Editing Candida albicans (section)

==Genetic and genomic tools==

Due to its nature as a model organism, being an important human pathogen and the alternative codon usage (CUG translated into serine rather than leucine), several specific projects and tools have been created to study ''C. albicans''.<ref name="Calderone"/> The diploid nature and the absence of a sexual cycle make the organism difficult to study, but in the last 20 years, many systems have been developed to observe its genetics.<ref name="Kabir"/>

===Selection markers===
The selection markers most used in ''C. albicans'' are the CaNAT1 resistance marker (confers resistance against [[nourseothricin]]) and MPAr or IMH3r (confers resistance to [[mycophenolic acid]]).<ref>{{cite journal | vauthors = Shen J, Guo W, Köhler JR | title = CaNAT1, a heterologous dominant selectable marker for transformation of Candida albicans and other pathogenic Candida species | journal = Infection and Immunity | volume = 73 | issue = 2 | pages = 1239–1242 | date = February 2005 | pmid = 15664973 | pmc = 547112 | doi = 10.1128/IAI.73.2.1239-1242.2005 }}</ref>
Next to the above-mentioned selection makers a few auxotrophic strains were generated to work with auxotrophic makers. The [[URA3]] marker (URA3 blaster method) is an often-used strategy in uridine auxotrophic strains; however, studies have shown that differences in URA3 position in the genome can be involved in the pathogeny of ''C. albicans''.<ref>{{cite journal | vauthors = Cheng S, Nguyen MH, Zhang Z, Jia H, Handfield M, Clancy CJ | title = Evaluation of the roles of four Candida albicans genes in virulence by using gene disruption strains that express URA3 from the native locus | journal = Infection and Immunity | volume = 71 | issue = 10 | pages = 6101–6103 | date = October 2003 | pmid = 14500538 | pmc = 201070 | doi = 10.1128/IAI.71.10.6101-6103.2003 }}</ref> Besides the URA3 selection one can also use the histidine, leucine and arginine autotrophy. The advantage of using those autotrophies lies in the fact that they exhibit wild-type or nearly wild-type virulence in a mouse model compared to the URA3 system.<ref>{{cite journal | vauthors = Noble SM, Johnson AD | title = Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans | journal = Eukaryotic Cell | volume = 4 | issue = 2 | pages = 298–309 | date = February 2005 | pmid = 15701792 | pmc = 549318 | doi = 10.1128/EC.4.2.298-309.2005 }}</ref> One application of the leucine, arginine and histidine autotrophy is for example the candida two-hybrid system.<ref name="stynen"/>

===Full sequence genome===

The full genome of ''C. albicans'' has been sequenced and made publicly available in a [http://www.candidagenome.org Candida database]. The heterozygous diploid strain used for this full genome sequence project is the laboratory strain SC5314. The sequencing was done using a whole-genome shotgun approach.<ref>{{cite journal | vauthors = van het Hoog M, Rast TJ, Martchenko M, Grindle S, Dignard D, Hogues H, Cuomo C, Berriman M, Scherer S, Magee BB, Whiteway M, Chibana H, Nantel A, Magee PT | display-authors = 6 | title = Assembly of the Candida albicans genome into sixteen supercontigs aligned on the eight chromosomes | journal = Genome Biology | volume = 8 | issue = 4 | pages = R52 | date = 2007 | pmid = 17419877 | pmc = 1896002 | doi = 10.1186/gb-2007-8-4-r52 | doi-access = free }}</ref>

===ORFeome project===

Every predicted ORF has been created in a gateway adapted vector (pDONR207) and made publicly available. The vectors ([[plasmid]]s) can be propagated in ''E.coli'' and grown on LB+[[gentamicin]] medium. This way every ORF is readily available in an easy to use vector. Using the gateway system it is possible to transfer the ORF of interest to any other gateway adapted vector for further studies of the specific ORF.<ref name="ReferenceB"/><ref>{{cite book | vauthors = Cabral V, Chauevl M, Firon A, Legrand M, Nesseir A, Bachellier-Bassi S, Chaudhari Y, Munro CA, d'Enfert C | chapter = Modular Gene Over-expression Strategies for Candida albicans | title = Host-Fungus Interactions | display-authors = 6 | series = Methods in Molecular Biology | volume = 845 | pages = 227–244 | year = 2012 | pmid = 22328378 | doi = 10.1007/978-1-61779-539-8_15 | isbn = 978-1-61779-538-1 | veditors = Brand AC, MacCallum DM }}</ref>

===CIp10 integrative plasmid===

Contrary to the yeast [[Saccharomyces cerevisiae|''S. cerevisiae'']] episomal plasmids do not stay stable in ''C. albicans''. In order to work with plasmids in ''C. albicans'' an integrative approach (plasmid integration into the genome) thus has to be used. A second problem is that most plasmid transformations are rather inefficient in ''C. albicans''; however, the CIp10 plasmid overcomes these problems and can be used with ease to transform ''C. albicans'' in a very efficient way. The plasmid integrates inside the RP10 locus as disruption of one RP10 allele does not seem to affect the viability and growth of ''C. albicans''. Several adaptations of this plasmid have been made after the original became available.<ref>{{cite journal | vauthors = Chauvel M, Nesseir A, Cabral V, Znaidi S, Goyard S, Bachellier-Bassi S, Firon A, Legrand M, Diogo D, Naulleau C, Rossignol T, d'Enfert C | display-authors = 6 | title = A versatile overexpression strategy in the pathogenic yeast Candida albicans: identification of regulators of morphogenesis and fitness | journal = PLOS ONE | volume = 7 | issue = 9 | pages = e45912 | year = 2012 | pmid = 23049891 | pmc = 3457969 | doi = 10.1371/journal.pone.0045912 | doi-access = free | bibcode = 2012PLoSO...745912C }}</ref><ref name="auto2">{{cite journal | vauthors = Walker LA, Maccallum DM, Bertram G, Gow NA, Odds FC, Brown AJ | title = Genome-wide analysis of Candida albicans gene expression patterns during infection of the mammalian kidney | journal = Fungal Genetics and Biology | volume = 46 | issue = 2 | pages = 210–219 | date = February 2009 | pmid = 19032986 | pmc = 2698078 | doi = 10.1016/j.fgb.2008.10.012 }}</ref>

===Candida two-hybrid (C2H) system===

Due to the aberrant codon usage of ''C. albicans'' it is less feasible to use the common host organism (''[[Saccharomyces cerevisiae]]'') for [[two-hybrid screening|two-hybrid studies]]. To overcome this problem a ''C. albicans'' two-hybrid (C2H) system was created. The strain SN152 that is auxotrophic for leucine, arginine and histidine was used to create this C2H system. It was adapted by integrating a HIS1 reporter gene preceded by five LexAOp sequences.
In the C2H system the bait plasmid (pC2HB) contains the ''[[Staphylococcus aureus]]'' LexA BD, while the prey plasmid (pC2HP) harbors the viral AD VP16. Both plasmids are integrative plasmids since episomal plasmids do not stay stable in ''C. albicans''. The reporter gene used in the system is the ''HIS1'' gene. When proteins interact, the cells will be able to grow on medium lacking histidine due to the activation of the ''HIS1'' reporter gene.<ref name="Calderone"/><ref name="stynen"/> Several interactions have thus far been detected using this system in a low scale set up.<ref name="stynen"/><ref>{{cite journal | vauthors = Legrand M, Bachellier-Bassi S, Lee KK, Chaudhari Y, Tournu H, Arbogast L, Boyer H, Chauvel M, Cabral V, Maufrais C, Nesseir A, Maslanka I, Permal E, Rossignol T, Walker LA, Zeidler U, Znaidi S, Schoeters F, Majgier C, Julien RA, Ma L, Tichit M, Bouchier C, Van Dijck P, Munro CA, d'Enfert C | display-authors = 6 | title = Generating genomic platforms to study Candida albicans pathogenesis | journal = Nucleic Acids Research | volume = 46 | issue = 14 | pages = 6935–6949 | date = August 2018 | pmid = 29982705 | pmc = 6101633 | doi = 10.1093/nar/gky594 }}</ref> A first high-throughput screening has also been performed.<ref>{{cite journal | vauthors = Schoeters F, Munro CA, d'Enfert C, Van Dijck P | title = A High-Throughput ''Candida albicans'' Two-Hybrid System | journal = mSphere | volume = 3 | issue = 4 | date = August 2018 | pmid = 30135223 | pmc = 6106057 | doi = 10.1128/mSphere.00391-18 }}</ref><ref name="frontiersin.org">{{cite journal | vauthors = Schoeters F, Van Dijck P | title = Protein-Protein Interactions in ''Candida albicans'' | journal = Frontiers in Microbiology | volume = 10 | pages = 1792 | date = 2019 | pmid = 31440220 | pmc = 6693483 | doi = 10.3389/fmicb.2019.01792 | doi-access = free }}</ref> Interacting proteins can be found at the [[BioGRID]].<ref>{{cite web|url=https://thebiogrid.org/|title=BioGRID - Database of Protein, Chemical, and Genetic Interactions|vauthors=Tyers M|website=thebiogrid.org|access-date=2018-08-25|archive-date=2017-09-11|archive-url=https://web.archive.org/web/20170911015109/https://thebiogrid.org/|url-status=live}}</ref>

===Bimolecular fluorescence complementation (BiFC)===

Besides the C2H system, a [[bimolecular fluorescence complementation|BiFC]] system has been developed to study protein-protein interactions in ''C. albicans''. With this systems protein interactions can be studied in their native sub cellular location contrary to a C2H system in which the proteins are forced into the nucleus. With BiFC one can study for example protein interactions that take place at the cell membrane or vacuolar membrane.<ref name="frontiersin.org"/><ref>{{cite journal | vauthors = Subotić A, Swinnen E, Demuyser L, De Keersmaecker H, Mizuno H, Tournu H, Van Dijck P | title = A Bimolecular Fluorescence Complementation Tool for Identification of Protein-Protein Interactions in ''Candida albicans'' | journal = G3 | volume = 7 | issue = 10 | pages = 3509–3520 | date = October 2017 | pmid = 28860184 | pmc = 5633398 | doi = 10.1534/g3.117.300149 }}</ref><ref>{{cite journal | vauthors = Mamouei Z, Zeng G, Wang YM, Wang Y | title = Candida albicans possess a highly versatile and dynamic high-affinity iron transport system important for its commensal-pathogenic lifestyle | journal = Molecular Microbiology | volume = 106 | issue = 6 | pages = 986–998 | date = December 2017 | pmid = 29030877 | doi = 10.1111/mmi.13864 | doi-access = free }}</ref>

===Microarrays===
Both DNA and protein microarrays were designed to study DNA expression profiles and antibody production in patients against ''C. albicans'' cell wall proteins.<ref name="auto2"/><ref>{{cite journal | vauthors = Mochon AB, Jin Y, Kayala MA, Wingard JR, Clancy CJ, Nguyen MH, Felgner P, Baldi P, Liu H | display-authors = 6 | title = Serological profiling of a Candida albicans protein microarray reveals permanent host-pathogen interplay and stage-specific responses during candidemia | journal = PLOS Pathogens | volume = 6 | issue = 3 | pages = e1000827 | date = March 2010 | pmid = 20361054 | pmc = 2845659 | doi = 10.1371/journal.ppat.1000827 | doi-access = free }}</ref>

===GRACE library===

Using a [[tetracycline-controlled transcriptional activation|tetracycline-regulatable promoter system]] a gene replacement and conditional expression (GRACE) library was created for 1,152 genes. By using the regulatable promoter and having deleted 1 of the alleles of the specific gene it was possible to discriminate between non-essential and essential genes. Of the tested 1,152 genes 567 showed to be essential. The knowledge on essential genes can be used to discover novel antifungals.<ref name="Large-scale essential gene identifi"/>

===CRISPR/Cas9===

[[CRISPR|CRISPR/Cas9]] has been adapted to be used in ''C. albicans''.<ref>{{cite journal | vauthors = Dean N, Ng H | title = Method for CRISPR/Cas9 Mutagenesis in ''Candida albicans'' | journal = Bio-Protocol | volume = 8 | issue = 8 | pages = e2814 | date = April 2018 | pmid = 34286028 | pmc = 8275232 | doi = 10.21769/BioProtoc.2814 | s2cid = 90620202 }}</ref> Several studies have been performed using this system.<ref>{{cite journal | vauthors = Vyas VK, Barrasa MI, Fink GR | title = A ''Candida albicans'' CRISPR system permits genetic engineering of essential genes and gene families | journal = Science Advances | volume = 1 | issue = 3 | pages = e1500248 | year = 2015 | pmid = 25977940 | pmc = 4428347 | doi = 10.1126/sciadv.1500248 | bibcode = 2015SciA....1E0248V }}</ref><ref>{{cite journal | vauthors = Min K, Ichikawa Y, Woolford CA, Mitchell AP | title = Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System | journal = mSphere | volume = 1 | issue = 3 | year = 2016 | pmid = 27340698 | pmc = 4911798 | doi = 10.1128/mSphere.00130-16 }}</ref>