Loss of cell viability in Cas9-expressing cells treated with Edit-R Lethal crRNA controls

Phase contrast microscopy clearly indicates a strong cell death phenotype with Edit-R crRNA control #1 and a slightly more moderate level of death with Edit-R crRNA control #2 when compared to NTC. The loss in cell viability was dose-dependent on the amount of DharmaFECT 4 transfection reagent used to deliver the crRNA:tracrRNA complex. Cas9-expressing U2OS-Proteasome cells were plated in 96-well plates at 10,000 cells per well. 24 h after plating, cells were transfected with 25 nM crRNA:tracrRNA using 0.02-0.13 µg/well of DharmaFECT 4 transfection reagent. NTC = Non-targeting control.

Optimal transfection conditions for cell death from lethal controls correlate with proteasome-dependent phenotype

A. Recombinant U2OS Ubi[G76V]-EGFP-Cas9 cells were analyzed for cell viability using a resazurin assay under a range of transfection reagent amounts. Lethal controls show a dose-dependent level of cell death. B. Cells were analyzed for EGFP expression as a readout of proteasome disruption. Optimal conditions indicated by cell viability reproduced optimal EGFP expression. All results were normalized to UT, 72 h after transfection. UT = untreated cells, NTC = Non-targeting control

Effective gene editing of PPIB in human and mouse cells

A human recombinant U2OS ubiquitin-EGFP proteasome cell line (Ubi[G76V]-EGFP) (A) and a mouse fibroblast (NIH/3T3) (B), were stably transduced with lentiviral particles containing Cas9 and a blasticidin resistance gene driven by the indicated promoters.. A population of cells with stably integrated Cas9-blastR was selected with blasticidin for a minimum of 10 days before transfections. Cells were transfected with 50 nM synthetic crRNA:tracrRNA targeting Human PPIB / mouse Ppib using DharmaFECT 1 and DharmaFECT 3 Transfection reagent, respectively. After 72 hours, the relative frequency of gene editing was calculated based on a DNA mismatch detection assay using T7EI on genomic DNA extracted from the transfected cells.

Comparable gene editing of PPIB and DNMT3B with either unmodified crRNA:tracrRNA or modified for nuclease resistance

HEK293T cells expressing Cas9 nuclease were transfected with unmodified crRNA:tracrRNA targeting PPIB or DNMT3B or with crRNA:tracrRNA carrying modifications to resist nuclease degradation (2'-O-methyl; 2'OMe) and backbone phosphorothioate linkages (PS) on the two nucleotides at the 5' end of the crRNA and on the two nucleotides at the 3’ end of the tracrRNA). A T7EI DNA mismatch assay was performed and the samples were separated on a 2% agarose gel. Percent indels was calculated and is shown at the bottom of the lanes.

1. R. Barrangou, A. Birmingham,et. al.Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference.Nucleic Acids Research,43(7) 3407-3419 (2015)

2. M.L. Kelley, Ž. Strezoska, et al. Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing. J. Biotechnol. 233, 74–83 (2016). doi:10.1016/j.jbiotec.2016.06.011