Efficient targeted integration directed by short homology in zebrafish and mammalian cells

Wierson, W.A., Welker, J.M., Almeida, M.P., Mann, C.M., Webster, D.A., Torrie, M.E., Weiss, T.J., Kambakam, S., Vollbrecht, M.K., Lan, M., McKeighan, K.C., Levey, J., Ming, Z., Wehmeier, A., Mikelson, C.S., Haltom, J.A., Kwan, K.M., Chien, C.B., Balciunas, D., Ekker, S.C., Clark, K.J., Webber, B.R., Moriarity, B.S., Solin, S.L., Carlson, D.F., Dobbs, D.L., McGrail, M., Essner, J.
eLIFE   9: (Journal)
Registered Authors
Balciunas, Darius, Chien, Chi-Bin, Clark, Karl, Ekker, Stephen C., Essner, Jeffrey, Kwan, Kristen, McGrail, Maura
CRISPR/Cas9, developmental biology, end joining, genetics, genomics, human, knock-in, pig fibroblasts, targeted integration, zebrafish
MeSH Terms
  • Animals
  • Animals, Genetically Modified
  • CRISPR-Associated Proteins/genetics*
  • CRISPR-Associated Proteins/metabolism
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Fibroblasts/metabolism
  • Gene Expression Regulation
  • Gene Knock-In Techniques*
  • Genes, Reporter*
  • Green Fluorescent Proteins/genetics*
  • Green Fluorescent Proteins/metabolism
  • Humans
  • K562 Cells
  • Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics
  • Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism
  • RNA, Guide/genetics
  • RNA, Guide/metabolism
  • Recombinational DNA Repair
  • Sequence Homology, Nucleic Acid
  • Sus scrofa
  • Transcription Activator-Like Effector Nucleases/genetics*
  • Transcription Activator-Like Effector Nucleases/metabolism
  • Zebrafish/genetics*
32412410 Full text @ Elife
Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22-100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.
Genes / Markers
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Engineered Foreign Genes
Errata and Notes