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

• Published in: Neuron (Cambridge, Mass.) Vol. 102; no. 1; pp. 105 - 119.e8
• Main Authors: Bäck, Susanne, Necarsulmer, Julie, Whitaker, Leslie R., Coke, Lamarque M., Koivula, Pyry, Heathward, Emily J., Fortuno, Lowella V., Zhang, Yajun, Yeh, C. Grace, Baldwin, Heather A., Spencer, Morgan D., Mejias-Aponte, Carlos A., Pickel, James, Hoffman, Alexander F., Spivak, Charles E., Lupica, Carl R., Underhill, Suzanne M., Amara, Susan G., Domanskyi, Andrii, Anttila, Jenni E., Airavaara, Mikko, Hope, Bruce T., Hamra, F. Kent, Richie, Christopher T., Harvey, Brandon K.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.04.2019; Elsevier Limited
• Subjects: AAV; Adult Germline Stem Cells - metabolism; Animal models; Animals; brain; Brain - metabolism; Brain research; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Cas Systems; CRISPR-Cas9; Deoxyribonuclease I - genetics; Deoxyribonucleic acid; Dependovirus; Disease Models, Animal; DNA; Dopamine; Dopamine Plasma Membrane Transport Proteins - genetics; Dopamine receptors; Dopaminergic Neurons - metabolism; Drug abuse; Gene Editing - methods; Gene Knock-In Techniques - methods; Gene Knockout Techniques; Gene targeting; Gene Targeting - methods; Genes; Genetic Vectors; genome editing; Genomes; gRNA; Hydroxylase; Integrases; lsl-Cas9; Luminescent Proteins - genetics; MANF; Mutation; Neurons; Neurons - metabolism; Nucleotide sequence; Promoter Regions, Genetic; Rats; Rats, Transgenic; Red Fluorescent Protein; RNA, Guide, CRISPR-Cas Systems; Rodents; spermatogonial stem cells; Statistical analysis; Stem cell transplantation; Stem cells; Transgenic mice; transgenic rat; Tyrosine 3-monooxygenase; Tyrosine 3-Monooxygenase - genetics; Vectors (Biology); transgenic rat; genome editing; spermatogonial stem cells; CRISPR-Cas9; AAV; brain; lsl-Cas9; MANF
• ISSN: 0896-6273; 1097-4199
• DOI: 10.1016/j.neuron.2019.01.035

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. [Display omitted] •Methods are described for cell-specific genome modification in the adult rat brain•Transgenic rats were engineered to express Cre-dependent Cas9 or Cas9 nickase•A Cre driver rat for dopaminergic neurons and an mCherry Cre reporter rat were made•The first transgenic rat made by CRISPR and spermatogonial stem cells is described Limited availability of transgenic rats has hindered their utility in modeling human behavior and cognition. Bäck, Necarsulmer, et al. describe new transgenic rats and viral vectors that enable neuron- and region-specific gene modifications within the adult rat brain.