Edit-R CRISPR knock-out

Edit-R algorithm scores correlate to cleavage efficiency

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.

Flow cytometry analysis of protein knockout in CD4+ T cells using a single predesigned synthetic sgRNA per gene target

Primary human CD4+ T cells were nucleofected with Cas9 RNP using an individual predesigned synthetic sgRNA per gene target or a non-targeting control (NTC), via a Lonza 96-well Shuttle system. After 72 hours, functional knockout of CD28, CD3E, and CXCR3 were assessed as a percent of cells not expressing the target gene by FACS analysis. Cells were stained and normalized for CD4 expression using an Alexa Fluor 488 conjugated antibody and compared to each target CD28, CD3E, and CXCR3 using APC conjugated primary antibodies.

Fluorescent Cas9 nuclease mRNA maintains editing efficiency

Fluorescent Cas9 mRNA U2OS cells were plated at 10,000 cells/well in a black 96-well plate 24 hours before transfection. At the time of transfection, EGFP Cas9 nuclease mRNA (200 ng, Cat #CAS11860) or non-fluorescent Cas9 Nuclease mRNA (200 ng, Cat #CAS11195) was co-transfected with Edit-R PPIB Synthetic crRNA Control (25 nM, Cat #U-007000-01-05) and tracrRNA (25 nM, Cat# U-002005-05) using DharmaFECT™ Duo transfection reagent (0.3 µL/well). After 24 hours, cells were imaged for EGFP fluorescence using the Nikon Eclipse Ti-S/L100 inverted microscope (A). At 72 hours after transfection, cells were harvested and gene editing was measured through a DNA mismatch detection assay (B).

Fluorescent Cas9 nuclease mRNA indicates optimal transfection conditions

U2OS cells expressing a Ubiquitin-tagged EGFP were plated at 10,000 cells/well in a black 96-well plate and co-transfected with Cas9 mRNA (200 ng, Cat #CAS11859) and synthetic tracrRNA (25 nM, Cat# U-002005-05) targeting PSMD11 (25 nM, Cat #CM-085161-03), a known proteasome component, or a Non-targeting control (NTC, Cat #U-007501-xx). Increasing amounts of DharmaFECT Duo Transfection Reagent were tested per well (0.1 µL to 0.7 µL). After 24 hours, cells were imaged for mKate2 fluorescence using the In Cell Analyzer 2200. At 72 hours after transfection, cells were imaged for EGFP fluorescence resultant from proteasome knockout. Based on mKate2 fluorescence, optimal transfection conditions occur at 0.3-0.4 µL of DharmaFECT Duo per well, which corresponds to an increased phenotypic effect. Above 0.4 µL of DharmaFECT Duo per well, significant cell death is observed (graph, red boxes). UT = untransfected sample.

Nucleofection of CD4+ primary human T-cells with Edit-R Cas9 Protein Hybrid NLS RNPs

CD4+ cells were stimulated with CD3/CD28 T-cell activation beads (BioLegend) at a 1:1 bead:T-cell ratio and grown for 8 days with 30 U/mL of IL-2. The Lonza 4D shuttle system was used to nucleofect RNPs composed of Edit-R Cas9 Protein Hybrid NLS and Edit-R synthetic guide RNA individual and target pools for CXCR3 (Gene ID: 2833) and CD3D (Gene ID: 915). Functional knockout was measured by flow cytometry analysis of target prevalence for CXCR3 (Clone ID: G025H7) and CD3D (Clone ID: OKT3) (BioLegend). Representative flow cytometry analysis plots of CD4+ T-cells shown after target knockdown by RNP nucleofection.

Fluorescent lentiviral Cas9 nuclease enables enrichment for high expression and improved CRISPR-Cas9 gene editing efficiency

U2OS cells were transduced at low multiplicity of infection (MOI 0.3) with Edit-R Lentiviral hCMV mKate2-Cas9 Nuclease particles (Cat #VCAS11869) so that transduced cells would have only one integration of Cas9. Cells were expanded for fluorescence activated cell sorting (FACS) where populations were sorted into negative, low, medium and high mKate2 fluorescence. These subpopulations were expanded and then plated at 10,000 cells/well in a 96-well plate. One day later, cells were transfected with tracrRNA (25 nM, Cat #U-002005-xx) and Edit-R PPIB Synthetic crRNA Control (Cat #U-007501-xx) or Edit-R MYC Predesign crRNA (Cat #CM-003282-01) using DharmaFECT 1 transfection reagent (0.3 µL/well, Cat #T-2001-01). After 72 hours, cells were imaged for mKate2 fluorescence using the In Cell Analyzer 2200 (GE Healthcare; A) and then harvested for DNA mismatch detection assay to estimate gene editing (B). High mKate2 expression can be associated with the highest levels of gene editing for both PPIB- and MYC-targeting crRNAs.