Supplemental Material
Targeted gene editing by transfection of in vitro reconstituted Streptococcus thermophilus
Cas9 nuclease complex
Monika Glemzaite1, Egle Balciunaite1, Tautvydas Karvelis2, Giedrius Gasiunas2, Mantvyda M. Grusyte2,
Gediminas Alzbutas1, Aiste Jurcyte1, Emily M. Anderson3, Elena Maksimova3, Anja J. Smith3, 4, Arvydas
Lubys1, Lolita Zaliauskiene*1 and Virginijus Siksnys*2
1
Thermo Fisher Scientific Baltics, Graiciuno 8, Vilnius, LT-02241, Lithuania.
Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius, LT-02241, Lithuania.
3
Thermo Fisher Scientific, 2650 Crescent Drive, Suite 100, Lafayette, CO 80026, US
4
Current address: Dharmacon GE Healthcare, 2650 Crescent Drive, Suite 100, Lafayette, CO 80026, US
2
Correspondence: Virginijus Siksnys or Lolita Zaliauskiene
E-mail: siksnys@ibt.lt or lolita.zaliauskiene@thermofisher.com
1
Supplementary methods
Dual reporter cassette construction
The dual reporter cassette contains RFP-GFP gene fusion. The eGFP reporter gene is split at the L67 codon
by GAPDH intron sequence. The intron fragment was PCR amplified from the genomic DNA of Jurkat cells.
iCreI nuclease target site was introduced into the intron by annealing oligonucleotides Int-D and Int-R (Table
S1), blunting the product and subcloning it into the intron (via PdmI site).
The split eGFP gene was inserted into the RFP gene at the Y196 codon position. The RFP gene carries 400
bp direct homologous repeats engineered on both sides of the eGFP insert. The repeats were generated by
digesting pMTC-RFP plasmid with restriction enzymes Eco91I (RE1) and Bsp1407I (RE2) or Eco31I (RE3).
RE1 target sequence is located on the plasmid, RE2 and RE3 target sites are located within the RFP gene.
The N-terminal RFP fragment was obtained by RE1 and RE3 double digestion; the C-terminal fragment was
generated by RE2 and RE1 double digestion. Each fragment was gel purified and ligated with intron-split
eGFP gene (see above) to yield the final pMTC-RFP/EGFP dual reporter plasmid (6644 bp).
Cas9 expression and purification
To engineer the pBAD-Cas9-NLS-6His plasmid, encoding S. thermophilus CRISPR3 Cas9 (StCas9) protein
fused with SV40T nuclear localization signal (NLS), the NLS sequence was PCR amplified using synthetic
pMTC-eCFPnuc vector as a template and a corresponding primer pair (Table S1). The product was
subsequently cloned into the pBAD-StCas9 plasmid1 via PstI and XhoI sites. The StCas9 protein containing
NLS and 6xHis-tag at the C-terminus was expressed and purified as described in1.
To engineer the pET21-SpCas9-NLS-6His plasmid encoding S. pyogenes Cas9 (SpCas9) protein, the
synthetic SpCas9 gene fused with NLS and 6xHis-tag (DNA2.0) was subcloned into pET-21 expression
vector via SmaI and NotI sites. Protein expression was induced by adding 0.5M IPTG for 20 hours at 16C.
Cells were harvested and disrupted by sonication. SpCas9 protein was purified by chromatography on
heparin-sepharose, hydroxyapatite, buthyl-sepharose and DEAE-sepharose columns.
Construction of plasmids for StCas9 and sgRNA transfection
For the Cas9 complex delivery by plasmid transfection phSpCas9-SapI and pU6-sp-sgRNA plasmids, which
are derivatives of pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (a gift from Feng Zhang (Addgene
plasmid # 42230)) were generated. The construction of phSpCas9-SapI plasmid was performed in two steps.
First, the Sp-sgRNA encoding sequence was eliminated by XbaI and PsiI double digestion of pX330-U6Chimeric_BB-CBh-hSpCas9 plasmid. Next, PCR fragment obtained using G1/G2 (Table S1) primers, was
ligated via BglII and EcoRI sites to obtain phSpCas9-SapI plasmid. To generate the phStCas9-NLS plasmid
containing human codon optimized cas9 (hStCas9), a synthetic hstCas9 gene was ligated into the phSpCas9SapI vector pre-cleaved via SapI and AgeI sites.
2
To engineer a pU6-St-sgRNA plasmid encoding St-sgRNA the backbone of St-sgRNA gene was amplified
using primers G3 and G4 (Table 1) and ligated into pU6-sp-sgRNA plasmid pre-cleaved via BpiI and XbaI
sites. The resulting pU6-st-sgRNA plasmid contains two SapI sites for the insertion of targeting sequence. To
obtain plasmids encoding targeting St-sgRNA, oligoduplexes containing targeting sequence (T1 or T2) and
SapI compatible overhangs were assembled by annealing complementary oligonucleotides (G5/G6 and
G7/G8, accordingly) (Table S1) and ligated into pU6-st-sgRNA via two SapI sites.
Generation and purification of anti-StCas9 polyclonal antibodies
Experimental mice were maintained in an animal facility at the Department of Biochemisty, Vilnius
University (Lithuania). Mice were immunized four times with 50 µg of recombinant StCas9 protein plus
complete (first immunization) or incomplete Freund’s adjuvant. Polyclonal antibodies were purified from
blood by ammonium sulfate precipitation. Antibody titers and specificity were determined by ELISA,
Western blot and immunofluorescense assays.
Cas9 complex reconstitution
The crRNA and tracrRNA were synthesized at Thermo Fisher Scientific (Lafayette). RNA sequences are
provided in Table S2. The crRNA-tracrRNA duplexes as well as Cas9 complexes were prepared as described
in Karvelis et al1.
To assess the functionality of reconstituted Cas9 complex, the NotI restriction enzyme linearized dual
reporter plasmid pMTC-RFP/EGFP was digested with Cas9 as described in Karvelis et al1.
I-CreI expression and purification
I-CreI DNA was generated by PCR using I-CreI plasmid (Cellectis) as a template. NLS and 6xHis-tag
sequences were engineered during the subsequent overlap extension PCR (for primer sequences see Table
S1). Resultant DNA was cloned into pLATE31 vector (aLICator LIC Cloning and Expression system
(Thermo Fisher) and used for transformation of ER2566 strain using TransformAid Bacterial Transformation
Kit (Thermo Fisher). Protein expression was induced with 1 mM IPTG for 5 hours at 30 C. Cells were
harvested and disrupted by sonication. The supernatant was loaded onto the Ni-NTA column (Thermo
Scientific) and eluted with 250 mM imidazole solution. The eluted I-CreI was dialyzed against 25 mM TrisHCl pH 8.0, 20 mM NaCl, 0.2 % Triton X-100, 1 mM DTT, 0.1 mM EDTA, 50% glycerol. The I-CreI
homogeneity and concentration were estimated by SDS-PAGE.
Cell culture and transfection
One day before the experiment, the CHO-K1 or HEK293T cells were seeded in a 24-well plate at the density
of 7x104 cells per 1 ml of RPMI-1640 culture medium supplemented with 10% fetal bovine serum (FBS).
For protein transfection, cells were transferred into serum-free medium just before the experiment. For DNA
transfection, plasmid DNA was mixed with TurboFect in vitro DNA transfection agent (Thermo Scientific)
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following manufacturer’s protocol. The transfection efficiencies were analyzed using “Guava EasyCyte
8HT” flow cytometer and Guava CytoSoft 5.3 cell acquisition/analysis software (Millipore).
For DNA/protein co-transfection experiments the dual reporter plasmid DNA (0.5 µg) and Cas9 complex (1
µg) plus extra 300 ng of crRNA-tracrRNA duplex (to ensure more efficient complex formation with cationic
polymer TurboFect) were diluted in separate tubes with serum-free medium to a final volume of 50 µl. Next,
0.5 µl of TurboFect transfection agent was added into each tube, samples were incubated for 15-20 min at
room temperature and added to the cell cultures at the same time. The serum-free cell culture medium was
changed into the complete growth medium 3 h later and cells were incubated for an additional 48 h.
For protein transfections, 1 µg of the recombinant Cas9 or Cas9 complex plus excess of crRNA-tracrRNA
duplex (300 ng) were diluted with serum-free medium to a athe final volume of 100 µl. The samples were
mixed with 1µl of TurboFect protein transfection agent, incubated for 15 min and added to the cell culture.
Three hours after transfection the serum-free cell culture medium was changed into the full growth medium.
Cells were analyzed 48 h later.
For plasmid co-transfection 0.5 µg of hStCas9 encoding plasmid phStCas9-NLS and 0.5 µg of sgRNA
encoding plasmid were diluted into serum-free medium to a final volume of 100 µl. The samples were mixed
with 2 µl of TurboFect transfection agent, incubated for 15-20 min at room temperature and added to the cell
culture. After 24 hours, the cell were additionally transfected with 0.5 µg of reporter plasmid diluted into a
serum-free medium to a final volume of 100 µl and mixed with 1 µl of TurboFect transfection agent.
Following 15-20 min incubation at room temperature, samples were added to the cell culture. Cells were
analyzed 48 h after the reporter plasmid delivery.
For multiplex gene editing of DNMT3B and PPIB genes, 2 µg of corresponding Cas9 complexes plus 300 ng
of each RNA duplex were diluted with PBS to a final volume of 100 µl, mixed with 2.5 µl of TurboFect and
following 15 min of incubation added to the cells. Serum-free medium was replaced with complete growth
medium 16h after transfection and the cells were incubated for another 48h.
The stable cell line expressing a dual reporter gene cassette was generated using “cGPS® CHO-κ1 Full Kit
DD“ (Cellectis Bioresearch). TurboFect in vitro transfection reagent was used for plasmid delivery into the
CHO-K1 cells.
Immunofluorescense
For immunofluorescense analysis CHO-K1 cells were grown on collagen coated coverslips. Transfections
were performed as described above. Samples were analyzed following the 24 h incubation at 37C in a 5%
CO2 incubator.
Cellular uptake of Cas9 was analyzed using mouse polyclonal anti-Cas9 antibodies followed by FITClabeled rat anti-mouse secondary antibodies (eBioscience). Cas9 bound RNA was detected via the 3’-biotintagged tracrRNA using streptavidin-Qdot®585 conjugate (Life Technologies). Cell nuclei were stained with
DAPI (“Vectashield® Mounting Medium with DAPI”).
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Imaging was performed on a Leica TCS SP8 system (Leica Microsystems, Exton, PA) using Leica DMI
6000 inverted microscope (63x objective); 405 Diode and argon lasers were used to excite DAPI, Qdot585
and FITC (405 and 488 nm laser lines, respectively). Samples were imaged by sequential scanning. Optical
sections (0.5 μm each) through the z-axis were generated; flattened maximum projections of image stacks
(cross-sections through the nuclei) were prepared using Leica application suite (LAS AF) confocal imaging
software.
Indel analysis
DNA was extracted from CHO-K1 or HEK293T cells using “GeneJET genomic DNA purification kit”, RNA
- using “GeneJET RNA purification kit”, cDNA was generated using “Maxima First Strand cDNA Synthesis
Kit for RTqPCR” (Thermo Scientific).
Regions surrounding Cas9 target sites were PCR amplified using Thermo Scientific Phusion Hot Start II
DNA polymerase. Corresponding primer sequences are provided in Table S1.
For Surveyor assay, PCR products were reannealed and analyzed using “SURVEYOR Mutation Detection
Kit for Standard Gel Electrophoresis“ (Transgenomic). Amplicon sizes and expected Surveyor nuclease
cleavage products are provided in Table S3.
For Cas9 complex digestion assay, 3 nM of PCR products were mixed with 50 nM of Cas9 complex and
incubated for 20 minutes at 37°C in a buffer described in Karvelis et al 1. Reaction was quenched with
phenol/chloroform and digestion products were examined in 2 % agarose gel.
Quantitative analysis was done using ImageJ program (National Institutes of Health) as described in2.
Next generation sequencing
For analysis of indels resulting from the StCas9 cleavage of the plasmid DNA, target amplicons were
generated from plasmid DNA extracted from transfected cell cultures. For analysis of indels in chromosomal
DNA, total RNA was extracted from transfected cells and amplicons were generated from cDNA. Samples
for the next generation sequencing (MiSeq, Illumina) were prepared using Thermo Scientific Phusion Hot
Start II DNA polymerase and “ClaSeek Library Preparation Kit, Illumina™ compatible” (Thermo Scientific)
kits and genomic DNA extracted from the reporter cell line (GeneJET genomic DNA purification kit,
Thermo Scientific). Reads were aligned to their corresponding templates by SHRiMP software package (v
2.2.3) 3 using increased sensitivity mode. Apart from the input parameters the following parameters were set
in the command line: “--pair-mode opp-in -N 12 --qv-offset 33 --trim-illumina -E --single-best-mapping -no-improper-mappings”. Indels were analyzed by Python program written using HTSeq v0.5.4p2 library
[http://www-huber.embl.de/users/anders/HTSeq] (to be published elsewhere and currently available upon
request from the authors). The program output provides data on indel type (insertion, deletion, and
substitution), position in respect to the reference sequence, indel length, and corresponding coverage.
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References
1.
Karvelis T, Gasiunas G, Miksys A, Barrangou R, Horvath P, Siksnys V. crRNA and tracrRNA guide
Cas9-mediated DNA interference in Streptococcus thermophilus. RNA Biol 2013; 10:841–51.
2.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, et al.
Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339:819–23.
3.
David M, Dzamba M, Lister D, Ilie L, Brudno M. SHRiMP2: sensitive yet practical SHort Read
Mapping. Bioinformatics 2011; 27:1011–2.
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Supplementary figures
Figure S1. StCas9-crRNA-tracrRNA complexes are localized in the nucleus and perinuclear region 24 h
after the transfection. CHO-K1 cells were transfected with in vitro reconstituted StCas9 complexes, 24 h
later the cells were fixed and nuclei were stained with DAPI. The StCas9 protein detected with polyclonal
anti-StCas9 antibodies, followed by the FITC-labeled anti-mouse secondary antibodies; biotinylated
tracrRNA was detected using streptavidin-coupled Q dots. Images were taken using Leica TCS SP8 system,
a composite picture of four z-stacks, cross-sections through the nuclei, is shown. Negative control – nontransfected cells.
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Figure S2. Target DNA hydrolysis by in vitro reconstituted StCas9 complexes. The reporter plasmid pMTCRFP/EGFP (6644 bp) was digested with NotI restriction enzyme or double digested with NotI and T1 or T2
specific or non-targeting (Ctrl) StCas9 complexes. 2345 and 876 bp digestion products correspond to the
distance between T1 or T2 target sites and the NotI restriction site, respectively. M – O’GeneRuler 1 kb Plus
DNA Ladder.
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Figure S3. Surveyor analysis of target gene indels in the reporter plasmid following StCas9 and sgRNA
plasmid co-transfection. CHOK-1 cells were co-transfected with StCas9 ± sgRNA encoding plasmids, 24 h
later cells were transfected with dual reporter plasmid pMTC-RFP/EGFP and analyzed by Surveyor assay 48
hours thereafter. Regions surrounding Cas9 target sites in the reporter plasmid were PCR amplified and
reannealed PCR amplicons (592 bp) were digested with Surveyor nuclease. Red arrows indicate 100+492 bp
and 363+229 bp hydrolysis products for T1 and T2, respectively. Numbers below indicate indel % calculated
by densitometric analysis of corresponding bands.
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Figure S4. HDR contributes to the repair of StCas9 generated DSB within the chromosomal targets T1 or
T2. Cas9 complexes were delivered into CHO-K1 cells with stable dual reporter gene expression. Cells were
harvested 48 h later and centrifuged onto the microscope slides. Cell nuclei were stained with DAPI.
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Figure S5. NHEJ-mediated deletions and insertions are centered on the T1 and T2 target sites. For analysis
of indels resulting from the StCas9 cleavage of the plasmid DNA, target amplicons were generated from
plasmid DNA extracted from transfected cell cultures. For analysis of indels in chromosomal DNA, total
RNA was extracted from transfected cells and amplicons were generated from cDNA. Cleavage specificity at
each target was determined by deep sequencing using MiSeq system (Illumina). “0” position on the x axis
indicates the StCas9 cleavage site.
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Figure S6. Surveyor analysis of endogenous gene modification following in vitro reconstituted S. pyogenes
Cas9 complex transfection. HEK293T cells were transfected with SpCas9 complexes specific for DNMT3B
and/or PPIB gene loci. Surveyor digestion was performed on reannealed PCR amplicons - 544 bp
(DNMT3B) or 505 bp (PPIB) yielding hydrolysis products of 335+209 bp or 330+174 bp, respectively (red
arrowheads). Calculated indel percentages indicate each gene modification extent from single or dual
transfection experiments.
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Supplementary tables
Supplementary Table S1. Oligonucleotides used in DNA manipulations.
Name
GAPDH-f
primer
GAPDH-r
primer
Int-D
Int-R
Name
Cre-f primer
Cre-r primer
Cre NLS-6His-f
primer
Cre NLS-6His-r
primer
Tail reverse
Name
Cas9 NLS-f
primer
Cas9 NLS-r
primer
Name
EGFP_T1/T2-f
primer
EGFP_T1/T2-r
primer
DNMT3B-f
primer
DNMT3B-r
primer
PPIB-f primer
PPIB-r primer
Name
G1
G2
G3
G4
G5
G6
G7
G8
Oligonucleotides for dual reporter cassette construction
Sequence (5’-3’)
GTAAATCAAAGAAGTGGGTTT
CTAAGAGACAAGAGGCAAGAA
CCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACC
TATAAGCTCGAATTCACCTGGGTTCAAAACGTCGTGAGACAG
CACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTG
TGGAAATTACGAATTCACCACCAAACTGTCTCACGACGTTTTGAACCCAG
Oligonucleotides for I-CreI nuclease cloning
Sequence (5’-3’)
GAATTTGTCCGGGGATTCTT
AGGGTTTGTAGACGGTGACG
AGCTCGAGATCTGTCGGCCGCCGGGGAGGATTTCTTCTT
AGAAGGAGATATAACTATGGCCAATACCAAATATAACAAA
GTGGTGGTGATGGTGATGGCCTCTAGATCCGGTGGATCCTACC
Oligonucleotides for Cas9-NLS-6His cloning
Sequence (5’-3’)
GAGATCTCGAGGCTGATC CAAAAA AGAAG
TGCCTGCAGTTAGT GATGGTGGTGGTGATGACCTCTAGATCC
GGTGGATCCTACC
Oligonucleotides for Surveyor nuclease assay
Sequence (5’-3’)
AGGGCGAGGAGCTGTTCACC
TAGTGGTTGTCGGGCAGCAG
TGAGAAGGAGCCACTTGCTT
GACCAAGAACGGGAAAGTCA
GAACTTAGGCTCCGCTCCTT
CTCTGCAGGTCAGTTTGCTG
Oligonucleotides for hStCas9 and st-sgRNA cloning into pX330 plasmid
Sequence (5’-3’)
AA AGATCT GCTCTTC C AAAAGGCCGGCGGCCACG
CGA GGC TGA TCA GCG AGC TC
AAGAAGACGGCACCAGAAGAGCAGCAAGCTCTTCTGTTTTAGAGCTGTGTTG
TTTCG GTTAAAACAACACAGCGAGTTAAAATAAG
AAT CTA GAA AAA ACA CCG AAT CGG TGC CAC CTT TTC AAG TTG AGT
ACG GAC TAA GCC TTA TTT TAA CTC GCT GTG TTG TTT TAA C
ACC GCTTCAGGGTCAGCTTGCCGT
AACACGGCAA GCTGACCCTGC AAG
ACC GCTGAAGGGCATCGACTTCA
AACTGAAGTC GATGCCCTTC AGC
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Supplementary Table S2. Synthetic tracrRNA and crRNA sequences.
Name
tracrRNA
NC crRNA
EGFP_T1 crRNA
EGFP_T2 crRNA
DNMT3B
crRNA
PPIB crRNA
Sequence (5’-3’)
AACAACACAGCGAGUUAAAAUAAGGCUUAGUCCGUACUCAACUUGAAAAG
GUGGCACCGAUUCGGUGUUUUUUU
CGCUAAAGAGGAAGAGGACAGUUUUAGAGCUGUGUUGUUUCG
CUUCAGGGUCAGCUUGCCGUGUUUUAGAGCUGUGUUGUUUCG
GCUGAAGGGCAUCGACUUCAGUUUUAGAGCUGUGUUGUUUCG
GCUGAAUUACUCACGCCCCAGUUUUAGAGCUGUGUUGUUUCG
GUGUAUUUUGACCUACGAAUGUUUUAGAGCUGUGUUGUUUCG
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Supplementary Table S3. DNA fragments in Surveyor nuclease assay.
Gene
EGFP_T1
EGFP_T2
DNMT3B
PPIB
Amplicon size (bp)
592
544
505
Expected size of Surveyor nuclease cleavage fragments (bp)
100+492
363+229
335+209
330+174
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