DNA-snipping technique inspired by bacteria shows therapeutic promise
Y. Niu et al/Cell 2014
The birth of two monkeys in China provides hope that a new type of gene therapy may one day help correct genetic defects in people.
The two cynomolgus monkeys, also known as crab-eating macaques, are the first primates to have their genes precisely edited using a gene-snipping tool borrowed from bacteria, a team of Chinese scientists reports January 30 in Cell. The work is part of an effort to genetically engineer monkeys to produce mutations like those seen in human diseases, especially ones involving the brain.
Other researchers have inserted foreign genes into primates (SN: 6/20/09, p. 13), but until now, no one has succeeded in altering the animals’ own genes, says Guoping Feng, a neurobiologist at the McGovern Institute for Brain Research at MIT who was not involved in the work.
To alter the monkeys’ genes, Jiahao Sha of Nanjing Medical University and his colleagues wielded molecular scissors first discovered in bacteria. The scissors are a DNA-cutting enzyme called Cas9. In bacteria, Cas9 is part of a primitive “immune system” — known as CRISPRs — that defends against viruses by chopping up ones that the bacteria have encountered before and recognize as threats.
The technique has been used to edit the genes of human cells growing in laboratory dishes and in rats, mice and other laboratory organisms, but never before in a living primate.
Sha, along with Xingxu Huang of Nanjing University and Weizhi Ji of the Yunnan Key Laboratory of Primate Biomedical Research and Kunming Biomed International, injected mRNA used to produce Cas9 into single-celled monkey embryos. At the same time, the researchers inserted other small RNA molecules that would guide the enzyme to three genes the scientists wanted to disrupt. Once the enzyme reached the genes, it would snip the DNA, leaving the cell to attempt a repair. In some cases, the cell would be unable to repair the break correctly, leading to disruption of the gene’s activity.
Researchers hope to use the technique to disrupt genes linked to human diseases so they can study how the disease develops and test treatments. For this study, the researchers chose three genes to disrupt: Nr0b1, which is involved in keeping embryonic stem cells flexible and for determining sex; Ppar-gamma, which helps regulate metabolism; and Rag1, an immune system gene.
The researchers found that two of the three targeted genes had been simultaneously altered in eight of 15 injected embryos. Eight embryos were transplanted into surrogate mothers. The researchers delivered the first two female babies, named Mingming and Ningning, from one of the surrogate moms on November 11, 2013. Both infants carry disrupted Ppar-gamma and Rag1 genes. Two of the other surrogates miscarried, and the researchers said in an e-mail that they are awaiting the birth of the remaining baby monkeys.
Only the targeted genes were disrupted, the researchers reported. That fact is encouraging, says Jennifer Doudna, a biochemist and Howard Hughes Medical Institute investigator at the University of California, Berkeley who is a pioneer of CRISPR techniques. It suggests that CRISPRs could be used to repair some human genes without inadvertently damaging others.
Feng agrees that the work suggests gene editing might one day fix some genetic defects in people by snipping out and replacing mutated DNA. “If you can put a mutation in, this suggests you can take a mutation out,” he said.
There are still problems to solve before the technology could ever be used in people, and even hurdles to using gene-edited monkeys as stand-ins for humans, he said. The technique was not as efficient as the researchers had hoped; they failed to disrupt one of the three targeted genes.
Another pitfall: Even though the researchers injected embryos at the single cell stage, the enzyme didn’t start snipping until the cells had divided, making the monkeys into mishmashes of cells with different mutations, and leaving some cells unaltered. Such mixed-up monkeys would confuse studies of any diseases they might be designed to mimic, so the researchers would need to wait years until the monkeys could breed and produce offspring with just one type of mutation in all their cells.
Y. Niu et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.Cell. Vol. 156, February 13, 2014, published online January 30, 2014. doi: 10.1016/j.cell.2014.01.027