ArticleNature biotechnology2018
Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.
Article in Nature biotechnology, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 195 papers.
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Who cites it
195 citing papers in PubMed, 336 citations in OpenAlex.
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- NFYA regulates two sequential genome-wide transcriptional activation events during oocyte to embryo transition.bioRxiv : the preprint server for biology · 2026Article
- Efficient multi-kilobase knock-ins in mice and cell lines using CRISPR/Cas9 and rAAV donors with unbiased whole-genome characterization by LOCK-seq.Nucleic acids research · 2026Article
- Degron models: a toolbox for rapid in vivo depletion of essential proteins regulating mRNA metabolism.Communications biology · 2026Article
- HUH-tagged Cas9 as a platform for efficient ssODN-mediated knock-in via embryo and adult injection in insects.Communications biology · 2026Article
- In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation.The Journal of experimental medicine · 2026Article
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- Strategies and considerations for the generation of ssDNA-Based HDR templates for CRISPR-based genome editing.BMC genomics · 2026Review
- Decoding the cell intrinsic and extrinsic roles of PRC2 in early embryogenesis.bioRxiv : the preprint server for biology · 2026Article
135 more citing papers are in PubMed but not listed here.
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Authors and funding
3 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Rapid, efficient generation of knock-in mice with targeted large insertions remains a major hurdle in mouse genetics. Here, we describe two-cell homologous recombination (2C-HR)-CRISPR, a highly efficient gene-editing method based on introducing CRISPR reagents into embryos at the two-cell stage, which takes advantage of the open chromatin structure and the likely increase in homologous-recombination efficiency during the long G2 phase. Combining 2C-HR-CRISPR with a modified biotin-streptavidin approach to localize repair templates to target sites, we achieved a more-than-tenfold increase (up to 95%) in knock-in efficiency over standard methods. We targeted 20 endogenous genes expressed in blastocysts with fluorescent reporters and generated reporter mouse lines. We also generated triple-color blastocysts with all three lineages differentially labeled, as well as embryos carrying the two-component auxin-inducible degradation system for probing protein function. We suggest that 2C-HR-CRISPR is superior to random transgenesis or standard genome-editing protocols, because it ensures highly efficient insertions at endogenous loci and defined 'safe harbor' sites.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.