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Updated: Mar 11, 2026
Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice
Published on: June 24, 2022
Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ
Tomomi Aida1,2,3, Shota Nakade4, Tetsushi Sakuma5
1Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo, Tokyo, 113-8510, Japan. aidat@mit.edu.
Background:
Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining.
Results:
Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes.
Conclusions:
Our results provide a technical platform for high-throughput knock-in.
