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Split selectable markers

Author

Listed:
  • Nathaniel Jillette

    (The Jackson Laboratory for Genomic Medicine)

  • Menghan Du

    (The Jackson Laboratory for Genomic Medicine
    University of Connecticut Health Center)

  • Jacqueline Jufen Zhu

    (The Jackson Laboratory for Genomic Medicine)

  • Peter Cardoz

    (The Jackson Laboratory for Genomic Medicine)

  • Albert Wu Cheng

    (The Jackson Laboratory for Genomic Medicine
    University of Connecticut Health Center
    University of Connecticut Health Center)

Abstract

Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple “unlinked” transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called “inteins” can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a “chaining” design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells.

Suggested Citation

  • Nathaniel Jillette & Menghan Du & Jacqueline Jufen Zhu & Peter Cardoz & Albert Wu Cheng, 2019. "Split selectable markers," Nature Communications, Nature, vol. 10(1), pages 1-8, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-12891-2
    DOI: 10.1038/s41467-019-12891-2
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    Cited by:

    1. Dixit, Yatika & Yadav, Preeti & Sharma, Arun Kumar & Pandey, Poornima & Kuila, Arindam, 2023. "Multiplex genome editing to construct cellulase engineered Saccharomyces cerevisiae for ethanol production from cellulosic biomass," Renewable and Sustainable Energy Reviews, Elsevier, vol. 187(C).
    2. Stanislav Anastassov & Maurice Filo & Ching-Hsiang Chang & Mustafa Khammash, 2023. "A cybergenetic framework for engineering intein-mediated integral feedback control systems," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    3. Yuanli Gao & Lei Wang & Baojun Wang, 2023. "Customizing cellular signal processing by synthetic multi-level regulatory circuits," Nature Communications, Nature, vol. 14(1), pages 1-14, December.

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