Split selectable markers

• Published in: Nature communications 2019-10, Vol.10 (1), p.4968-8, Article 4968
• Main Authors: Jillette, Nathaniel, Du, Menghan, Zhu, Jacqueline Jufen, Cardoz, Peter, Cheng, Albert Wu
• Format: Article
• Language: English
• Subjects: 13/44; 631/1647/1511; 631/337/4041/3196; 631/61/2300; Cell Line, Tumor; Chain scission; Cinnamates; CRISPR; CRISPR-Cas Systems; Drug Resistance, Bacterial - genetics; Fluorescence; Gene Editing; Gene Transfer Techniques; Genes; Genetic Engineering - methods; Genetic modification; Genetic Vectors; Genome editing; Genomes; Genotypes; HEK293 Cells; HeLa Cells; Humanities and Social Sciences; Humans; Hygromycin; Hygromycin B - analogs & derivatives; Induced Pluripotent Stem Cells; Inteins; Lentivirus; Luminescent Proteins - genetics; Markers; multidisciplinary; Neomycin; Nucleosides; Protein Splicing; Proteins; Puromycin; Science; Science (multidisciplinary); Splicing; Trans-Splicing; Transgenes; Transgenes - genetics; Vectors
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-12891-2

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. Selectable markers are widely used in cell engineering but there is only a limited variety to choose from. Here the authors split markers using inteins, allowing up to six transgene integration events to be selected for with one marker.