• Multiplexed HDR knock-in? Yes you can!

    Simultaneously insert two different fluorescent reporters

    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):

    • 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 Technical Support team.

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

    Additional Resources

  • Webinar: A virtual workshop for precise HDR-mediated genome engineering with CRISPR-Cas9