Efficient and flexible tagging of endogenous genes by homology-independent intron targeting

Genome Res. 2019 Aug;29(8):1322-1328. doi: 10.1101/gr.246413.118. Epub 2019 Jun 25.

Abstract

Genome editing tools have simplified the generation of knock-in gene fusions, yet the prevalent use of gene-specific homology-directed repair (HDR) templates still hinders scalability. Consequently, realization of large-scale gene tagging requires further development of approaches to generate knock-in protein fusions via generic donors that do not require locus-specific homology sequences. Here, we combine intron-based protein trapping with homology-independent repair-based integration of a generic donor and demonstrate precise, scalable, and efficient gene tagging. Because editing is performed in introns using a synthetic exon, this approach tolerates mutations in the unedited allele, indels at the integration site, and the addition of resistance genes that do not disrupt the target gene coding sequence, resulting in easy and flexible gene tagging.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism
  • Calnexin / genetics
  • Calnexin / metabolism
  • Cell Line, Tumor
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism
  • Exons
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Gene Editing / methods*
  • Gene Expression
  • Genome, Human*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Introns*
  • Mutagenesis, Insertional*
  • Plasmids / chemistry
  • Plasmids / metabolism
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics*
  • Vimentin / genetics
  • Vimentin / metabolism

Substances

  • CANX protein, human
  • Chromosomal Proteins, Non-Histone
  • RNA, Guide
  • Recombinant Fusion Proteins
  • VIM protein, human
  • Vimentin
  • heterochromatin-specific nonhistone chromosomal protein HP-1
  • Calnexin
  • CRISPR-Associated Protein 9