Journal: Nucleic Acids Research

Article Title: Optimized CRISPR-Cas9 system for efficient engineering of ecDNA in cancer cells

doi: 10.1093/nar/gkag005

Figure Lengend Snippet: ecDNA knock-in efficiency of standard and safeguard sgRNAs. ( A ) Schematic showing insertion of a 96-mer TetO repeat sequence into ecDNA of CORL23 cells. See Material and Methods for details of the knock-in system. ( B ) Quantification of CORL23 cell numbers after expression of all-in-one CRISPR plasmids with the indicated sgRNAs and the knock-in template plasmid. Data represent mean ± SD for n = 3 biological replicates. Statistical significance was assessed using a two-sided Student’s t -test. ( C ) Experimental scheme for DNA FISH analyses shown in panels (D–F). (D–F) Box-and-whisker plots showing copy numbers of ( D ) MYC ecDNA and ( E ) TetO knock-in ecDNA in cells transfected with all-in-one CRISPR plasmids with the indicated sgRNAs and the knock-in template plasmid. Panel ( F ) shows the fraction of MYC ecDNA carrying the TetO knock-in sequence. Sample sizes ( n ) are shown below sgRNA labels. Statistical significance was assessed using a Wilcoxon rank-sum test.

Article Snippet: Tracking of indels by decomposition (TIDE) analysis ( https://tide.nki.nl ) was performed using Sanger sequencing traces generated from target-specific PCR products by Eurofins [ ].

Techniques: Knock-In, Sequencing, Expressing, CRISPR, Plasmid Preparation, Whisker Assay, Transfection

Journal: Nucleic Acids Research

Article Title: Optimized CRISPR-Cas9 system for efficient engineering of ecDNA in cancer cells

doi: 10.1093/nar/gkag005

Figure Lengend Snippet: Application of the safeguard sgRNA system in ecDNA-positive CORL23 cells. ( A ) AmpliconArchitect analysis showing the presence of ecDNA structures involving the MYC and PVT1 loci in CORL23 cells.( B ) IGV snapshot showing genome sequence profiles at the MYC and PVT1 loci. ( C ) Representative FISH images of CORL23 cells using an MYC locus probe (red) together with a chromosome 8 probe (green; control for the MYC -containing chromosome). Two representative single cells with different MYC ecDNA copy numbers are shown. Scale bar, 10 µm. ( D ) Schematic illustration of the proposed molecular mechanism by which safeguard sgRNAs fine-tune Cas9 activity. Cytosine extensions decrease Cas9 activity and attenuate Cas9-mediated DNA cleavage via multiple mechanisms, including reduced sgRNA synthesis and impaired target loading efficiency of Cas9–sgRNA RNP complexes (left). When using potent sgRNAs, [C]-extended sgRNAs exhibited decreased bi-allelic indels on chromosomal targets and increased mono-allelic indels in a length-dependent manner (right). This figure was adapted from Kawamata et al. , licensed under the CC BY 4.0. ( E ) Schematic of all-in-one CRISPR plasmids expressing standard and safeguard sgRNAs. ( F ) Western blot analysis of FLAG-tagged Cas9 proteins expressed from the indicated all-in-one plasmids in CORL23 cells. ( G ) Quantification of sgRNA expression levels by RT-qPCR. Left, normalized expression values; right, normalized values multiplied by 1000 and log 2 -transformed. Data represent mean ± SD for n = 3 technical replicates. Statistical significance was assessed using a two-sided Student’s t -test.

Article Snippet: Tracking of indels by decomposition (TIDE) analysis ( https://tide.nki.nl ) was performed using Sanger sequencing traces generated from target-specific PCR products by Eurofins [ ].

Techniques: Sequencing, Control, Activity Assay, CRISPR, Expressing, Western Blot, Quantitative RT-PCR, Transformation Assay