVCMV: NLS-SaCas9-NLS-3xHA-bGHpA; U6: BsaI-sgRNA (pX601, #61591, Addgene).

Techniques: Expressing, Injection, Infection, Plasmid Preparation, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: bioRxiv

Article Title: Allele-specific gene editing approach for vision loss restoration in RHO -associated Retinitis Pigmentosa

doi: 10.1101/2022.11.16.516784

Figure Lengend Snippet: Identification of single-nucleotide variants (SNVs, A) and indels (C) in 293T cells transfected with pX601-EFS-SaCas9-U6-17-Sg2 (17-Sg2) plasmid at the WGS level. The type of SNVs (B) and indels (D) in 293T cells transfected with 17-Sg2 plasmid and untreated cells at the WGS level.

Article Snippet: The selected sgRNA was cloned into the plasmid pX601-AAVCMV: NLS-SaCas9-NLS-3xHA-bGHpA; U6: BsaI-sgRNA (pX601, #61591, Addgene).

Techniques: Transfection, Plasmid Preparation

Journal: Cell reports

Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection

doi: 10.1016/j.celrep.2019.05.103

Figure Lengend Snippet: Key Resources Table

Article Snippet: A plasmid derivative of pX601 (Addgene #61591) containing a nanoluciferase cassette was digested with HindIII and ligated to the pGEM stuffer sequence to generate pX601-long.

Techniques: Recombinant, Software