CRISPR/Cas9-Mediated Genetic Engineering In Vivo

Request Consultation The ability to directly and precisely modify the genome of mouse and rat embryos using CRISPR/Cas9 technology has revolutionized rodent model generation. The first CRISPR-modified mice were created in 2013. While this technology has simplified the process of creating genetically engineered mouse and rat models, there are still many important details to consider to maximize the successful creation of a model that meets project goals. Taconic Biosciences has generated CRISPR-modified mice and rats for years and has produced hundreds of different models. Our experience and expertise ensure that your project will meet your needs.

CRISPR/Cas9 technology is rapidly evolving. Taconic uses the latest CRISPR/Cas9 genome modification methodologies to generate genetically engineered mice and rat models. For example, Taconic was the first commercial provider to license Easi-CRISPR technology from the University of Nebraska. Taconic also holds CRISPR licenses from both ERS Genomics and the Broad Institute.

Three key advantages of CRISPR/Cas9 technology:

1. Reduced timelines compared to ES cell-based gene targeting methods
2. Reduced cost compared to traditional methods
3. Ability to modify a broad range of genetic backgrounds including existing genetically engineered models and models on complex genetic backgrounds

The CRISPR/Cas9 system uses a small RNA molecule called a guide RNA (gRNA) that complexes with Cas9 nuclease and guides it to a specific target site in the genome using sequence homology where Cas9 induces a double-stranded DNA break (DSB). The DSB can be repaired by either of two major DNA repair pathways: non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone process that often leads to mutations including small insertions or deletions (indels). As illustrated in the diagram below, a large deletion can be engineered by the use of two gRNAs to induce two DSBs resulting in the generation of a knockout allele. HDR is a high-fidelity repair process that occurs less frequently than NHEJ and relies on the presence of a homologous DNA repair template. This process can be exploited to insert DNA segments with targeted alterations to create knock-in alleles.

Easi-CRISPR

Easi-CRISPR is an improved genetic engineering technology that facilitates the specific insertion of large segments of exogenous DNA into the genome to generate knock-in mice or rats. Some examples where Easi-CRISPR has proven to be effective and efficient include the following genetic modifications:

Recombinase Knock-ins

The expression of site-specific recombinases is used to delete a genomic region flanked by specific recognition sites (e.g., loxP sites for Cre recombinase and FRT for Flp recombinase). The sequence coding for the recombinase is inserted into a specific genomic locus to achieve tissue-specific expression (one example with Cre recombinase inserted at the 3' end is shown below).

In this example, a self-cleaving 2A peptide coding sequence is inserted between the Cre sequence and the target gene sequence to ensure proper expression of the recombinase without disrupting the function of the gene. However, Taconic employs multiple strategies to generate recombinase knock-in models.

Reporter Gene Knock-ins

Reporter genes are used to identify cells expressing specific genes. The coding sequence for a wide variety of reporter proteins (e.g., EGFP, Luciferase, LacZ, etc.) is inserted in a specific genomic locus to achieve tissue-specific expression (one example with EGFP inserted at the 3' end is shown below).

In this example, a self-cleaving 2A peptide coding sequence is inserted between the EGFP sequence and the target gene sequence. However, Taconic employs multiple strategies to generate reported gene knock-in models.

Epitope Tag Knock-ins

Proteins can be tagged with short amino acid sequences to facilitate antibody-dependent assays (e.g., Western blot, ELISA, immunohistochemistry, immunofluorescence, FACS, etc.). The sequence coding for the tag (e.g., HA, Flag™, V5, etc.), is inserted in a specific location within the gene, thus adding the desired amino acids to the endogenous protein sequence. The tag sequence can be inserted either at the 5' or the 3' end of the gene (one example shown below).