Journal: Nature biotechnology
Article Title: Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo
Figure Lengend Snippet: Delivery of Cas9:sgRNA, Cas9 D10A nickase, and dCas9-VP64 transcriptional activators to cultured human cells. ( a ) Green entries: U2OS EGFP reporter cells were treated with 100 nM of the Cas9 protein variant shown, 0.8 µL of the cationic lipid shown, and either 50 nM of the sgRNA shown for Cas9 protein treatment, or 125 nM of the sgRNA shown for (+36)dGFP-NLS-Cas9 and (−30)dGFP-NLS-Cas9 treatment. The fraction of cells lacking EGFP expression was measured by flow cytometry. Blue entries: plasmid DNA transfection of 750 ng Cas9 and 250 ng sgRNA expression plasmids using 0.8 µL Lipofectamine 2000. ( b ) T7 endonuclease I (T7EI) assay to measure modification of EGFP from no treatment (lane 1), treatment with EGFP-targeting sgRNA alone (lane 2), Cas9 protein alone (lane 3), Cas9 protein + VEGF-targeting sgRNA + RNAiMAX (lane 4), DNA transfection of plasmids expressing Cas9 and EGFP-targeting sgRNA (lane 5), or Cas9 protein + EGFP-targeting sgRNA + RNAiMAX (lane 6). ( c ) T7EI assay of simultaneous genome modification at EGFP and three endogenous genes in U2OS cells 48 hours after a single treatment of 100 nM Cas9 protein, 25 nM of each of the four sgRNAs shown (100 nM total sgRNA), and 0.8 µL RNAiMAX. ( d ) Delivery of Cas9 D10A nickase and pairs of sgRNAs either by plasmid transfection or by RNAiMAX-mediated protein:sgRNA complex delivery under conditions described in ( a ) with 50 nM EGFP -disrupting sgRNAs (25 nM each) for protein delivery, and 250 ng sgRNA-expressing plasmids (125 ng each) for DNA delivery. EGFP -disrupting sgRNAs g1 + g5, or g3 + g7, are expected to result in gene disruption, while g5 + g7 target the same strand and are expected to be non-functional. ( e ) Delivery of dCas9-VP64 transcriptional activators that target NTF3 either by DNA transfection or RNAiMAX-mediated protein delivery. Error bars reflect s.d. from six biological replicates performed on different days.
Article Snippet: The re-annealed DNA was incubated with 1 µl of T7 Endonuclease I (10 U/µl, NEB) at 37 °C for 15 min. 10 µL of 50 % glycerol was added to the T7 Endonuclease reaction and 12 µL was analyzed on a 5 % TBE 18-well Criterion PAGE gel (Bio-Rad) electrophoresed for 30 min at 200 V, then stained with 1x SYBR Gold (Life Technologies) for 30 min. Cas9-induced cleavage bands and the uncleaved band were visualized on an AlphaImager HP (Alpha Innotech) and quantified using ImageJ software .
Techniques: Cell Culture, Variant Assay, Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection, T7EI Assay, Modification, Functional Assay