# CRISPR/Cas9-compatible plasmids enabling seven dominant genetic selection methods for the human fungal pathogen Cryptococcus neoformans

## TL;DR

This paper introduces new genetic selection tools for studying the fungus Cryptococcus neoformans, enabling more efficient genetic experiments.

## Contribution

The study introduces blasticidin S resistance as a new selection method and validates phleomycin resistance for genetic manipulation in C. neoformans.

## Key findings

- Blasticidin S resistance via BSD or BSR is a novel dominant selection method for C. neoformans.
- Phleomycin resistance via BLE is validated as an additional selection method.
- A vector series for CRISPR/Cas9-mediated genome modification was developed and deposited at Addgene.

## Abstract

Cryptococcus neoformans is the most common cause of human
fungal meningitis and an important model system for studying fundamental
eukaryotic biology. Genetic manipulation of this organism relies on three
dominant drug resistance markers (nourseothricin acetyltransferase
[NAT], neomycin phosphotransferase II
[NEO], and hygromycin B phosphotransferase
[HYG]) and the recyclable dominant prototrophic marker
amdS. With ongoing technological advances that are
expanding our ability to explore cryptococcal gene function, contemporary
studies often require multiple genetic manipulations in the same strain.
Additional dominant selection methods would maximize the utility of these
tools by facilitating their combinatorial use. Here, we identify blasticidin
S resistance via the blasticidin S deaminase (BSD) or
blasticidin S resistance (BSR) markers as a novel dominant
selection method for C. neoformans. We further validate
phleomycin resistance via the bleomycin resistance gene
(BLE) marker as an additional selection method,
confirming a study that first established this marker 25 years ago (J. Hua,
J. D. Meyer, and J. K. Lodge, Clin Diagn Lab Immunol 7:125–128, 2000,
https://doi.org/10.1128/cdli.7.1.125-128.2000). To enable
highly efficient CRISPR/Cas9-mediated genome modification, we incorporated
these markers, as well as the newly established dominant prototrophic marker
ptxD (M. Khongthongdam, T. Phetruen, and S. Chanarat,
Microbiol Spectr 13:e01618-24, 2025, https://doi.org/10.1128/spectrum.01618-24), into a vector series
that enables the construction of fused marker-sgRNA products via PCR.
Altogether, this work expands the number of dominant genetic selection
methods for C. neoformans to seven, including five drug
selection regimes and two prototrophic methods. The vector series has been
deposited at Addgene.

Cryptococcus neoformans is the top-ranked World Health
Organization priority fungal pathogen due to its widespread distribution
and inadequate treatment options. Additionally, as a basidiomycete yeast
occupying an underexplored branch of the fungal kingdom, this organism
is a powerful system for deciphering core eukaryotic biology that is
absent in classic model fungi. Defining functions for novel cryptococcal
genes is a crucial priority, and the availability of additional genetic
selection methods would facilitate these efforts. In this study, we
establish blasticidin S resistance as a novel genetic selection method
for C. neoformans, and we validate a previous report
using phleomycin resistance as such. This work expands the number of
reliable dominant selection methods to seven, providing flexibility for
the introduction of sequential genetic modifications into single
strains.