Zeng2014

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protocol
464 | VOL.9 NO.2 | 2014 | nature protocols
INTRODUCTION
C. albicans is one of the most prevalent fungal pathogens in
humans1,and it has long been considered an obligate diploid yeast.
The completion of the C. albicans genome database (http://www.
candidagenome.org/) has made it possible to systematically inter-
rogate genes for their roles in the biology and virulence of this
pathogen2–6. A common and straightforward approach to assess
the function of a gene is to delete it from the genome2,3. Owing to
the diploid nature of all previous laboratory strains of C. albicans,
the construction of a homozygous null mutant required two
rounds of transformation to delete both alleles of a gene, which
is tedious and more time-consuming than in organisms with a
haploid genome or phase such as Saccharomyces cerevisiae.
A standard method for generating null mutants in C. albicans
relies on homologous recombination to replace the target gene
with a selection marker that can rescue an auxotrophic defect
in the host strain7–9. A gene-deletion cassette is normally con-
structed by flanking a selection marker gene with DNA segments
homologous to regions in the 5′ and 3′ ends of the target gene.
Commonly used selection marker genes include URA3, HIS1 and
ARG4 (refs. 7,10–13).A gene-deletion cassette can be constructed
by using either cloning-based7 or PCR-mediated methods10,14,15,
and then it can be transformed into host cells by a lithium acetate/
heat-shock method16–18 or electroporation19,20.
Hickman et al.21 recently discovered that C. albicans can exist
in a viable haploid state, and that such haploids have essentially
the same characteristics as the diploids, except for slower growth
and reduced virulence. For example, haploids are fully competent
to undergo the yeast-hyphae growth transition, the white-opaque
phenotypic switch, the formation of chlamydospores and mat-
ing21. Although the genomes of most of the haploids isolated
were somewhat unstable, spontaneously duplicating at different
frequencies, one highly stable haploid strain was successfully iso-
lated, from which a set of tool strains with multiple auxotrophic
markers was constructed21. The availability of these new haploid
tool strains opened up new opportunities for developing and
applying experimental approaches and resources that are difficult
to achieve in the diploids. One example is targeted gene deletion,
which can now be performed in a single step. This breakthrough
will greatly enhance functional analysis of genes and advance our
understanding of C. albicans biology and pathogenicity.
The first stable haploid we isolated, GZY792 (ref. 21), bears
a mutant allele of HIS4 (ref. 22), and thus it is auxotrophic for
histidine.We used the wild-type HIS4 gene as a selectable marker
to delete the most commonly used auxotrophic marker gene
URA3 from GZY792, which yielded strain GZY803 (ref. 21). In
GZY803, the entire coding region of URA3 was precisely replaced
with the HIS4 gene21 without affecting its upstream gene IRO1,
which occurred in the construction of the diploid tool strain CAI4
and compromised its virulence23.
Compared with diploids, C. albicans haploids have much lower
transformation efficiency, for unknown reasons. In addition, the
propensity of the haploids to autodiploidize21 during transforma-
tion gives rise to heterozygous diploid transformants, thus further
reducing the chance of obtaining the desired haploid gene-deletion
mutants. To deal with these issues, we modified several steps in
previous transformation protocols. Here, we describe in detail
a protocol for the construction of gene-deletion mutants in
C. albicans haploids. The transformation protocol can also be used
in other genetic manipulations such as epitope tagging of genes.
Experimental design
Here, we use the deletion of the ARL3 gene (ORF19.2297), which
encodes a putative Ras superfamily GTPase with unknown func-
tion, in GZY803 as an example of the standard protocol (Fig. 1)
for one-step gene deletion in C. albicans haploids.
We chose the URA3 flipper (UFP)11 as the selection marker to
construct the ARL3-deletion cassette (Fig.2), because it allows the
recycling of the URA3 marker for future use. In the UFP, the URA3
gene follows an inducible FLP1 gene encoding a recombinase,
and the FLP1-URA3 gene pair is flanked by short direct repeats
(flippase recognition targets or FRTs) that are substrates of Flp1.
Once inserted into the genome, the FLP1-URA3 region can be
excised together via Flp1-mediated recombination between the
flanking FRTs upon Flp1 induction, leaving only one FRT at the
One-step targeted gene deletion in Candida albicans
haploids
Guisheng Zeng1,Yan-Ming Wang1, Fong Yee Chan1 & Yue Wang1,2
1Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore. 2Department of Biochemistry,Yong Loo Lin School of
Medicine, National University of Singapore, Singapore. Correspondence should be addressed to Y.W. (mcbwangy@imcb.a-star.edu.sg).
Published online 30 January 2014; doi:10.1038/nprot.2014.029
The recent discovery of haploids in Candida albicans and the construction of tool strains carrying multiple auxotrophic
markers have enabled, for the first time, performing one-step gene deletions in this fungal human pathogen. This breakthrough
promises to greatly facilitate the molecular and genetic study of C. albicans biology and pathogenicity. However, the construction
of gene-deletion mutants in C. albicans haploids involves many technical difficulties, particularly low transformation efficiency
and autodiploidization. Here we describe a highly effective protocol for designing and performing one-step gene deletion in
C. albicans haploids, which takes ~11 d to complete (not including plasmid construction, which may take ~2 weeks). A gene
deletion cassette is constructed on a plasmid and subsequently released for transformation by lithium acetate incubation or
electroporation. Desired gene-deletion mutants are identified and their ploidy is assessed simultaneously by colony PCR
before final confirmation by flow cytometry.

protocol
nature protocols | VOL.9 NO.2 | 2014 | 465
insertion site11.The URA3 marker can then be reused,for example,
to reintroduce a wild-type copy of the deleted gene at its endog-
enous promoter region for complementation experiments.
We use a cloning-based method to flank the UFP with endog-
enous sequences for gene targeting (Fig. 2). Although the addi-
tion of flanking sequences of a target gene to UFP by PCR is
time- and cost-effective, the short homologous sequences used
for gene targeting in this approach reduce the chance of correct
homologous recombination and often result in nonhomologous
integration of the cassette14. Thus, many transformants must be
screened to identify a correct insertion event. In contrast, we use
a gene-deletion cassette generated by cloning that can harbor
much longer homologous regions (typically 400–500 bp long),
which greatly increases the chance of correct gene targeting and
reduces the number of transformants that must be screened.
A map of the gene-deletion cassette bearing plasmid is schemati-
cally described in Figure 2. Construction of this plasmid using
two sets of primers (P01F/P02R and P03F/P04R) follows standard
gene cloning procedures (Fig. 2). One limitation with the use of
UFP is the position effects of URA3, as demonstrated in a previous
study24. Integration of URA3 at certain ectopic loci reduced its
expression level and resulted in a number of phenotypic changes.
To overcome this problem, other positive selection markers such
as the SAT1 flipper25 are suitable, and they can be used for the
construction of the gene-deletion cassette.
We tested three transformation procedures: the lithium acetate/
heat-shock method17, electroporation19 and a lithium acetate
incubation method provided by the supplier of the fast yeast
transformation kit (G-Biosciences). We found that the lithium
acetate/heat-shock method, but not the other two, frequently
produces autodipoidized transformants, probably owing to the
higher temperature (45 °C) applied to cells (G.Z., unpublished
observations, and J. Berman (University of Minnesota), personal
communication), and is therefore unsuitable for our purpose. The
lithium acetate incubation method, performed at 30 °C, is simple
but requires long incubation time; the electroporation method is
labor intensive but requires less time. Nevertheless, both lithium
acetate incubation and electroporation are effective for targeted
gene deletion in C. albicans haploids, with minimal autodiploid
formation. Moreover, these methods can also be applied in other
molecular manipulations such as insertion of a controllable
promoter in front of a target gene or integration-mediated gene
tagging. It is noteworthy that to achieve similar transformation
efficiencies in C. albicans haploids as in the diploids, up to ten
times more DNA may be required.
We adopted a colony PCR method26 to quickly identify
transformants carrying the correct gene deletion and to simulta-
neously assess their ploidy. To this end, two pairs of oligonucle-
otide primers (Fig. 2) are used in the PCR. One pair includes a
marker-specific forward primer (URA3F, targeting a site within
the selection marker) and a target gene–specific reverse primer
(P06R, targeting a site downstream of the 3′ homologous region
used in the cassette) for the detection of a specific ‘knockout’
band (KO band, 600 bp), which can only be amplified when
the deletion cassette has correctly replaced the target gene. The
second pair of primers includes a forward primer (P05F) specific
to a site within the target gene and the reverse primer P06R. This
pair of primers is used to detect a band specific to the target
gene (TG band, 1,100 bp), which is produced when an intact
target gene exists. Haploid transformants are expected to pro-
duce only the KO band (in case of correct deletion) or TG band
(in case of the cassette integration at a wrong locus), whereas
autodiploidized transformants may produce both the KO and
TG bands, or only the TG band.
Finally, we use flow cytometry to confirm the ploidy of
transformants deemed to be correct on the basis of the results
of the colony PCR screen.
(Steps 1 and 2)
(Step 7)
(Steps 16–26) (Steps 10–15)
Characterization of
transformants by colony PCR
Verification of disruptants
by flow cytometry analysis
(Step 8)
Preparation of gene
deletion cassette
Growth of haploid
host strain
Preparation of
competent cells
Yeast transformation
(lithium acetate
incubation
or electroporation)
Digestion of the
plasmid construct
(Steps 3–6)
(Step 9)
Figure 1 | Experimental workflow for one-step targeted gene deletion in
C. albicans haploids.
pBluescript II KS+
3.0 kb
LacZ
MCS
f1(+)ori
T3 T7
~600 bp
~450 bp
~1.1 kb
Notl
URA3F
P03F P04R
P05F P06R
Sacll
~500 bp
A
P01F P02R
Promoter Target gene Terminator
B C D
UFP (~4.2 kb)
Kpnl Xhol
Amp r
ColE1origin
Figure 2 | Schematic diagram of the gene deletion construct. DNA
sequences at the promoter and terminator regions of the target gene are
PCR-amplified by using the primer pairs P01F/P02R (for fragment AB) and
P03F/P04R (for fragment CD). To clone fragment AB, digest both the insert
(fragment AB) and the plasmid (pBKS containing UFP between XhoI and
NotI sites; available upon request) with KpnI and XhoI, followed by ligation.
Transform the ligated plasmid into competent E. coli cells and pick 8–10
colonies for inoculation and mini-prepping. Identify the desired clones with
the correct insert by digesting each colony plasmid with KpnI and XhoI,
followed by gel electrophoresis. Subsequently, use the resulting construct
as the vector to clone fragment CD into the NotI and SacII sites with the
same procedure. Vector-derived primers T3 and T7 are used for sequencing
to verify successful cloning. The construct is then digested with KpnI and
SacII to release the gene deletion cassette for transformation. The primers
used for colony PCR characterization of the transformants (URA3F, P05F and
P06R) and the expected sizes of PCR products are indicated.

protocol
MATERIALS
REAGENTS
XL-Blue electroporation Escherichia coli–competent cells (Stratagene,
cat. no. 200228)
C. albicans haploid GZY803 and its derived strains21 (available from our
laboratory upon request)
Plasmid pYGS1109 (ARL3∆::UFP/pBluescript II KS (pBKS) construct)
(available from our laboratory upon request)
Primers (restriction enzyme cleavage sites are underlined): P01F
5′-CGGGGTACCGGGTTATTATATTGCAACAAAC-3′; P02R 5′-C
CGCTCGATACGAATATCGGTTAGGAG-3′; P03F 5′-TGCGGCCGC
CCTGAGTATAAGTGAAAATAG-3′; P04R 5′-TCCCCGCGGCAATTGAAT
ATGTTGGGTACG-3′; P05F 5′-GATTTTACCCACAGTTGGTC-3′; P06R
5′-CGAAATATCTCCTTAACAACG-3′; URA3F 5′-CAATCAAAGGTGGTCC
TTCTGCAG-3′; T3 5′-AATTAACCCTCACTAAAGGG-3′; T7 5′-TAATACG
ACTCACTATAGGGC-3′
KOD hot start DNA polymerase (Merck-Novagen, cat. no. 71086-3)
DreamTaq DNA polymerase (Thermo Scientific, cat. no. EP0702)
dNTP mix, 10 mM each (Thermo Scientific, cat. no. R0192)
MgCl2, 25 mM (Qiagen, cat. no. 201205)
KpnI-HF (New England Biolabs, cat. no. R3142L)
XhoI (New England Biolabs, cat. no. R0146L)
NotI-HF (New England Biolabs, cat. no. R3189L)
SacII (New England Biolabs, cat. no. R0157S)
T4 DNA ligase (Thermo Scientific, cat. no. EL0011)
EZNA cycle-pure kit (Omega Biotek, cat. no. D6492-02)
EZNA gel extraction kit (Omega Biotek, cat. no. D2500-02)
EZNA plasmid DNA mini kit (Omega Biotek, cat. no. D6942-02)
Agarose (Invitrogen, cat. no. 15510-027)
Tris-acetate-EDTA (TAE) buffer, 50×, pH 8.0 (1st BASE, cat. no.
BUF-3000-50 × 4L)
Ethidium bromide solution, 10 mg ml−1 (Promega, cat. no. H5041)
! CAUTION Ethidium bromide is a carcinogen. Wear gloves to avoid direct
skin contact and follow standard protocols for proper waste disposal.
Bromophenol blue (Bio-Rad Laboratories, cat. no. 161-0404)
Glycerol (Invitrogen, cat. no. 15514-011)
DNA ladder, 1 kb (New England Biolabs, cat. no. N3232L)
NaCl (Sigma-Aldrich, cat. no. S9888)
Tryptone (BD Bacto, cat. no. 211705)
Yeast extract (BD Bacto, cat. no. 212750)
Agar (BD Bacto, cat. no. 214010)
Yeast nitrogen base without amino acids (BD Difto, cat. no. 291940)
Uridine (Sigma-Aldrich, cat. no. U3003)
Histidine (Sigma-Aldrich, cat. no. H8000)
Lysine (Sigma-Aldrich, cat. no. L5501)
Arginine (Sigma-Aldrich, cat. no. A-5506)
Glucose (Sigma-Aldrich, cat. no. G8270)
Ampicillin sodium salt (Sigma-Aldrich, cat. no. A9518)
Fast yeast transformation kit, containing three solutions: wash solution, com-
petent solution and transformation solution (G-Biosciences, cat. no. GZ-1)
Tris-HCl, 1 M, pH 7.4 (1st BASE, cat. no. BUF-1415-1L-pH7.4)
EDTA (Bio-Rad Laboratories, cat. no. 161-0729)
Lithium acetate (Sigma-Aldrich, cat. no. 517992)
DTT (Sigma-Aldrich, cat. no. 43815)
Sorbitol (Sigma-Aldrich, cat. no. S-1876)
Salmon sperm DNA (Agilent Technologies, cat. no. 201190)
Ethanol (Sigma-Aldrich, cat. no. 459844)
Tris-HCl, 1 M, pH 7.5 (1st BASE, cat. no. BUF-1415-1L-pH7.5)
RNase A (Sigma-Aldrich, cat. no. R4875)
Propidium iodide (Sigma-Aldrich, cat. no. 81845)
Na2HPO4 (Sigma-Aldrich, cat. no. 255793)
KH2PO4 (Sigma-Aldrich, cat. no. P0662)
KCl (Sigma-Aldrich, cat. no. P9541)
EQUIPMENT
Clear PCR tubes, 0.5 ml (Axygen Scientific, cat. no. PCR-05-C)
Clear microtubes, 1.7 ml (Axygen Scientific, cat. no. MCT-175-C)
Polystyrene inoculating loops, 1 µl (Nunc, cat. no. 254410)
Polystyrene round-bottom tubes, 5 ml, 12 × 75 mm (BD Falcon,
cat. no. 352058)
Polypropylene round-bottom tubes, 14 ml, 17 × 100 mm (BD Falcon,
cat. no. 352059)
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Polypropylene round-bottom tubes, 50 ml, 30 × 115 mm (BD Falcon,
cat. no. 352070)
Sterile Petri dishes, 90 × 15 mm (Sterilin, cat. no. 101VR20)
Lazy-L-Spreaders (Sigma-Aldrich, cat. no. SPR-L-S10)
Filter unit, 500 ml, 0.20 µm (Nalgene, cat. no. 566-0020)
Polystyrene spectrophotometer cuvettes, 0.5–2 ml (LP Italiana Spa,
cat. no. 112117)
Serological pipettes, 10 ml (BD Falcon, cat. no. 357551)
Gene Pulser cuvettes, 0.2-cm gap (Bio-Rad, cat. no. 165-2086)
Pipette aid (Thermo Scientific, cat. no. 9541)
Elite dual block dry bath (heating block; Major Science, cat. no. EL-02)
Accumet AB15 basic pH meter (Fisher Scientific, cat. no. 13-636-AB15BC)
Eppendorf centrifuge 5424 (Eppendorf, cat. no. 5424000.410)
Genie 2 vortex mixer (Scientific Industries, cat. no. SI-0266)
Mini-Sub cell GT system (Bio-Rad, cat. no. 170-4467)
PowerPac basic power supply (Bio-Rad, cat. no. 164-5050)
T3000 Thermocycler (Biometra, cat. no. 050-724)
UV-visible spectrophotometer (GE Healthcare, cat. no. 80-2112-21)
Gel documentation system (Viber Loumat E-box system)
Transilluminator (UV light box) (Viber Loumat, model TF-35L)
! CAUTION UV is harmful to skin and eyes. Wear face shield for protection.
Gene Pulser with pulse controller (Bio-Rad, model nos. 1652076 and
1652098)
Incubators (Jouan EB115 and EB170)
Orbital shaker (New Brunswick G10 Gyrotory Shaker)
Water bath shaker (New Brunswick Gyrotory Water Bath Shaker)
Benchtop swinging bucket centrifuge with temperature control (Hettich
Lab Rotina 420R)
Revolver tube mixer (Labnet International, cat. no. H5600-230V-UK)
Sonics sonicator (Vibra Cell VCX500)
Flow cytometer system (BD FACSCalibur)
REAGENT SETUP
DNA loading buffer,10× Mix 50 ml of glycerol, 2 ml of 0.5 M EDTA (pH 8.0)
and 0.25 g of bromophenol blue; top up the mixture with distilled water to
100 ml. Store it at 4 °C for up to 1 year.
DNA ladder, 1 kb Mix 0.5 ml of 1-kb DNA ladder with 0.5 ml of 10× DNA
loading buffer and 4 ml of distilled water. Divide the solution into aliquots
and store them at −20 °C for up to 1 year.
Agarose gel, 1% (wt/vol) Mix 1.5 g of agarose with 150 ml of TAE buffer
and boil the solution. Allow the solution to cool and add 3 µl of ethidium
bromide to the dissolved agarose. Mix the solution and pour the mixture into
a gel tray with a comb placed, and then let the gel cool to room temperature
(25 °C) before use.
Ampicillin stock, 1,000× Prepare a 100 mg ml−1 solution of ampicillin with
distilled water and sterilize it by filtration by using a 0.20-µm filter unit.
Divide the solution into aliquots and store them −20 °C for up to 1 year.
LB medium and plates Dissolve 10 g of tryptone, 5 g of yeast extract and
10 g of NaCl into 900 ml of distilled water; adjust the pH to 7.5. To prepare
LB liquid medium, top up the mixture with distilled water to 1 liter and
sterilize by autoclaving. Allow the autoclaved medium to cool before adding
1 ml of ampicillin stock. Store it at 4 °C for up to 6 months. To prepare LB
plates, add 20 g of agar to the mixture, and top up with distilled water to
1 liter for autoclaving. Allow the autoclaved medium to cool before adding
ampicillin and pour it into 90 × 15 mm sterile Petri dishes. Store the plates
at 4 °C for no more than 1 month.
Glucose minimal medium (GMM) Dissolve 6.7 g of yeast nitrogen base
without amino acids and 20 g of glucose in 1 liter of distilled water, and then
autoclave to sterilize. Store it at 4 °C for up to 2 months. To make GMM
plates, add 20 g of agar before autoclaving, allow the autoclaved medium to
cool and pour it into 90 × 15 mm sterile Petri dishes. Store the plates at 4 °C
for no more than 1 month.
Amino acids stocks Dissolve each of the following amino acids with
distilled water to make stock solutions: 1,000× uridine (80 mg ml−1),
1,000× arginine (40 mg ml−1), 1,000× lysine (50 mg ml−1) and 500× histidine
(20 mg ml−1). Sterilize the stock solutions by filtration with a 0.20-µm filter
unit and store the solutions at 4 °C for up to 6 months. To supplement a
GMM plate with amino acids, spread 20 µl of uridine, 20 µl of arginine, 20 μl
of lysine, or 40 µl of histidine stock solution onto the plate.
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protocol
TE-lithium acetate (LiAc) solution Dissolve 372 mg of EDTA and 6.6 g
of lithium acetate in 800 ml of distilled water, mix with 10 ml of 1 M
Tris-HCl (pH 7.4) and top up the mixture with distilled water to
1 liter. Autoclave the solution to sterilize, and store it at 4 °C for up to
1 year.
Solution I Solution I contains 200 mM Tris-HCl (pH 7.5) and 20 mM
EDTA. Dissolve 7.44 g of EDTA in 700 ml of distilled water, mix it with
200 ml of 1 M Tris-HCl (pH 7.5) and top up the mixture with distilled
water to 1 liter. Sterilize the solution by filtration with a 0.20-µm filter unit
and store it at 4 °C.
PBS, 10× Dissolve 80 g of NaCl, 2 g of KCl, 14.4 g of Na2HPO4 and 2.4 g
of KH2PO4 in 800 ml of distilled water, and adjust the pH to 7.4. Top up the
mixture with distilled water to 1 liter and sterilize it by autoclaving. Store
PBS at 4 °C for up to 1 year.
PROCEDURE
Preparation of the gene deletion cassette ● TIMING 3 d
1| Transform the plasmid (pYGS1109) carrying the gene-deletion cassette (Fig. 2) into E. coli–competent cells and grow a
15–20-ml LB culture supplemented with 100 µg ml−1 ampicillin overnight.
2| Purify the plasmid from the overnight culture by using a column-based method such as the EZNA plasmid DNA
mini kit. The typical yield from a 1.5-ml culture is ~10–15 µg of plasmid DNA (eluted in 50 µl of sterile distilled H2O).
 CRITICAL STEP The purity and concentration of the plasmid DNA are crucial for the success of gene deletion; hence, a
column-based miniprep method is recommended.
3| Set up a digestion reaction by mixing 50 µl of the plasmid DNA (~10–15 µg) with 2 µl of each of the restriction
enzymes KpnI and SacII, and 6 µl of the 10× NEB buffer 4. Incubate the reaction at 37 °C for 3–4 h or overnight for
complete cleavage of the plasmid.
4| Take 2 µl of the digested DNA and mix it with 16 µl of H2O and 2 µl of 10× DNA loading buffer. Run the mixture on a
1% (wt/vol) agarose gel containing ethidium bromide at 150 V for 15–18 min. Include 10 µl of 1-kb DNA ladder.
5| Visualize the DNA bands with a gel documentation system or on a UV light box.
! CAUTION Follow standard protocols for proper disposal of ethidium bromide–containing agarose gel and buffer. Use proper
protection of skin and eyes to avoid exposure to UV light.
? TROUBLESHOOTING
6| Keep the remaining digested plasmid at 4 °C before using it for yeast transformation.
 PAUSE POINT The digested DNA can be stored at −20 °C for 1–2 weeks.
Growth of haploid C. albicans cells ● TIMING 3–4 d
7| Refresh the C. albicans haploid strain (GZY803 or other tool strains) from a −80 °C stock by streaking frozen cells
onto a GMM plate supplemented with uridine or appropriate amino acids and incubating them at 30 °C for 2–3 d to obtain
single colonies.
 PAUSE POINT The refreshed haploid cells can be stored on plates at 4 °C for up to 2–3 weeks.
8| Inoculate 8–10 haploid colonies with a sterile inoculation loop into 15 ml of GMM medium supplemented with uridine or
appropriate amino acids in a 50-ml Falcon tube, and then grow the cells at 30 °C on a rotary shaker at 220 r.p.m. until they
reach an OD600 of 1.0–1.5.
Transformation of haploid C. albicans cells ● TIMING 3.5–5 d
9| For transformation using the lithium acetate incubation method, follow option A. For transformation by electroporation,
follow option B.
(A) Transformation by lithium acetate incubation
(i) Transfer 0.5–1.0 ml of the yeast culture into a microcentrifuge tube, spin it at 20,000g for 1 min at room temperature
to pellet the cells, and then discard the supernatant.
 CRITICAL STEP To achieve an optimal transformation efficiency, the volume of the cell pellet should be around
15–20 µl.
(ii) Add 1 ml of wash solution (from the fast yeast transformation kit) to the pellet and vortex to resuspend the cells.
Re-pellet the cells by centrifugation at 20,000g for 1 min at room temperature and discard the supernatant.
(iii) Add 50 µl of competent solution (from the fast yeast transformation kit) to the pellet, and vortex to resuspend the cells.
(iv) Mix the cell suspension gently with the digested plasmid from Step 6. Slowly add 500 µl of the viscous transformation
solution (from the fast yeast transformation kit) to the mixture and vortex vigorously to mix thoroughly.

protocol
(v) Incubate the mixture at 30 °C on a heating block for 6–10 h. Vortex it once every 1–2 h to avoid cell sedimentation.
 CRITICAL STEP A longer incubation time of yeast cells with the plasmid at 30 °C improves the transformation
efficiency. If the transformation mixture is set up in the afternoon, it can be incubated at 30 °C overnight.
(vi) Centrifuge the transformation mixture at 20,000g for 1 min at room temperature to pellet cells, and then discard
the supernatant.
(vii) Add 60 µl of H2O to the pellet and vortex it vigorously to resuspend the cells. Spread the cell suspension evenly onto
a GMM plate supplemented with the appropriate amino acids.
(viii) Incubate the plate at 30 °C for 3–4 d to allow for growth of transformants.
? TROUBLESHOOTING
(B) Transformation by electroporation
(i) Pellet the cells from the 15-ml culture from Step 8 in a benchtop centrifuge at 4,500g for 5 min at 4 °C, and then
discard the supernatant.
(ii) Add 20 ml of H2O to the pellet, and vortex to resuspend the cells. Re-pellet the cells by centrifugation at 4,500g for
5 min at 4 °C and discard the supernatant.
(iii) Add 4 ml of TE-LiAc solution to the pellet and vortex to resuspend the cells. Incubate the cell suspension at 30 °C on
a shaker at 220 r.p.m. for 30–45 min.
(iv) Add 100 µl of 1 M DTT to the cell suspension and continue the incubation at 30 °C for 0.5–2 h.
 CRITICAL STEP The incubation of cells with DTT is crucial for high transformation efficiency.
(v) Pellet the cells by centrifugation at 4 °C at 4,500g for 5 min at 4 °C, and discard the supernatant.
(vi) Add 20 ml of ice-cold H2O to the pellet, and vortex to resuspend the cells. Re-pellet the cells by centrifugation at 4 °C
at 4,500g for 5 min and discard the supernatant.
(vii) Repeat Step 9B(vi).
(viii) Add 2 ml of ice-cold 1 M sorbitol to the pellet and vortex to resuspend the cells. Re-pellet the cells by centrifugation
at 4 °C at 4,500g for 5 min, and then discard the supernatant.
(ix) Add ice-cold 1 M sorbitol at 1–1.5 times the volume of the cell pellet (~0.5–1 ml) and vortex to resuspend the cells.
Keep the cell suspension on ice.
(x) Purify the digested plasmid DNA from Step 6 by using the EZNA cycle-pure kit (or any other column-based kit)
according to the manufacturer’s instructions, and then elute the DNA with 10 µl of H2O.
 CRITICAL STEP The digested plasmid DNA must be purified to remove salts before electroporation.
(xi) Add 40 µl of the prepared cell suspension (Step 9B(ix)) and 2 µl of 10 mg ml−1 salmon sperm DNA (optional) to the purified
DNA and mix gently. Transfer the mixture to a prechilled (on ice) Gene Pulser cuvette carefully to avoid generating bubbles.
(xii) Perform electroporation by using the Bio-Rad Gene Pulser with the following setting: output 1.65 KV, resistance
200 Ohms and capacitance 25 µFD. The time constant for electroporation is usually between 4.1–4.6 s.
(xiii) Transfer the electroporated cells to a GMM plate supplemented with appropriate amino acids and spread out evenly.
(xiv) Incubate the plate at 30 °C for 3–4 d to allow transformants to grow.
? TROUBLESHOOTING
Characterization of transformants by colony PCR ● TIMING 4–5 h
10| Randomly choose 5–10 transformant colonies of different sizes, and then transfer roughly one-third of each colony with
a 200-µl pipette tip to the bottom of a 0.5-ml PCR tube. Use about the same amount of cells from the host strain (GZY803)
as a control. Prepare duplicates for each colony.
 CRITICAL STEP Be careful not to transfer any agar, as it inhibits the PCR.
11| Prepare the PCR mix according to the following table to a final sample volume of 100 µl, with the first pair of primers
(URA3F and P06R). Vortex the mixture briefly to mix, and spin the mixture at 20,000g for 15–30 s at room temperature.
Compound Amount (ml) Final concentration
DreamTaq buffer, 10× 10 1×
dNTP mix (10 uM each) 2 0.2 mM each
MgCl2 (25 mM) 6 1.5 mM
Forward primer (100 µM) 1 1 µM
Reverse primer (100 µM) 1 1 µM
DreamTaq polymerase (5 U µl−1) 1 0.05 U µl−1
Sterile distilled water 79

protocol
12| Add 16 µl of the PCR solution to each PCR tube from Step 10, and pipette it gently to mix with the cells at the bottom.
13| Use the same set of colonies as above to set up a parallel PCR with the second pair of primers, P05F and P06R.
 CRITICAL STEP Keep the remaining one-third of each colony for use in Step 16.
14| Perform the PCR with the following program:
Cycle number Denature Anneal Extend Hold
1 96 °C, 6 min
2–31 94 °C, 1 min 52 °C, 1 min 72 °C, 1 min 15 s
32 72 °C, 10 min
33 4 °C
15| Mix each of the PCR products with 2 µl of 10× DNA loading buffer for electrophoresis on a 1% (wt/vol) agarose gel, and
visualize the PCR products with a gel documentation system or on a UV light box. Analyze the results as described in
Experimental design section.
! CAUTION Follow standard protocols for proper disposal of ethidium bromide–containing agarose gel and buffer. Use proper
protection of skin and eyes to avoid exposure to UV light.
? TROUBLESHOOTING
Ploidy verification of mutants by flow cytometry ● TIMING 2.5–3 d
16| Inoculate the remaining one-third of each colony (Step 10) into 2 ml of GMM medium supplemented with the appropri-
ate amino acids, and grow the cells at 30 °C on a shaker at 220 r.p.m. overnight. Also grow both nontransformed haploid
cells (GZY803 or other tool strains) and diploid cells (SC5314) to serve as controls.
17| Add 500 µl of the culture to a microcentrifuge tube, pellet the cells by centrifugation at 20,000g for 1 min at room
temperature and discard the supernatant.
 CRITICAL STEP Keep the remaining 1.5 ml of culture at 4 °C. It will be used for producing strain stock once haploidy of
the cells is verified by flow cytometry.
18| Add 1 ml of 70% (vol/vol) ethanol to the pellet, vortex it to resuspend the cells and leave it at room temperature for 1 h.
 PAUSE POINT The ethanol-fixed cells can be stored at 4 °C for 2–3 weeks.
19| Pellet the cells by centrifugation at 20,000g for 1 min at room temperature and discard the supernatant. Resuspend the
cells with 1 ml of solution I and pellet them again. Add 500 µl of solution I and 5 µl of RNase A (10 mg ml−1) to the pellet,
and then vortex to resuspend the cells.
20| Incubate the cell suspension at 37 °C on a rotary mixer for 6–7 h or overnight.
21| Pellet the cells by centrifugation at 20,000g for 1 min at room temperature and discard the supernatant. Add 500 µl of
PBS to the pellet and vortex to resuspend the cells.
22| Pellet the cells again by centrifugation at 20,000g for 1 min at room temperature, and discard the supernatant.
Add 100 µl of PBS and 1 µl of propidium iodine (5 mg ml−1) to the pellet, and vortex to resuspend the cells.
23| Keep the cell suspension in the dark by wrapping the tubes with aluminum foil and incubate at 4 °C overnight.
24| Add 900 µl of PBS to the cell suspension and transfer the mixture to a 5-ml polystyrene round-bottom tube (12 × 75 mm)
suitable for use with the BD FACSCalibur flow cytometer.
25| Sonicate the cells by using a sonicator with appropriate output for 5–10 s.
! CAUTION Wear ear protection during sonication.
 CRITICAL STEP It is necessary to separate cell clusters by sonication, as haploid cells tend to form clumps in liquid
culture and result in abnormally high DNA content peaks in flow cytometry analysis.

protocol
26| Briefly vortex the sonicated SC5314 diploid cells, load the sample to BD FACSCalibur and follow the manufacturer’s
instructions to optimize the instrument settings for DNA content analysis. Vortex each sample briefly before loading it to the
cytometer, and perform data acquisition with 10,000 cells for each sample by using the CellQuest Pro software.
! CAUTION Follow standard protocols for proper disposal of propidium iodine–containing samples.
? TROUBLESHOOTING
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
Table 1 | Troubleshooting table.
Step Problem Possible reason Solution
5 Detection of a DNA band
larger than the vector band
and gene-deletion cassette
(Fig. 3)
Digestion of the plasmid is
incomplete
Use new restriction enzymes and increase their amount up
to 10% of the total reaction volume
Extend the digestion time up to 20 h
Concentration of the plasmid is
too high
Use less plasmid or increase the volume of the reaction to
dilute the plasmid (≥0.2 µg µl−1)
9A(viii),
9B(xiv)
No growth of transformant
colonies on the plate
Amino acids required for the
growth of the transformants were
not supplied
According to the auxotrophic deficiency of the host strain,
supplement the GMM plate with the required amino acids
The target gene is essential for
cell viability
Insert a controllable promoter to replace the native
promoter of the target gene
Transformation efficiency is
too low
See below for measures to improve transformation
efficiency
The colony size of trans-
formants is very small
Deletion of the target gene
results in slower cell growth
Extend the time for colony growth
No or few colonies of
transformants on the plate
Transformation efficiency is
too low
Use a newly refreshed host strain to prepare
competent cells
Increase the amount of plasmid DNA (up to 15 µg) used
in transformation
Perform transformation by electroporation
Extend the incubation time of DNA with yeast cells up to
20 h in Step 9A(v)
Wash the cell pellet once with H2O before spreading cells
onto the selection plate (Step 9A(vii))
Extend the incubation time of cells with TE-LiAc and DTT to
up to 8 h at 30 °C in Steps 9B(iii, iv)
15 No PCR band is detected PCR failed Ensure that all PCR reagents work by testing them using
purified genomic DNA as a template
Design the primers to ensure that the size of the diagnostic
PCR fragment is 1,100 bp
Avoid picking up any agar when transferring cells to PCR
tubes in Step 10
Multiple PCR bands are
detected
Primer annealing temperature
is too low
Increase the primer annealing temperature to up to 60 °C
Primers have poor specificity Increase the primer annealing temperature
Re-design the primers to target different sites on the locus
Avoid sequences with nucleotide runs in primer design
Avoid using primers containing long inverted repeats
(continued)

protocol
Table 1 | Troubleshooting table (continued).
Step Problem Possible reason Solution
Only the TG band is detected High probability of integration of
the gene deletion cassette at a
wrong genomic loci
Screen more transformants by colony PCR
Reconstruct the gene deletion cassette by using longer
flanking homologous sequences
Both the KO and TG bands
are detected in most
transformants
The transformants have become
diploid
Use newly refreshed haploid cells to reduce the chance of
autodiploidization during transformation
Confirm that the host strain is in the haploid status before
using it for transformation
The target gene is duplicated in
the haploid genome
Perform another round of gene deletion
26 No clear 1N and 2N DNA
peaks detected
High background signal from
propidium iodine–stained RNA
Use freshly made RNase A and increase its concentration to
15 mg ml−1; extend the incubation time up to 24 h in
Step 20 to completely remove RNA
Peaks of 2N DNA content
are detected
Clustering of cells due to defects
in cell separation (Step 16)
Extending the sonication time to 1 min may help if the
phenotype is weak; but if it is a strong phenotype, flow
cytometry analysis will not be applicable
Cells have become filamentous Shortening the culture time in Step 16 to ~15 h may help
in some cases; but if it is a phenotype of gene deletion,
skip flow cytometry analysis
● TIMING
Steps 1–6, preparation of the gene deletion cassette: 3 d
Steps 7 and 8, growth of haploid cells for transformation: 3–4 d
Step 9A(i–vii), yeast transformation by lithium acetate incubation: 1 d
Step 9A(viii), growth of transformants: 3–4 d
Step 9B(i–xiii), yeast transformation by electroporation: 0.5–1 d
Step 9B(xiv), growth of transformants: 3–4 d
Steps 10–15, characterization of transformants by colony
PCR: 4–5 h
Step 16, growth of disruptants for flow cytometry: 1 d
Steps 17–25, staining of cells with propidium iodine for flow
cytometry: 1–1.5 d
Step 26, analysis of cell ploidy with a flow cytometer: 2–3 h
ANTICIPATED RESULTS
pBKS, a simple plasmid commonly used in molecular biology,
is used as the vector to construct the gene-deletion cassette.
Depending on the auxotrophic defect of a haploid host strain,
different selection markers (e.g., URA3, ARG4 and HIS1) can
be used. Because the homologous flanking sequences are typi-
cally 400–500 bp in length, the size of a gene-deletion cas-
sette is mainly determined by the size of the selection marker
gene used. In the example experiment (Fig. 2), the URA3
flipper (4.2 kb) was used to make the ARL3-deletion cassette.
Therefore, complete digestion with KpnI and SacII is expected
to produce a 5.2-kb-long DNA band corresponding to
the ARL3-deletion cassette in addition to a 3-kb-long vector
band (Fig. 3). If a shorter marker gene (e.g., the 2.0-kb-long
ARG4) is used, the released gene-deletion cassette and the
vector could be similar in size and may overlap on the agarose
Figure 3 | Visualization of the gene deletion cassette. The plasmid
construct containing the gene deletion cassette (pYGS1109) was digested
with KpnI and SacII, and a small aliquot (1–2 µl) was analyzed by agarose
gel electrophoresis to confirm the complete release of the gene deletion
cassette. M: 1-kb DNA ladder; P: digested plasmid construct.
Deletion
cassette
10.0
8.0
5.0
6.0
4.0
3.0
2.0
1.5
1.0
0.5
(kb)
M P
Vector
band

protocol
gel. However, the disappearance of the full-length plasmid
band will indicate complete cleavage.
The lithium acetate incubation and electroporation transformation methods have comparable transformation efficiencies.
The number of colonies obtained from each transformation may vary over a wide range from a few to 100 (Fig. 4). So far,
for the ~50 genes we have successfully deleted, we usually obtained 20 colonies per transformation. Notably, even when
only a few colonies were obtained, the chance of finding the correct haploid mutant was high. The transformant colonies
usually become visible after 2 d of incubation at 30 °C and reach a colony size of 1–1.5 mm in diameter after 1–2 more
days. Sometimes, the transformants may take a longer time (5–6 d) to grow, possibly owing to the effect of gene deletion on
growth. In some cases, the majority of the transformants produced colonies of similar sizes; in other cases, colonies of mark-
edly different sizes were visible. It is difficult to judge which transformant is likely to be correct on the basis of colony size,
and thus colonies of different sizes should be picked for verification by colony PCR.
Once the gene-deletion cassette is integrated into the genome of a host cell, the selection marker, regardless of its
genomic locus, will allow the transformant to grow on the selection plate. Therefore, characterization of transformants is
crucial to distinguish correct mutants from false positives. Colony PCR is a fast and reliable method for this purpose. With
the use of a forward primer targeting a site within the marker gene (URA3F) and a reverse primer targeting a sequence
outside the 3′ flanking region (P06R), only the colonies with the target gene correctly replaced by the selection marker will
produce the expected KO band in PCR (Fig. 5a). In a separate PCR, a different forward primer P05F recognizing a sequence
within the target gene is paired with P06R to allow detection of the TG band, indicating the presence of the target gene
(Fig. 5b). If a colony produces only the TG but not the KO PCR band, it is a false positive. If a colony produces both the TG
and KO PCR bands, it means that either the transformed cells have autodiploidized before the target gene was deleted or the
cells remains haploid but have a duplicated copy of the target gene. If a colony produces only the KO band but not the TG
band, it is highly likely to be a correct gene-deletion mutant. Autodiploidization after deletion of the target gene is a rare event.
We used flow cytometry to validate the effectiveness of using colony PCR to assess the ploidy of transformants. On the
basis of analysis of a large number of transformants that produced different PCR bands, we found 95% of the transformants
that produced only the KO band to have a 1N DNA content
(Fig. 6), whereas all transformants that produced both the
KO and TG bands had a 2N DNA content, indicating autodip-
loidization (Fig. 6). It is, however, important to be aware
that disruption of some target genes may cause a strong cell
separation defect or filamentous growth, which renders flow
cytometry analysis inapplicable.
LiAc incubation Electroporation
ba
Figure 4 | Examples of transformants on plates. (a,b) Transformants obtained
using LiAc incubation (a) and electroporation (b) are shown. The plates were
incubated at 30 °C for 4 d.
URA3F + P06R P05F + P06Ra b
M Ctrl T1 T2 T3 T4 T5 M CtrlT1 T2 T3 T4 T5
-KO
-TG
Figure 5 | Characterization of transformants by colony PCR. (a,b) Five
transformants (T1–T5) were randomly chosen for colony PCR with the primer
pairs URA3F/P06R (a) and P05F/P06R (b). M, 1-kb DNA ladder; KO, knockout
band; TG, target gene band; Ctrl, the control haploid host strain GZY803.
Colonies T1 and T3-5 produced only the KO band, indicating correct gene
deletion and haploidy; and T2 produced both KO and TG bands, indicating
autodiploidization before one copy of the target gene was deleted.
T1
Propidium iodide
CellcountCellcount
CellcountCellcount
Cellcount
Propidium iodide Propidium iodide
Transformants
1N control
2N control
Propidium iodide Propidium iodide
T2
T4 T5
T3
Figure 6 | Ploidy analysis of transformants by flow cytometry. The PCR-
characterized transformants (T1–T5) were cultured and cells were stained
with propidium iodide for flow cytometry. Raw data were analyzed by the
WinMDI (version 2.8) software. The DNA content of each transformant
(purple) was compared with that of haploid (GZY803, blue) and diploid
(SC5314, red) control strains. The results confirm the verification of the
transformants by colony PCR in Figure 5. Colonies T1 and T3–T5 are the
desired mutants and are haploid, whereas T2 has autodiploidized.

protocol
The summary of the results from the successful deletion of 40 genes by using the GZY803 strain is as follows: the rate of
correct targeting is 90%, that of mistargeting is 10% and that of autodiploidization is 20%. However, we observed that
the rate of autodiploidization and mistargeting is higher in the haploid strains with multiple auxotrophic markers, including
GZY815, GZY822 and GZY823 (ref. 21), thus reducing the rate of obtaining correct transformants to ~40–50%.
Acknowledgments We thank J. Berman and members of the Wang lab for
critical reading of the manuscript. This work was funded by the Agency for
Sciences, Technology and Research of Singapore.
AUTHOR CONTRIBUTIONS G.Z. designed and performed the experiments
and wrote the first draft of the manuscript. F.Y.C. contributed to
plasmid construction, and Y.-M.W. helped with flow cytometry analysis.
Y.W. discussed and commented on the results at all stages and revised
the manuscript.
COMPETING FINANCIAL INTERESTS The authors declare no competing
financial interests.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html.
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