Next week, Transgenic Animal Model Core director Thom Saunders, Ph.D., will be presenting on CRISPR/Cas9 at the 16th Annual Complex Trait Community and Rat Genome & Models Conference at the University of Glasgow, UK. Read his abstract below to learn more about CRISPR/Cas9 and current uses by TAMC, as will be discussed in his talk.
CRISPR/Cas9 is a RNA guided nuclease that produces double strand breaks in DNA. Rat and mouse rat zygotes repair chromosome breaks with non-homologous endjoining (NHEJ) or homology directed repair (HDR). The resulting founder animals are typically genetic mosaics that carry multiple edits of the targeted allele. NHEJ repair can produce deletions incritical regions to knockout gene expression or remove coding exons from transcripts.
HDR repair is used to introduce new information into the genome so as cause the expression of proteins with mutant amino acid codons or to introduce reporter proteins that are expressed with exacting specificity and development timing from endogenous genes. Guide RNAs (gRNA) predicted to be active are tested in zygotes or cells. gRNAs that target Cas9 to the gene of interest and induce chromosome breaks are used to engineer genes in rat or mouse zygotes to produce genetically engineered animals.
CRISPR/Cas9 was used to produce rat gene knockout models, point mutations, reporter knockins, and conditional genes (floxed genes). The same range of mutations was produced in mouse models, additionally, deletions up to 100 kb were produced. Results show that the use of enhanced specificity Cas9 protein is highly effective in producing chromosome breaks to stimulate repair by NHEJ and HDR.
The efficiency of oligonucleotide knockins is lower than the production of simple indels and the introduction of reporters is more efficient that the generation of floxed genes. Compared to preceding embryonic stem cell techniques and other genome editing tools (zinc finger nucleases and transcription like effector nucleases) we find that CRISPR/Cas9 significantly increases access to mouse and rat genomes for the generation of biomedical research models.
Thom Saunders1,2, Wanda E. Filipiak1, Galina B Gavrilina1, Anna K. LaForest1, Corey E. Ziebell1, Elizabeth D. Hughes1
1Biomedical Research Core Facilities, Transgenic Animal Model Core; 2Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan Medical School, Ann Arbor, USA