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

Susanne Bäck; Julie Necarsulmer; Leslie R Whitaker; Lamarque M Coke; Pyry Koivula; Emily J Heathward; Lowella V Fortuno; Yajun Zhang; C Grace Yeh; Heather A Baldwin; Morgan D Spencer; Carlos A Mejias-Aponte; James Pickel; Alexander F Hoffman; Charles E Spivak; Carl R Lupica; Suzanne M Underhill; Susan G Amara; Andrii Domanskyi; Jenni E Anttila; Mikko Airavaara; Bruce T Hope; F Kent Hamra; Christopher T Richie; Brandon K Harvey

doi:10.1016/j.neuron.2019.01.035

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

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

Authors

Susanne Bäck¹, Julie Necarsulmer², Leslie R Whitaker³, Lamarque M Coke¹, Pyry Koivula¹, Emily J Heathward², Lowella V Fortuno², Yajun Zhang², C Grace Yeh³, Heather A Baldwin¹, Morgan D Spencer¹, Carlos A Mejias-Aponte⁴, James Pickel⁵, Alexander F Hoffman⁶, Charles E Spivak⁶, Carl R Lupica⁶, Suzanne M Underhill⁷, Susan G Amara⁷, Andrii Domanskyi⁸, Jenni E Anttila⁸, Mikko Airavaara⁸, Bruce T Hope³, F Kent Hamra⁹, Christopher T Richie², Brandon K Harvey¹⁰

Affiliations

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.

PMID: 30792150
DOI: 10.1016/j.neuron.2019.01.035

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.

Published by Elsevier Inc.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adult Germline Stem Cells / metabolism*
Animals
Brain / metabolism*
CRISPR-Associated Protein 9 / genetics
CRISPR-Cas Systems*
Deoxyribonuclease I / genetics
Dependovirus
Disease Models, Animal
Dopamine Plasma Membrane Transport Proteins / genetics
Dopaminergic Neurons / metabolism*
Gene Editing / methods*
Gene Knock-In Techniques / methods
Gene Knockout Techniques
Gene Targeting / methods*
Genetic Vectors
Integrases
Luminescent Proteins / genetics
Neurons / metabolism
Promoter Regions, Genetic
RNA, Guide, CRISPR-Cas Systems
Rats
Rats, Transgenic
Red Fluorescent Protein
Tyrosine 3-Monooxygenase / genetics

Substances

Dopamine Plasma Membrane Transport Proteins
Luminescent Proteins
RNA, Guide, CRISPR-Cas Systems
Tyrosine 3-Monooxygenase
Cre recombinase
Integrases
CRISPR-Associated Protein 9
Deoxyribonuclease I