Next Article in Journal
A Systematic Review on the Therapeutic Potentiality of PD-L1-Inhibiting MicroRNAs for Triple-Negative Breast Cancer: Toward Single-Cell Sequencing-Guided Biomimetic Delivery
Previous Article in Journal
The Phenotypic and Genetic Spectrum of Glycogen Storage Disease Type VI
Previous Article in Special Issue
Delivery Approaches for Therapeutic Genome Editing and Challenges
Article

Unlocking loxP to Track Genome Editing In Vivo

1
Virology and Gene Therapy Graduate Program, Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN 55905, USA
2
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA
3
Department of Medicine, Division of Infectious Diseases, Mayo Clinic, Rochester, MN 55905, USA
4
Department of Immunology, Mayo Clinic, Rochester, MN 55905, USA
5
Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Salvador F. Aliño
Genes 2021, 12(8), 1204; https://doi.org/10.3390/genes12081204
Received: 15 May 2021 / Revised: 5 July 2021 / Accepted: 22 July 2021 / Published: 3 August 2021
The development of CRISPR-associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly quantify and monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses. Here we report a new CRISPR “fingerprinting” approach to activating luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior cre recombinase (cre)-detector system and is designed for Cas editors able to target loxP including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within loxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL-luciferase and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to cre, demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated loxP animal models. View Full-Text
Keywords: CRISPR; SaCas9; ErCas12a; loxP; Cre; luciferase reporter; fluorescent reporter; gene editing; naked DNA injection; in vivo CRISPR; SaCas9; ErCas12a; loxP; Cre; luciferase reporter; fluorescent reporter; gene editing; naked DNA injection; in vivo
Show Figures

Graphical abstract

MDPI and ACS Style

Gendron, W.A.C.; Rubin, J.D.; Hansen, M.J.; Nace, R.A.; Simone, B.W.; Ekker, S.C.; Barry, M.A. Unlocking loxP to Track Genome Editing In Vivo. Genes 2021, 12, 1204. https://doi.org/10.3390/genes12081204

AMA Style

Gendron WAC, Rubin JD, Hansen MJ, Nace RA, Simone BW, Ekker SC, Barry MA. Unlocking loxP to Track Genome Editing In Vivo. Genes. 2021; 12(8):1204. https://doi.org/10.3390/genes12081204

Chicago/Turabian Style

Gendron, William A.C., Jeffrey D. Rubin, Michael J. Hansen, Rebecca A. Nace, Brandon W. Simone, Stephen C. Ekker, and Michael A. Barry 2021. "Unlocking loxP to Track Genome Editing In Vivo" Genes 12, no. 8: 1204. https://doi.org/10.3390/genes12081204

Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop