The CRISPR/Cas (clustered regularly interspaced short palindromic repeat and CRISPR-associated nucleases) system has revolutionized the way we generate animal models. While it is very efficient, fast, and less expensive than standard techniques; it is not a well characterized system. One limitation of this system was thought be the difficulty to generate animal models by targeting genes that may be essential. If both alleles of an essential gene are disrupted, the embryos would die. We wanted to test if CRISPR/Cas9 system can be use to target a potential essential genes, Diexf. We used two sgRNAs to target exon1 of Diexf, one of which yielded live animals with deleted allele. Even though both alleles were targeted in most animals, one or both alleles were always in-frame resulting in embryo survival. We observed that all alleles were transmitted via germline to the next generation. After germline transmission of the Diexf allele with frame-shift deletion, we observed that Diexf null animals are not viable. A major concern of the CRISPR/Cas9 system is the modifications (insertion or deletions) at off-target sites, however, no off-target effect was observed. In summary, we learned that Diexf is essential for the viability of the embryos and that CRISPR/Cas9 can be used to target essential genes.

Acknowledgements: This presentation is supported by the National Cancer Institute through the U54 CA096297/CA096300: UPR/MDACC Partnership for Excellence in Cancer Research Training Program.

Researchers should cite this work as follows:

• Raisa Reyes Castro (2016), "CRISPR/Cas9 system can be used to target essential genes," https://ncihub.cancer.gov/resources/1707.

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