Going green in Cryptococcus neoformans: The recycling of a selectable drug marker

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen that primarily affects immunocompromised individuals. Reverse genetics is commonly used to identify and characterize genes involved in a variety of cellular processes. In C. neoformans there is a limited set of positive selectable markers available to make gene deletions or other genetic manipulations. This has hampered the application of reverse genetics in this organism. We have adapted the Bacteriophage P1 Cre-loxP system for use in C. neoformans and successfully excised and reused the same drug marker, G418, to make two sequential gene deletions, lac1Δ and cap59Δ, in the same strain. This tool will allow investigators to make multiple sequential gene deletions in the same strain, which should facilitate the analysis of multigene families.

amdS as a dominant recyclable marker in Cryptococcus neoformans

“…The molecular genetic manipulations possible in C. neoformans have until now been limited, largely as a result of relying on markers dependent on auxotrophies or conferring resistance to just a select few of antibiotics. Improvements have been made in the form of Cre-loxP mediated recycling, enabling marker removal following a second biolistic transformation event to just leave a loxP site behind, or through the heterologous expression of CRISPR-Cas9 (Arras et al, 2016;Patel et al, 2010).…”

“…Typically, most selectable markers can only be used once to genetically manipulate a specific strain, but there are strategies by which they can be recycled. One approach is using the bacteriophage P1 site-specific Cre recombinase to excise markers flanked by loxP sites (Patel et al, 2010;Sauer, 1987), however this requires heterologous expression of recombinase, and repeated recycling introduces additional loxP sites that can lead to chromosomal rearrangements as typified by SCRaMbLE (Dymond et al, 2011). A second, simpler recycling approach employs markers that can be selected both for and against, such as the orotidine-5'-phosphate decarboxylaseencoding URA3 gene.…”

Cryptococcus neoformans: historical curiosity to modern pathogen

“…Several proteins have also been epitope‐tagged or fused to fluorescent proteins for visualization. Although these techniques can be successfully applied to C. neoformans (Ding et al ., ; Haynes et al ., ; Liu et al ., ; Patel et al ., ; Reilly et al ., ; Waterman et al ., ), such studies are still often confounded by the difficulty of permeabilizing these well‐protected cells (our unpublished observations), inadequate expression of vector‐encoded proteins (see above), or the instability of modified proteins. This situation may be improved by the recent development of several fluorescent proteins that have been codon‐optimized for Cryptococcus and are robustly expressed, e.g.…”

Simple and efficient recycling of fungal selectable marker genes with the Cre-loxP recombination system via anastomosis

“…However, the ability to disrupt multiple members of extensive gene families or to eliminate redundancy is restricted by the limited number of selectable marker genes (SMGs) that are available for filamentous fungi. This limitation has been addressed by the adaption of yeast (FLP/ FRT ) and bacteriophage (Cre- loxP ) recombination systems to recycle SMGs (Kopke et al, 2010; Dennison et al, 2005; Florea et al, 2009; Forment et al, 2006; Krappmann et al, 2005; Patel et al, 2010). In the latter, more widely used recombination system, expression of an integrated Cre recombinase results in excision of an integrated SMG that is flanked by loxP sites, allowing a subsequent round of transformation with the same SMG lacking the flanking loxP sites.…”