Journal: Molecular Therapy

Article Title: Targeted Knockout of the Vegfa Gene in the Retina by Subretinal Injection of RNP Complexes Containing Cas9 Protein and Modified sgRNAs

doi: 10.1016/j.ymthe.2020.09.032

Figure Lengend Snippet: sgRNA Design and Effect of sgRNA Modification (A) Part of the human and murine vascular endothelial growth factor A ( Vegfa ) exon 3 sequence. Homologous sequence used for design of single guide RNAs (sgRNAs) targeting both human and murine Vegfa shown in bold. Mismatches are indicated in red font. Three sgRNAs targeting the Vegfa gene were designed and obtained with and without chemical modifications of the first and last three nucleotides of the sgRNA. sgRNAs were named according to CRISPRko on-target prediction scores, with sgRNA1 ranked highest. sgRNA sequences were indicated with underlined protospacer adjacent motif (PAM) sequences. (B) Fluorescence analysis of EGFP expression 1 day post-electroporation of K562 cells. Electroporation of approximately 2 × 10 5 K562 cells with 2 μg EGFP mRNA was used to evaluate electroporation efficacy. As judged from the EGFP and phase contrast images, almost all of the cells expressed EGFP. Representative images were shown (n = 3). Scale bar, 200 μm. (C) Tracking of indels by decomposition (TIDE) analysis of indels following electroporation of modified and unmodified synthetic sgRNAs. A total of 3 μg Cas9 protein and 1.5 μg sgRNA were used for electroporation of approximately 2 × 10 5 K562 cells. Two days post-electroporation, genomic DNA (gDNA) was isolated and used as the template for PCR amplification. PCR products were isolated following gel electrophoresis and sequenced. Sequencing results were analyzed for indels by TIDE. sgRNA-Irr (irrelevant) was used as negative control (CTRL) for TIDE analysis. Indel formation frequencies are shown as mean ± SD (n = 3). ∗Statistically significant. (D) Representative sequencing chromatogram for each modified sgRNA (msgRNA) and sgRNA. Cut site 3 bp upstream to the PAM was indicated by the red dotted line. PC, phase contrast.

Article Snippet: K562 (CLL-243; ATCC) cells were maintained in RPMI 1640 medium (Lonza, Basel, Schweiz).

Techniques: Modification, Sequencing, Fluorescence, Expressing, Electroporation, Isolation, Amplification, Nucleic Acid Electrophoresis, Negative Control

Journal: Molecular Therapy

Article Title: Targeted Knockout of the Vegfa Gene in the Retina by Subretinal Injection of RNP Complexes Containing Cas9 Protein and Modified sgRNAs

doi: 10.1016/j.ymthe.2020.09.032

Figure Lengend Snippet: Assessment of Off-Target Effects Mediated by sgRNA1 (A) Predicted off-target loci using the Benchling in silico bioinformatics tool. , Benchling score presents the target compatibility of sgRNA1 (ON), with 100.0 representing a perfect match, and declining values decrease targeting probability. The top three computationally predicted off-target sites (OFF1–3) are highlighted in bold. Mismatches indicated by red font and PAM sequences are underlined. (B and C) Specificity of targeted cleavage in K562 cells mediated by RNPs containing 3 μg Cas9 protein and 1.5 μg of either msgRNA1 or sgRNA1 using ICE (B) and TIDE (C) analysis. Indel frequencies were measured following Sanger sequencing of PCR amplicons of the targeted genomic locus ( VEGFA ) and the top three bioinformatically predicted off-target sites. Bars represent average values + SD (n = 3). OFF1, off-target 1.

Article Snippet: K562 (CLL-243; ATCC) cells were maintained in RPMI 1640 medium (Lonza, Basel, Schweiz).

Techniques: In Silico, Sequencing