Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system

Among Candida species, the opportunistic fungal pathogen Candida glabrata has become the second most common causative agent of candidiasis in the world and a major public health concern. Yet, few molecular tools and resources are available to explore the biology of C. glabrata and to better understand its virulence during infection. In this study, we describe a robust experimental strategy to generate loss-of-function mutants in C. glabrata. The procedure is based on the development of three main tools: (i) a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system, (ii) an online program facilitating the selection of the most efficient guide RNAs for a given C. glabrata gene, and (iii) the identification of mutant strains by the Surveyor technique and sequencing. As a proof-of-concept, we have tested the virulence of some mutants in vivo in a Drosophila melanogaster infection model. Our results suggest that yps11 and a previously uncharacterized serine/threonine kinase are involved, directly or indirectly, in the ability of the pathogenic yeast to infect this model host organism.

! 2! 200V, 1.5 kW, 25 mF. Recovery was carried out by adding 950µL of YPD and incubation at 30 °C for 1! 4h at 130 rpm. After that, cells were harvested at 3,000 xg for 5 min, resuspended in 100µL of deionized 2! water and plated onto appropriate media.

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Design and synthesis of donor DNAs. XTAG donor DNA was produced by PCR using the pU1-and 4! pD1-XTAG primers (Supplementary Table S3). Addition of 20bp flanking region of the ADE2 locus at 5! sgADE2.1 cut site was done by PCR using the previous XTAG PCR as matrix and the pAU1-and pAD1-6! ADE2 primers. Synthesis of the 200bp flanking regions of ADE2 cut site were achieved by two 7! separated PCR reactions using genomic DNA as template and the primer pairs pAU2-ADE2 and pAS-8! HD ADE2 for the 5' upstream region and pAD2-ADE2 and pS1-HD ADE2 for the 3' upstream region.

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The resulting PCR products were added to the XTAG-20bp cassette by Gibson assembly (NEB) 10! following manufacturer's instruction.

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HIS3 donor DNA bearing 20bp of HD was produced in a two steps experiment: first we amplified two 12! fragments of HIS3 with 20bp of overlapping nucleotides with the primer pair pAU3-ADE2 and pAS1-

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HIS3 and the pair pS1-HIS3 and pAD3-ADE2. These fragments were then combined by Gibson

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assembly. HIS3-200bp HD was synthesized by association of the HIS3-20bp cassette with the previous 15! 5' upstream and the 3' upstream region used for XTAG-200bp HD synthesis.

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HIS3 donor DNA used for VPK1 disruption was generated by PCR reaction using the VPK1-HIS3-Fwd

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and Rev primers and CBS138 genomic DNA as matrix. Both primers contain 20bp of homology with 18! the VPK1 cutting site for homologous recombination.

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Each donor DNA were either purified by the Wizard SV Gel and PCR clean-up system (Promega) when 20! no aspecific bands were amplified during PCR, or the band of interest was cut out from the gel and 21! DNA was extracted by electrolysis in dialysis membrane (Carl Roth GmbH) at 110V for 30 min and 30 22! sec with reverse voltage. DNA was then subjected to phenol-chloroform extraction, precipitated with 23! 0.2 volume of 3M AcNH 4 and 1 mL of isopropanol, and the pelleted DNA resuspended in deionized 24! water.

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Homologous recombination in C. glabrata. Transformations were done as described above with a 26! slight modification. During the 10 min incubation on ice, a mix of 1µg of donor DNA and 1µg of pRS314-

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CAS9 was added to the ∆HTL + sgADE2.1 strain. Insertion of the donor DNA at the ADE2 locus was 28! checked by colony-PCR using the ADE2-Fwd and either pD1-XTAG (for XTAG insertion) or pSeqHIS3-

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For sequencing, PCR was performed with the Phusion DNA polymerase using different couples of 24! primers described in Supplementary Table S3 and purified on the Wizard SV Gel and PCR clean-up 25! system (Promega). For ADE2, we used the ADE2-Fwd and Rev primers, for XTAG insertion ADE2-