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Harvard Team Demonstrates Methods for Gene Drive Containment, Reversal

NEW YORK (GenomeWeb) – While synthetic gene drives — systems that significantly increase the likelihood that a particular gene will be passed on to an organism's progeny — have the potential to address a range of ecological problems, concerns exist about the possibility that they may have unexpected and uncontrolled side effects.

But in a paper appearing in Nature Biotechnology this week, two scientists who are pioneering synthetic gene drives detailed new methods for preventing lab-based gene drives from escaping into wild populations, as well as reversing gene drive effects.

"The gene drive research community has been actively discussing what should be done to safeguard shared ecosystems, and now we have demonstrated that … proposed safeguards work extremely well and should therefore be used by every gene drive researcher in every relevant lab organism," Kevin Esvelt, a Harvard Medical School researcher and one of the study's senior authors, said in a statement.

Last year, Esvelt, along with Harvard Medical School's George Church and others, published paper discussing how the gene-editing technology CRISPR/Cas9 could be used to overcome the technical constraints that have kept gene drives largely theoretical.

CRISPR/Cas9 involves the use of the nuclease Cas9 to induce double-strand DNA breaks. These breaks are targeted to specific locations in the genome using a synthetic RNA, known as a guide RNA, that directs Cas9. According to Church and Esvelt, Cas9 can be used to not only cut target genes, but copy itself and the genes it is driving in their place.

In their 2014 paper, the scientists also suggested safeguards to prevent gene drives from altering entire wild populations, and therefore ecosystems, and a way to reverse their effects. This week, they presented data validating these approaches. 

Working in the model yeast Saccharomyces cerevisiae, the investigators showed how CRISPR/Cas9 gene drives can bias inheritance over successive generations at efficiencies of over 99 percent. Such robustness, they wrote, "demands caution," they wrote. 

"A single escaped organism carrying a synthetic CRISPR-Cas9 gene drive system could alter a substantial fraction of the wild population with unpredictable ecological consequences," they added. "It is therefore imperative for scientists performing experiments with gene drive constructs to use stringent confinement measures to minimize the risk of accidentally altering wild populations."

To that end, Church and Esvelt developed and demonstrated two molecular confinement approaches. The first, called a split drive, involves separating Cas9 and guide RNAs so they are not encoded in the same organism.

In their experiments, Cas9 was encoded on an unlinked episomal plasmid and the gene drive element contained only the guide RNA. "Because the gene encoding Cas9 is required and is unlinked from the drive, and wild yeast populations do not encode Cas9, the [guide] RNA-only drive is unable to spread in wild organisms lacking Cas9," they explained.

In the second containment strategy, Cas9 is designed to target genes in which a DNA sequence not found in wild-type organisms has been inserted. As expected, gene drive-containing yeast was unable to affect yeast lacking the synthetic target sequence.

Lastly, the research group showed that a trait imposed on yeast using a gene drive could be reversed by using another gene drive to overwrite the initial change. In doing so, the gene drive machinery remained in place, but was rendered inactive.

Overall, this work is expected to "inform, safeguard, and accelerate efforts to build CRISPR-Cas9 gene drives in other organisms," Church and Esvelt concluded. "Given the considerable potential for this technology to address global problems in health, agriculture, and conservation, these results underscore the urgent need for formal guidelines specifying requisite safeguards, inclusive public discussions, and regulatory reform to build a reliable foundation for future humanitarian applications."