Viable Mice with Extensive Gene Humanization (25-kbp) Created Using Embryonic Stem Cell/Blastocyst and CRISPR/Zygote Injection Approaches.

Journal Article (Journal Article)

Here, we describe an expansion of the typical DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus by zygotic injection. Driving our efforts was the desire to create a whole animal model that would replace 17 kilobase pairs (kbp) of the mouse Bcl2l11 gene with the corresponding 25-kbp segment of human BCL2L11, including a conditionally removable segment (2.9-kbp) of intron 2, a cryptic human exon immediately 3' of this, and a native human exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic embryonic stem cell (ESC) targeting, dual selection, and recombinase-driven cassette removal (ESC/Blastocyst Approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with redundant single guide RNAs (sgRNAs) and Cas9 mRNA into mouse zygotes (CRISPR/Zygote Approach). In both instances, we were able to achieve humanization of Bcl2l11 to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable, loxP-flanked, 2.9-kbp intronic segment.

Full Text

Duke Authors

Cited Authors

  • Leidy-Davis, T; Cheng, K; Goodwin, LO; Morgan, JL; Juan, WC; Roca, X; Ong, ST; Bergstrom, DE

Published Date

  • October 9, 2018

Published In

Volume / Issue

  • 8 / 1

Start / End Page

  • 15028 -

PubMed ID

  • 30301924

Pubmed Central ID

  • PMC6177426

Electronic International Standard Serial Number (EISSN)

  • 2045-2322

Digital Object Identifier (DOI)

  • 10.1038/s41598-018-33408-9


  • eng

Conference Location

  • England