Versatile 3\xe2\x80\xb2 Functionalization of CRISPR Single Guide\nRNA
Specific applications of CRISPR/Cas genome editing systems benefit from chemical modifications of the sgRNA. Herein we describe a versatile and efficient strategy for functionalization of the 3\xe2\x80\xb2-end of a sgRNA. An exemplary collection of six chemically modified sgRNAs was prepared containing crosslinkers, a fluorophore and biotin. Modification of the sgRNA 3\xe2\x80\xb2-end was broadly tolerated by Streptococcus pyogenes Cas9 in an in vitro DNA cleavage assay. The 3\xe2\x80\xb2-biotinylated sgRNA was used as an affinity reagent to identify IGF2BP1, YB1 and hnRNP K as sgRNA-binding proteins present in HEK293T cells. Overall, the modification strategy presented here has the potential to expand on current applications of CRISPR/Cas systems.
Introduction<!>Chemical modification of sgRNA at the 3\xe2\x80\xb2-end<!>Impact of 3\xe2\x80\xb2-end modifications of sgRNA on Cas9 cleavage efficiency in\nvitro<!>Crosslinking modified sgRNA to Cas9<!>Capture of sgRNA-binding proteins with 3\xe2\x80\xb2-biotinylated sgRNA<!>Discussion<!>Conclusions<!>Modification of 30-nt fragment of sgRNA:<!>Splint ligation of modified sgRNAs:<!>In vitro cleavage assay:<!>General crosslinking sgRNAs to Cas9 procedure:<!>Pulldown of endogenous sgRNA binding proteins:<!>Verification of protein candidates by western blot:<!>sgRNA variants binding to IGF2BP1:
<p>The CRISPR/Cas9 genome editing system uses guide RNAs (gRNAs) and the Cas9
endonuclease to scan and cleave target DNA with high specificity.[1,2] Cas9 derives its specificity by first scanning the DNA for a
protospacer adjacent motif (PAM).[3] Once the PAM is located, Cas9 stalls and begins interrogation
by local DNA melting, then probing for gRNA–DNA base
complementarity.[4,5] If full complementarity exists between the
DNA and the gRNA, R-loop formation occurs, and the two nuclease domains are engaged
resulting in double-stranded breaks (DSBs).[6,7] Naturally a part of
the bacterial adaptive immune system, CRISPR-Cas9 has been repurposed for genome
editing.[8] Improvements to
editing efficiencies or repurposing Cas9 for new applications have largely been
accomplished by engineering Cas9.[9–13] The first
significant modification of the gRNAs involved fusing the CRISPR RNA (crRNA) and the
trans-activating crRNA (trRNA) via a stem loop creating the
chimeric single guide RNA (sgRNA).[1] The sgRNA simplifies the assembly of the ribonuclear protein
(RNP) complex as well as the transcription of the gRNA from a delivered plasmid.
Essential parts of the sgRNA have been investigated[14] and many studies have been carried out to
optimize the sgRNA sequence.[15–17] However,
for therapeutic applications of the CRISPR/Cas9 system that involve delivery of an
intact RNP, chemical modifications of the sgRNA will be required, similar to other
existing therapeutic oligonucleotides (i.e., antisense oligonucleotides,
siRNAs).[18–21] Notably, chemically synthesized
sgRNAs have been shown to have increased gene editing efficiency relative to in
vitro transcribed versions.[22,23] Additional chemical modifications
to the sgRNA could increase metabolic stability, help tissue-specific delivery and
improve editing efficiencies directly or indirectly through interactions with Cas9.
Importantly, modifications to the sgRNA could also prevent unintended sgRNA
interactions with endogenous proteins if the sgRNA is orphaned from Cas9.</p><p>While chemically modified gRNAs have been explored in some detail, most
studies investigate modifications to the sugar or use the dual guide RNA system
(e.g., crRNA + trRNA).[21,22,24–31]
Generally, these modifications (e.g., 2′-O-methyl) are
intended for increasing or maintaining on-target potency while reducing off-target
gene editing or sgRNA stability to nucleases[21,22,27,28,31] and less envisioned for
application-based experiments although some examples exist.[24–26] Hence, many of these modifications have been incorporated into
the 5′-end target sequence. Furthermore, introduction of chemical
modifications to the sgRNA have heavily relied on the limited set of commercially
available chemical modifications[27] such as 2′-O-methyl or
phosphorothioate linkages, amongst others. Although some modifications have been
reported to be incorporated in the 5′-end of the crRNA (e.g., strained
cyclooctyne or fluorophore),[24,25] modification at this location
could result in disruption of DNA unwinding and ultimately disable cleavage of the
target DNA. Thus, we sought a single modification strategy for the sgRNA at the
3′-end that was high yielding and versatile enough to be compatible with a
variety of different functional groups. Here, we describe a strategy for the
efficient and adaptable modification of the sgRNA 3′-end. We rationalize the
3′-end being more suitable for a variety of modifications that were
introduced including a fluorophore, covalent crosslinkers and a 3′-end
biotin. We describe two different approaches for covalent crosslinking Cas9 to its
sgRNA. In addition, the biotinylated sgRNA was used along with mass spectrometry to
identify endogenous human proteins capable of sgRNA binding. This provides a
powerful new method for identifying cellular factors that may affect sgRNA stability
or availability.</p><!><p>sgRNAs are typically 90–115-nt in length and challenging to
produce in high yield and purity via solid-phase synthesis alone, hence we aimed
to synthesize the sgRNA in two parts and use splint ligation to form our desired
sgRNA.[24,29,30,32] To
accomplish this, we chemically synthesized a 30-nt fragment of the sgRNA
modified at the 3′-end with a primary amine (Figure 1A and Table S1 in the Supporting
Information). The amine was then allowed to react with several
different functionalized NHS esters to produce chemically modified 3′
fragments of the sgRNA (Figure 1B). The
amine/NHS ester reactions proceeded efficiently, and identity of product was
confirmed by MALDIMS (Table
S2). To produce the full-length sgRNA, the functionalized 30-nt RNA
bearing a 5′-phosphate and a chemically synthesized 51-nt RNA was
hybridized to a complementary DNA and ligated using T4 DNA ligase. It should be
noted that the 51-nt fragment contains a 20-nt target sequence complementary to
the promoter region of the SNURF1 gene previously
described.[33] The
ligation reaction proceeded efficiently with minimal starting materials observed
for all sgRNAs produced (Figure S1). The modification reaction and ligation efficiently
created a collection of six chemically modified sgRNAs that have a variety of
functional groups including iodoacetamide, maleimide, strained cyclooctyne,
fluorophore and biotin (Figure 1C).</p><!><p>We wished to determine whether these sgRNA modifications were detrimental
for Cas9-mediated cleavage of a dsDNA target. End-point in vitro cleavage was
performed with purified Streptococcus pyogenes NLS-Cas9
protein[1] that was
preincubated with sgRNA and then mixed with a dsDNA that contained a single
target site. The asymmetric cleavage products were resolved by gel
electrophoresis and percent cleavage was determined (Figure 2A and B).
We demonstrated that only sgRNA bearing the free amine and the BMPS modified
sgRNA decreased the overall cleavage reaction relative to the transcribed sgRNA
standard (Figure 2B). To our surprise, the
3′-iodine modification of the sgRNA had a small stimulatory effect.
Overall, results from the in vitro cleavage assay indicated these modifications
were well tolerated at the sgRNA 3′-end.</p><!><p>We generated Cas9 mutants (Figure S2) that positioned a
reactive handle near the RNA-protein interface such that covalent crosslinking
of the sgRNA to the protein could occur (Figure
3A). With the sgRNAs generated, we envisioned that this crosslinking
could occur between a cysteine on Cas9 and the sgRNA bearing an iodoacetamide or
maleimide (Figure 3B). Similarly, Cas9
functionalized with azido-phenylalanine could react with a sgRNA containing a
strained cyclooctyne (Figure 3B). To
decrease unwanted crosslinking, the two native cysteines present in S.
pyogenes Cas9 were mutated to serine (C80S, C574S) and the
resulting mutant was used as a control in crosslinking experiments. Two triple
mutants of a maltose binding protein (MBP)-Cas9 fusion were generated. Both had
the two native cysteines mutated to serine, but one triple mutant had a K31C
mutation and the other had K31Stop mutation for incorporation of the unnatural
amino acid p-azido-l-phenylalanine (pAzF) by stop
codon suppression in the presence of a pAzF-charged tRNA.[35] To assay for crosslinking between the
sgRNA and Cas9, the two components were incubated in order for Cas9 to bind the
sgRNA. The sgRNA-Cas9 complex was then allowed to incubate at room temperature
overnight. After incubation, the reaction was quenched, and the products were
resolved by denaturing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel
electrophoresis). A protein-RNA crosslink was observed only when the triple
mutants of Cas9 were in the presence of an appropriately modified sgRNA (Figure 3C). The extent of crosslinking varied
among the sgRNAs tested (Figure 3D). The
sgRNA modified with AMAS produced the highest extent of crosslinking giving
about 29%. However, this was the highest yield obtained despite further attempts
at optimization.</p><!><p>The sgRNA bearing a 3′ biotin provided a reagent useful for
studying sgRNA-binding proteins present in human cells. Thus, we designed a
pull-down assay using HEK293T cell lysate (see the Experiment Section in the Supporting
Information) and the 3′-biotin-modified sgRNA (Figure 4A).</p><p>First, we wanted to identify possible sgRNA binding protein candidates
from human lysates. To accomplish this, HEK293T cell lysate was incubated in the
presence of the 3′-biotinylated RNA before mixing with streptavidin
magnetic beads. The beads were washed extensively to remove any nonspecific
binding proteins and were further eluted by heating in SDS loading buffer under
reducing conditions. The eluted samples were resolved by SDS-polyacrylamide gel
electrophoresis and protein bands were visualized. To avoid identifying abundant
HEK293T proteins, cell lysate was used as a negative control. Similarly, protein
cell lysate treated in the absence of biotinylated RNA was used to avoid
proteins that interact with the streptavidin beads. Lastly, a sample was
prepared with 100-fold excess of tRNA to determine which protein bands
corresponded to generic RNA-binding proteins. In total, five bands were excised
from the gel (Figure 4B) and subjected to
mass spectrometric analysis for protein identification (see the Experiment Section in the Supporting
Information).</p><p>From the mass spectrometric analysis, insulin-like growth factor 2 mRNA
binding protein 1 (IGF2BP1), heterogenous nuclear ribonucleoprotein K (hnRBP K),
heterogeneous nuclear ribonucleoprotein C1/C2 (hnRBP C1/C2), Y-box-binding
protein 1 (YB1), and Matrin 3 (MATR3) were determined to be candidates (Table S3). To confirm
these proteins were present in the final elution, the pull-down assay was
repeated, and bound proteins were detected by western blot analysis with
commercially available antibodies to the candidate proteins. Of the five
candidates, three were confirmed (IGF2BP1, hnRBP K, and YB1; Figure 4C). As the RNA binding requirements for
IGF2BP1 binding are not well understood, we tested the effect of scrambling the
20-nt targeting sequence on IGF2BP1 binding. However, we found that scrambling
the targeting sequence did not lead to an appreciable change in IGF2BP1 detected
by the western blot analysis (Figure 4B).
Similarly, we wished to determine whether the 30-nt biotinylated fragment of the
sgRNA was sufficient for detection of IG2BP1. Interestingly, we found that this
fragment was sufficient for significant IGF2BP1 binding (Figure 4D). Furthermore, IGF2BP1 could be competed
with excess tRNA or an in vitro transcribed 85-nt sgRNA (Figure 4D). Overall, these experiments identified
IFG2BP1, YB1, and hnRBP K as human sgRNA-binding proteins and indicate that the
IFG2BP1 binding determinants reside in the 3′ fragment of the sgRNA.</p><!><p>In this paper, we report a simple, efficient and versatile method for
chemically modifying the 3′-end of the sgRNA. Given the buried location of
the 5′-end of the sgRNA within the complex with Cas9, we suspected that
modification of the 5′-end could have unintended effects on Cas9 cleavage
activity or sgRNA binding. Therefore, we chose to modify the sgRNA at the
3′-end. This method allowed for the creation of a small collection of six
chemically modified sgRNAs, generated from a single 3′-propylamine-modified
30-nt fragment. Although only a sample library of bifunctional NHS-esters was used
here, we expect a wide variety of functionalized NHS-esters would be compatible with
this method. This work expands the types of modifications that can be attached to
the sgRNA and location of these functional groups.</p><p>Once our modified sgRNAs were generated, we sought to determine if the
location or type of modification was detrimental to Cas9′s ability to cleave
a double stranded DNA target. For this purpose, we used an sgRNA bearing an RNA
scaffold that had been shown previously to support cleavage and was used for
structural studies of Cas9 complexes.[34] We chose to study the impact of modification at this location
using the in vitro DNA cleavage assay,[36] which is known to show a strong correlation with cleavage
in a cellular context.[29,37,38] None of the chemically modified sgRNAs disabled
Cas9′s ability to cleave the target DNA in vitro, suggesting this location is
suitable for a wide variety of modifications. However, the 3′-amine modified
sgRNA showed a small decrease in cleavage efficiency, possibly due to repulsion with
positively charged amino acids at the RNA binding interface (i.e., K30, K31, K33).
Smaller functional groups like 3′-iodine and 3′-methyl-maleimide
showed a small increase in cleavage, whereas larger and more flexible appendages
were not significantly different from the unmodified, transcribed sgRNA. We believe
that the slight stimulation of the 3′-iodine modified sgRNA could arise from
an interaction between the adjacent lysine residues and the haloalkyl group, however
further studies are needed to validate this hypothesis.</p><p>Tight regulation of Cas9 expression and activity is highly desirable in
genome editing so unwanted off-target edits do not occur.[39] For this reason, delivery of an intact RNP
containing Cas9 and sgRNA is considered potentially advantageous over continuous
expression from a plasmid or mRNA.[40–42]
Expression of Cas9 from a transfected plasmid also poses spatial and temporal
control issues. If the timing and location of Cas9 and sgRNA expression are not
tightly coupled, free Cas9 might bind other endogenous RNAs, which could have
unintended effects. Likewise, unbound sgRNA might interact with endogenous
RNA-binding proteins and affect their function or be degraded by RNase present in
the cell. Assembly of the RNP in vitro, followed by delivery, would mitigate these
issues and artificially stabilizing the Cas9–sgRNA complex could be
beneficial in this context. As a potential means of advancing this idea, we sought
to introduce a covalent crosslink into the Cas9–sgRNA complex. To do so, we
positioned a reactive amino acid (K31C or K31pAzF), near the 3′-end of the
sgRNA modified with a compatible reactive handle. Only when the appropriately
modified sgRNA was incubated in the presence of the Cas9 mutant with the reactive
amino acid positioned near the 3′-end was a crosslinked band observed. We
demonstrated crosslinking using a Cas9 cysteine mutant and different electro-philes
including iodoacetamide and maleimide. We also showed that a Cas9 protein expressed
with the unnatural amino acid pAzF crosslinked to sgRNA bearing a cyclooctyne. The
81 AMAS sgRNA combined with the K31C mutant gave the highest yield of crosslinked
Cas9. However, this yield never surpassed ≈30% with additional attempts at
optimization. Further improvements in covalent crosslinking yields will be needed
for this to be a practical approach to stabilizing a sgRNA-Cas9 RNP. While the
3′-end of the sgRNA was fairly amendable to the crosslinking reaction, the
close proximity of the sgRNA to the protein at this location might influence the
fluorescence of 81 3′-Cy5 and could be a potential limitation to imaging
inside cells.</p><p>Although there are reports that sgRNAs that have a 5′-tri-phosphate
activate the RIG-I and MDA5 pathways of the innate immune response,[23,43] there are no reports of endogenous human proteins that
bind directly to the sgRNA. Moreover, removal of the 5′-triphosphate
abolishes inflammatory signaling,[23,43] suggesting that
this interaction is a result of the 5′-triphosphate and not specific to the
secondary structure of the sgRNA. Therefore, we sought to identify human proteins,
not associated with the RIG-I or MDA5 pathway, that are capable of binding to the
unique structure of the sgRNA in the absence of a 5′-triphosphate. It is
possible that sgRNA orphaned from Cas9 could bind to endogenous proteins causing
disruption of normal cellular functions. Similarly, temporal or spatial issues may
arise from delivered plasmids that constitutively express Cas9 and the sgRNA
component.[40–42] This could result in unbound
sgRNA inside cells with the potential to interact with endogenous human proteins.
Thus, we used our 3′-biotinylated sgRNA to reveal human sgRNA-binding
proteins. From this experiment, we observed five protein bands that were enriched in
the presence of the biotinylated sgRNA (Figure
4B). Protein identification by tandem mass spectrometry for these five
bands gave five possible protein candidates (Table S3). Protein candidates were
further validated by western blot and IGF2BP1, YB1, and hnRNP K were confirmed to be
sgRNA-binding proteins (Figure 4C).
Interestingly, MATR3 was not confirmed by western blot analysis despite literature
evidence that MATR3 and hnRNP K co-immunoprecipitate in the presence of
RNA.[44] We decided to
further investigate the structural requirements of IGF2BP1 because of reports in the
recent literature on this protein.[45,46] These reports
demonstrated that IGF2BP1 preferably binds highly structured RNAs,[46] and a consensus sequence was
established.[45]
Scrambling the target sequence on the sgRNA did not abolish binding, suggesting the
sequence and structure in this region is not critical for IGF2BP1 binding to the
sgRNA. Interestingly, the 30-nt biotinylated sgRNA 3′ fragment, comprising
stem loops 1 and 2, was sufficient for detection of IGF2BP1. Intriguingly, we were
able to pull down IGF2BP1 despite the fact that the 30-nt biotinylated 3′
fragment does not contain any known IGF2BP1 consensus sequence.[45,47,48] Because most sgRNAs employed
today contain the stem loop 1 and 2 structures, binding affinity for IGF2BP1 is
likely a feature common to all these sgRNAs. Additional experiments are needed to
determine the precise binding location between IGF2BP1 and the sgRNA. Further
studies following the strategy reported here could also help identify other
endogenous human sgRNA-binding proteins from other cell lines, tissues or under
different conditions (e.g., after inter-feron treatment).</p><!><p>In this paper, we have described a versatile, efficient and generalizable
method for functionalizing the 3′-end of the CRISPR sgRNA. We constructed an
example collection of six modified sgRNAs and showed that the modifications do not
interfere with the cleavage activity of Cas9 in vitro. We demonstrated crosslinking
of the sgRNA to Cas9 in a site-specific manner and used a biotinylated sgRNA to
identify three endogenous human sgRNA-binding proteins (IGF2BP1, YB1, and hnRNP K).
Lastly, we showed the 30-nt 3′ fragment of the sgRNA was sufficient for
binding IGF2BP1 indicating this is likely a property of most sgRNAs.</p><!><p>N-α-maleimidoacetoxysuccinimide ester (AMAS),
N-β-maleimidopropyloxysuccinimiide ester (BMPS),
succinimidyl iodoacetate (SIA), and EZ-link NHS biotin were purchased from
Thermo Fisher Scientific.
Dibenzocycloocytne-N-hydroxysuccinimidyl ester (DBCO) was
purchased from Sigma Aldrich. 3H-Indolium,
2-[5-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)-1,3-pentadien-1-yl]-1-[6-[(2,5-dioxo-1-pyrrolidinyl)-oxy]-6-oxohexyl]-3,3-dimethyl-,
tetrafluoroborate (Cy5) was purchased from Lumiprobe. A 30-nt RNA was
synthesized with a 5′-phosphate and a 3′-propylamine
(N3–3′) from (Horizon-Dharmacon). In a microcentrifuge tube, 30-nt
RNA (20 μL, 10 nmol), 10× PBS pH 7.4 (5 μL), and the
heterobifunctional NHS-ester (25 Î¼L, 20 mm dissolved in DMSO)
were added and thoroughly mixed. The reaction was allowed to incubate at
30°C overnight. To remove excess of the reagents, the reaction was
applied to a 3000 MWCO Amicon Ultra 0.5 mL (Millipore) centrifuge filter
followed by 400 Î¼L of high-purity nuclease-free water. The reaction was
concentrated to ≈100 μL by centrifugation (16200g
for 8 min). The flow through was discarded. Nuclease free water (400 Î¼L)
was added to the centrifugation unit and concentrated again to ≈ 100
Î¼L by centrifugation (16200g for 8 min). This process
was repeated a total of five times and concentrated to a final volume of 50
Î¼L. Reaction completion and identity of the oligonucleotides was
confirmed by MALDI-TOF mass spectroscopy. Mass spectra were recorded in negative
ion mode and calibrated to an external DNA standard mass (6999.9 Da; Supporting Information, Table
S2). We assumed the reaction between the NHS ester and the amine
modified RNA proceeded quantitively because an ion corresponding to the
unmodified sgRNA was not found in the mass spectrum for any of the modified
sgRNAs synthesized.</p><!><p>Full-length 81-nt sgRNAs were generated by splint ligation of two RNA
fragments: a synthetic 51-nt RNA (Integrated DNA Technologies) and the modified
30-nt RNA bearing a 5′-phosphate. In a microcentrifuge tube, the two RNA
fragments (3.5 nmol) and the complementary DNA splint (3.5 nmol, Integrated DNA
Technologies) were combined and lyophilized to dryness. The pellet was
resuspended in 10× ligation buffer (50 mm Tris·HCl pH
7.5, 10 mm MgCl2, 1 mm ATP) and 90 Î¼L of
water. To generate a DNA:RNA hybrid the solution was heated at 95°C for 5
min then slow cooled to 25°C. T4 DNA ligase (10 μL, 400000
UmL−1, New England Biolabs) was added to the solution and
the solution was incubated at 30°C overnight. The sample was
phenol-chloroform extracted and ethanol precipitated. The sample was lyophilized
to dryness. To remove the DNA template, the sample was resuspended in 10×
DNase buffer (5 Î¼L, New England Biotechnologies), water (45 Î¼L)
and DNase I (5 μL, 2 UμL−1, New England Biolabs)
and incubated at 37°C for 1 h. The reaction was quenched in 2× RNA
loading dye (New England Biolabs) and heated at 95°C for 5 min. RNA was
further purified by 10% denaturing PAGE gel and visualized by UV shadowing and
excised. To elute the sgRNA, gel slices were crush and soaked overnight at room
temperature in 500 mm NH4OAc and 100 mm EDTA.
Polyacrylamide fragments were removed by 0.2-Î¼m filter (Costar)
followed by ethanol precipitation and lyophilized to dryness. The modified
sgRNAs were resuspended in nuclease free water and stored in
−20°C.</p><!><p>Recombinant S. pyogenes Cas9-NLS was purchased from QB3
Macrolabs (University of California Berkeley). The purchased protein was
received at a stock concentration of 40 Î¼m in 20 mm
HEPES-KOH pH 7.5, 150 mm KCl, 10% glycerol, 1 mm DTT and
stored at −80°C. Prior to the reaction, a sample of Cas9 was
thawed on ice and diluted in 1x cleavage buffer (20 mm HEPES-KOH pH
7.5, 100 mm KCl, 5 mm MgCl2, 5% glycerol) to a
concentration of 6 Î¼m. sgRNAs were diluted to a concentration of
7.2 μm and heated at 95°C for 5 min then cooled to room
temperature. The sgRNA and Cas9 samples (1 Î¼L each) were incubated
together at room temperature for 15 minutes. Cleavage reactions were initiated
by the addition of the Snurf1 (S1) DNA (the sequence can be
found in the Supporting
Information), mixed thoroughly and incubated at 37°C for 45
min. Cleavage assays were performed in a final volume of 15 Î¼L with a
final concentration of 400 nm Cas9. The final reaction solution contained 150 ng
Snurf1 DNA target, 20 mm HEPES-KOH pH 7.5, 100
mm KCl, 5 mm MgCl2, 5% glycerol. Reactions were
quenched by the addition of proteinase K (1 Î¼L, 20
mgmL−1, New England Biolabs) and RNase A (0.3 μL,
10 mgmL−1, Thermo Fischer Scientific) and incubated at room
temperature for 20 min. Loading dye (6×; New England Biolabs) was added
to the reaction, and the cleavage products were separated on a 1.2% agarose gel
prestained with SYBR gold (Thermo Fischer Scientific). Cleavage reactions were
carried out in triplicate and values reported are averages ±standard
deviations.</p><!><p>Cas9 mutant (K31C C80S C574S or C80S C574S or K31pAzF C80S C574S) was
thawed on ice and then diluted to 6 Î¼m in 1x cleavage buffer
containing 20 mm HEPES·KOH pH 7.5, 100 mm KCl, 5
mm MgCl2, 5% glycerol, supplemented with HALT protease
inhibitor (Thermo Fischer Scientific). sgRNAs were diluted to 7.2
μm in water, hybridized at 95°C for 5 min and slowly
cooled to room temperature. To each reaction was added sgRNA (1 Î¼L, 7.2
Î¼m) and Cas9 mutant (1 Î¼L, 6 Î¼m) in a
PCR tube and incubated at room temperature for 15 min. The reaction mixture was
then diluted with additional 1fl cleavage buffer to a volume of 10 Î¼L and
incubated at room temperature overnight. The reaction was quenched by the
addition of 2fl SDS loading buffer and heated at 95°C for 5 min.
Covalently crosslinked sgRNA:Cas9 product was resolved from unreacted Cas9 by
4–12% SDS-polyacrylamide gel (200 V, 2 h). The resulting gel was stained
in SYPRO Orange stain and visualized on a Typhoon imager (Molecular Dynamics).
Cas9 bands were quantified using volume integration using ImageQuant software
(Molecular Dynamics). Percent crosslinking was determined by the equation: %
cross-linking=100×(volume covalent sgRNA:Cas9/[volume covalent
sgRNA:Cas9+volume unreacted Cas9].</p><!><p>Pulldown reactions were prepared by combining 375 Î¼L of 2flTENT
buffer (20 mm Tris·HCl pH 8, 2 mm EDTA, 500 mm
NaCl, 1% (v/v) Trion X-100, 7.5 Î¼L
100fl HALT protease, 7.5 Î¼L RiboLock RNase inhibitor, 300 Î¼g of
cell lysate, 100 picomoles of biotinylated sgRNA, and 100 Î¼L of water in
a total volume of 750 Î¼L. To account for endogenous proteins interacting
with the streptavidin beads, a reaction was prepared with no biotinylated RNA.
Another reaction was prepared by replacing the volume of water with yeast tRNA
(10 mgmL−1, Invitrogen). This created a 100-fold excess of
tRNA relative to biotinylated RNA as bait for endogenous proteins. Another
sample was prepared with a 10-fold excess of unmodified 85-nt sgRNA. Reactions
were mixed by pipetting and incubated at room temperature for 30 min.</p><p>Streptavidin-coupled M280 Dynabeads (Thermo Fischer Scientific; 75
μL) were washed three times in 1× TENT buffer (1 mL) and separated
using a BD IMag magnetic stand (BD Biosciences). Each RNA-protein mixture
prepared above was added to the washed beads and mixed thoroughly by pipetting.
The reactions were incubated at room temperature for an additional 30 min,
mixing intermittently by tapping the tube every 5–10 min. Tubes were
applied to the magnetic stand for 1 min to allow the beads to settle and the
supernatant was discarded. The reaction was removed from the magnetic stand and
1 mL of ice-cold 1× TENT buffer was added, resuspending the magnetic
beads and applied to the magnetic stand again. In total, the beads were washed
three times with 1 mL of ice-cold 1× TENT buffer. To elute proteins, 40
μL of 1× SDS loading buffer supplemented with DTT was added to the
beads and heated for 5 min at 95°C. The eluted volume containing the RNA
interacting proteins was resolved on a 4–12% BOLT Bis-Tris Plus gel
(Thermo Fischer Scientific). Proteins were visualized with Imperial Protein Gel
Stain (Thermo Fischer Scientific) following the manufacturer′s protocol
for overnight staining. Bands were chosen for further analysis by the intensity
of the band and competition with added tRNA (Figure 4B).</p><!><p>Cells were harvested, lysed and pull-down reactions were performed
according to the protocol above. Proteins were eluted from the Dynabeads in
1× SDS loading buffer (40 μL). The eluted protein sample (10
Î¼L) was separated by SDS-PAGE. Transferred. Proteins were transferred to
an Immobilon-P polyvinylidene fluoride (PVDF) membrane (Millipore). Blots were
blocked in a 5% skim milk solution in Tris·HCl (25 mm, pH 7.4),
3 mm KCl, 140 mm NaCl, 0.05% Tween 20 (TBST, Affymetrix) and
then probed with primary rabbit IgG antibodies for IGF2BP1 (18H5L31, 1:5000
dilution, Invitrogen), hnRBP K (D9A8, 1:1000 dilution, Cell Signaling
Technology), hnRNP C1/C2 (D6S3N, 1:1000 dilution, Cell Signaling Technology),
YB1 (D2B12, 1:1000 dilution, Cell Signaling Technology), MATR3
(A300–591A, 1:1000 dilution, Bethyl Laboratories) respectfully. This was
followed by incubation with a secondary goat anti-rabbit IgG antibody (1:5000
dilution; Santa Cruz Biotechnology). PageRuler Plus (10 Î¼L, Thermo
Fischer) was loaded into each gel to estimate protein sizes. As a negative
control, a pulldown reaction without the biotinylated RNA (10 Î¼L) was
loaded onto the gel. The protein candidates were detected using ECF substrate
(GE Healthcare) on a Typhoon Trio Variable Mode Imager (GE Healthcare).</p><!><p>A 51-nt RNA (Integrated DNA technologies) was obtained with the
Snurf1 targeting sequence randomly scrambled (Table S1). The scrambled
51-nt RNA was ligated to the 30-nt biotinylated fragment of the sgRNA using the
splint ligation protocol above. Cell lysate from three different passages were
used as biological replicates. Western blots were conducted according to the
same protocol as above. IGF2BP1 was quantified using volume integration and
reactions were normalized to the unscrambled Snurf1 targeted 81
3′-biotin sgRNA sample.</p>