dc.contributor.authorLeidy-Davis, Tiffany
dc.contributor.authorCheng, Kai
dc.contributor.authorGoodwin, Leslie O.
dc.contributor.authorMorgan, Judith L.
dc.contributor.authorJuan, Wen Chun
dc.contributor.authorRoca, Xavier
dc.contributor.authorOng, S. Tiong
dc.contributor.authorBergstrom, David E.
dc.identifier.citationLeidy-Davis, T., Cheng, K., Goodwin, L. O., Morgan, J. L., Juan, W. C., Roca, X., ... Bergstrom, D. E. (2018). Viable Mice with Extensive Gene Humanization (25-kbp) Created Using Embryonic Stem Cell/Blastocyst and CRISPR/Zygote Injection Approaches. Scientific Reports, 8(1), 15028-. doi:10.1038/s41598-018-33408-9en_US
dc.description.abstractHere, 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.en_US
dc.description.sponsorshipNMRC (Natl Medical Research Council, S’pore)en_US
dc.description.sponsorshipMOH (Min. of Health, S’pore)en_US
dc.format.extent17 p.en_US
dc.relation.ispartofseriesScientific Reportsen_US
dc.rights© 2018 The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.en_US
dc.subjectDRNTU::Science::Biological sciencesen_US
dc.titleViable mice with extensive gene humanization (25-kbp) created using embryonic stem cell/blastocyst and CRISPR/zygote injection approachesen_US
dc.typeJournal Article
dc.contributor.schoolSchool of Biological Sciencesen_US
dc.description.versionPublished versionen_US

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