PUBLICATION
GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish
- Authors
- Welker, J.M., Wierson, W.A., Almeida, M.P., Mann, C.M., Torrie, M.E., Ming, Z., Ekker, S.C., Clark, K.J., Dobbs, D.L., Essner, J.J., McGrail, M.
- ID
- ZDB-PUB-210817-7
- Date
- 2021
- Source
- Bio-protocol 11: e4100 (Journal)
- Registered Authors
- Clark, Karl, Ekker, Stephen C., Essner, Jeffrey, McGrail, Maura
- Keywords
- CRISPR/Cas9, Homology mediated-end joining, Knock-in, Targeted integration, Zebrafish
- MeSH Terms
- none
- PubMed
- 34395736 Full text @ Bio Protoc
Citation
Welker, J.M., Wierson, W.A., Almeida, M.P., Mann, C.M., Torrie, M.E., Ming, Z., Ekker, S.C., Clark, K.J., Dobbs, D.L., Essner, J.J., McGrail, M. (2021) GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish. Bio-protocol. 11:e4100.
Abstract
Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for germline recovery of integration alleles. The GeneWeld protocol and pGTag (plasmids for Gene Tagging) vector series provide a set of resources to streamline precision gene targeting in zebrafish. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at a CRISPR/Cas induced DNA double-strand break. The pGTag vectors contain reporters flanked by a universal CRISPR sgRNA sequence to liberate the targeting cassette in vivo and expose homology arms for homology-driven integration. Germline transmission rates for precision-targeted integration alleles range 22-100%. Our system provides a streamlined, straightforward, and cost-effective approach for high-efficiency gene targeting applications in zebrafish. Graphic abstract: GeneWeld method for CRISPR/Cas9 targeted integration.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping