CRISPR
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CRISPR technology has led to innumerable advances in our understanding of the human genome and genetically inherited diseases, however it is not without imperfections.  A newer technology, TARE (Toxin-Antidote Recessive Embryo), is being tested out in animal studies, which have thus far been very promising. TARE genomic modification technology improves on CRISPR in two aspects: genetic expression of the modified gene and the ability to pass on the mutation to future generations.

TARE was developed for modifying the epigenetics of non-human species, but as it is a genomic editing technology, can be used to test and modify any genome through the same mechanisms, including human.

CRISPR uses gene drives from genetic engineering to create a desired mutation in a few individuals.  To spread this mutation, these individuals mates with a larger population, thus modifying the epigenetics of that species by passing on their DNA. In humans, it could remove and replace an allele that causes a disease.

In theory, such a mechanism could be used to prevent malarial mosquitoes from transmitting disease, or possibly wipe out an invasive species by disabling its ability to reproduce.

Though scientists have had success proving the concept in the lab, they have found that wild populations invariably adapt and develop resistance to the scheme. And when gene drives work, they are and all or nothing, meaning either the gene is modified or not.

A new Cornell study titled, “A toxin-antidote CRISPR gene drive system for regional population modification,” published Feb. 27 in the journal Nature Communications, describes a new type of gene drive with the potential to delay this resistance.

In a classic gene drive, called a homing drive, an offspring inherits one set of genes, or genome, from the mother and another from the father. If an offspring inherits a gene with a drive from one parent and not the other, the drive copies itself into the genome from the parent without the drive.

“Those are two things that this new drive that we developed here addresses to some extent,” said Philipp Messer, an assistant professor of computational biology, and the paper’s senior author. Jackson Champer, a postdoctoral researcher in Messer’s lab, is the first author.

“Now that individual has that drive in both of its genomes and it will pass it on to every offspring,” Messer said.

The drives are engineered with CRISPR-Cas9 gene-editing technology, so when the drive are copied into a new genome, the CRISPR machinery will cut into the chromosome without the drive, and paste in the new code. However, occasionally cells will repair the incision and, in doing so, randomly delete DNA letters. When this happens, the CRISPR gene drive can no longer find a genetic sequence it recognizes in order to make the incision, which creates resistance to the gene insertion and stops the gene drive from spreading.

Natural genetic variation—another source of changes in DNA sequences—can also create resistance to editing technology, since CRISPR gene drives must recognize short genetic sequences in order to make the genomic incision.

“We were among the first labs to show that this is a tremendous problem,” Messer said.

The paper describes a new gene drive, called TARE (Toxin-Antidote Recessive Embryo), which works by targeting a gene that is essential for an organism to function. Because the organism can survive with only one intact copy of this essential gene, instead of cutting and pasting DNA as homing drives do, the TARE drive simply cuts the other parent’s gene, disabling it.

Meanwhile, the engineered TARE drive gene has a DNA sequence that has been recoded; the gene works but it won’t be recognized or cut in future generations. This way, if an offspring inherits two disabled genes, those individuals won’t survive, thereby removing those copies from the population. Meanwhile, as viable individuals mate, more and more surviving offspring will carry TARE drive genes.

This technology allows for a more smooth genetic modification, and one that can last for several generations without worries of the gene being “lost” after insertion.

Because TARE drives do not cut and paste a drive into a target gene, instead they destroy one of the target gene copies in the offspring, the drive requires a higher frequency of engineered individuals in the population to spread. For this reason, TARE drives are less likely to transfer from one distinct population to another.

Fruit Flies with fluorescent protein
A CRISPR gene drive that carries a red fluorescent protein as payload is spread through a population of fruit flies in laboratory experiments. [Jackson Champer/Cornell University]
In lab experiments, when fruit flies with TARE gene drives were released in cages of wild-type fruit flies, all the flies in the cage had the TARE drive in just six generations.

The researchers pointed out that resistance can indeed evolve with a TARE drive in the wild, especially in very large populations, but they believe it will take longer and evolve at a much lower rate, Messer said. The implications of using this technology to study human disease remain to be seen.

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