Generation of Large Fragment Knock-In Mouse Models by Microinjecting into 2-Cell Stage Embryos

  • Bin GuEmail author
  • Marina Gertsenstein
  • Eszter Posfai
Part of the Methods in Molecular Biology book series (MIMB, volume 2066)


Large fragment knock-in mouse models such as reporters and conditional mutant mice are important models for biological research. Here we describe 2-cell (2C)-homologous recombination (HR)-CRISPR, a highly efficient method to generate large fragment knock-in mouse models by CRISPR-based genome engineering. Using this method, knock-in founders can be generated routinely in a time frame of about two months with high germline transmission efficiency. 2C-HR-CRISPR will significantly promote the advancement of basic and translational research using genetic mouse models.

Key words

CRISPR-Cas9 2-Cell stage Knock-in Homologous recombination 



This work was supported by CIHR (FDN-143334) and Genome Canada and Ontario Genomics (OGI-099). The authors wish to thank Dr. Janet Rossant for her guidance and support during the development of 2C-HR-CRISPR and critical discussion and comments.


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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Program in Developmental and Stem Cell BiologyHospital for Sick ChildrenTorontoCanada
  2. 2.The Centre for Phenogenomics (TCP)TorontoCanada
  3. 3.Department of Molecular BiologyPrinceton UniversityPrincetonUSA

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