CRISPR/Cas9 technology enables the targeting of mutations to specific DNA sequences within an organism’s genome. Cas9 is a nuclease that interacts with a guide RNA (gRNA) whose sequence determines the target DNA site for the Cas9 to create a double-strand break in the genomic DNA. The endogenous repair mechanisms lead to mutant DNA sequence once a double-stranded DNA break has been made. To facilitate genome modification in the rat, we generated and characterized a strain of transgenic Long Evans rats with Cre-dependent expression of Cas9 nuclease I (LE-Rosa26Tm1(LSL-Cas9)Ottc). The RAT enables cell/tissue-specific genome modification in the rat when it is combined with a Cre recombinase via viral vector or Cre-driver rat and by providing gRNAs via viral or non-viral vectors.
Status and Availability
As of date, this strain is available as line #00833 at the RRRC.
This rat is registered at the Rat Genome Database (RGD) ID# 13208224.
Figure 1: Schematic of Rosa26-targeted Cre-dependent Cas9 transgene. The coding region for FLAG-tagged SpCas9 with nuclear-localization signals was amplified from pX458 (Addgene #48138), then inserted downstream of a CAG promoter between a flox-stop cassette and the polyadenylation signal from the gene for bovine growth hormone (pOTTC1080). The final construct was used as a donor template for homology directed repair of a CRISPR-Cas9 double-strand break targeted by the following guide RNAs:
rRosa26 A GCAGATCACGAGGGAAGAAG
rRosa26 B GAGTCTTTCTGGAAGATAGG
The donor template (OTTC1080), the gRNAs (produced by in vitro transcription), and the mRNA encoding the Cas9-nickase (Tri-Link), were injected into fertilized Long Evans rat embryos by NIMH Transgenic Core. Founder rats were identified then genotypically and phenotypically verified.
Assay for the presence of Rosa26-targeted LSL-Cas9 insert, click here
References that cite this rat
In: Neuron, vol. 102, no. 1, pp. 105–119, 2019, ISSN: 1097-4199 (Electronic); 0896-6273 (Linking).
No comments or reviews are available at this time.
Other related rats
Registered Symbol: LE.Cg-(ROSA)26 em1(CAG-Cas9*D10A)Ottc
RGD ID: 13602097
Proposed Name: LE-Rosa26Tm1(LSL-Cas9D10A)ottc
Susanne Back, YaJun Zhang, Julie Necarsulmer, Pyry Koivula, Emily Heathward, Christopher T. Richie, Brandon Harvey, Janette Lebron