Materials and Methods

Two Distinct Approaches for CRISPR-Cas9-Mediated Gene Editing inCryptococcus neoformansand Related Species

MATERIALS AND METHODSFungal strains and plasmid DNA. Wild-type strains, including C. neoformans H99 (serotype A) and C. deneoformans JEC21 (serotype D) and C. gattii/C. deuterogattii WM268 (serotype B, VGIIa), were used in this study (29,–31). All strains were cultured in yeast extract-peptone-dextrose (YPD) medium for 3 days at 30°C. Construction of the gib2::NAT mutant allele was previously described (19, 20). Briefly, the nourseothricin resistance gene cassette was inserted into the coding region of the GIB2 gene that encodes the atypical Gβ/RACK1-like adaptor protein of Cryptococcus neoformans (22,–24). The mutant allele was used as donor DNAs for the Cas9-mediated gene deletions described in this work. The gib2::NAT fragment was amplified by PCR with primers PW166 and PW167 and purified using the Qiagen gel extraction kit (Qiagen). The ~3.2-kb fragment contains the NAT cassette flanked by ~900-bp 5′ and ~800-bp 3′ homologous regions of the GIB2 gene.To replace the 75-nt U. oryzae tRNA sequence of pmCas9:tRNAp-gRNA with an endogenous U6 promoter, primers PW2051 and PW2052 were used to link the U6 promoter from C. deneoformans (10) with the BsmBI/Esp3I restriction sites followed by the tracrRNA sequence. The KpnI-EcoRI fragment was used to replace the original fragment within pmCas9:tRNAp-gRNA resulting in pCnCas9:U6-gRNA. The plasmid and the sequence are available upon request while the deposition into public domains is in progress.Construction of plasmids expressing Cas9 and gib2-specific gRNAs was made possible by an insertion of annealed, complementary 20-nt oligonucleotide gRNA oligonucleotides as gRNA sequence plus a 4-nt linker at the BsmBI/Esp3I site. Insertion of the gRNA sequence eliminates the BsmBI/Esp3I restriction enzyme site so that positive clones can be easily screened. Primer names and their sequences are listed in Table 1.Design of crRNA and preparation of the ribonucleoprotein complex. To test the CRISPR-Cas9 ribonucleoprotein complex-mediated gene disruption, two Cas9-gRNA constructs were employed. First crRNA was designed to target the region closer to the 5′ end of the Gib2 gene, while the second crRNA was designed to be outside the coding region near the stop codon (Fig. 1). The same crRNAs were used to target the GIB2 gene of H99 (C. neoformans) and JEC21 (C. deneoformans). For C. gattii/C. deuterogattii, a new 3′ crRNA (crRNA3) was needed, while the crRNA targeting 5′ of the GIB2 gene (crRNA1) remains the same. gRNA sequences were designed using the resources at Integrated DNA Technologies, Inc. (IDT; Coralville, IA).To prepare the ribonucleoprotein complex, two crRNAs were hybridized to the equal molar amounts of tracrRNA in nuclease-free duplex buffer (100 mM KCH3COO, 30 mM HEPES, pH 7.5 IDT) to ~20 µM. The mixture was annealed by heating at 94°C for 5 min and then slowly cooling down to room temperature (20°C). The resulting gRNA was used immediately or stored at −20°C. To generate the Cas9 ribonucleoprotein complex, 1 µl of gRNA, 8 µl of nuclease-free reaction buffer (150 mM KCl, 20 mM HEPES, pH 7.5), and 1 µl of diluted Cas9 (1 µg/µl in reaction buffer IDT) were mixed. The mixture was incubated at 30°C for 5 min to allow ribonucleoprotein complex formation.Electroporation. Electroporation-mediated transformation of Cryptococcus species was performed in a Bio-Rad electroporation system (Gene Pulser Xcell) according to a previously published protocol (21, 25). Briefly, fungal cells grown overnight in liquid YPD were used to start fresh cultures the next day. When cultures reached an OD of ~1.0, the cells were precipitated and washed three times with distilled water. Cells were washed once in electroporation buffer (EB: 10 mM Tris-HCl, 1 mM MgCl2, 270 mM sucrose, pH 7.5) and resuspended in EB with 1 mM DTT for 30 min. Cells were washed again, resuspended in 1/10 of the original volume of EB, and incubated on ice until use. For electroporation, we mixed the ribonucleoprotein complexes described above and 2 µg of gib2::NAT donor DNA with 100 µl fungal cells on ice. Electroporation was performed within 5 min of mixing using either the exponential protocol (0.450 kV, 125 µF, and 500 Ω) or the time constant protocol (1.8 to 2.0 kV, 5 ms). Following electroporation, 1 ml of cold YPD with 0.5 M sorbitol and the contents of the cuvette were transferred to a 1.5-ml microcentrifuge tube for recovery by incubation at 30°C for 90 min with shaking (150 rpm). Cells were then pelleted and plated onto YPD plates (one cuvette to one plate) with nourseothricin (70 µg/ml). The plates were incubated at 30°C for 3 days or until colonies emerged.Oligonucleotide annealing and biolistic transformation. To anneal complementary oligonucleotide primers prior to ligation into pCnCas9:U6-gRNA, oligonucleotides were dissolved in the aforementioned nuclease-free duplex buffer. Complementary primer pairs were heated by being submerged in boiling water for 4 min and then allowed to gradually cool down to room temperature. Annealed oligonucleotides (25 nmol) were diluted 50× with nuclease-free water prior to ligation.For biolistic transformation, two pCnCas9:U6-gRNA plasmid constructs, each expressing an individual gib2 gRNA, and donor DNA (gib2::NAT) were mixed with gold particles. Processing of the particles and biolistic transformation were carried out as described previously (32). For both electroporation and biolistic transformation, the 3.2-kb gib2::NAT fragment alone was used as a control.Verification of gib2::NAT mutants. Putative transformants were streaked onto fresh selective medium and grown in liquid cultures for genomic DNA extraction. Primers PW166 and PW2055 were used in PCR amplification to generate an ~3-kb gib2::NAT fragment, while primers PW166 and PE167 were used to amplify the 1.7-kb wild-type GIB2 allele. For phenotype verification, transformants and control strains were serially diluted and spotted onto YPD medium for testing at 20, 30, and 37°C as described previously (23).Accession number(s). The complete nucleotide sequence of pCnCas9:U6-gRNA can be found in Fig. S1 and has also been deposited in GenBank under accession no. {"type":"entrez-nucleotide","attrs":{"text":"MH220818","term_id":"1394975609","term_text":"MH220818"}}MH220818.FIG S1 pCnCas9:U6-gRNA nucleic acid sequence. Download FIG S1, docx file, 0.02 MB.Copyright © 2018 Wang.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Article TitleTwo Distinct Approaches for CRISPR-Cas9-Mediated Gene Editing inCryptococcus neoformansand Related Species

Abstract

Electroporation-mediated transformation ofCryptococcusspecies was performed in a Bio-Rad electroporation system (Gene Pulser Xcell) according to a previously published protocol (21,25). Briefly, fungal cells grown overnight in liquid YPD were used to start fresh cultures the next day. When cultures reached an OD of ~1.0, the cells were precipitated and washed three times with distilled water. Cells were washed once in electroporation buffer (EB: 10 mM Tris-HCl, 1 mM MgCl2, 270 mM sucrose, pH 7.5) and resuspended in EB with 1 mM DTT for 30 min. Cells were washed again, resuspended in 1/10 of the original volume of EB, and incubated on ice until use. For electroporation, we mixed the ribonucleoprotein complexes described above and 2 µg ofgib2::NATdonor DNA with 100 µl fungal cells on ice. Electroporation was performed within 5 min of mixing using either the exponential protocol (0.450 kV, 125 µF, and 500 Ω) or the time constant protocol (1.8 to 2.0 kV, 5 ms). Following electroporation, 1 ml of cold YPD with 0.5 M sorbitol and the contents of the cuvette were transferred to a 1.5-ml microcentrifuge tube for recovery by incubation at 30°C for 90 min with shaking (150 rpm). Cells were then pelleted and plated onto YPD plates (one cuvette to one plate) with nourseothricin (70 µg/ml). The plates were incubated at 30°C for 3 days or until colonies emerged.


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