ATTO™ 550 labeled Alt-R™ Cas9 tracrRNA allows for FACS sorting and intracellular RNP-complex visualization Mollie S. Schubert1*, Ashley M. Jacobi1, Rolf Turk1, Jennifer Y. Barr2, Michel Cannieux1, Scott M. Lieberman2, Mark A. Behlke1

Integrated DNA Technologies, Inc., Coralville, IA, 52241 USA1

Stead Family Department of Pediatrics, University of Iowa, Iowa City, IA, 52242 USA2

Enrichment of sorted cells leads to higher editingefficiencies. Jurkat cells or HEK293 cells were electroporatedusing the Neon™ Transfection System in the presence of Alt-R™ Cas9 Electroporation Enhancer with 1.5 or 0.5 µM RNPconsisting of labeled tracrRNA (ATTO™ 550). Cells subjectedto RNP but without electroporation were used as backgroundcontrols and were used to set the gates. Cells were sorted 24hr post-transfection, and positive cells were re-plated andgrown for an additional 48 hr. A population of the cells was notsorted, 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 wasmeasured using a T7 Endonuclease I assay. FACS sortingenriched the detected editing at a sub-optimal dose of 0.5 µM.

Effect of post-transfection time on flow cytometricresolution. Jurkat and HEK293 cells were electroporatedusing the Neon™ Transfection System in the presence ofAlt-R™ Cas9 Electroporation Enhancer with 1.5 or 0.15µM RNP, consisting of unlabeled crRNA and ATTO™ 550labeled tracrRNA. Cells were sorted 24, 48, or 72 hr post-transfection. Prior to sorting, cells were washed 1 timewith PBS + 1% FBS. Histogram plots were generated aspreviously described to show fluorescence intensities andpercent positive cells. Optimal flow cytometric resolutionoccurs at 24 hr post-transfection, with a slight decreaseat 48 hr, and significant decrease of positively sortedcells 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

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Detection of fluorescently-labeled ATTO™ 550 tracrRNAby fluorescence microscopy. HEK293 cells stablyexpressing Cas9 were reverse transfected using RNAiMAXwith unlabeled crRNA and ATTO 550 labeled tracrRNA at afinal concentration of 10 nM. Detection of ATTO 550 dyeshows cellular localization.

T7EI editing efficiencies of labeled guide RNAcomplexes. Labeled crRNAs were complexed to unlabeledtracrRNAs, whereas labeled tracrRNAs were complexed tounlabeled crRNAs. crRNA oligos were fluorescently labeledon the 3’ end. tracrRNA oligos were fluorescently labeled onthe 5’ end. These guide RNA complexes were bound toCas9 protein as RNP, and the RNP (10 nM) were reversetransfected into HEK293 cells using RNAiMAX™ (ThermoFisher). Genomic DNA was isolated using QuickExtract™(Epicentre) after cells were incubated for 48 hr. Total editingefficiency was measured using a T7 Endonuclease I assay.

Flow cytometric resolution of positively transfected cells.Jurkat cells were electroporated using the Neon™ TransfectionSystem (Thermo Fisher) in the presence of Alt-R™ Cas9Electroporation Enhancer (single-stranded, non-targeting DNA)with 1.5 µM RNP consisting of either labeled crRNA or labeledtracrRNA. Jurkat cells subjected to the same RNP but withoutelectroporation were used as background controls and wereused to set the gates. The frequency of cells within each gateis reported on each histogram. Plots shown were previouslygated on FSC-A x SSC-A and FSC-A x FSC-W. Dye-labeledtracrRNAs gave better results than dye-labeled crRNAs, withATTO™ 550 labeled tracrRNA yielding the best results.

Effect of washing cells prior to flow cytometricresolution. Jurkat cells were electroporated using theNeon Transfection System in the presence of Alt-R™Cas9 Electroporation Enhancer with 1.5 or 0.15 µMRNP consisting of unlabeled crRNA and ATTO™ 550 orATTO™ 647 labeled tracrRNA. Histogram plots weregenerated as previously described to showfluorescence intensities and percent positive cells.Cells were sorted 24 hr post-transfection. Prior tosorting, cells were washed 0, 1, or 2 times with PBS +1% FBS. Histogram plots show fluorescenceintensities. The wash step does not impact ATTO 550,but FACS resolution of ATTO 647 is best with 1 wash.

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Functional testing of labeled gRNA complexes

Positive cells by flow cytometric analysis (%)

Cas9 protein is increasingly being used for genome editing bydirect transfection of an active guide RNA:Cas9ribonucleoprotein (RNP) complex into cells. The RNP complexcan be delivered via lipofection, electroporation, or microinjectioninto cells of interest, but the efficiency of delivery can vary widelybetween cell types. We investigated the use of dye-labeledsynthetic crRNAs and tracrRNAs as an aid to assess RNPcomplex delivery efficiency and the ability to enrich fortransfected cells via FACS (fluorescence-activated cell sorting).

Introduction Washing cells increases positive fraction by flow cytometry

Alt-R™ crRNA:tracrRNARibonucleoprotein Complex (RNP)

Of the variety of dyes tested, some suffered from non-specific binding to the cells, making evaluation oftrue transfection efficiency and sorting imprecise. Others had low fluorescence intensity overbackground. Optimal results were obtained using the ATTO™ 550 dye, with placement at the 5’-end ofthe tracrRNA. We describe an optimized protocol for use of the dye-labeled reagents to monitortransfection efficiency and for use in FACS to enrich for transfected cells. In addition, intracellularlocation of the dye-labeled complexes can be visualized using fluorescence microscopy.

Optimal flow cytometric resolution occurs at 24 hr post-transfection

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* Corresponding author: mschubert@idtdna.com

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