px459v2 plasmid  (Addgene inc)

Journal: Nature Communications

Article Title: Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics

doi: 10.1038/s41467-026-68774-w

Figure Lengend Snippet: a Schematic overview of the CHyMErA system illustrating dual-gene targeting and precise exon deletion. Graphical elements were created in BioRender. b , c Overview of the CHyMErA platform and the development of scCHyMErA-Seq. b Diagram of the CHyMErA system enabling multiplexed combinatorial CRISPR screening using Cas9 and Cas12a, including elements added for single-cell profiling. c Modifications introduced to enable efficient, simultaneous detection of Cas9 and Cas12a guide (g)RNAs in single cells, including capture sequences (CS), the direct repeat variant 10 (DR v10 ), and the incorporation of the tevopreQ1 riboswitch. d Bar plots showing the proportions of cells with detectable Cas9 and/or Cas12a gRNAs using 10x Genomics droplet-based single-cell profiling. Different hgRNA designs (Fig. 1c) were tested using a standard 10x CRISPR library or a custom Cas12a library preparation workflow. Data are normalized to the total number of cells with detectable gRNAs (see Supplementary Data 1). Data represent mean ± SEM from three biological replicates. e CD46 exon-3 deletion measured by flow cytometry in HAP1 cells transduced with three independent spacer sequence pairs, using the indicated hgRNA designs. Data represent mean ± SEM from biological replicates ( n = 3–9). **** p < 0.0001, *** p < 0.001, n.s. not significant; two-way ANOVA. f Bar plots quantifying the relative expression levels of unprocessed hgRNAs and processed Cas12a gRNAs across different hgRNA designs assessed by northern blotting (see Supplementary Fig. 2a). Data represent mean ± SEM from three biological replicates. **** p < 0.0001, *** p < 0.001, ** p < 0.01, n.s. not significant; one-way ANOVA. g Comparison of As Cas12a DR sequences. The wild-type (WT) DR is shown with the T-rich region highlighted in yellow; DR variant 10 (v10) is shown below, with nucleotide substitutions indicated in red.

Article Snippet: To generate the pX462-ABE8e-n Sa Cas9 base editor backbone plasmid (Addgene #237462), we first replaced the Sp Cas9 tracrRNA sequence in pX459v2 (Addgene #62988) with the Sa Cas9 tracrRNA sequence.

Techniques: CRISPR, Variant Assay, Flow Cytometry, Transduction, Sequencing, Expressing, Northern Blot, Comparison

Journal: Nature Communications

Article Title: Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics

doi: 10.1038/s41467-026-68774-w

Figure Lengend Snippet: a Schematic representation of the lentiviral scCHyMErA-Seq library targeting 224 alternative cassette exons for deletion and 161 genes for knockout. Blue and orange triangles indicate Cas9 and Cas12a target sites, respectively, for each hgRNA category. b Overview of the scCHyMErA-Seq experimental pipeline. Graphical elements were created in BioRender. c , d UMAP visualization of HAP1 cells expressing hgRNAs inducing gene-level knockouts ( c ) or exon deletions ( d ) following linear discriminant analysis (LDA). Insets show UMAPs recalculated after removal of cells associated with the highlighted perturbations in the original plots. Distinct clusters are labeled by the targeted gene or exon. Black dots denote cells carrying hgRNAs targeting other genes, exons, or intergenic controls.

Article Snippet: To generate the pX462-ABE8e-n Sa Cas9 base editor backbone plasmid (Addgene #237462), we first replaced the Sp Cas9 tracrRNA sequence in pX459v2 (Addgene #62988) with the Sa Cas9 tracrRNA sequence.

Techniques: Knock-Out, Expressing, Labeling