Keeping transformation on target
Biolistic genetic transformation in C. neoformans produces few off-target side effects.
While genome editing is a staple of genetics research, there remains anxiety about unintended side effects of genetic transformation, one of the most common longstanding genome-editing techniques. Some researchers fear that the process of introducing exogenous DNA into a cell may cause unwanted mutations, adding confounding variables to their experiments—but others aren’t content to accept this lore.
In G3, Friedman et al. report their study of the off-target effects of transformation in the common fungal pathogen Cryptococcus neoformans. They created 23 new strains using biolistic transformation, a standard procedure for this organism that involves shooting gold beads coated with DNA into cells, to add a marker to a neutral site in the strains’ genomes. They then sequenced the genomes of these new strains. Across all 23 strains, they found only four point mutations; of these, just one changed an amino acid in the encoded protein. They also found one case of insertion of a second, partial copy of the drug resistance marker.
They used the same transformation method to create more than 100 strains, each with a single gene replaced by a marker gene. By carrying out RNA-Seq on this group, they identified six strains that expressed the marker at unusually high or low levels. On average, these outlier strains had 1.67 off-target point mutations, and three of them (50%) carried multiple copies of the marker. The greater number of mutations in these six strains compared to the first set of 23 likely reflects selection for mutations that compensate for the genes the researchers replaced. Nonetheless, the overall number of off-target effects was still low, and the authors write that the mutations would be unlikely to have consequences as drastic as deleting the intended gene would. Therefore, they argue, effects observed when a gene is deleted using this protocol are likely most often due to the deletion and not to off-target effects, although additional confirmation of any deletion’s effects is still prudent.
The study illustrates the importance of testing conventional wisdom, and it will be important to investigate whether these findings apply to other species used in research and other transformation techniques. In the process of conducting this study, the researchers also sequenced a frequently used C. neoformans laboratory strain’s genome—a vital resource because this fungus is estimated to kill hundreds of thousands of people each year.
Unintended Side Effects of Transformation Are Very Rare in Cryptococcus neoformans
Ryan Z. Friedman, Stacey R. Gish, Holly Brown, Lindsey Brier, Nicole Howard, Tamara L. Doering and Michael R. Brent
G3: GENES|GENOMES|GENETICS 2018 8: 815-822; https://doi.org/10.1534/g3.117.300357