Simultaneously insert two different fluorescent reporters

Generation of a fluorescent reporter cell line using homology-directed repair (HDR) is a challenging and time-consuming process, even for experienced researchers. First, it requires design of a guide RNA to cut the genomic DNA near the insertion site, then design of a donor plasmid with appropriate homology arms to facilitate the knock-in and finally assembly of the donor plasmid. Following transfection of all the components necessary for knock-in, you’ll need to do clonal cell isolation. After weeks of waiting for your clonal cell lines to grow, and the additional time required to isolate and sequence each clone, your patience pays off, and you hopefully have your new reporter cell line.

That’s a lot to go through even for one fluorescent insert; what if you had to repeat this procedure again because you need two proteins tagged, not just one? If performed sequentially, this entire process can take months. Luckily, we have found that you can save time by combining CRISPR-Cas9 reagents targeting two different gene targets along with two corresponding HDR donor plasmids (one red and green), in a single transfection, to generate a double knock-in!

Simultaneous knock-in of two fluorescent proteins

We wanted to create a U2OS reporter cell line with EGFP-tagged LMNA and mKate2-tagged SEC61B. To accomplish this, we simultaneously transfected our U2OS cells with LMNA and SEC61B-targeting custom synthetic crRNAs (complexed with tracrRNA), along with fluorescent donor plasmids and Cas9 mRNA (Figure 1). Design of the custom guide RNAs and the fluorescent donors was relatively straightforward using the CRISPR Configurator and HDR Donor Designer, along with the Edit-R™ HDR plasmid donor kits for both the mKate2 and EGFP inserts. Donor plasmids were generated, and the co-transfection of all components was performed as directed in the kit protocol. At the seventh day post-transfection, we subjected the cells to flow cytometry and identified the percent of the cell population expressing each fluorescent reporter (Figure 1):

U2OS cell line with EGFP-LMNA and mKate2-SEC61B fluorescent reporters generated through multiplexed CRISPR-Cas9-mediated knock-in

u2os cell line egfp lmna mkat2 sec61b fluorescent reporters multiplex

Figure 1: Briefly, synthetic crRNAs targeting LMNA and SEC61B were designed to generate double-strand breaks near the N-termini of each of the proteins using the CRISPR RNA Configurator. The Edit-R HDR Donor Designer was used to design reagents to generate EGFP and mKate2 donor plasmids specific to the N-termini of LMNA and mKate2, respectively. Both donor plasmids contained ~1000 base pairs of homology per homology arm. U2OS cells were plated at 10,000 cells per well in a 96-well plate the day before transfection. Cells were transfected using 0.3 µL/well DharmaFECT Duo transfection reagent according to optimized conditions determined in previous experiments. The following reagents were transfected into the cells simultaneously: 200 ng Edit-R™ Cas9 Nuclease mRNA, 25 nM synthetic crRNA:tracrRNA targeting LMNA, 25 nM synthetic crRNA:tracrRNA targeting SEC61B, 200ng EGFP-LMNA donor plasmid and 200 ng mKate2-SEC61B donor plasmid. Cells were passaged three times and imaged 7-days post transfection on a Nikon inverted epifluorescent microscope to identify single cells that contained both EGFP (green) and mKate2 (red) insertion.

  • EGFP-LMNA: 3%
  • mKate2-SEC61B: 3%
  • Both EGFP-LMNA and mKate2-SEC61B: 2.7%

By using this strategy to multiplex your HDR knock-in of fluorescent tags, you can generate clonal cell lines in half the time, allowing you to focus on experiments instead of generating assay components! If you would like to talk to an expert on whether this strategy might work for you, just call our Scientific Support team.

Author: John A Schiel, R&D Scientist at Dharmacon

Additional Resources

A virtual workshop for precise HDR-mediated genome engineering with CRISPR-Cas9
  • Design guide RNAs and DNA oligo or plasmid repair templates using online tools for HDR-mediated CRISPR-Cas9 applications.
CRISPR RNA Configurator Tool for HDR - video
  • Quickly learn how to use the CRISPR RNA Configurator tool to specifically design and order custom guide RNAs for your precise HDR-mediated experiment.
Edit-R HDR Donor Designer - plasmid
  • Design and order a plasmid DNA donor kit for insertion of an mKate2 or EGFP fluorescent marker, or a custom insert
Edit-R HDR plasmid donor kit for fluorescent reporter knock-in - Technical Manual
  • Easily generate a fluorescent reporter knock-in for any gene. This protocol guides the user through HDR donor design, cloning, and transfection.