Scientists in China have successfully inserted a tuberculosis-resistance gene into the bovine genome, without any off-target genetic effects. The researchers, at the College of Veterinary Medicine, Northwest A&F University in Shaanxi, China, used CRISPR (clustered regularly interspaced short palindromic repeats) Cas9n gene-editing technique to introduce the new gene to 11 calves.
Success in this process involves identifying the appropriate resistance gene and inserting it into the right location to transfer resistance without introducing off-target effects. The researchers identified a bovine tuberculosis (Mycobacterium bovis) resistance gene, called NRAMP1, which they inserted into the genome of bovine fetal fibroblast donor cells. Using somatic cell nuclear transfer, they inserted the nucleus of the donor cells into bovine ova, which were cultured in-vitro for embryo transfer into recipient cows.
The researchers used genetic analysis to confirm that the NRAMP1 gene had successfully integrated into the genetic code at the targeted region in the 11 resulting calves. They also compared results using an older CRISPR/Cas9 technique, and found that the older method resulted in off-target genetic effects while the novel CRISPR/Cas9n method did not.
The researchers also exposed the transgenic calves to Mycobacterium bovis, and found increased resistance to the tuberculosis pathogen as measured by standard blood markers.
In their conclusions, the scientists say their experiment demonstrated that a single Cas9n can be used for gene insertion at a selected target site in the cattle genome and that this method is advantageous in terms of avoiding additional indel mutations. The resulting transgenic cattle exhibited increased resistance to M. bovis infection. “Our study provides an avenue to develop the CRISPR/Cas9 system for agriculture applications.”
The research report is published in the journal Genome Biology.