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3897
Efficie
nt Transient Gene Knock
-down in Tobacco Plants Using Carbon Nanocarriers
Goz
de S
. Demirer
1, $ ,
* and Markita P. Landry
1, 2, 3, 4,
*
1
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720,
USA;
2
California Institute for Quantitative Biosciences, QB3, University of California, Berkeley, CA 94720,
USA;
3
Innovative Genomics Institute, Berkeley,
CA 94702, USA;
4
C ha n-
Zuckerberg Biohu
b, Sa
n
Francisco, CA 94158, USA;
$
Current address: Department of Plant Biology and Genome Center,
University of California, Davis, 451 Health Sciences Drive, Davis, CA 95616, USA
*For correspondence: gdemirer@berkeley.edu
; landry@berkeley.edu
[Abstract]
Gene knock
-down in plants is a useful approach to study genotype-
phenotype relationships,
render disease resistance to crops, and enable efficient biosynthesis of molecules in plants. Small
interfering RNA (siRNA)
-mediated gene silencing is one of the most
common ways to achieve gene
knock-
dow n in plants. Traditionally, siRNA is delivered into intact plant cells by coding the siRNA
sequences
into DNA vectors, which are then delivered through viral and/or bacterial methods. In this
protocol, we provide an alternative direct delivery method of siRNA molecules into intact plant cells for
efficient transient gene knock
-down in model tobacco plant,
Nicotiana benthamiana
,
leaves. Our
approach uses one dimensional carbon
-based nanomaterials, single-
walled carbon
na not ubes (SWNTs),
to deliver siRNA, and does not rely on viral/bacterial delivery. The distinct advantages of our method are
i) there is no need for DNA coding of siRNA sequences, ii) this abiotic method could work in a broader
range of plant species than
biotic methods, and iii) there are fewer regulatory complications when using
abiotic delivery methods, whereby gene silencing is transient without permanent modification of the plant
genome.
Graphic abstract:
Keywords:
Pla nt gene s ile nc i ng, R NA i nter
ference, siRNA delivery, Gene knock
-down,
Nicotiana
benthamiana
,
Carbon nanotubes, Single-
walled carbon nanotubes (SWNT), Nanomaterials
[Background]
Gene silencing through RNA interference (RNAi) was discovered in the early 1990s by
plant researchers studying petunia flower coloring ( Van d
er Krol
et al.
, 1990)
. In RNAi, specifically in
post
-transcriptional gene silencing (PTGS), gene expression level is reduced through mRNA
degradation caused by small RNA molecules –
micro
(miRNA) or small interfering (siRNA) RNA. RNAi
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has been a breakthrough technology, not only in plant research and biotechnology applications, but also
for many other organisms, including human therapy applications (Sierakowska
et al.
, 1996)
.
The first step of siRNA
-mediated RNAi in plants is the delivery of siRNA molecules into plant cells.
Delivery is a big bottleneck in plant biotechnology, given the presence of plant cell wall that acts as a
physical barrier for the delivery of biotechnology
-relevant cargoes such as DNA, RNA, and protein. In
plants, siRNA delivery is most commonly accomplished through viral vector delivery
via
Agrobacterium
tumefaciens
. However, most plant viruses are limited in their host range (Silva
et al.
, 2010)
and the size
of cargo they can efficiently deliver (
Burc h-
Smit h
et a
l.
, 2004)
.
Agrobacterium
-
mediated delivery
is also
limited in terms of plant host species, causes uncontrolled DNA integration into the plant nuclear genome,
and results in constitutive expression of siRNA, which limits temporal control over gene silencing (Baltes
et al.
, 2017)
.
Carbon nanotubes are one dimensional high
-aspect
-ratio nanomaterials that have many
advantageous features for siRNA delivery in plants. First, given their needle-
like structure with a small
diameter (~1 nm), long length (~500 nm) and high stiffness, single-
walled carbon nanotubes (SWNTs)
have show n to transport across the plant cell wall and localize inside plant cells (Demirer
et al.
, 2019b)
.
Second, high surface area and diverse surface chemistry options of SWNTs enable delivery of diverse
biological cargoes (Beyene
et al.
, 2016; Del Bonis
-O’Donnell
et al.
, 2017; Demirer
et al.
, 2020)
. Lastly,
SWNTs have the ability to delay the intracellular degradation of biomolecular cargoes (Demirer
et al.
,
2019a and 2020)
, which is especially valuable when working with fragile molecules like RNA.
Recently, we have developed a method to deliver siRNA molecules targeting the silencing of a
transge nic GFP ge ne i n
Nicotiana benthamiana
leaves, and an endogenous stress gene, ROQ
1, usi ng
S W NTs
(Demirer
et al.
, 2020)
. In this approach, we first load sense and antisense strands of siRNA onto
two separate SWNT nanoparticle solutions
via
pi-pi interactions that form between the sp
2
carbon
nanotube surface lattice and the aromatic bases of single stranded RNA (ssR
NA). Next, we introduce
an equimolar mixture of these RNA
-SWNT solutions into intact plant leaves for GFP silencing. Our
results demonstrate efficient silencing of GFP as assessed by confocal microscopy imaging, quantitative
PCR (qPCR), and W
estern blotting, both for transgenic GFP and also for the endogenous ROQ1 gene,
with disease-
resistance applications (Demirer
et al.
, 2020). This transient gene knock
-down approach
could be applied to other plant species, tissues, and target genes with minimal modifications. Additionally,
t he R NA l
oading method used in this study is not specific to siRNA, and thus, it can be adapted for the
delivery of other types of nucleic acids with some optimization (
e.g.
, guide RNA or messenger RNA for
CRISPR genome editing applications).
Below, we provide a step-
by-step protocol for the synthesis and characterization of siRNA loaded
SWNTs, and the measurement of gene silencing efficiency in tobacco leaves through confocal imagi
ng,
qPCR and W
estern blotting
(Figure 1)
.
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Figure 1.
Overview of the siRNA
-SWNT
gene silencing procedure
Materials and Reagents
1. SunGro Sunshine LC1 Grower soil mix (SUN52128CFLP)
2. Delicate task wipes (Kimberly
-Clark,
catalog number:
06-
666)
3. 100K MWCO Amicon spin filters (MilliporeSigma, catalog number: UFC510024)
4. PVDF Membrane, Precut, 7
× 8.4 cm (Bio-
Rad,
catalog number: 1620174)
5. Sterile syringe filter (0.45
μ
m;
VWR, catalog number: 28145-
481)
6. Microcentrifuge tubes (1.5 ml; VWR, catalog number: 89000-
028)
7. Conical tubes (50 ml; Olympus, catalog number: 28-
106)
8. Pipette tips (Low retention 10
μ
l, 200
μ
l, 1,000
μ
l filter tips; USA Scientific, catalog numbers:
1181-
3710, 1180
-8710, 1182-
1730)
9. Extended-
length pipette tips (1,000
μ
l; Eppendorf, catalog number: 0030073614)
10.
#1 Microscopy cover glass (Fisher Scientific,
catalog number:
12-
542B)
11.
Microscope slides (VWR, catalog number: 16004-
422)
12.
Syringe (1 ml; BD, catalog number: 14-
823-
434)
13.
Mini Trans
-Blot Filter paper (Bio-
Rad, catalog number: 1703932)
14.
EasyStrip‚ Plus PCR Tube (Thermo Scientific, catalog number
: AB2005)
15.
Plant seeds (
mGFP5
Nicotiana benthamiana
is obtained from the Staskawicz lab at UC Berkeley,
mGFP5
plants constitutively express GFP targeted to the ER under the control of the Cauliflower
mosaic virus 35S promoter)
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16.
Goat anti
-rabbit horseradish peroxidase-
conjugated antibody (Abcam, catalog number:
ab205718)
17.
Anti
-GFP antibody
, ChIP Grade (Abcam,
catalog number:
ab290)
18.
HiPCO SWNTs (NanoIntegris, Super purified, catalog number: HS28-
037)
19.
MilliQ water
20.
Nuclease-
free water (Qiagen, catalog number: 129114)
21.
Sodium chloride, NaCl (Sigma-
Aldr ich,
catalog number: S9888-
500G)
22.
Hydrochloric acid, HCl (37% [
vol/vo l
]; Sigma,
catalog number:
320331)
23.
Single
-stranded RNA oligonucleotides, including sense and antisense siRNA strands
– 21
nucleotides (Integrated DNA Technologies, IDT)
24.
Sodium dodecyl sulfate,
molecular biology grade
(Sigma
-Aldrich,
catalog number:
436143-
100G)
25.
Tris/ HCl (Sigma
-Aldrich,
catalog number: 10812846001)
26.
EDTA (Sigma
-Aldrich,
catalog number:
E9884-
100G)
27.
NP
-40 (Sigma-
Aldrich,
catalog number:
492016-
100ML)
28.
Glycerol (Sigma
-Aldrich,
catalog number: G5516-
500ML)
29.
Pierce 660 nm Protein Assay (Thermo, catalog number: 22660)
30.
iScript cDNA synthesis kit (Bio
-Rad,
catalog number: 1708891)
31.
PowerUp SYBR
green master mix (Applied Biosystems, catalog number: A25742)
32.
Qubit Protein Assay (ThermoFisher Scientific, catalog number: Q33211)
33.
RNeasy plant mini kit (QIAGEN, catalog number: 74904)
34.
BSA (Sigma
-Aldrich,
catalog number:
A4737-
25G)
35.
TWEEN20 (Sigma-
Aldrich,
catalog number: P9416-
100ML)
36.
Ammonium persulphate, AP
S (Sigma, catalog number: 248614-
100G)
37.
Low range ultra agarose (Bio-R
ad,
catalog number: 1613107)
38.
ECL Prime Western Blotting System (MilliporeSigma, catalog number:
GERPN2232)
39.
TEM ED ( N, N, N, N'
-tetramethylethylenediamine; Sigma, catalog number: T9281)
40.
Glycin
e (Sigma, catalog number: G8898)
41.
Methanol (Sigma, catalog number: 179957)
42.
4 × Laemmli sample Buffer (Bio-R
ad, 10 ml, catalog number: 1610747)
43.
Liquid nitrogen
44.
SYBR Gold Nucleic Acid Gel Stain (Invitrogen, catalog number: S11494)
45.
30% Acrylamide/Bis solution
19:1 (Bio-R
ad,
catalog number: 1610154)
46.
Prote
ase inhibitor cocktail (
Sigma,
catalog number:
P9599-
1ML)
47.
0.1 M NaCl (see Recipes)
48.
10%
(wt/vol)
Ammonium persulphate solution (APS) (see Recipes)
49.
10×
Transfer buffer (see Recipes)
50.
1 × Transfer buffer
(see Recipes)
51.
10×
Tris
-Buffered Saline (TBS) buffer (1 M Tris, 1.5 M NaCl, pH 7.4) (see Recipes)
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52.
1 × TBST
buffer
(see Recipes)
53.
Lysis buffer
(see Recipes)
Equipment
1. Analytical balance (Radwag, model: AS 60/220.R2)
2. Ultrasonic bath (Branson, model: 15-
336-
100)
3. Ultrasonic homogenizer with 6-
mm tip (Cole
-Parmer, model
s: UX
-04711-
70, UX
-04712-
14)
4. Vortex mixer (Fisher Scientific, model: 02-
215
-365)
5. pH meter (Spectrum, model: 242-
97839)
6. Orbital shaker (Waverly, model: S1CE)
7. NanoVue Plus spectrophotometer (GE Life Sciences, model: 28-
9569
-61)
8. Visible spectrophotometer (Thermo Scientific, model: 14-
385
-445)
9. Near
-infrared spectrometer (Princeton Instruments IsoPlane 320 coupled to a liquid nitrogen
-
cooled Princeton Instruments PyLoN
-IR 1D array of InGaAs pixels)
10.
UV
-Vis
-NIR Spectrophotometer (Shimadzu, model:
UV
-3600 Plus)
11.
Tabletop centrifuge (Eppendorf, catalog number: 5418000017)
12.
Centrifuge (Eppendorf, model: 5424R)
13.
Tweezers (VWR, catalog number: 63042-
518)
14.
Scissors (VWR, catalog number: 82027-
582)
15.
Mortar and pes
tle (Cole
-Parmer,
catalog number
: EW
-63100
-54)
16.
Pl
ant growth chamber (HiPoint, model: 740 FHLED)
17.
Gel image-
analysis system (Typhoon FLA 9500, GE Healthcare Services)
18.
Electrophoresis power supply (PowerPac basic power supply; Bio-
Rad,
catalog number
:
1645050)
19.
Mini Trans
-Blot Cell (Bio
-R ad,
catalog number: 1703811)
20.
Mini
-Protein TGX gels (Bio-R
ad,
catalog number: 456-
1094)
21.
ChemiDoc XRS+ System (Bio
-R ad,
catalog number: 1708265)
22.
Confocal Microscope (Zeiss, model:
LSM 710)
23.
Thermal Cycler CFX96 Touch Rea
l- Time PCR Detection System (Bio
-R ad,
catalog number
:
1855195)
24.
Thermal Cycler PCR (Applied Biosystems Veriti 96
-Well,
catalog number: 4375786)
Software
1. GraphPad Prism 7.0a (
https://www.graphpad.com/scientific
-software/prism/
)
2. Fiji ImageJ 2.0.0 (
https://imagej.net/Fiji/Dow nloads
)
3. Zen Blue 2.6 (
https://www.zeiss.com/microscopy/us/downloads.html
)
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Procedure
A. Plant growth
Germinate transgenic
mGFP5
Nicotiana benthamiana
seeds (see Note 1) and grow seeds in
SunGro Sunshine LC1 Grower soil mix
in a growth chamber for four to six weeks before experiments.
Use 12
h light at 24
°C and 12
h dark at 18
°C cycle
s for growing plants.
Note: Different plant species may require different germination and growth conditions.
B. siRNA design and generation
1. Currently, there are many software
programs
to design gene specific siRNA sequences with
minimal off
-target effects. A recently developed software called “siRNA
-Finder (si
-Fi) Software”
can be used in plants (Lück
et al.
, 2019)
.
2. After the design of siRNA sequences, sense and antisense RNA strands can be purchased from
Integrated DNA Technologies (IDT) as single-
stranded oligonucleotides.
C. R NA
-SWNT preparation
1. Dissolve sense and antisense siRNA strands in 0.1 M NaCl
at a concentration of 100 mg/ml
.
2. Add
1 mg dry HiPCO SWNTs to 20 μl
of dissolved sense RNA, and complete the solution volume
to 1
ml with 0.1 M NaCl (see Note 2).
3. Bath so nic
ate the mixture for 10 min at room temperature in Ultrasonic bath (Branson).
4. Probe-
tip sonicate the mixture with a 3
mm tip at 50% amplitude (~7W) for 30 min in an ice bath.
Renew ice bath if it starts melting during the sonication to prevent heating.
5. Rest the solution at room temperature for 30 min.
6. Centrifuge the solution at 16,
100
×
g
for
1 h in room temperature to
remove bundled SWNTs.
The supernatant contains the individually suspended sense-
R NA
-SWNTs. Keep the
supernatant and discard the SWNT pellet to the hazardous nanomaterials waste.
7. Repeat the same protocol for the antisense RNA strand (see Note 3). Store RNA
-SW NT
sol utio ns at 4
°C.
D. Removal of unbound siRNA
1.
Add 500 μl sense
-
RNA
-
SWNT and 500 μl antisense
-
RNA
-SWNT into two separate 100K
Amicon spin filters that are placed in 2 ml collection tubes. Centrifuge 4 min at 8,
000
×
g
in room
temperature.
2. Collect the flow
-thro ug hs from se nse
-RNA
-SWNT and antisense
-RNA
-SW NT i n
separate
tubes
and place the spin filters into the same collection tubes.
3. Add 0.1 M NaCl into the spin filters
until the volume reaches 500 μl
. Repeat the wash step.
4. Perform a total of 8 washes to remove all unbound RNA molecules. Merge and accumulate all
flow
-through solutions (separately for sense-
RNA
-SW NT a nd a ntise nse
-RNA
-SWNT
) for later
measurement of removed RNA amount
.
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5. Calculate the SWNT concent
ratio n
by measuring the carbon nanotube absorbance at 632 nm
using a spectrophotometer (use 2 μl for NanoVue Plus spectrophotometer or dilute to 1 ml for
Thermo Scientific). Divide the absorbance value by SWNT extinction coefficient of 0.036 to
obtain SWNT concen
tration in the unit of μg/ml (If the sample is diluted, multiply the absorbance
va lu
e also by the dilution factor).
6. Calculate the concentration of RNA loaded on SWNTs by measuring the absorbance of collected
flow
-
through solutions at 260 nm (use 2 μl for
NanoVue Plus spectrophotometer), and
subtracting the total amount of RNA removed from the total amount of RNA added (2 mg in this
case).
7. (Optional) For additional characterization, record sense-
RNA
-SWNT and antisense-
R NA
-
SWNT absorbance spectra with Shimad
zu UV
-3600 Plus, and fluorescence spectra with a near
-
infrared spectrometer (Princeton Instruments IsoPlane 320 coupled to a liquid nitrogen
-cooled
Pri nceton I nstrume nts PyLo N
-IR 1D array of InGaAs pixels). See Demirer
et al.
, 2020
for
representative examples of SWNT absorbance and fluorescence spectra.
E. Infiltration of leaves with RNA
-S W N Ts
1. Select
healthy and fully
-developed leaves from
mGFP5
Nicotiana benthamiana
(4-6 weeks old)
plants for experiments.
2. Merge 100
μl
of 200 nM sense
-R NA
-SWNTs with 100
μl
of 200 nM antisense-
RNA
-S W NTs i n a
1.5
ml Eppendorf tube (see Note 4). Mix well.
3. Immediately after mixing, make a small puncture on the abaxial (bottom) surface of the leaf with
a pipette tip, and infiltrate ~100-
200
μl
of the siRNA
-SWNT mixture from the hole with a 1
ml
needleless syringe with caution not to damage the leaf (see Note 5).
4. Use a Kimwipe tissue to remove the excess siRNA
-SWNT solution on the leaf surface. Mark
the infiltrated area with a Sharpie pen without damaging the leaf.
5. Infiltrate a negative control solution, such as the free siRNA without SWNTs or scrambled RNA
suspended SWNTs that does not target the gene of interest. If possible, also infiltrate a positive
control solution, such as viral siRNA delivery sample (see Note 6)
.
6. Return the infiltrated plant(s) into the growth chamber until the measurement of gene silencing.
F. Gene silencing determination through quantitative PCR (qPCR)
1. 24
h after infiltration, cut the infiltrated leaf areas (maximum of 100 mg leaf tissue per sample)
and extract total RNA with a
n RNeasy plant mini kit. After cutting the leaf, immediately proceed
with the first step of the RNA extraction protocol (
i .e.
, grindi ng t he tissue i n li q uid nitroge n us i ng
mortar and pestle) to make sure gene expression levels do not change after cutting. Follow the
protocol of the RNeasy plant mini kit (see Note 7).
2. Following RNA extraction, measure the RNA concentration and pur
ity with a spectrophotometer
.
Reverse transcribe 1
μ
g total RNA into complementary DNA (cDNA) using an iScript cDNA
synthesis kit. Follow the protocol of the iScript cDNA synthesis kit.
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3. Use PowerUp SYBR green master mix for the qPCR step with 2
μ
l cDNA fro
m Step 2 and
specific primers for the target and housekeeping genes. Follow the protocol of the PowerUp
SYBR green kit for relative quantification of target mRNA in the siRNA
-SWNT infiltrated leaf
compared to negative controls of free siRNA without SWNTs or scrambled RNA
-S W N Ts .
Example:
The target gene in our qPCR was
mGFP5
(GFP transgene inserted into
Nb
), and
EF1
(elongation factor 1) as our housekeeping (reference) gene.
4. Analyze the qPCR data using the ddCt method (Rao
et al.
, 2013). See Figure 2B for
representative
qPCR results.
G. Gene silencing determination through confocal fluorescence microscopy imaging
1. If silencing a fluorescent protein, such as GFP, confocal fluorescence microscopy can be used
to detect approximate silencing efficiency.
2. After infiltration, leave the plants with intact infiltrated leaves in the growth chamber for 72 h.
3. 72 h
after infiltration
, cut a small flat piece (0.5-
1 cm
× 0.5-
1 cm) of the infiltrated leaf around the
infiltration point and prepare a glass slide with cover slip (thickness #1). Add 50-
100
μ
l water in
between the glass slide and cover slip for imaging. Image samples before the leaf piece dries
out (optimally within 15 to 30 min).
4. Image the plant tissue with 488 nm laser excitation with a GFP filter cube
(in the case of GFP
silencing), and also capture brightfield with a transmitted light if available (see Note 8)
.
5. Capture at least 10 to 15 fields of view with same optical settings per sample, including non
-
treated leaf, and any negative and positive control samples.
6. For each sample, compare the mean fluorescence intensity value with the mean GFP
fluoresce nce i nte nsity of a no n-
treated leaf, which can be used to determine silencing efficiency
of siRNA
-SWNTs. Pay attention to use the same imaging parameters and quantification
analyses for samples imaged on different days. See Figure 2A for representative confocal
imaging results.
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Figure 2.
Representative gene silencing results.
A. Confocal microscopy images of non
-
treated and free-
siRNA treated control leaves and siRNA
-SWNT infiltrated sample leaves of
Nicotiana benthamiana.
Imaged after 3 days post
-infiltration, scale bars are 50
μm. B.
Quantitative PCR analysis results for GFP gene silencing in
Nicotiana benthamiana
leaves with
siRNA
-S W NTs .
H. Gene silencing det
ermi natio n thro ug h W
estern blotting
1. 72
h after infiltration,
harvest infiltrated leaves and grind them in liquid nitrogen using mortar and
pestle to recover dry frozen leaf powders.
2. Transfer the frozen leaf powder into a tube with 400
μl
pre-
chilled lysis buffer (see R
ecipes).
3. Lyse tissue on ice for 1 h. Centrifuge
the tubes at 10,000
×
g
at 4
°C for 20 min. Following
centrifugation, gently transfer the supernatants containing whole proteins to a new clean tube.
Quantify total extracted proteins with a Pierce 660 nm Protein Assay.
4. Mix the samples with th
e appropriate loading buffer for gel electrophoresis, and boil the mixture
at 95-
100 °C for 5 min either using a heat block or thermal cycler. Load 0.5 μg of normalized
total proteins from each sample and analyze with SDS
-PAGE gel (Bio
-R ad precast tris/glyci
ne
gel, 4-
20% gradient). Run the gel at 120 V for 60 min.
5. Transfer the gel to a PVDF membrane
in cold transfer buffer (see R
ecipes) and run at 400 mA
i n 1
× transfer buffer with methanol for no more than 60 min in a cold room with an ice block.
6. Block the membrane for 1 h usi ng 7.5% BSA in 1
× TBST buffer (see
Recipes) followed by
overnight incubation at 4
°C with the primary GFP antibody. After extensive washing, probe the
corresponding protein bands with a goat anti
-rabbit horseradish peroxidase-
conjugated