bioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Quantitative assaying of SpCas9-NG with fluorescent reporters
Alexandre Baccouche1†, Kevin Montagne2†, Nozomu Yachie3, Teruo Fujii1, Anthony Genot 1*
1

Laboratory for Integrated Micro and Mechatronic Systems, CNRS-IIS UMI 2820, The University of Tokyo, Tokyo, 153-8505,
Japan
2

Department of Mechanical Engineering, School of Engineering, The University of Tokyo, Tokyo, 113-8656, Japan3

School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia,
Vancouver, British Columbia, BC V6T 1Z3 Canada†

Joint Authors* To whom correspondence should be addressed. Tel: +81-3-5452-6036; Email: genot@iis.u-tokyo.ac.jp

ABSTRACTThe Cas9 enzyme has revolutionized biology in less than a decade. Engineering Cas9 to expand its
functionality has become a major research goal, yet assaying variants of Cas9 remains a laborious
task that is commonly performed with gel electrophoresis. Fluorescence assays have been reported
for Cas9 but their utility for assaying variants of Cas9 has not been investigated in detail. Here we use
a simple fluorescent assay to resolve differences of activity between the wild type Streptococcus
pyogenes Cas9 (SpCas9) and SpCas9-NG, a variant with an expanded PAM repertoire. We compare
the kinetics of the two enzymes on dozens of mutated RNA guides – highlighting the benefits offluorescence such as quantitativity, sensitivity, multiplexing, non-invasiveness and real-timeness. Thisvalidates fluorescence as a tool for engineering Cas9 and lays the groundwork for directly evolving
Cas9 in microfluidic compartments.
INTRODUCTION
In less than a decade, the CRISPR-Cas9 system1,2 has upended many fields of life sciences. Its
programmability has enabled genome editing, synthetic gene regulation, or live imaging ofchromosomes3–6, and it augurs groundbreaking advances for human healthcare7, agricultural
biotechnology8 or molecular diagnosis9.
The CRISPR-Cas9 system comprises a Cas9 enzyme and a guide RNA (gRNA), itself composed of aconserved region (the scaffold, which initiates binding with Cas9), and a variable region (the spacer,
which directs Cas9 to the target region for cleavage). Upon loading the guide RNA, the Cas9:gRNAcomplex first scouts double-stranded DNA (dsDNA) for a specific sequence (the Proto Adjacent Motif
or PAM). Once it finds one, it unwinds the DNA nearby and attempts to match it with the spacer of theguide RNA by forming an R-loop. When the two domains match, Cas9 cleaves both sides of the target
DNA (3 base pairs upstream of the PAM), leaving a double-stranded break in the genome that is theentry point for genome editing.
Protein engineers have expanded the functionality of Cas910–14. The simplest and most common
mutation consists in deactivating one of the endonuclease lobes (producing a programmable nicking
enzyme) or both lobes (producing the so-called dead Cas9, which has lost its ability to cut DNA but
retains its ability to lock onto target DNA). Splitting Cas9 is also a common strategy to control theactivity of Cas915,16. The enzyme is split in two nonfunctional parts that become functional whenreunited by a chemical or physical signal (small molecule, light, temperature..).
A highly sought-after variant is a Cas9 with an expanded PAM repertoire. Wild type (WT) Cas9
recognizes a 3-letter motif NGG, which means that a GG sequence must be located 1 bp upstream ofthe domain recognized by the spacer. This constraint on the PAM limits the repertoire of targettablesequences, as for instance GG/CC motifs comprise only ~ 5% of the human genome. Thanks torational protein engineering, Cas9 enzymes with an expanded NG PAM have been reported 7–9. In thebioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

SpCas9-NG variant, the base-specific interactions between Cas9 and the third G of the PAM weredeleted, and the surrounding amino residues were tuned to compensate for the loss of stability. TheseNG variants considerably expand the repertoire of targetable sequences, since G/C nucleotides
comprise about ~41% of the human genome17.
Assaying the activity of Cas9 is a bottleneck in the cycle of protein engineering. It is commonly doneby incubating together the Cas9 variant, a guide RNA, and a target plasmid, and then resolving thecleaved plasmid by gel electrophoresis1. But this method is laborious, poorly adapted to the ubiquitous
96-well format (a gel slab accommodates at most a dozen wells) and prone to failures (smeared
bands, buffer leakage, gel tearing or uneven staining occur commonly in the hands of unskilled
operators). Gel electrophoresis also lacks multiplexing: one separate tube and gel lane must be
allocated for each PAM to be tested. Additionally gel electrophoresis is an end-point method that lacks
temporal resolution, sensitivity and quantitativity. Lastly the readout of electrophoresis is incompatible
with modern tools for protein evolution, like the droplet-based directed evolution of protein withmicrofluidics, which use fluorescence in their screening step18. These drawbacks make gelelectrophoresis poorly adapted to high-throughput and automated screening of variants of Cas9.
Fluorescence assays have many benefits that gel electrophoresis lacks. Fluorescence is real-time,
multiplexed, sensitive and quantitative. An unskilled operator can easily collect hundreds offluorescent traces in a day with automated instruments (plate reader, spectrophotometer,
thermocyclers...), standardised plasticware and pipettes (e.g. 96-well or 384-well plates), as well as
commercial services for synthesis of fluorescent DNA probes (hundreds of commercial dyes are now
available). This throughput is crucial for quantitative fields like enzymology (which tests dozens or
hundreds of combinations of experimental conditions like concentration, salinity, temperature...), or
molecular diagnostics (which requires testing hundreds of patient samples per day). Fluorescence has
revolutionised many areas of life science - replacing venerable but hazardous methods likeradioactive quantification. Real-time measurement has also enabled PCR in its modern andquantitative form (qPCR). At the forefront of development, new methods like DNA paint takeadvantage of fluorescently labelled nucleic acids to push forward super-resolution microscopy 19.
In the past years, a few groups have introduced fluorescent assays for Cas9. Mekler and colleagues
reported a simple yet robust displacement-based assay to follow in real-time the binding of Cas9 to its
target and the formation of the R-loop 20–22. They used this assay to evaluate the competitive bindingof nonspecific RNA to Cas9 - which was hypothesised to be one reason why Cas9 usually shows
decreased activity when operated in vivo. This fluorescent assay was used to prototype allostericRNA guides 23,24. Two other reports used high-throughput fluorescent assays to sift through massive
chemical libraries with hundreds of thousands of compounds and identify small molecule inhibitors of
Cas925,26. Lastly, indirect fluorescent assays have also been reported - though they do not directly
yield the kinetics of Cas9 27,28.
Fluorescence assays could speed up the engineering or evolution of Cas9 proteins. The throughput offluorescence could massively accelerate the assaying of variants, while the quantitativity offluorescence could resolve incremental changes in activity, which would otherwise be lost withsemi-quantitative methods like gel electrophoresis. Lastly, fluorescence is more sensitive thanconventional gel electrophoresis, which could reduce the amount of enzyme needed down to scales
that are easily produced with cell-free protein expression27. Yet reports on the utility of fluorescence
for assaying variants of Cas9 have been elusive. Here we use a fluorescence assay to systematicallycompare the activity of the SpCas9-NG variant against the wild type enzyme. We compare theresponse of the two enzymes on four PAMs, and with RNA guides of varying lengths and secondary
structures. Lastly, we multiplex the assaying of Cas9 - comparing in one pot the activity of the Cas9

bioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

enzymes on four PAMs. Overall, our results show that fluorescence assays are a powerful tool toassay the activity of Cas9 mutants, and their dependence on guide sequence.

MATERIALS AND METHODS
Reagents
SYBR Green II RNA gel stain was purchased from Thermo Fisher Scientific (Eugene, OR, USA).
Propan-2-ol and dithiothreitol were from Sigma-Aldrich (Saint-Louis, MO, USA). Tris-EDTA buffer (TE)and glycerol were from Nacalai Tesque (Kyoto, Japan). Ribonucleotide triphosphates (rNTP, N0450),
T7 RNA polymerase (M0251) and murine RNase inhibitor (M0314) were obtained from New England
Biolabs (NEB, Ipswich, MA, USA). gBlock Gene Fragments were purchased from Integrated DNA
Technologies (IDT, Coralville, IA, USA). Other oligonucleotides were HPLC-purified and obtained from
Biomers (Ulm, Germany). All sequences are listed in Supplementary Table 1. To enable specificmonitoring of beacon cleavage by Cas9, the bottom strands of the beacons were conjugated at their
3’ end to the fluorophores Atto 532, Atto 590, Atto 647N or DY-681, whose maximum
absorption/emission wavelengths are respectively 532/554, 594/624, 646/664, and 691/708 nm. The
top strands were conjugated at their 5’ end to various quenchers: BMN-Q535 for Atto 532, BMN-Q620
for Atto 590, BBQ-Q650 for Atto 647N and BMN-Q620, BMN-Q650 or BBQ-650 for DY-681. All
nucleotides were dissolved in TE.
Cas9 synthesis and purification
The Cas9 proteins were expressed as previously reported 11.
Guide RNA transcription
RNA guides were transcribed from gBlock Gene Fragments. Transcription was carried out in a 25
μL volume containing 40 mM Tris-HCl, pH 7.9, 6 mM MgCl2, 1 mM DTT, 2 mM spermidine, 5000
units.mL-1 T7 RNA polymerase, 500 μM of each rNTP, 1000 units.mL-1 murine RNase inhibitor, SYBRGreen II 10 nM (1x), and 4 nM gBlock. The reaction was run for 7 h at 34 ºC on a CFX96 real-time
PCR detection system (Biorad) and monitored thanks to the SYBR Green II signal.
Guide RNA purification
Transcribed RNA was purified by adding 280 μL of nuclease-free water to the 20 μL transcription
reaction, followed by 30 μL of 3 M sodium acetate, mixing, and adding 330 μL of propan-2-ol, thus
precipitating the RNA. The RNA was then pelleted by centrifugation at 10.000 g for 10 min at 4ºC. The
RNA pellet was washed once in 75% ethanol, centrifuged at 10.000 g for 5 min at 4ºC, air dried andresuspended in nuclease-free water before quantification with a Biodrop spectrophotometer (Berthold
Technologies).
Reaction assembly
Reactions were assembled in a buffer containing 20 mM HEPES, pH 7.5, 100 mM KCl, 2 mM
MgCl2, 1 mM DTT, 5% glycerol, and 1000 units.mL-1 murine RNase inhibitor. All reagents except the
double-stranded beacon were first assembled in a total volume of 9 μL and run at 37 °C in a CFX96
real-time PCR detection system (Biorad). Both WT SpCas9 and SpCas9-NG are highly active at thistemperature; the activity is relatively stable between 30 and 45°C but drops suddenly above 46°C for
SpCas9-NG and above 48°C for WT SpCas9 (Supplementary Figure 4). After about 5 minutes toallow stabilization of the temperature, 1 μL of beacon solution was injected in each tube, giving a final

bioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

volume of 10 μL, while the machine was paused for a minimal period. Unless specified otherwise, thefinal concentration of beacon was 40 nM, that of Cas9 (WT or NG) was 200 nM, and that of guideRNA was 400 nM. After cleavage of the beacon, the cleaved strands separate thus releasing thefluorophore on the bottom strand from the vicinity of the quencher. Cleavage of the beacon was thus
monitored specifically by measuring the fluorescence signal of the beacon’s bottom strand fluorophore(Fig.1 top). Fluorescence signals were acquired every 15 s by the CFX96 real-time PCR detection
system in the channels corresponding to the fluorophore of the beacon’s bottom strand .

RESULTS
We use the fluorescent reporter of Mekler et al. to assay the activity of Cas9 and its variants 20 .
Briefly, the reporter is made of a DNA duplex (called beacon here) with a PAM sequence and two
moieties at one of its ends (a fluorophore and a quencher). A Cas9 loaded with a guide recognizes
the PAM sequence of the reporter and initiates an R-loop - attempting to match the sequence of thespacer with that of the reporter. This invasion by the spacer separates the two ends of the duplex,
increasing fluorescence. Of note, this reporter does not directly report on the cleavage of the dsDNA
beacon by Cas9, but on its invasion by the RNA spacer (as demonstrated by the fact that it also
reports activity with dCas9).
We first verified that the fluorescent assay corroborates the cleavage kinetics of WT SpCas9 and
SpCas9-NG as measured by gel electrophoresis (Fig. 1 of Nishimasu et al.11). Both gels andfluorescence measurements rank the kinetics of WT SpCas9 on PAM as TGG>>TGA>TGC>TGT.
Gels and fluorescence measurements also agree that SpCas9-NG is active on all presented PAMs,
and that SpCas9-NG is slightly slower than WT SpCas9 on the canonical PAM TGG. This agreement
between gel and fluorescence kinetics suggests that finding a PAM and forming an R-loop are the rate
limiting steps, rather than the cleavage of DNA (which is not directly measured by the fluorescentreporter 20.
We used the fluorescent assay and the thermal gradient of a thermocycler to quickly evaluate thetemperature stability of the Cas9 enzymes (Supplementary Fig. 4). Both enzymes are stable up to~45°C, at which point their activities drop precipitously with increasing temperatures (although WT
SpCas9 was marginally more stable than SpCas9-NG over 45°C). This thermal stability is in line witha previous study with differential scanning calorimetry which found that WT SpCas9 denatured around
45°C 29.
The direct delivery to cells of RNA guides complexed with Cas9 is one of the main modalities forediting genes. In order to improve delivery, there is interest in minimizing the size of this cargo, eitherwith more compact CRISPR nucleases30–32, or by removing superfluous domains in the guide33. It isessential to know how much of the guide can be truncated while retaining its function. Truncation in
the variable domain of the guide (the spacer) has been extensively studied - mainly for the purpose of
reducing off-target effect33. Yet, truncation in the constant domain of the guide (the scaffold, i.e theregion located downstream of the spacer) has been comparatively less studied.
We used the fluorescent assay to test guides of various lengths, progressively truncating the guidefrom its 3’ end (Fig. 2). SpCas9-NG and WT SpCas9 show a similar, yet slightly distinct, dependence
on the length of the guide. The activity of both Cas9 enzymes is suppressed for guides shorter than
62 bases, i.e. both enzymes require the presence of the stem and the nexus - a small but crucial
secondary structure that contacts the α-helical and nuclease lobes in Cas9 15. In WT SpCas9, those
two structural domains are the minimal requirement for activity34, and guides longer than 66 bases
show strong activity with WT SpCas9 (Fig. 2). However SpCas9-NG needs an extra domain, the firstbioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

3’ hairpin (located in the 70-80 nt range) to show an appreciable activity (Fig. 2). Once these 3
domains (stem, nexus and first 3’ hairpin) are present in the guide, SpCas9-NG is strongly active on
all 4 PAMs - while WT SpCas9 is only fully active on the TGG PAM, and to a lesser extent on the TGA
PAM. Similar results were obtained using another beacon with a different spacer sequence(Supplementary Figure 1).
We then assayed how the Cas9 enzymes responded to mutations in the guide. There is an increasing
interest in engineering the secondary structure of the guide to control the activity of Cas9. For
instance, the decoration of the upper stem with an aptamer helps to recruit transcription factors converting Cas9 into a synthetic transcription factor35. DNA nanotechnologists have also explored theallosteric control of the guide with strand displacement - which converts the guide between active andinactive secondary structures24,36,37. Thus it is important to know which mutations in the guide aretolerated by Cas9 enzymes in order to expand the design space.
To that end, we selected a range of representative mutations that have been tested in vitro and in vivo
34
. We mutated the lower stem, the bulge, the nexus (Fig.3) or the 3’ hairpins (Fig. 4) - and measured
the activity of SpCas9-NG and WT SpCas9, ten minutes after beacon injection. Overall, the assay
confirms that SpCas9-NG and WT SpCas9 tolerate and reject the same kind of mutations in theguide. Mutations that conserve the secondary structure of the guide are tolerated to a certain degree(e.g. guide v3 coding for a mutation in the stem, and guide v15 coding for a mutation in the nexus).
Mutations or deletions that disrupt crucial secondary structures like the bulge (guide v12) or the nexus(guide v18) completely abolished the activity of both enzymes. Similar results were obtained on a
beacon with a different sequence (Supplementary Figure 3) or with shorter guides with mutations inthe stem or nexus (though Cas9 seemed more sensitive to stem mutations with shorter guides,
Supplementary Figure 2). On the other hand, deletions in the terminal 3’ hairpins, or even deletion of
the first hairpin, had little effect on the activity of WT SpCas9 or SpCas9-NG (Fig. 4). Interestingly,
mutations are symmetric with respect to the PAM: all PAMs are similarly affected by mutations, which
confirms that the contacts of Cas9 with the guide are essentially uncoupled from the contacts of Cas9
with the PAM11.
We then showcased the benefits of fluorescence for multiplexing measurements and speeding up
workflows in protein engineering. We simultaneously measured the activity of Cas9 enzymes on four
competing beacons (Fig .5). Each beacon bears a distinct PAM and reports its invasion by Cas9 in its
own fluorescence channel. Multiplex measurements confirm the trends observed with singlex
measurements (i.e. on single PAM). SpCas9-NG is equally active on all PAMs, as shown by the even
distribution of activity in the four different fluorescence channels (right bars of Fig. 5). And as
expected, WT SpCas9 is mostly active on the TGG PAM, and to a lesser extent on the TGA PAM (leftbars). The pattern also suggests some non-linear competition between the PAMs for WT SpCas9.
Overall, these results confirm that multiplex fluorescence measurements can be performed on Cas9,
although no more than 4-6 targets can in principle be followed by fluorescence due to spectral
overlap.
DISCUSSION
We have explored fluorescence as a modality for assaying an engineered variant of Cas9.
Fluorescence reporting not only corroborates conventional gel electrophoresis, but it also gives acrispier picture of enzymatic kinetics. The sensitivity and quantitativity of fluorescence help to capture
fine variations in activity which would otherwise be lost with gel electrophoresis. The resolution ofincremental changes of activity should enable finer cycles in protein engineering, and help to home inon mutations that drive those changes in activity. Fluorescence measurements also requiresignificantly less labour or starting materials than gel electrophoresis - providing a turnkey method toquickly screen Cas9 variants or more generally other types of CRISPR enzymes (Cas12, Cas13....).

bioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fluorescent assays could also be essential for the directed evolution of Cas9 in droplet microfluidics,
whose massive parallelism and low consumption of reagents make it a powerful tool for protein
engineers 38–41.

DATA AVAILABILITY
The data that support the findings of this study are available on request from the corresponding
author..

ACKNOWLEDGEMENT
We warmly thank Pr Nishimasu and Pr Nureki (University of Tokyo) for kindly providing us with sampleof wild type Cas9 and Cas9-NG. This work was supported by the JSPS Core-to-Core Program A(grant number:JPJSCCA20190006）

FUNDING
This project is funded by the Japanese Society for Promotion of Science [JSPS KAKENHI
JP17F17796], the French National Research Agency [ANR-17-CE18-0013], and by the Japanese
Ministry of Education, Culture, Sports, Science and Technology through the Global Leader Programfor Social Design and Management.

CONFLICT OF INTEREST
AJG, TF and AB have filed a patent on Smart Guide RNA WO 2021/111641 A1.

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bioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

TABLE AND FIGURE LEGENDS

Figure 1. Fluorescence assaying of Cas9. (Top) Workflow of the cleavage assay. The reporter is a
dsDNA strand bearing a pair of fluorophore and quencher, and which is targeted by the spacer of an
RNA guide. After pre-incubation of the guide and Cas9, this complex is mixed with the reporter. After
recognizing the PAM, Cas9 unwinds the adjacent domain and matches it with the spacer. This R-loop
separates the fluorophore from its quencher, and thus increases the fluorescence. (Bottom) Assayingof SpCas9-NG or WT SpCas9 (right) against four distinct PAMs.

bioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2. Effect of the guide’s length on the Cas9 activity. (top) anatomy of the guide RNA
showing the spacer, upper and lower stem, bulge, nexus and hairpins with the RNA sequence.
(bottom) WT SpCas9 (thick line, filled diamond) or SpCas9-NG (thin line, open diamond) was first
incubated with guide RNAs of different lengths. The top schematic diagram shows the full-length
guide RNA sequence; the shorter guide RNAs all contain the spacer (in mauve) and finish at one ofthe positions indicated in the diagram. After stabilization of the fluorescence signal, the beacon(beacon 3 containing one of four PAMs) was then injected and the signal amplitude was measured 10

bioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

min after injection. The signal amplitudes were normalized, for each Cas9, to the value obtained withthe full-length 103 bp-long guide RNA and the canonical beacon (with the PAM TGG).

Figure 3. Effect of mutations in the guide RNA’s stem or nexus on the Cas9 activity. WT SpCas9
or SpCas9-NG was first incubated with full-length non-mutated guide RNA (top) or guide RNAs
containing various mutations shown in blue in the schematic diagrams. The beacon (beacon 3
containing one of four PAMs) was then injected and the signal amplitude was measured 10 min afterbioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

injection. The signal amplitudes were normalized, for each Cas9, to the value obtained with thefull-length non-mutated guide RNA and the canonical beacon (with the PAM TGG). The radar chartsshow the normalized activity (signal amplitude) of WT SpCas9 (purple) or SpCas9-NG (orange) in thepresence of each guide RNA depending on the PAM of the injected beacon. The nomenclature
follows Briner et al.34

bioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

bioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 4. Effect of mutations in the guide RNA’s 3’ hairpins on the Cas9 activity. WT SpCas9 orSpCas9-NG was first incubated with full-length non-mutated guide RNA (top) or guide RNAs
containing various deletions in the 3’ hairpins (hp1 or hp2) as shown in the schematic diagrams (thedeletion sites are indicated with blue arrows). After stabilization of the fluorescence signal, the beacon(beacon 3 containing one of four PAMs) was injected and the signal amplitude was measured 10 min
after injection. The signal amplitudes were normalized, for each Cas9, to the value obtained with thefull-length non-mutated guide RNA and the canonical beacon (with the PAM TGG). The radar chartsshow the normalized activity (signal amplitude) of WT SpCas9 (purple) or SpCas9-NG (orange) in thepresence of each guide RNA depending on the PAM of the injected beacon.

bioRxiv preprint doi: https://doi.org/10.1101/2022.08.04.502727; this version posted August 4, 2022. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 5. Multiplexed assessment of Cas9 activity on different PAMs. WT SpCas9 or SpCas9-NG
was first incubated with full-length guide RNA. Then, four beacons - each with a different PAM andconjugated to a different fluorophore - were injected simultaneously. Cas9 activity was assessed bymeasuring the signal shift from each fluorophore 10 min after injection. The signal amplitudes werenormalized to the value obtained after injecting on its own 40 nM of beacon 2 with the respective
fluorophore (singleplex measurement). The stacked bar graph shows the activity of WT SpCas9 orSpCas9-NG on the different beacons present in solution at different concentrations.