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G3 (Bethesda). 2018 Mar 2;8(3):815-822. doi: 10.1534/g3.117.300357.

Unintended Side Effects of Transformation Are Very Rare in Cryptococcus neoformans.

Author information

1
Center for Genome Sciences and Systems Biology, Washington University School of Medicine, Saint Louis, Missouri 63110.
2
Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri 63110.
3
Center for Genome Sciences and Systems Biology, Washington University School of Medicine, Saint Louis, Missouri 63110 brent@wustl.edu.
4
Department of Computer Science and Engineering, Washington University, Saint Louis, Missouri 63130.
5
Department of Genetics, Washington University School of Medicine, Saint Louis, Missouri 63110.

Abstract

Received wisdom in the field of fungal biology holds that the process of editing a genome by transformation and homologous recombination is inherently mutagenic. However, that belief is based on circumstantial evidence. We provide the first direct measurement of the effects of transformation on a fungal genome by sequencing the genomes of 29 transformants and 30 untransformed controls with high coverage. Contrary to the received wisdom, our results show that transformation of DNA segments flanked by long targeting sequences, followed by homologous recombination and selection for a drug marker, is extremely safe. If a transformation deletes a gene, that may create selective pressure for a few compensatory mutations, but even when we deleted a gene, we found fewer than two point mutations per deletion strain, on average. We also tested these strains for changes in gene expression and found only a few genes that were consistently differentially expressed between the wild type and strains modified by genomic insertion of a drug resistance marker. As part of our report, we provide the assembled genome sequence of the commonly used laboratory strain Cryptococcus neoformans var. grubii strain KN99α.

KEYWORDS:

Cryptococcus neoformans strain KN99; Reverse genetics; gene deletion; genome sequence; yeasts

PMID:
29305388
PMCID:
PMC5844303
DOI:
10.1534/g3.117.300357
[Indexed for MEDLINE]
Free PMC Article

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