Genome engineering of Yarrowia lipolytica has created strains that convert sugars to lipid that make up >90% of their dry cell weight. Other strains have been engineered to produce high titers of omega-3 fatty acids and carotenoids; none the less, genetic modifications in Y. lipolytica are challenging, and the available genome editing tools are limited to low-efficiency homologous recombination (HR) and sequential integrations by Cre/Lox using a selectable marker.

The widespread adoption of type II CRISPR−Cas9 systems for genome editing has made less genetically tractable organisms more accessible. Targeted Cas9 nuclease creates double-stranded breaks at genomic loci defined by a 20 bp region of single guide RNA (sgRNA). Repair of the breaks by nonhomologous end joining or HR can be used to introduce insertions and deletions (indels), disrupt gene function, and integrate larger heterologous DNA sequences. This genome editing strategy has been implemented in Escherichia coli, Saccharomyces cerevisiae, and mammalian cells and has enabled similar capabilities in the yeasts Schizosaccharomyces pombe and Kluyveromyces lactis as well as bacteria including Streptomyces. Here, we add to the genome engineering tools available for Y. lipolytica by developing a high-efficiency CRISPR−Cas9 system for markerless gene disruption and insertion.

—Schwartz et al.

Described in 2012, CRISPR-Cas9 is a groundbreaking technique that enables scientists to make precise targeted changes in living cells. Unlike traditional gene-editing methods, it is cheap, easy to use and effective in almost any organism.

Traditionally, researchers have focused on model organisms that are relatively easy to manipulate at the genetic level, and those working on less tractable species have had to go through long and tedious processes to create new strains. Our work with Y. lipolytica is a good example of how the CRISPR-Cas9 system is facilitating research in organisms that are biologically interesting but historically difficult to work with.

—Ian Wheeldon, the study’s principal investigator

In the paper, the team adapted CRISPR-Cas9 for Y. lipolytica, showing that the system could be used to knock genes out and introduce new genes, both useful tools in bio-manufacturing.

Wheeldon said the current work was the first step in a National Science Foundation-funded project to create long chain hydrocarbons—used to make specialty polymers, adhesives, coatings and fragrances—from yeast rather than synthetically.

Other researchers may use the system to create precursors for biofuels, reducing the current reliance on edible plant oils, Wheeldon said.

The work was done by Wheeldon, Cory Schwartz, a graduate student in the Department of Chemical and Environmental Engineering at UCR, and Murtaza Shabbir Hussain and Mark Blenner from Clemson University in South Carolina. The research was supported by the National Science Foundation.

Resources

• Cory M. Schwartz, Murtaza Shabbir Hussain, Mark Blenner, and Ian Wheeldon (2015) “Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR−Cas9-Mediated Genome Editing in Yarrowia lipolytica” ACS Synthetic Biology doi: 10.1021/acssynbio.5b00162