ATTO™ 550 labeled Alt-R™ Cas9 tracrRNA allows for FACS sorting and intracellular RNP-complex visualization Mollie S. Schubert 1 *, Ashley M. Jacobi 1 , Rolf Turk 1 , Jennifer Y. Barr 2 , Michel Cannieux 1 , Scott M. Lieberman 2 , Mark A. Behlke 1 Integrated DNA Technologies, Inc., Coralville, IA, 52241 USA 1 Stead Family Department of Pediatrics, University of Iowa, Iowa City, IA, 52242 USA 2 Enrichment of sorted cells leads to higher editing efficiencies. Jurkat cells or HEK293 cells were electroporated using the Neon™ Transfection System in the presence of Alt- R™ Cas9 Electroporation Enhancer with 1.5 or 0.5 μM RNP consisting of labeled tracrRNA (ATTO™ 550). Cells subjected to RNP but without electroporation were used as background controls and were used to set the gates. Cells were sorted 24 hr post-transfection, and positive cells were re-plated and grown for an additional 48 hr. A population of the cells was not sorted, but simply re-plated, to serve as the unsorted control. Genomic DNA was isolated using QuickExtract™ (Epicentre) after cell incubation for 72 hr. Total editing efficiency was measured using a T7 Endonuclease I assay. FACS sorting enriched the detected editing at a sub-optimal dose of 0.5 μM. Effect of post-transfection time on flow cytometric resolution. Jurkat and HEK293 cells were electroporated using the Neon™ Transfection System in the presence of Alt-R™ Cas9 Electroporation Enhancer with 1.5 or 0.15 μM RNP, consisting of unlabeled crRNAand ATTO™ 550 labeled tracrRNA. Cells were sorted 24, 48, or 72 hr post- transfection. Prior to sorting, cells were washed 1 time with PBS + 1% FBS. Histogram plots were generated as previously described to show fluorescence intensities and percent positive cells. Optimal flow cytometric resolution occurs at 24 hr post-transfection, with a slight decrease at 48 hr, and significant decrease of positively sorted cells at 72 hr. ATTO 550 tracrRNA as a marker for transfection efficiency Enrichment of editing in positive cells by FACS sorting Conclusions • Dye-labeled tracrRNAs gave better results than dye-labeled crRNAs, with ATTO™ 550 labeled tracrRNA giving the best results • It is possible to visualize cellular localization and transfection efficiency by fluorescence microscopy using ATTO 550 labeled tracrRNA • A wash step before FACS analysis is recommended to reduce non-specific binding to the cells • Optimal FACS sorting occurred at 24 hr post-transfection • FACS sorting allowed for enrichment of editing by selecting positive cell fraction 0 10 20 30 40 50 60 70 80 90 100 unlabeled ATTO550 ATTO655 Cy3 Cy5 ATTO550 ATTO647 ATTO655 Cy3 labeled crRNA labeled tracrRNA T7EI editing (%) 0 10 20 30 40 50 60 70 80 90 100 ATTO550 ATTO647 ATTO655 Cy3 Cy5 ATTO550 ATTO647 ATTO655 Cy3 crRNA tracrRNA Positive cells (%) Detection of fluorescently-labeled ATTO™ 550 tracrRNA by fluorescence microscopy. HEK293 cells stably expressing Cas9 were reverse transfected using RNAiMAX with unlabeled crRNA and ATTO 550 labeled tracrRNA at a final concentration of 10 nM. Detection of ATTO 550 dye shows cellular localization. T7EI editing efficiencies of labeled guide RNA complexes. Labeled crRNAs were complexed to unlabeled tracrRNAs, whereas labeled tracrRNAs were complexed to unlabeled crRNAs. crRNA oligos were fluorescently labeled on the 3’ end. tracrRNA oligos were fluorescently labeled on the 5’ end. These guide RNA complexes were bound to Cas9 protein as RNP, and the RNP (10 nM) were reverse transfected into HEK293 cells using RNAiMAX™ (Thermo Fisher). Genomic DNA was isolated using QuickExtract™ (Epicentre) after cells were incubated for 48 hr. Total editing efficiency was measured using a T7 Endonuclease I assay. Flow cytometric resolution of positively transfected cells. Jurkat cells were electroporated using the Neon™ Transfection System (Thermo Fisher) in the presence of Alt-R™ Cas9 Electroporation Enhancer (single-stranded, non-targeting DNA) with 1.5 μM RNP consisting of either labeled crRNA or labeled tracrRNA. Jurkat cells subjected to the same RNP but without electroporation were used as background controls and were used to set the gates. The frequency of cells within each gate is reported on each histogram. Plots shown were previously gated on FSC-A x SSC-A and FSC-A x FSC-W. Dye-labeled tracrRNAs gave better results than dye-labeled crRNAs, with ATTO™ 550 labeled tracrRNA yielding the best results. Effect of washing cells prior to flow cytometric resolution. Jurkat cells were electroporated using the Neon Transfection System in the presence of Alt-R™ Cas9 Electroporation Enhancer with 1.5 or 0.15 μM RNP consisting of unlabeled crRNA and ATTO™ 550 or ATTO™ 647 labeled tracrRNA. Histogram plots were generated as previously described to show fluorescence intensities and percent positive cells. Cells were sorted 24 hr post-transfection. Prior to sorting, cells were washed 0, 1, or 2 times with PBS + 1% FBS. Histogram plots show fluorescence intensities. The wash step does not impact ATTO 550, but FACS resolution of ATTO 647 is best with 1 wash. 0 10 20 30 40 50 60 70 80 1.5 μM 0.5 μM 1.5 μM 0.5 μM HEK293 Jurkat T7EI editing (%) Unsorted Sorted FACS enrichment of cell population at suboptimal dose Functional testing of labeled gRNA complexes Positive cells by flow cytometric analysis (%) Cas9 protein is increasingly being used for genome editing by direct transfection of an active guide RNA:Cas9 ribonucleoprotein (RNP) complex into cells. The RNP complex can be delivered via lipofection, electroporation, or microinjection into cells of interest, but the efficiency of delivery can vary widely between cell types. We investigated the use of dye-labeled synthetic crRNAs and tracrRNAs as an aid to assess RNP complex delivery efficiency and the ability to enrich for transfected cells via FACS (fluorescence-activated cell sorting). Introduction Washing cells increases positive fraction by flow cytometry Alt-R™ crRNA:tracrRNA Ribonucleoprotein Complex (RNP) Of the variety of dyes tested, some suffered from non-specific binding to the cells, making evaluation of true transfection efficiency and sorting imprecise. Others had low fluorescence intensity over background. Optimal results were obtained using the ATTO™ 550 dye, with placement at the 5’-end of the tracrRNA. We describe an optimized protocol for use of the dye-labeled reagents to monitor transfection efficiency and for use in FACS to enrich for transfected cells. In addition, intracellular location of the dye-labeled complexes can be visualized using fluorescence microscopy. Optimal flow cytometric resolution occurs at 24 hr post-transfection 24 hour 72 hour 48 hour untransfected transfected ATTO™ 550 0.15 μM 1.5 μM 0.15 μM 1.5 μM ATTO™ 550 Jurkat HEK293 * Corresponding author: [email protected] Functional testing of dye-labeled RNAs crRNA tracrRNA Normalized to mode ATTO550 ATTO550 ATTO647 ATTO™ 550 ATTO™ 647 ATTO™ 550 ATTO™ 647 ATTO™ 550 0.15 μM 1.5 μM Normalized to mode ATTO™ 647 0 wash 1 wash 2 wash 0.15 μM 1.5 μM untransfected transfected Normalized to mode Flow cytometric analysis of dye-labeled RNAs crRNA tracrRNA untransfected transfected Normalized to mode ATTO647 ATTO647 ATTO550