bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

License to cut: Smart RNA guides forconditional control of CRISPR-Cas9
Alexandre Baccouche1, Arman Adel 2, Nozomu Yachie2, Teruo Fujii1, Anthony J.
Genot1
1 Laboratory for Integrated Micro and Mechatronic Systems, CNRS-IIS UMI 2820, The University ofTokyo, Tokyo, 153-8505, Japan
2 School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The
University of British Columbia, Vancouver, British Columbia, BC V6T 1Z3 Canada

Abstract:
The Cas9 enzyme is a programmable endonuclease, whose target sequence isdirected by a companion RNA guide. Cas9 and RNA guides have revolutionizedbiology, enabling facile editing of the genome in almost all organisms. Controllingwhere and when Cas9 and the guide operate is indispensable for many fields
ranging from developmental biology to therapeutics, but it remains a challenge. Most
methods focus on controlling Cas9 with physico-chemical means (which lack finesse,
precision or multiplexing), or transcriptional tools (which are slow and difficult todesign). Rather than directly engineering Cas9, engineering the RNA guide itself has
emerged as a more general and potent way to manage the activity of Cas9. Here we
report smart RNA guides that are conditionally activated by the presence of a
specific RNA opener. Contrary to most previous approaches, the design affords
ample freedom as spacer and the opener are independent. We demonstrate thisflexibility by operating SmartGuides activated by a panel of miRNA relevant forhuman health, and by composing SmartGuides in Boolean logic circuits. Lastly, we
test the SmartGuides in mammalian cells - validating the basics tenets of the design,
but also highlighting the challenges that remain to be lifted for in-vivo operation.

Introduction
Cas9 is a programmable endonuclease that is directed by a RNA guide to cut
a target DNA sequence1–3. The ease of programming Cas9 has enabled a range of
new applications, ranging from base and gene editing to genetic and epigenetic
regulation4,5. As Cas9 has expanded to all walks of life sciences, so has the need forfiner control of its activity6. Once Cas9 and a RNA guide are present in a cell, thebioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

tandem will inevitably cleave its DNA target, irrespective of the type, environment,internal state or developmental stage of the cell. But mistimed, misplaced or
mismatched cleavage is detrimental, and researchers are looking for methods todecide when, where and how Cas9 is activated.
Temporal control of Cas9 is desirable for instance in developmental biology
because premature knockout of a vital gene causes embryonic lethality7–9.
Controlling the timing of Cas9 is also important for improving the efficacy and safetyof gene editing. Prolonged activity of Cas9 after germline editing has been linked tomosaicism in embryos10,11. Synchronizing Cas9 with specific cellular cycles promotes
homology directed repair over non-homologous end joining - improving the precisionof gene editing12,13.
Spatial control of Cas9 is also needed in multicellular organisms to confine its
action to specific tissues or organs. The conventional method for conditional
knockout in mice (gene floxing controlled by a cell-specific transcription factor14) islaborious and delicate, and only a handful of institutions can establish and maintain arepository of floxed mice. Gene knockout would be considerably simplified if theaction of Cas9 could be conditioned to the presence of a transcript that is specific toa certain cellular type. More generally, Cas9 bears strong promises as a therapeutic
tool, but concerns linger about off-target effects - limiting the tolerable dose of Cas9
that can be administered15. A tool that mutes Cas9 in healthy cells and activates it indiseased cells could help to administer stronger doses of Cas9 - improving its
efficacy
Conditioning Cas9 to specific cellular states may enable new applications
ranging from the scale of a single-cell to a whole population 16–19. At the single-celllevel, cellular recorders log molecular events into DNA memories with Cas9. But
their generality remains limited without a tool that couples a specific cellular event(e.g. the transcription of a given mRNA) to the editing of the memory register by
Cas9. The majority of events that have been recorded so far with Cas9 are either
random mutations by Cas9 (which is the basis for reconstructing cell lineages20), or
externally triggered signals (small molecules, light...21). Conditioning Cas9 to thepresence of specific endogenous RNA (messenger RNAs, long noncoding RNA,
microRNA...) would widen the repertoire of recordable events, and enable thetime-resolved recording of transcription profiles.
At a much larger scale, gene drives based on Cas9 could remodel
exponentially fast the genetic makeup of a whole population22. In the future, gene
drives could, for instance, eradicate mosquito-borne pathologies, but for the moment
they raise serious environmental issues, because a copy of Cas9 is left lurking in the
genome of the mutated population, opening the unpalatable prospect ofbioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

genomico-ecological catastrophes. These concerns would be alleviated if thisgenetic circuit could carry a kill switch, an instruction that would force Cas9 to erase
itself from the genome after a given number of generations has passed23,24. This
self-erasure of Cas9 could be achieved by combining a heritable DNA ledger (thataccumulates mutations at a constant rate), and a Cas9 that is conditioned to knockout its own gene once the ledger has been fully mutated.
Engineers have followed four complementary routes to control Cas9: chemicalengineering, protein engineering, genetic engineering and RNA engineering.
Chemical engineering designs or discovers chemical compounds that inhibit Cas9,such as Anti-CRISPR proteins25,26. This goes hand in hand with protein engineering,which designs variants of Cas9 that are susceptible to chemical signals or physicalsignals (e.g. light or temperature)27,28. But physico-chemical control of Cas9 lacks
single-cell precision and multiplexing. The signal can be toxic to cells, and its
delivery is a limiting factor: optical methods are hindered by the low penetrability ofvisible light into deep tissues, whereas chemical methods are limited by thechallenge of timely and localized delivery. Most importantly, those methods areunconditional. The signal is exogenous and activates Cas9 irrespective of the stateof the host cell. Many applications such as cellular recording call for an endogenous
control of Cas9, where the signal inducing Cas9 is produced by the cell itself rather
than by an external operator.
Genetic engineering - one of the main tools of synthetic biology - offers thisendogenous control and has been explored for the CRISPR-Cas9 system. It places
the transcription or translation of the Cas9 gene, an RNA guide, or even ananti-CRISPR gene, under the control of a factor that is only expressed under certain
conditions29, or inside specific cells 30–32. But transcriptional control of Cas9 suffersfrom some of the classical drawbacks of genetic circuits: delayed response, poor
predictability, paucity of actionable transcription factors and overloading of the host’s
machinery33.
Rather than engineering the transcription, translation or activity of Cas9, a
promising alternative is to engineer the RNA guide itself. Firstly, RNA strands are far
simpler to sequence, express or modify than proteins. Secondly, RNA sensors canbe installed in RNA guides to interface them with the transcriptome - providing
access to a rich trove of cellular information. A natural approach to control the RNA
guide is to sequester its spacer domain with a complementary RNA sequence, whichis removed in the presence of a specific inducer - activating the guide34–36. Anotherapproach is to stabilize or destabilize portions of the RNA guide with ligands37, or toprocess the guide with an enzymatic machinery. Wang et al. designed a precursor of
an RNA guide which is constitutionally inactive. But in the presence of a specificmicroRNA, it is processed by the RISC machinery into a competent RNA guide38.

bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

This opens numerous possibilities as miRNA are overexpressed in many tissues andpathologies, which make them ideal biomarkers to infer the state or type of a cell.
However this method is not universal: it is limited to organisms like plants or animals
with a miRNA machinery (which prokaryotes lack), and is only applicable to miRNA(excluding the vast majority of the transcriptome). Lastly, it is unclear if the method
can be scaled up to meet the multiplexing needs of methods like transcriptional
recording. In the current design, the precursor guide (~100 bp) needs a large
promoter (~2000 bp). Assembling vectors for dozens or hundreds of miRNA would
be a herculean effort, and would undoubtedly burden the host’s RNAi machinery.
In parallel, several groups have leveraged the tenets of DNA nanotechnology
to allosterically regulate RNA guides with a general and non-enzymatic mechanism:
toehold-mediated strand displacement39,40. The mechanism is based solely on
Watson-Crick base pairing and does not require external factors (such as a Dicer
protein), so it could be deployed in any organism. Most designs start by sequestering
the spacer of the RNA guide with a complementary RNA domain, which abolishes
the activity of Cas9. The presence of a trigger RNA initiates a toehold-mediatedstrand displacement, and releases the spacer. Some of the initial designs imposed astrong homology between the spacer and the trigger RNA. This excludes the mostsought-after applications where the trigger RNA and the DNA target are endogenous
and unrelated. For instance, for targeted gene editing,the trigger could be a miRNA
marker of a given cellular type, while the DNA target is the loci of the gene to beedited. Subsequent designs have improved orthogonality between the trigger RNA
and the spacer41–43. Li and colleagues proposed an elegant design in which a tailappended to the guide folds back and binds to the loop of the upper stem, preventing
the guide from interacting with Cas944. Addition of a target RNA removes this folding
and restores functionality. Interestingly, this design removes homology constraints
and in principle could link unrelated input RNA and output DNA. However the lengthof the input must be in the 50 nt range, which prevents operation with short RNA likemiRNAs.
Here we report smart RNA guides for Cas9 that are allosterically modulated
by a small and arbitrary RNA input. We modulate the affinity of the guide for Cas9 bycontrolling the folding of a crucial secondary structure: the nexus. Using an in vitrotranscriptional circuit, we demonstrate orthogonal and multiplexed regulation of theSmartGuides with a panel of miRNAs relevant for human health. We thendemonstrate higher-order computation with the SmartGuide, such as non-monotonic
Boolean logic. Lastly, we coupled the SmartGuide to a base-editor and explored theiractivity in mammalian cells - laying out challenges that will need to be overcome fora future deployment in complex organisms.

bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Results
We benchmarked the SmartGuides in vitro with a transcriptional circuit basedon a fluorescent reporter for Cas945. Compared to the conventional workflow forassessing the activity of RNA guides in vitro (overnight transcription, gel purification,cleavage assay and finally gel electrophoresis1), this one-pot assay not only reduces
the hands-on time from a few hours to a dozen minutes, but also provides
quantitative, multiplexed and time-resolved kinetic measurements - allowing us totest ~300 designs of guides over the course of two years. A one pot assay is also amore realistic model of the cell than a conventional cleavage assay, because allprocesses occur concurrently (transcription and cotranscriptional folding of the guide,
opening of the guide by the miRNA, loading of the opened guide by Cas9, and finallycleaving).
The key element of our control strategy is the nexus of the RNA guide, a short
hairpin located near the first stem of the scaffold46. In the Cas9:guide complex, thenexus makes extensive contacts with Cas9, and sits at the interface of α-helical lobe
(used for recognition) and the nuclease lobe of Cas9 (used for cleavage)47,48. This
makes the nexus an ideal target for allosteric regulation, because small changes tothe nexus have an outsized influence on the activity of Cas9. For instance, mutating
the two nucleotides that hold the nexus suppressed the hairpin structure andcompletely abolished the activity of the guide46.
Informed by these structural and functional insights about the criticality of thenexus, we installed two modifications on the canonical RNA guides to control thefolding of the nexus. Firstly, we appended a tail fully complementary to the cognate
miRNA. Secondly, we mutated the sequence of the nexus and the bridge - while
preserving the secondary structure of the nexus - to make this region partiallycomplementary to the tail and form a hairpin. It prevents the folding of the nexus andthe loading by Cas9, resulting in a guide that natively folds in an inactive state. The
presence of a cognate miRNA rescues the activity of the SmartGuide. The miRNAbinds to the sequestering hairpin by a long toehold, unzips it by toehold-mediatedstrand displacement (ref), and releases the nexus. This guide presents a canonical though mutated - secondary structure which is loaded by Cas9 for enzymatic
processing.
We tested two designs of the guide: one with a toehold located outside theblocking hairpin (external toehold), and one with a toehold located inside (internal
toehold). Both SmartGuides display. In the ON state (opener present), thefluorescence of the reporter rises in about 60-120 min to ~50-100% of thefluorescence of the control guide. Various factors could explain this discrepancy
between control guides and SmartGuides : secondary structures in the miRNA or thebioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

SmartGuide, lower transcription efficiency of the SmartGuide, or altered contacts ofthe mutated nexus of the SmartGuide with Cas9. In the OFF state of the guide (noopener present), the reporter displays a leak activity corresponding to ~ 10-20% of
the ON state. Interestingly, the time trace informs about the possible origin of theleak. If the leak was caused by a subpopulation of guides that were incorrectlytranscribed or folded (thus failing to turn OFF), we would observe a slow upward driftof fluorescence which would eventually reach the same level as the control (because
the guides are continuously transcribed over the course of the experiment, and theirconcentrations will eventually vastly exceed that of the reporter). Rather we observea quick jump in fluorescence followed by a plateau. This suggests that the leak may
instead be caused by a faulty subpopulation of reporters, which is incorrectly
synthesized or assembled.

We then benchmarked the SmartGuides against a panel of 6 representative
human miRNAs. They are are enriched in endothelial cells (mir-2149), hypertrophic
scarring tissues (mir-9850), cancer cells (mir-45151), psoriasis skin cells (mir-3152),
neutrophils following traumatic injury (mir-394553) or in the liver (mir-12254). When the
SmartGuides and the miRNA matched (traces on the diagonal), the fluorescence ofthe reporter showed strong and fast activation. When they were mismatched,
activation remained negligible (at ~5-10% of the ON level). Half of the ON guides in
the panel displayed a kinetic and end-point fluorescence similar to the control(namely mir-98, mir-451 and mir-21). For the other half, the kinetic was slower thanthe control, and finished at a lower level (mir-3945, mir-31 and mir-122). However, allguides showed robust levels of activation on a timescale compatible with in-vivo
application (minutes to hours).
In view of making Cas9 safer and more precise, we composed SmartGuides
to demonstrate Boolean computations. In real-world settings, simply testing thepresence or absence of a single miRNA may not be sufficient, and one may
condition Cas9 on the presence of a panel of miRNAs that form a signature of acellular state. This would improve the specificity of Cas9, by ensuring that it is only
activated inside cells or in response to situations that need it.
We first installed a NOT gate in the guide by mutating the hairpin loop after
the upper stem. The mutated loop binds to the target RNA, which disrupts thesecondary structure of the guide and abolishes its function (Fig. 3b). We then
combined conditional activation and inhibition, and computed a NOT AND gate. The
gate only fires in the presence of target input and in the absence of another one (Fig.
3b). Lastly, we computed a XOR gate, one of the most complex Boolean functions
on two inputs. The circuit comprises two symmetrical guides, each computing a NOT
AND gate on the same pair of inputs. As expected, the circuit only fires when one -bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

and only one - input is present. The activation level, while robust, is noticeably lower
than the control guide. Lastly, we multiplexed two SmartGuides with distinct spacers,
demonstrating their concurrent and individualized operation in the same mixture (Fig.
3b). This multiplexing is an added benefit compared to physical or chemical methods
to control Cas9 (e.g. with small molecules, light or temperature), as it is difficult to
expand the repertoire of their actionable signals.
Encouraged by the robustness and versatility of the SmartGuides in vitro, we
tested them in human cells. We placed a Cas9 base editor4 under the control of a
SmartGuide - using a miRNA or a synthetic RNA as input (Figure 4). When theguide is ON, it directs the base editor to edit a base in a mutated GFP gene,rescuing its activity and causing the cell to fluoresce green. All components of thisgenetic circuit (the input RNA, the guide, the GFP gene and the Cas9-Target AID
fusion gene) are genetically encoded in plasmids and transfected in the cells.
Surprisingly, we found that when operated in vivo, Cas9 did not tolerate mutations in
the guide that it tolerated in vitro. A control guide, lacking the blocking tail but
harboring in the nexus and the linker, failed to light cells up (Fig. 4c left). We
surmised that some mutations in the nexus disrupted contacts with Cas9, thus
lowering the affinity of the guide with Cas9. While this lower affinity may not havebeen problematic in vitro, it has been hypothesized that in vivo, the RNA guide mustcompete with non-specific RNA for binding with Cas9, and thus require a strong
affinity to overcome this competition45. To test this hypothesis, we rescued mutationsin the nexus, but not the linker of the SmartGuide. In turn, this required us to modify
the design of the SmartGuide, because the unmutated nexus breaks thecomplementarity of the tail with the input RNA. We moved the toehold internally,
putting it inside the loop and at the base of the hairpin neck. Opening of hairpins withinternal toeholds has been studied in vitro55, and though it progresses at a slower
pace than for external toeholds, it remains sufficiently fast for biological timescales.
We tested the design with an internal toehold in vitro and confirmed that it worked
satisfactorily (Fig. 1c).
We then attempted to operate the SmartGuide by triggering them in presenceof microRNA. Satisfyingly, we observe that the control ON guide has a basal activity
similar to that of the wild type guide (Fig. 4c right) - validating our hypothesis about
the in vivo tolerance to mutations in the linker but not in the nexus. Additionally, theSmartGuide in its OFF state displays little fluorescence (Fig. 4e left), validating thetight control exerted by the tail on the nexus. However, expression of the opener
RNA led to a modest increase of fluorescence - indicating that the SmartGuides do
not fully switch ON in presence of their target. We tested various lengths of hairpins
with two different openers, but could not observe a significant increase of green
fluorescence in presence of the opener.

bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

This lack of switching in vivo is not likely due to design: guides designed to bein the ON states are ON, and guides designed to be in the OFF state are OFF. We
surmise that the failure to completely switch in vivo could be due to the complexity of
nuclear biology: the guide and the opener may be expressed far away in the nucleus
and never meet, or they could be exported outside of the nucleus and meet in thecytoplasm, but never traffic back to the nucleus. In addition, the secondary structuresof small RNA are known to be highly regulated 56,57. Further studies with live cell
imaging could illuminate the fate of the guide and the opener RNA. Studies inprokaryotes are also warranted to ascertain the workings of the SmartGuide in aliving organism.

DISCUSSION
We have reported smart RNA guides for Cas9 that are conditionally activatedby the presence of an arbitrary RNA. The spacer and the input RNA are unrelated,which expands the range of circuits that can be designed. While we have used
miRNA as a proof-of-principle, our design does not rely on miRNA-processing
machinery, and could in principle apply to any RNA sequence. Operation of theSmartGuide in mammalian cells confirms the ON and OFF states of the SmartGuide,
but the switching between those states remains incomplete and warrants further
studies.
In the future, such SmartGuides may perform programmatic transactions on
the genome based on the state of the in which they operate. This could make genetherapy safer and more potent, but also enable the recording of transcription eventsin a DNA memory.
DATA AVAILABILITY
The data that support the findings of this study are available on request fromthe corresponding author.
CONFLICT OF INTEREST
A patent has been filed by A.B, A.J.G and T.F. based on the results presented
here.

FUNDING
This project was funded by the Japanese Society for Promotion of Science[JSPS KAKENHI JP17F17796 and JSPS Core-to-Core Program A(JPJSCCA20190006) and the French National Research Agency[ANR-17-CE18-0013].

bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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638–642 (1985).

FiguresFigure 1: Smart RNA guides for conditional control of Cas9. a. The SmartGuides(SG) are benchmarked with a transcriptional circuit. The guide is transcribed from a
dsDNA template and bears a tail in 3’ that folds back onto the nexus and linker
domain - preventing loading of the guide by Cas9. But the presence of a target
miRNA opener rescues the nexus by invading the terminal hairpin through asingle-stranded toehold domain. This invasion releases the nexus and exposes a
canonical guide that is loaded by Cas9. This guide:Cas9 complex invades andcleaves a fluorescent reporter, generating an increase in fluorescence. The process
is one-pot and all reactions occur concurrently. b. Testing of a SmartGuide with atoehold located outside the terminal hairpin loop (external toehold). The plot shows
the fluorescence level of the beacon. gRNA is the control trace (canonical guide thatis constitutively ON). c Testing of a SmartGuide with a toehold located inside theterminal hairpin loop (internal toehold). gRNA is the control trace (canonical guidethat is constitutively ON).

bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2: Activation of SmartGuides against a panel of miRNA, measured in a
one-pot assay. Each plot shows the fluorescence timetrace of the reporter following
mixing of one guide (top row) against one miRNA input (left column). Traces on the
diagonal correspond to ON-target activation, while traces off the diagonal correspondto OFF-target activation. The traces are normalized with respect to their control
guide (i.e. the SmartGuide lacking the blocking tail but harboring a mutation in thenexus).

bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 3: Logical operation and multiplexing of the SmartGuides. A Scheme forcomputing a NOT gate by mutating a RNA guide. The guide is constitutively ON.

bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Addition of the input RNA opens the stem loop (which has been appropriately
mutated to bind the RNA) and abolishes functionality of the guide. B Computation ofvarious logic gates by composing the conditional activation of the SmartGuide with
the NOT gates. C Multiplexing: orthogonal operation of two SmartGuides, each with
its own opener, spacer and fluorescence reporters. (Note that we adjusted thereadout time according to the composition of the logic circuit.). The fluorescencelevel is defined with respect to the maximum level of fluorescence reached by thecontrol.

bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 4 Arman’s In-vivo section

Figure 4: a) CRISPR-Cas9 base-editing reporter system b) Gene architecture of
SmartGuide, small RNA opener and Target-AID cassettes c) gRNA activity for theoriginal miR-31 responsive SmartGuide and redesign without nexus mutations in theabsence of the toehold sequence d) Fluoroscope images of HEK293Ta base-editing
reporter cells after transfection with miR-31 sequence responsive SmartGuide withTarget-AID in the presence or absence of a short opener sequence e) Fluoroscope
images of HEK293Ta base-editing reporter cells after transfection with redesigned
miR-31 sequence responsive SmartGuide with Target-AID in the presence orabsence of a short opener sequence f) Fluoroscope images of HEK293Ta
base-editing reporter cells after transfection with redesigned miR-451 or synthetic
sequence responsive SmartGuide of varying hairpin length (in orange) withTarget-AID in the presence or absence of a short opener sequence.

bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Methods

Plasmid cloning
Plasmid cloning was carried out by conventional cloning methods such as PCRamplification, restriction enzyme digest, ligation and golden gate assembly. PCR
amplification products were all generated using Phusion High-Fidelity Polymerase(NEB) and all restriction digests were carried out using NEB High-Fidelity restriction
enzymes unless stated otherwise.
Lentiviral Base-editing reporter
Lentiviral C→T base-editing reporter plasmid (LV-CS-sg1-GTG-EGFP-Pur) reporter
cassette was constructed by amplifying the eGFP gene fragment from pLV-SI-112(Addgene 131127) using the primer set
AA-sg1-BC-CS-eGFP-FW/AA-eGFP-BamHI-RV followed by restriction enzyme
digest by EcoRI/BamHI of the eGFP fragment and pLV-SI-112, respectively. The
digested fragment was then cloned into the EcoRI and BamHI sites of the pLV-SI-112
using T4 DNA ligase (Nippon Gene).
SmartGuide plasmid backbone and Golden Gate cloning of SmartGuides
The PB4 plasmid backbone used to construct the SmartGuide plasmid backbone
was a generous gift from Dr. Yangming Wang of Peking University. The SmartGuide
backbone plasmid (PB4-sg1-scaf-cloning) was constructed by first generating theinsert fragment by fusion PCR: The hU6 promoter fragment was generated byamplifying the U6 promoter with primer set
AA-MluI-U6-gib-FW/AA-U6-sg1-scaf-short-RV from the plasmid backbone pSI-359(Addgene 131131) and the dsRed filler fragment was generated by amplifying a
portion of the dsRed gene from CMVp-dsRed2-Triplex-HHRibo-gRNA1-HDVRibo-pA(Addgene 55201) with primer set AA-sg1-scaf-short-filler-FW/AA-filler-SpeI-gib-RV in
separate PCR reactions. After gel purification of each fragment, the second PCR
reaction was carried out with equimolar amounts of the hU6p and dsRed fragments
to a total input mass of 10ng and using primer set
AA-MluI-U6-gib-FW/AA-filler-SpeI-gib-RV. The fusion PCR product and PB4
backbone were then respectively digested by KpnI/SpeI and the fusion PCR productwas cloned into the PB4 backbone using T4 DNA ligase (Nippon Gene).
Control scaffold sequences and SmartGuide toehold sequences were cloned into
SmartGuide backbone cassette by oligonucleotide annealing and Golden Gate
Assembly (Supplementary Methods).
Small RNA Opener plasmids

bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

In order to construct the SmartGuide opener plasmid, first an mCherry gene wasamplified from a pcDNA3.1 plasmid (pSI-177) using the primer set
AvrII-1x-AUG-XFP-FW/BstB1-mCherry-RV. The mCherry gene fragment and the
backbone pcDNA3.1 plasmid (pSI-166) were then respectively digested by
AvrII/BsrGI and then the mCherry fragment was cloned into the pcDNA3.1 backbone
using T4 DNA ligase (Nippon Gene). Following this, the opener sequences werecloned in by amplification of the hU6p promoter from pSI-359 and then the opener
fragment and backbone plasmid were respectively digested by MluI/XbaI and theopener fragment was cloned into the backbone plasmid using T4 DNA ligase(Nippon Gene).

Cell culture
HEK293Ta cells were purchased from GeneCopoeia and maintained in Dulbecco’s
Modified Eagle’s Medium (DMEM) (Sigma) supplemented with 10% fetal bovine
serum (FBS) (Thermo Fisher Scientific) and 1% penicillin–streptomycin (Sigma) at
37 °C with 5% CO2. Cells were routinely tested for mycoplasma contamination bynested PCR using culture medium as a template.

Generating base-editing reporter HEK293Ta cell line
The C→T base-editing reporter HEK293Ta cell line was generated by lentiviraltransduction. HEK293Ta cells were first seeded in one well of a 6 well plate at aseeding density of 3x105 cells in 2ml of DMEM 1 day prior to transfection. Plated
cells were then transfected with 489 ng of lentiviral plasmid, 366ng of psPAX2(Addgene 12260), 122ng of pMD2.G (Addgene 12259) and 9.38 µl of 1 mg/ml
polyethyleneimine MAX (PEI) (Polysciences) in 200 µl Opti-MEM (Gibco). 18 hours
after transfection, the cell culture media (DMEM) was exchanged. 4 days after
transfection, lentiviral particles were harvested by removing the supernatant from thewell and aliquoting in 1.5 ml tubes.
After harvesting lentiviral particles, a 6 well plate was seeded with HEK293Ta cells at
a seeding density of 3x105 cells in DMEM supplemented with 8mg/ml Polybrene(Sigma). Each well was then transduced with either 5µl, 10µl , 50µl, 100µl, 200µl or
no virus 6 hours after seeding when cells had annealed to plate. 24 hours after
transduction, the cell culture media was then exchanged for DMEM supplemented
with 3µg/ml Puromycin (Thermo). Selection was carried out for 3 days to select forsuccessfully transduced cells.

bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fluorescence microscopy
All transfections were carried out with 1µl of PEI reagent in a total volume of 50µl of
Opti-MEM. Base-editing reporter HEK293Ta cells were plated 2 days prior totransfection on a 24-well tissue culture plate with a seeding density of 5x104 cells per
well. Two days later, wells were subsequently transfected with 250ng of Target-AID(Addgene 131125) and 250ng of SmartGuide vector with or without 250ng of Opener
vector or cells were transfected with 250ng of Control vectors with 250ng ofTarget-AID.
4 days after transfection, cells were stained with 5µg/ml Hoechst and were imaged
using a Keyence BZ-X800 high-resolution imaging system.
In-vitro transcription of SgRNA.
All experiments were performed at 34 °C in a CFX96 TouchTM (BioRAD) thermal
cycler, and activity monitored upon fluorescence level shifts. Transcription was
initiated upon addition of 4 nM of dsDNA template (gblocks Gene Fragment, IDT) toa solution of 5 U.μL-1 of T7 RNA polymerase (NEB, M0251), 1 U.μL-1 of murine
RNase Inhibitor (NEB M0314), 500 μM of rNTPs (NEB, N0450) and 10 nM (1x) ofSYBR™ Green II RNA Gel Stain (Invitrogen) in the reaction buffer containing 40 mM
Tris-HCl, 6 mM MgCl2, 1 mM 1,4-Dithiothreitol, 2 mM spermidine with a final volumeof 20 μL and incubated for 6 to 8 hours. We work in low yield conditions to reduce
the proportion of transcription byproducts (ref). After transcription, RNA was isolated
by precipitation in isopropanol- ethanol 75% and resuspended in 10 μL nuclease freewater (NEB, B1500) before quantification by BioDrop μLITE (Biodrop). Transcripts
were then stored at - 25 °C. Cognate miRNAs were chemically synthetized from IDT(standard desalting), resuspended in 10 mM Tris-HCl (pH 8), 1 mM EDTA andstored at -25 °C prior to use.

Fluorescence cleavage assay
200 nM of isolated smgRNA were injected in a pre-incubating solution of 200 nM
Cas9 (S. pyogenes, NEB, M0386), 120 nM fluorescent beacon and 1 U.μL-1 murine
RNase Inhibitor (NEB M0314) in the reaction buffer containing 50 mM Tris-HCl (pH7.5), 5.5 mM MgSO4, 75 mM NaCl, 100 μg.μL-1 BSA (NEB, B9000S) to a final
volume of 10 μL per condition.

bioRxiv preprint doi: https://doi.org/10.1101/2022.10.26.513620; this version posted October 27, 2022. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

One-pot assay
Reunion of 4-6 nM of dsDNA template (gblocks ®️ Gene Fragment, IDT) to a solutionof 5 U/μL of T7 RNA polymerase (NEB, M0251), 200 nM Cas9 (S. pyogenes, NEB,
M0368), 120 nM fluorescent beacon, 1 U.μL-1 of murine RNase Inhibitor (NEB
M0314), 500 μM of rNTPs (NEB, N0450) and 10 nM (1x) of SYBR™ Green II RNA
Gel Stain (Invitrogen) in the reaction buffer -containing 50 mM Tris-HCl (pH 7.5), 5.5mM MgSO4, 75 mM NaCl, 100 μg.μL-1 BSA (NEB, B9000S) to a final volume of 10
μL per smgRNA.