Simple growth conditions improve targeted gene deletion in Cryptococcus neoformans

Abstract

Cryptococcus neoformans infections are a significant cause of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. The cryptococcal cell wall is very dynamic and can be modulated depending on growth conditions. It was reported that when C. neoformans is grown in unbuffered yeast nitrogen base (YNB) for 48 hours, the pH of the media drastically drops, and the cells start to shed their cell walls. With this observation, we sought to determine if YNB-grown cells could be used directly for genetic transformation. To test this, we targeted ADE2 using TRACE (transient CRISPR-Cas9 coupled with electroporation) in YNB-grown or competent cells. Deletion of the ADE2 gene results in red-pigmented colonies, allowing visual confirmation of disruption. We were able to successfully delete ADE2 in YNB-grown cells with better efficiency compared to competent cells. Recent studies have shown that gene deletion can be accomplished using short (50 bp) homology arms in place of the normal long arms (~1 kb). However, it was inefficient, leading to more insertions and gene disruption than gene deletions. We tested short homology with YNB-grown cells vs. competent cells and found that gene deletion was significantly improved in YNB-grown cells, at around 60% compared to 6% in competent cells. This was also observed when we deleted LAC1 with the short arms. Altogether, using simple growth conditions, we have greatly improved the speed and efficiency of cryptococcal genetic transformations.IMPORTANCEThe World Health Organization recently ranked C. neoformans as the highest-priority fungal pathogen based on unmet research and development needs and its public health importance. Understanding cryptococcal pathogenicity is key for developing treatments. We found that using simple growth conditions can greatly improve the speed and efficiency of cryptococcal genetic transformations. This finding will advance the field by expanding the ease of cryptococcal genetic manipulations.

Keywords: Cas9; Cryptococcus neoformans; growth media; molecular genetics.

Fig 1

Graphical synopsis of cell culture, preparation, and transformation protocol. Brief workflow diagram depicting the timeline and key steps for preparation of (A) competent cells and (B) YNB-grown cells. Precise details may be consulted in Materials and Methods. The image was created with Biorender.

Fig 2

YNB-grown cells can be used directly for genetic transformation. Transformation plates for deletion of ADE2 using competent cells or YNB-grown cells using TRACE. Transformation without the Cas9 cassette was also included as a CRISPR control. The transformations were plated and selected on YPD plates supplemented with nourseothricin (NAT).

Fig 3

YNB-grown cells improve deletions using short (50  bp) homology arms. (A) Transformation plates for deletion of ADE2 using competent cells or YNB-grown cells using short-homology deletion cassette. Transformation without the Cas9 cassette was also included as a CRISPR control. The transformations were plated and selected on YPD plates supplemented with nourseothricin (NAT). (B) Image of a LAC1 deletion transformation using short homology deletion cassette plate patched onto L-DOPA plates. The wild-type (WT) parental strain (red box) and a known confirmed lac1Δ strain (green box) were also added to each plate as controls.

Fig 4

Short arm homology-directed repair is significantly improved in YNB-grown cells. Genotyping of (A) ADE2 and (B) LAC1 transformants. Representative gels from PCR genotyping of transformants. Ten red colonies from the ADE2 plates or ten white colonies from the LAC1 plates were randomly selected from each condition and screened. Black arrowheads indicate transformants with HDR at both ends. Data was collected from three independent experiments.