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In Vivo Validation of CRISPR Reagents in Preimplantation Mouse Embryos

  • Melissa A. Larson
  • Katelin A. Gibson
  • Jay L. VivianEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2066)

Abstract

The CRISPR/Cas9 system has enjoyed enormous success and has now become the standard method of generating gene-modified mouse models. The tools for predicting the activity of CRISPR reagents in the mouse embryo are currently limited and not particularly accurate in predicting if a given reagent will be active. Given the time and cost of generating genetically modified mice, it is highly desirable to use CRISPR reagents that are known to be active in the mouse embryo. In this chapter, we provide a detailed procedure for empirically testing the activity of CRISPR reagents via electroporation into cultured preimplantation mouse embryos. This platform has proven to be rapid, efficient, and applicable to a variety of mouse strains, and can be used for assessing on- and off-target activity through a variety of molecular assays.

Key words

CRISPR Validation crRNA gRNA Indels Cas9 Electroporation NEPA21 Embryo Zygote 

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Copyright information

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

Authors and Affiliations

  • Melissa A. Larson
    • 1
    • 2
  • Katelin A. Gibson
    • 1
  • Jay L. Vivian
    • 1
    • 3
    Email author
  1. 1.Transgenic and Gene-Targeting Institutional FacilityUniversity of Kansas Medical CenterKansas CityUSA
  2. 2.Department of Molecular and Integrative PhysiologyUniversity of Kansas Medical CenterKansas CityUSA
  3. 3.Department of Pathology and Laboratory MedicineUniversity of Kansas Medical CenterKansas CityUSA

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