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Neuron. 2019 Apr 3;102(1):105-119.e8. doi: 10.1016/j.neuron.2019.01.035. Epub 2019 Feb 18.

Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

Author information

1
Molecular Mechanisms of Cellular Stress and Inflammation Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA.
2
Optogenetics and Transgenic Technology Core/Genetic Engineering and Viral Vector Core, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA.
3
Neuronal Ensembles in Drug Addiction Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, MD 21224, USA.
4
Histology Core, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA.
5
Transgenic Technology Core, Intramural Research Program, National Institute of Mental Health, Bethesda, MD 20892, USA.
6
Electrophysiology Research Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA.
7
Laboratory of Molecular and Cellular Neurobiology, Intramural Research Program, National Institute of Mental Health, Bethesda, MD 20892, USA.
8
Institute of Biotechnology, HiLIFE, University of Helsinki, 00014 Helsinki, Finland.
9
Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
10
Molecular Mechanisms of Cellular Stress and Inflammation Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA; Optogenetics and Transgenic Technology Core/Genetic Engineering and Viral Vector Core, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA. Electronic address: bharvey@mail.nih.gov.

Abstract

Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.

KEYWORDS:

AAV; CRISPR-Cas9; MANF; brain; genome editing; lsl-Cas9; spermatogonial stem cells; transgenic rat

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