Tild-CRISPR Allows for Efficient and Precise Gene Knockin in Mouse and Human Cells

Dev Cell. 2018 May 21;45(4):526-536.e5. doi: 10.1016/j.devcel.2018.04.021.

Abstract

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.

Keywords: CRISPR/Cas9; Tild-CRISPR; gene editing; genetically modified mice; human embryo; knockin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Cells, Cultured
  • DNA / administration & dosage*
  • Electroporation
  • Embryo, Mammalian / cytology
  • Embryo, Mammalian / metabolism*
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / metabolism*
  • Female
  • Fertilization in Vitro
  • Gene Knock-In Techniques / methods*
  • Homologous Recombination
  • Humans
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Inbred DBA
  • Mice, Inbred ICR
  • RNA, Guide / administration & dosage*
  • Zygote / growth & development
  • Zygote / metabolism

Substances

  • RNA, Guide
  • DNA