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. A mutant form of Cas9 (D10A nickase) only cuts one strand of DNA. Using Cas9 nickase and two gRNAs targeting same region of DNA will create a functional double-stranded break but with less chance of off target mutations than the Cas9 wild-type nuclease. 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-Cas9D10A)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 two proximal gRNAs via viral or non-viral vectors.
Status and Availability
As of date, this strain is available as line #834 at the RRRC.
This rat is registered at the Rat Genome Database (RGD) ID# 13602097.
Figure 1: Schematic of Rosa26-targeted Cre-dependent Cas9n transgene. The coding region for FLAG-tagged SpCas9n with nuclear-localization signals was amplified from pX461 (Addgene #48140), then inserted downstream of a CAG promoter between a flox-stop cassette and the polyadenylation signal from the gene for bovine growth hormone (pOTTC1076). 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 (OTTC1076), and plasmids encoding the gRNAs to rat Rosa26 and Cas9 were electroporated into rat spermatogonial stem cells by the lab of Kent Hamra (UTSW), and selected by growth on geneticin-containing media. Geneticin-resistant clones were pooled and transplanted into the testes of an azoospermic male rat, which was subsequently used to sire transgenic LSL-nickase progeny.
Assay for the presence of NeoR and Rosa26, click here
References that cite this rat
In: Neuron, vol. 102, no. 1, pp. 105–119, 2019, ISSN: 1097-4199 (Electronic); 0896-6273 (Linking).
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