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.

CITATION:

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
http://www.g3journal.org/content/8/3/815

, , ,

Nicole Haloupek is a freelance science writer and a recent graduate of UC Berkeley's molecular and cell biology PhD program.

View all posts by Nicole Haloupek »

Read more in

  • Ed Grell at his dissecting microscope using ether to anesthetize his flies.

    In Memoriam: Ellsworth Herman Grell (1932–2023), a pioneer of Drosophila genome engineering and annotation

    Ellsworth (Ed) Grell blessed the Drosophila community through three enduring legacies: as a pioneer of chromosome mechanics, as a primary organizer and synthesizer of genetic knowledge in Drosophila, and as a graceful mentor to those fortunate to have known him personally. Ed grew up in rural Nebraska, completed his undergraduate studies at Iowa State, and…

  • Congratulations to the #Fungal24 Poster Award winners!

    We are pleased to announce the recipients of the GSA Poster Awards for posters presented at the 32nd Fungal Genetics Conference! Undergraduate and graduate student members of GSA were eligible for the awards, and a hard-working team of judges made the determinations. Congratulations to all! Felicia Ebot Ojong, The University of Georgia My research is focused…

  • Poster presentation tips for TAGC 2024

    You’ve been selected to present a poster at The Allied Genetics Conference 2024 in March—you’ve celebrated, made plans to attend, now what? This is an exciting opportunity to showcase your research and engage with fellow members of the genetics community, so you want to make sure you’re prepared. We wanted to offer you some tips…

  • Maximize your TAGC 2024 experience 

    A guide to all that National Harbor & DC have to offer Are you joining us for The Allied Genetics Conference 2024 in March? Make the most of your #TAGC24 experience in National Harbor! We know the science will keep you busy, but you deserve to unwind and have some fun, so we’ve curated a…

  • Early Career Leadership Spotlight: Sarah Petrosky

    We’re taking time to get to know the members of the GSA’s Early Career Scientist Committees. Join us to learn more about our early career scientist advocates. Sarah PetroskyMultimedia SubcommitteeUniversity of Pittsburgh Research Interest I am interested in understanding adaptation that has been happening recently in populations by dissecting the ways that genes underlying an adaptation…

  • TAGC 2024 Early Career Award Winners

    GSA is pleased to announce the winners of the early career awards presented at The Allied Genetics Conference 2024. These awards are specific to particular TAGC communities and recognize early career scientists’ outstanding work on their respective research organisms. The awardees will present their talks in keynote sessions at TAGC 2024.  Don’t miss the opportunity…

  • Preeminent geneticists recognized with revamped GSA Awards

    In 2022, GSA’s Board of Directors launched an audit to review the five major awards conferred by the Society. Today, we are thrilled to announce the recipients of the reimagined GSA Awards, including the new Genetics Society of America Early Career Medal. The scientists honored this year are recognized by their peers for their outstanding…

  • Fly Board funds outreach programs to spread the word about Drosophila research

    In 2020, the Fly Board voted to use part of its reserve fund to support efforts to increase trainee participation as well as equity and diversity in the Drosophila community. An awards committee decides how the money will be spent each year, and from 2020–2022, the committee posted a very broad call for applications from…

  • New members of the GSA Board of Directors: 2024–2026

    We are pleased to announce the election of four new leaders to the GSA Board of Directors: 2024 Vice President/2025 President Brenda Andrews Professor, University of Toronto It’s an honor to continue my association with the Society by serving as Vice President of the Board of Directors. I have broad knowledge of the ongoing activities…

  • A rose-ringed parakeet, one of three parrot species used in the study.

    Parrot plumage study aids breeders and endangered natural populations

    Yellow coloration is maladaptive in the wild but prized by breeders. People have long been fascinated with birds, which exhibit one of the widest ranges of coloration among vertebrates. Parrots, in particular, have captivated humans by their ability to mimic human speech and spectacular plumage. Brightly colored feathers are used primarily to attract mates, intimidate…

  • Early Career Leadership Spotlight: Sarah Gilmour

    We’re taking time to get to know the members of the GSA’s Early Career Scientist Committees. Join us to learn more about our early career scientist advocates. Sarah GilmourMultimedia SubcommitteeStowers Institute for Medical Research Research Interest Questions of evolution have always fascinated me. I am extremely fortunate to be starting out my research career in a…