Split selectable markers

Nat Commun. 2019 Oct 31;10(1):4968. doi: 10.1038/s41467-019-12891-2.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • CRISPR-Cas Systems
  • Cell Line, Tumor
  • Cinnamates
  • Drug Resistance, Bacterial / genetics*
  • Gene Editing
  • Gene Transfer Techniques*
  • Genetic Engineering / methods*
  • Genetic Vectors
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Hygromycin B / analogs & derivatives
  • Induced Pluripotent Stem Cells
  • Inteins*
  • Lentivirus
  • Luminescent Proteins / genetics*
  • Neomycin
  • Nucleosides
  • Protein Splicing*
  • Puromycin
  • Trans-Splicing
  • Transgenes / genetics

Substances

  • Cinnamates
  • Luminescent Proteins
  • Nucleosides
  • Hygromycin B
  • hygromycin A
  • Puromycin
  • blasticidin S
  • Neomycin