10 crRNAs with high functional scores for 10 genes (blue bars) and 10 crRNAs with low functional scores for the same genes (yellow bars) were tested for editing by Next Generation Sequencing. 93% of the high-scoring crRNAs and 32 % of the low scoring crRNAs showed > 40% of editing (indel formation). The Cas9-HEK293T cell line was transfected with 50 nM crRNA:tracrRNA, using 0.25 µL/well of DharmaFECT 1. Seventy-two hours post-transfection, cells were lysed and Nextera transposon-adapted amplicons spanning each crRNA site were generated for every treated sample as well as for a matched control amplicon from untransfected samples. Samples were indexed using the Nextera 96-well index kit and pooled for sequencing on a MiSeq instrument (paired end reads, 2 x 300 length). Reads that passed NGS quality filtering criteria were aligned to the reference file (Bowtie2 v2.1.0). Percent perfect reads were calculated and normalized to the control untransfected samples (Samtools v0.1.12a); the data is presented as normalized percent edited.
HEK293T cells stably expressing Cas9 nuclease were transfected with the indicated guide RNA targeting PPIB or DNMT3B. Edit-R unmodified products (crRNA:tracrRNA) and those modified to resist degradation (2xMS crRNA:2xMS tracrRNA) both maintain cell viability and stimulate virtually no immune response. 3xMS modified crRNA:tracrRNA reagents are more toxic to cells. Cellular viability was assessed with a Resazurin metabolism assay (red dots) and the levels of five immune response genes were quantified by qRT-PCR. MS= 2’-O-Methyl ribose and phosphorothioate backbone linkage.
A. Position of modifications for nuclease resistance on Edit-R synthetic crRNA and tracrRNA. Edit-R synthetic guide RNAs contain 2ʹ-O-methyl (2ʹ-OMe) and backbone phosphorothioate linkages (PS) on the two nucleotides at the 5ʹ end of the crRNA (green strand) and on the two nucleotides at the 3’ end of the tracrRNA (blue strand). B. Structure of the 2ʹ-OMe and PS backbone phosphorothioate linkages.
U2OS cells were plated at 10,000 cells/well one day prior to transfection. Cells were transfected with either Edit-R Cas9 Nuclease plasmid (200 ng), Edit-R Cas9 Nuclease mRNA(200 ng) or Cas9 nuclease protein (25 nM) and crRNA:tracrRNA (25 nM) targeting PPIB using DharmaFECT Duo transfection reagent (0.4 uL/well) in biological triplicates.
A. Modifications to block degradation by nucleases are required for successful co-electroporation of Cas9 mRNA and crRNA:tracrRNA. B. Co-electroporation of Cas9 protein and crRNA:tracrRNA does not require stabilizing modifications, but results may be improved with their addition.
NIH/3T3 cells were stably transduced with lentiviral particles containing Cas9 and a blasticidin resistance gene driven by the indicated promoters. A population of stably integrated cells were selected with blasticidin for a minimum of 10 days before transfections. Cells were transfected with 50 nM Edit-R synthetic crRNA:tracrRNA targeting PPIB using DharmaFECT 1 transfection reagent. After 72 hours, the relative frequency of gene editing was calculated based on a DNA mismatch detection assay with T7 Endonuclease I.
U2OS-Proteasome cells with integrated Cas9 (under CAG promoter) 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 0.2 µg/well of DF4. Cell were analyzed for apoptosis 48 h after transfection using the ApoONE homogeneous assay (Promega). For the box plot, the crRNAs were divided into bottom half (H1) and top half (H2) based on their Edit-R algorithm functional score (110 total data points represented). The medians, distribution of data between the lower and upper halves and the minimum and maximum values demonstrate that high-scoring crRNAs have increased functionality.
T7E1 mismatch analysis for a crRNA showing the intended target site (GGTCATCTGGGAGAAAAGCG) and a predicted off-target site that was identified by the Edit-R alignment tool but no other online tool, containing one gap and one mismatch (GGT-ATCTGGGAGAAAAGCa) Many commonly used web-based crRNA specificity tools do not fully account for gaps when performing alignments.
A recombinant U2OS ubiquitin-EGFP proteasome cell line (Ubi[G76V]-EGFP) was stably transduced with lentiviral particles containing Edit-R plasmid vectors expressing Cas9 nuclease and blasticidin resistance gene under the control of the indicated promoters. A population of cells with stably integrated Cas9-BlastR was selected with blasticidin-treatment for a minimum of 10 days before transfections. Cells were transfected with 50 nM Edit-R synthetic crRNA:tracrRNA complex targeting VCP, a gene required for proteasome function, using DharmaFECT 3 Transfection Reagent. After 72 hours, transfected cells were examined for EGFP+ cells (upper panel) and the relative frequency of gene editing was calculated (lower panel) based on a DNA mismatch detection assay with T7 Endonuclease I.