Journal
DEVELOPMENTAL CELL
Volume 45, Issue 4, Pages 526-+Publisher
CELL PRESS
DOI: 10.1016/j.devcel.2018.04.021
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Funding
- National Science and Technology major project [2017YFC1001302]
- Shanghai City Committee of Science and Technology project [16JC1420202]
- NSFC [31522037, 31500825, 81671413]
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetic [17DZ2271100]
- National Research and Development Plan [2016YFC1000604]
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The targeting efficiency of knockin sequences via homologous recombination (HR) is generally low. Here we describe a method we call Tild-CRISPR (targeted integration with linearized dsDNA-CRISPR), a targeting strategy in which a PCR-amplified or precisely enzyme-cut transgene donor with 800-bp homology arms is injected with Cas9 mRNA and single guide RNA into mouse zygotes. Compared with existing targeting strategies, this method achieved much higher knockin efficiency in mouse embryos, as well as brain tissue. Importantly, the Tild-CRISPR method also yielded up to 12-fold higher knockin efficiency than HR-based methods in human embryos, making it suitable for studying gene functions in vivo and developing potential gene therapies.
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