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CRISPR Researchers Create TB-Resistant Cattle

Cows in Hainan, China

NEW YORK (GenomeWeb) – Researchers in China have reported creating tuberculosis-resistant cattle using CRISPR/Cas9 genome editing.

Led by senior author Yong Zhang of Northwest Agricultural & Forestry University, the team used a single Cas9 nickase – a single-strand cutting Cas9 variant — to induce genome editing using the homology-directed repair pathway, inserting a gene for natural resistance-associated macrophage protein-1 (NRAMP1) into bovine fetal fibroblasts. As the researchers reported today in Genome Biology, they used somatic nuclear transfer to get the edit into an egg cell, creating 11 cows in vitro with NRAMP1 (nine using Cas9 nickase) and demonstrating that the gene provided increased resistance to tuberculosis.

Moreover, they said that while the Cas9 nickase did not completely eliminate off-target edits, it did reduce them, especially when compared to standard Cas9 which creates double-strand breaks and is much more likely to create indel mutations via the non-homologous end-joining DNA repair pathway.

"Our work has led to the discovery of a useful position in the bovine genome that can be targeted with this gene editing technology to successfully insert new genes that benefit agricultural livestock," Zhang said in a statement.

While the team said that this was the first instance of gene insertion into cattle using a single nicking Cas9, it was far from the first application of genome editing to livestock, or even cows. In May 2016, researchers led by Minnesota-based gene editing firm Recombinetics reported substituting an allele in dairy cattle to eliminate horns, using transcription activator-like effector nucleases (TALENs) to induce HDR. And last fall, another team of scientists from Northwest A&F University reported editing cashmere goats using CRISPR to produce more of the fine hairs used in wool production.

For this new study, finding the right spot to insert the gene was critical and required a "meticulous and methodological approach," Zhang noted. "When you want to insert a new gene into a mammalian genome, the difficulty can be finding the best place in the genome to insert the gene. You have to hunt through the genome, looking for a region that you think will have the least impact on other genes that are in close proximity."

The researchers settled on a site within the F-A locus. They chose to insert the NRAmP1 gene, which is associated with innate resistance to several bacterial pathogens such as Mycobacterium bovis and Salmonella, pursuing editing strategies with both standard Cas9 and Cas9 nickase editing.

To create the cows, the researchers did not directly edit cow embryos. Rather, they edited bovine fetal fibroblasts and used somatic nuclear transfer to create almost 2,400 embryos. They were then able to grow the embryos and transfer 173 Cas9-edited and 248 Cas9 nickase-edited blastocysts into cows to develop. Of those hundreds of blastocysts, 20 calves were born and 11 survived more than three months. The researchers validated the edits using junction PCR and Southern blot.

The team looked at 15 sites to assess genomic collateral damage. "Importantly, our method produced no off-target effects on the cow genetics, meaning that the CRISPR technology we employed may be better suited to producing transgenic livestock with purposefully manipulated genetics," Zhang said.

Standard Cas9 editing led to three observed indels, the authors reported.

They assessed M. bovis resistance by measuring growth rate in cells taken from the cows, with transgenic cows showing a lower growth rate. Moreover, the rate of apoptosis after infection, as opposed to necrosis, was about 30 percent in the edited cows.

Zhang and his co-authors characterized the study as a win for the single-nick approach to genome editing. While often less efficient for inserting DNA sequences than wild-type Cas9, based on their results, it is at least "a valid alternative to the classical [wild-type] Cas9 method," they wrote, adding that the prospect of fewer off-target indel mutations could be "advantageous."