Cell Reports, Volume 15

Supplemental Information

Rapid Protein Depletion in Human Cells

by Auxin-Inducible Degron Tagging

with Short Homology Donors

Toyoaki Natsume, Tomomi Kiyomitsu, Yumiko Saga, and Masato T. Kanemaki

Cell Reports

Supplementary Information

Rapid protein depletion in human cells by auxin-inducible degron

tagging with short homology donors

Toyoaki Natsume, Tomomi Kiyomitsu, Yumiko Saga, and Masato T. Kanemaki

Supplemental Inventory

1. Supplementary Experimental Procedures

2. Supplementary Figure Legends

3. Supplementary Figures

Figure S1, related to Figure 1

Figure S2, related to Figure 1

Figure S3, related to Figure 3

Figure S4, related to Figure 4

Figure S5, related to Figure 5

Figure S6, related to Discussion

Supplementary Experimental Procedures

Construction of HCT116 cells expressing OsTIR1

One day before transfection, 3.2 x 105 cells resuspended in 2 mL of medium

were plated in one well of a 6-well plate. In a microtube, 800 ng of the AAVS1

T2 CRISPR/Cas plasmid (based on pX330, and encoding gRNA and SpCas9)

and 1000 ng of pMK232 (CMV-OsTIR1) or pMK243 (Tet-OsTIR1) were mixed

in 10 µL of TE. Subsequently, 90 µL of OPTI-MEM (Gibco) and 8 µL of Fugene

HD (Promega) were added. The mixture was incubated for 15 min at RT before

applying to the cells. Two days after transfection, the cells were removed and

diluted at 10 to 200 times in 10 mL of medium containing 1 µg/mL of puromycin

before being transferred to 10 cm dishes. The selection medium was

exchanged every 3 to 4 days. After 10 to 12 days, colonies were washed with

PBS, picked using a yellow tip under a stereomicroscope, and subsequently

transferred to a 96-well plate containing 10 µL of trypsin-EDTA. After a few

minutes, 100 µL of the selection medium was added. Two to three days later,

the cells were transferred to a 24-well plate. When the cells grew full, they

were removed and resuspended in 250 µL of medium without puromycin. Fifty

µL of the cells were frozen in a microtube by adding the equal volume of

Bambanker Direct (Nippon Genetics) and the rest were used for genomic DNA

preparation.

Construction of AID mutants

The CMV-OsTIR1 or Tet-OsTIR1 cells were plated one day before transfection

as described above. In a microtube, 800 ng of a pX330-based CRISPR/Cas

plasmid (encoding gRNA and SpCas9) and 600 ng each of two donors (Neo

and Hygro) were mixed. Subsequently, 90 µL of OPTI-MEM and 8 µL of

Fugene HD were added. The mixture was incubated for 15 min at RT before

applying to the cells. Two days after transfection, the cells were removed and

diluted at 10 to 200 times in 10 mL of medium containing 700 µg/mL of G418

and 100 µg/mL of HygroGold (InvivoGen) before being transferred to 10 cm

dishes. The following procedures are the same as above except for using

G418 and HygroGold instead of puromycin.

Cell culture and transfection of mouse ES cells

Mouse ES cells (TT2) were cultured with mitomycin-treated feeders derived

from embryonic primary fibroblast (Yagi et al., 1993). One day before

transfection, 1 x 105 cells were plated in one well of a 24-well plate containing 1

mL of ES medium. One microgram each of a pX330-based CRISPR/Cas

plasmid (encoding gRNA and SpCas9) and a donor vector was diluted in

OPTI-MEM (the total volume is 50 µL). Subsequently, 2 µL of Lipofectamin

2000 (Thermo Fisher Scientific) was added. The DNA mixture was applied to

the ES cells after incubation for 20 min. After 4 h, the treated cells were

removed using trypsin-EDTA and were plated on a 6 cm dish with feeder cells.

Two days later, medium was exchanged with fresh ES medium containing 300

µg/mL of G418. The ES cells were cultured for 6 days with exchanging the

selection medium every day. G418-resistant ES clones were picked using

yellow tip and subjected to detection of homologous recombination by PCR

analyses with specific primers.

Supplementary Figure Legends

Figure S1

GFP tagging of the MCM8 protein using PCR-amplified linear DNA. (a)

Experimental scheme used to tag the C terminus of MCM8 with GFP. A

PCR-amplified linear DNA harbouring 175 bp homology arms at both ends was

transfected together with a CRISPR/Cas vector to target the MCM8 locus. (b)

Genomic PCR used to check the genotype of clones after selection with 700

µg/mL of G418.

Figure S2

GFP tagging of the MCM8 protein using donor plasmids harbouring homology

arms of varying length. The experimental scheme is shown at the top. The

table shows the results obtained after genotyping using genomic PCR.

Figure S3

Growth and cell-cycle distribution of WT and CMV-OsTIR1 cells in the

presence or absence of auxin. (a) Indicated cells were grown with or without

500 µM of IAA for 72 h counting the cell number at every 24 h. (b) Indicated

cells treated with or without 500 µM of IAA for 48 h were analysed by flow

cytometry. (c) Cell-cycle distribution was quantified using the data obtained in

(b).

Figure S4

RAD21-mAC depletion in cells with or without CMV-OsTIR1. The indicated

cells were treated with 500 µM of IAA (+ auxin) or DMSO (– auxin). Time-lapse

images were taken at 0 and 90 min after treatment. The scale bars represent

16 µm.

Figure S5

Colony formation by DHC1-mAC cells in the CMV-OsTIR1 and Tet-OsTIR1

background. The DHC1-mAC donors shown in Figure 5a were transfected with

or without a CRISPR/Cas plasmid, to target the DHC1 locus. After double

selection with G418 and hygromycin, colonies were stained with crystal violet.

Figure S6

(a) Table showing template plasmids containing the indicated fluorescent,

purification and AID tags with three selection markers, Neo, Hygro and Bsr.

Note that the plasmids containing the Neo marker do not contain the LoxP

sites, for removal of the marker. (b) Experimental schemes used for the

construction of donor vectors by PCR or conventional cloning via gene

synthesis. All plasmids listed in (a) can be processed in the same manner for

the construction of donor vectors.

PCR

NeoGFP

NeoGFP

T/F

Figure S1

a b

MCM8 exon GFP NeoMCM8-GFP

0.75 kbp

175 bp 175 bp

NeoGFP

NeoGFP

NeoGFP

the MCM8 gene

✂

Last coding exonExon 1

Last coding exonExon 1 NeoGFP

CAS9

the MCM8 gene

1 2 3 4 5 6 7WT

1 kb

ladd

er

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

0.75 kbp

the MCM8 gene

markertag

✂

Last coding exonExon 1

Last coding exonExon 1 markertag

CAS9

X bp X bp

Number of analyzed clonesDonorLength of homology Heterozygous Homozygous Targeting efficiency

175 bp (exp. 1) GFP-Neo 28 3 1 14 %

GFP-Neo80 bp (exp. 1) 26 0 0 0 %

175 bp w/o CRISPR GFP-Neo 33 0 0 0 %

175 bp (exp. 2) GFP-Neo 33 8 0 24 %

GFP-Neo150 bp 34 3 1 12 %

GFP-Neo125 bp 36 8 0 22 %

GFP-Neo80 bp (exp. 2) 35 1 0 3 %

S•tag-3FLAG-Neo936 bp (L), 740 bp (R) 27 11 1 44 %

Figure S2

the MCM8 gene

Figure S3

a

b

-24 0 24 48 72

1

10

Time (h)

Rel

ativ

e ce

ll nu

mbe

r (lo

g)

WT - auxinWT + auxinCMV-OsTIR1 - auxinCMV-OsTIR1 + auxin

+ auxin

2C 4C

CMV-OsTIR1

WT

+ auxin

+ auxin

- auxin

- auxin

c

0 50 100

- auxin

+ auxin

- auxin

+ auxin

% cell cycle

G1 S G2/M

CMV-OsTIR1

WT

0

RAD21-mAC

90 (min)auxin

+

-

RAD21-mAC+ CMV-OsTIR1

+

-

16 µm

16 µm 16 µm

16 µm

16 µm

16 µm 16 µm

16 µm

Figure S4

HCT116 CMV-OsTIR1background

HCT116 Tet-OsTIR1background

Figure S5

- CRISPR/Cas + CRISPR/Cas

DHC1 tagging with mAID-mClover

PCR

MarkerTagLinker Terminator Terminator

promoterLoxP LoxP

MarkerTagLoxP LoxP

MarkerTagLoxP LoxP

MarkerTagLoxP LoxP

MarkerTagLoxP LoxP

Cloning to a plasmid(TA or Topo cloning)

BamHI digestion

MarkerTagLoxP LoxP

BamHIBamHI

3’ homology arm(125 to 250 bp)

Figure S6

BamHI

Gene synthesis

5’ homology arm(125 to 250 bp)

Ligation

Selection marker

Attached tag at the C-terminus

mClover mCherry2 mAID mAID-mClovermAID-mCherry2S•tag-3FLAG

Neo

Hygro

Bsr

pMK277

pMK279

pMK278

pMK280

pMK282

pMK281

pMK283

pMK284

pMK285

pMK286

pMK287

pMK288

pMK289

pMK290

pMK291

pMK292

pMK294

pMK293

a

b