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OPEN

CRISPR/Cas9‑mediatedbase‑editing enables a chain
reaction through sequential repairof sgRNA scaffold mutations
Tsuyoshi Fukushima1, Yosuke Tanaka1*, Keito Adachi1, Nanami Masuyama2,3,
Akiho Tsuchiya1, Shuhei Asada1,4, Soh Ishiguro2,3, Hideto Mori2, Motoaki Seki2,
Nozomu Yachie2,3, Susumu Goyama5* & Toshio Kitamura1*
Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered
diverse roles of individual genes, it has been challenging to record or control sequential genetic eventsin living cells. In this study, we designed two cellular chain reaction systems that enable sequential
sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide
deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold
region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting incellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA
scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match
those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized inappropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction
systems that have a broad utility in the future biomedical research.
A chain reaction is a sequence of related events or reactions in which each event/reaction causes the next. Sincemany biological processes consist of sequential chemical or cellular reactions, methods and platforms that allow
molecular recording or intervention of sequential events in living cells have broad applications in cell biology,
bioengineering, and therapeutics. Several systems have been developed for recording chain reactions in mammalian cells. Friedland et al. developed two cellular chain reaction systems using T7/T3 RNA polymerases and
Cre/Flippase (Flp) recombinases. In the first system, T7 RNA polymerase induced the expression of T3 RNApolymerase and T3 RNA polymerase triggered the next r­ eaction1. In the second system, recombination of the
flippase recognition target (FRT) by Flp induced Cre expression, and Cre-induced LoxP recombination triggered
the next reaction. Since the number of RNA polymerases and recombinases is limited, the number of chain
reactions in these systems is also limited. Recently, Farzadfard et al. developed an elegant chain reaction systemcalled ­DOMINO2, using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA), resulting in
efficient substitution of the cytosine nucleotide (C) with a thymidine nucleotide (T) in the genomic ­sequence3.
In the DOMINO system, the C > T modification in the target DNA locus by sgRNA and nCas9-CDA triggered
the next modification. Farzadfard et al. also achieved the conversion of inactive sgRNA to active sgRNA byintroducing C > T substitutions into the protospacer sequence of nonfunctional sgRNA. One limitation of thissystem is the difficulty of generating multiple active sgRNAs during chain reactions because the protospacer
sequence is strictly dependent on the sequence of target DNA.
To establish platforms that enable sequential activation of multiple sgRNAs, we developed two nCas9-CDAbased chain reaction systems by sequential repair of mutations in sgRNA scaffolds or TATAloxP sequences. We
generated PAM-inserted sgRNAs without loss of function and successfully inactivated them by introducing T > C
1Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Minato‑ku,
Tokyo 108‑8639, Japan. 2Research Center for Advanced Science and Technology, The University of Tokyo,
Tokyo 153‑8904, Japan. 3School of Biomedical Engineering, The University of British Columbia, Vancouver, BC V6T
1Z3, Canada. 4The Institute of Laboratory Animals, Tokyo Women’s Medical University, Tokyo, Japan. 5Divisionof Molecular Oncology, Graduate School of Frontier Sciences, The Institute of Medical Science, The University ofTokyo, Minato‑ku, Tokyo 108‑8639, Japan. *email: ytims@ims.u-tokyo.ac.jp; goyama@edu.k.u-tokyo.ac.jp;
kitamura@ims.u-tokyo.ac.jp
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B

sgRNA-1

sgRNA-2 MTSpecific toeach sgRNA

A

sgRNA-3 MT

Same T>C mutation

sgRNA-2 WT

sgRNA-2 MT

sgRNA-3 WT
Next Reaction

XX
N
X
G
X
G
X

Stem-loop region

PAM

T>C Mutant for sgRNA
inactivation

sgRNA-3 MT
Y Y
N
Y
G
Y
G
Y

Stem-loop region

PAM

T>C Mutant for sgRNA
inactivation

C

13bp

8bp

13bp

A T AACT TCGT A T AGT A T AAA T T A T ACGAAGT T A T
13 12 11 10 9 8 7 6 5 4 3 2 1D

sgRNA-1

Transcription

sgRNA-1

Mutation

Mutation
U6promoter

TATAloxP

sgRNA-1

stop

TATAloxP

XXXXXNGG sgRNA-2

Mutant loxP repairing
U6promoter

sgRNA-1

TATAloxP

stop

TATAloxP

stop

PAM

XXXXXNGG sgRNA-2

stop

sgRNA-1

YYYYYNGGsgRNA-3
PAM

Mutationstop

Transcription
TATAloxP

TATAloxP

TATAloxP

YYYYYNGG sgRNA-3

sgRNA-2

Cre recombinase
sgRNA-2

Mutation
U6promoter

TATAloxP

XXXXXNGG sgRNA-2

stop

TATAloxP

YYYYYNGG sgRNA-3
PAM

Mutant loxP repairing
U6promoter
TATAloxP

sgRNA-2

stop

TATAloxP

XXXXXNGG sgRNA-2

stop

TATAloxP

YYYYYNGG sgRNA-3

Cre recombinase
Transcription
U6promoter

TATAloxP

YYYYYNGG sgRNA-3

sgRNA-3
Next Reaction

Figure 1.  Screening of inactive sgRNAs with scaffold mutations in yeast. (A) Schematic representation of
the chain reaction with nCas9-CDA and sgRNAs with scaffold mutations. (B) Schematic representation of
the sgRNA structure with the T > C mutation and specific sequences in the stem-loop region. (C) Schematic
representation of TATAloxP structure and sequences. (D) Schematic representation of chain reaction withnCas9-CDA and TATAloxP with T > C mutations.

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A

B

AA

A
G
A U 17
U A
C G
G CA
A
A
G
G UG
A U
A U
G C
5 U A
C G
4 U A
C G
3 U A
A U
2 U A
C G
1 G U
G C U U UU UAGA GU UAU C A A U
C G
G C
52 U A
G C
U
U A
C G
G A
UA
AA

D

-

A
G
A U 17
U A
C G
G CA
A
A
G GG
A U
5 U A
4 C A
3 U A PAM insertion
2 U A
1 G U

+

Active sgRNACan1
mutation

Yeast

Canavanine
resistance

Can1

Inactive sgRNA

Can1

Canavanine
sensitive

T>C Mutantation

E

nCas9-CDA
Canavanine
Yeast dose

C

AA

Cas9
Canavanine
Yeast dose

-

+

WT

WT

nd

2 T WT+PAM

2 T WT+PAM

2 T>C mut+PAM

2ndT>C mut+PAM

ndnd

3rdT WT+PAM

3rdT WT+PAM

3rdT>C mut+PAM

3rdT>C mut+PAM

4thT WT+PAM

4thT WT+PAM

4thT>C mut+PAM

4thT>C mut+PAM

17thT WT+PAM

17thT WT+PAM

17thT>C mut+PAM

17thT>C mut+PAM

5thT>C mut+PAM

5thT>C mut+PAM

52ndT>C mut+PAM

52ndT>C mut+PAM
NT

NT

Figure 2.  Screening of inactive sgRNAs with scaffold mutations in yeast. (A) Schematic representation of thescaffold region of sgRNAs. (B) Schematic representation of part of the scaffold region of sgRNAs with T > C
mutations and PAM insertion. (C–E) Schematic representation (C) and results of the canavanine assay withnCas9-CDA (D) or Cas9 (E). Yeasts transduced with the indicated sgRNA were grown on plates with (rightcolumn) or without (left column) canavanine.
mutations that can be repaired by nCas9-CDA. As a proof of concept, we established a cellular chain reaction
by repairing sgRNA scaffold mutations in 293 T cells. Our system could form the basis for the construction ofsophisticated chain reaction systems in living cells.

Results

Two strategies to generate cellular chain reactions using nCas9‑CDA.

We designed two systems
to generate chain reactions using nCas9-CDA. In the first system, the T > C mutations are introduced in thescaffold region of sgRNA to disrupt its structure. First sgRNA without mutation (sgRNA-1) and nCas9-CDA
correct the T > C mutations in the second sgRNA (sgRNA-2). The corrected sgRNA-2 in turn corrects the T > C
mutations in the third sgRNA (sgRNA-3) in cooperation with nCas9-CDA (Fig. 1A). Because the sequence andthe length of the stem-loop region in the sgRNA scaffold are not ­restrictive4,5, we can design multiple sgRNAs
by introducing various sequences in the stem-loop region (XXXXX in sgRNA-2-MT, YYYYY in sgRNA-3-MT)(Fig. 1B).
In the second system, the T > C mutations are introduced in the TATAloxP sequence (TATAloxP-MT). TATAloxP forms a 13-8-13 structure, which is composed of an 8-bp spacer sequence containing TATA box flanked on
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A

B

mutation in start codon
PAM
17 bp
CCG
GTG
G GC ࣭࣭࣭࣭࣭࣭ C A C EGFP

mutant EGFP expressing 293T
Lentiviral infection
pLV-mutant EGFP

293T

Transfection
pLV-nCas9-CDA-U6-sgRNA

nCas9-CDA+ sgRNA
PAM
17 bp
CCG
A TG
G GC ࣭࣭࣭࣭࣭࣭ T A C EGFP
Repairing start codon

C

Imaging or FACS analysis

D

EGFP

AA
A
G
A U
U A
C G
G CA
A
A
7 G࣭UG
A U
U A
4 U A
U A
U A
G࣭U
࣭࣭࣭

23.5 %

WT sgRNA

3.21 %+PAM sgRNA

0.42 %

No sgRNA

E

EGFP

EGFP
21.2 %

WT sgRNA

Optimized+PAM
sgRNA

No sgRNA

EGFP

WT

F

+PAM

Optimized+PAM

AA
A
G
A U
U A
C G
G CA
A
A
PAM
7 G GG
A U
U A
4 U A
U A
U A
G࣭U
࣭࣭࣭

EGFP

AA
A
G
G C
PAM
C G
C G
G C
U A
A U
U A
C G
G CA
A
A
7 G࣭UG
A U
A U
4 U A
U A
U A
G࣭U
࣭࣭࣭

WT sgRNA

23.1 %

17.9 %

4thT>C mut

23.3 %

0.17 %

2/4thT>C mut

0.63 %

3/4thT>C mut

4/5thT>C mut

No sgRNA

1.22 %

0.43 %

0.18 %

EGFP

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www.nature.com/scientificreports/◂Figure 3.  Screening of inactive sgRNAs with scaffold mutations in mammalian cells. (A) Schematic

representation of wild-type and mutant (ATG to GTG) EGFP with PAM sequence insertion. (B) Experimental
scheme used in (C,E–F) and Fig. 4A. 293 T cells were transduced with the EGFP mutant. The mutant EGFPexpressing 293 T cells were then transfected with nCas9-CDA and sgRNAs targeting the T > C mutation. (C)Fluorescence images (left) and FACS plots (right) of 293 T cells expressing mutant EGFP and nCas9-CDA
together with wild-type (top), PAM-inserted (middle), or no (bottom) sgRNA. (D) Schematic representationof wild-type (left) and PAM-inserted sgRNAs (original version: middle, optimized version: right). Note that theG-U interaction at the 7th base is lost in the original PAM-inserted sgRNA. The optimized version of the PAMinserted sgRNA contains additional sequences in the stem-loop region to preserve the G-U interaction at theseventh base. (E) Fluorescence images (left) and FACS plots (right) of 293 T cells expressing mutant EGFP andnCas9-CDA together with wild-type (top), PAM-inserted (middle), or no (bottom) sgRNA. (F) Fluorescence
images (left) and FACS plots (right) of 293 T cells expressing mutant EGFP and nCas9-CDA together withdifferent sgRNAs. The indicated T > C mutations were introduced into the control sgRNA. mut; mutation.

either side by two 13-bp inverted repeats (Fig. 1C). Cre recombinase binds to the 13 bp repeats of the TATAloxP,and importantly, this TATA box containing sequence can be incorporated into the U6 promoter to induce sgRNA­expression6. The T > C mutations in the TATAloxP are corrected by the nCas9-CDA and the sgRNA (sgRNA-1)
targeting the mutations and adjacent sequences (XXXXX in Fig. 1D). After the correction, a loxP flanked stop
cassette is excised by Cre recombinase, which drives expression of the second sgRNA (sgRNA-2). The sgRNA-2
targets the next mutation and adjacent sequences (YYYYY in Fig. 1D), resulting in expression of the third sgRNA(sgRNA-3) (Fig. 1D). Because there is no restriction for the adjacent sequences, we can design multiple sgRNAs
with various adjacent sequences that can be activated during the chain reaction.

Generation of inactive sgRNAs with T > C mutations in the scaffold region. To establish thefirst system, we introduced T > C mutations into the template sequence of the sgRNA scaffold region to inactivate it. Because nCas9-CDA was shown to efficiently induce C > T substitution in DNA when the cytosine was
located 18 bp upstream of PAM ­sequence3, we also introduced PAM sequence (NGG) 18 bp downstream of
the T > C mutation in each sgRNA template (Fig. 2A,B). We then evaluated the activity of each sgRNA with the
T > C mutations and PAM insertion using the canavanine assay. Canavanine is a toxic analog of arginine andis imported into yeast cells via a transporter Can1. Therefore, expression of Cas9 and Can1-targeting sgRNA
results in depletion of Can1 and decreases the sensitivity of yeast to canavanine (Fig. 2C)7. We used this assay
to evaluate the effects of each T > C mutation on sgRNA inactivation and found that the T > C mutation at the
fourth base resulted in sgRNA inactivation in yeast. Insertion of the PAM sequence did not inhibit sgRNA function in yeast (Fig. 2D,E).
We then examined the effects of T > C mutations on sgRNA function in mammalian cells. An EGFP mutant
was used in which the start codon was mutated from ATG to GTG​8. In this EGFP mutant, the template strand
has a T > C mutation at the start codon and a PAM sequence at its 18 bp downstream. Therefore, nCas9-CDA
and active sgRNA can correct the T > C mutation in the EGFP mutant and induce EGFP expression (Fig. 3A,B).
However, the sgRNA did not work when a PAM sequence was inserted into the scaffold region of sgRNA, in
contrast to the results of the canavanine assay (Fig. 3C). Because the weak interaction between G and U is
known to be important for the maintenance of RNA s­ tructure9, we suspected that the loss of G-U interaction
at the seventh base caused by PAM insertion might affect sgRNA function (Fig. 3D). Therefore, we introduced
additional sequences near the PAM sequence to maintain the G-U interaction at the 7th base, because extending
the stem-loop sequences in the sgRNA scaffold does not affect sgRNA f­ unction4,5. As expected, this optimized
PAM-inserted sgRNA maintained the G-U interaction at the 7th base (Fig. 3D) and efficiently induced EGFP
expression when expressed with nCas9-CDA and mutant EGFP in 293 T cells (Fig. 3E). We next examined
whether the T > C mutation at the 4th base, which decreased sgRNA activity in yeast, also inactivated sgRNA in
mammalian cells. Again, we obtained different results in mammalian cells from those in the canavanine assay.
The sgRNA with a single T > C mutation at the fourth base did not inactivate GFP expression, indicating thatit retained its normal function. Therefore, in addition to the 4th base, we introduced T > C mutations at 2, 3, or
5 bases into the sgRNA template. These sgRNAs with double T > C mutations at 2/4th, 3/4th, 4/5th bases lostthe function to restore GFP expression in 293 T cells (Fig. 3F). We then performed similar experiments with
the PAM-inserted sgRNAs and confirmed that the double T > C mutations at 3/4 bases, but not the single T > C
mutation at the 4th base, resulted in loss of sgRNA function (Fig. 4A). Thus, we generated an inactive sgRNAwith T > C mutations and PAM sequence in the scaffold region whose function can theoretically be restored bynCas9-CDA-induced base editing in mammalian cells.

Establishment of a chain reaction by repair of sgRNA scaffold mutations. Next, we investigatedwhether the inactive sgRNA with T > C mutations can be converted to the active form by nCas9-CDA-induced
base editing in mammalian cells. First, we transduced nCas9-CDA, the EGFP mutant, a PAM-inserted control
or inactive sgRNA targeting the EGFP mutation, with or without the second sgRNA in 293 T cells. The second
sgRNA was designed to correct the T > C mutations in the EGFP-targeting sgRNA and restore it to an active
form (Fig. 4B,C). As expected, the optimized version of the PAM-inserted sgRNA efficiently corrected the EGFP
mutation and induced GFP expression, whereas the version with T > C mutation did not. Importantly, coexpression of the second sgRNA restored the function of the inactive sgRNA and resulted in robust GFP expression in
293 T cells (Fig. 4D). Thus, the chain reaction was successfully established in mammalian cells with nCas9-CDA
and two sgRNAs targeting the T > C mutation or EGFP mutation.
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A

B

Optimized+PAM sgRNAT>C mutation-targeting sgRNA (sgRNA-1)

EGFP
20.1 %

No mutation

31.6 %

4thT>C mutant
Inactive sgRNA

Active sgRNA7.07 %

3/4thT>C mutant

mutant EGFPWT EGFPEGFPCTransfection
PCRblant-U6-T>C mutation-targeting sgRNA (sgRNA-1)

Mutant EGFP expressing 293T

Imaging or FACS analysis
Lentiviral infection
pLV-nCas9-CDA-U6-EGFP-targeting sgRNA (sgRNA-2)

EGFP

D

FSC

90.1%
Optimized+PAM sgRNA

0 103

104 105

EGFP

FSC

1.97%
Optimized +PAM sgRNAwith 3rd/4th T>C mutations(sgRNA-2)

0 103

104 105

EGFP

9.10%
FSC

Optimized +PAM sgRNAwith 3rd/4th T>C mutations(sgRNA-2)+
T>C mutation-targeting sgRNA(sgRNA-1)

0 103

104 105

EGFP

Figure 4.  Cellular chain reaction systems through sequential repair of sgRNA scaffold mutations. (A)Fluorescence images (left) and FACS plots (right) of 293 T cells expressing mutant EGFP and nCas9-CDA
together with different sgRNAs. The indicated T > C mutations were introduced into the optimized PAMinserted sgRNA. mut; mutation. (B) Schematic representation of the chain reaction by repair of sgRNA scaffold
mutations to express EGFP in 293 T cells. (C) Experimental scheme as in (D). 293 T cells were transduced withmutant EGFP, nCas9-CDA and an EGFP-targeting sgRNA, then transfected with another sgRNA targeting the
T > C mutation. (D) Fluorescence images (left) and FACS plots (right) of 293 T cells expressing mutant EGFPand nCas9-CDA together with the indicated sgRNAs. The sgRNA-1 converted the inactive sgRNA-2 to an active
form, and the sgRNA-2 corrected the EGFP mutation to induce EGFP expression.

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A

3500bp
Cre recombinase

TATAloxP

TATAloxP

TATAloxP

7000bp3500bpB

position of T>C mutant

NC WT 1st 3rd 4th 11th 13th
NC WT 1st 3rd 4th 11th 13th PC

Recombined DNA
Original DNA

7000bp3500bpposition of T>C mutant WT
WT
Recombined DNA
Original DNAC

NC WT WT WT WTNC NC WT 1st 13th

1st
1st

WT
WT
7000bp3500bpWT
1st7000bp3500bpFigure 5.  In vitro Screening of non-responsive TATAloxP with T > C mutations. (A) Schematic representationof the in vitro recombination assay with TATAloxP sequences. (B,C) Linear DNAs with different TATAloxP
sequences containing the indicated mutations were incubated with Cre recombinase. Note that TATAloxP with
the T > C mutation at the first base probably did not efficiently produce recombined DNA in the first experimentbecause of technical errors (B), but did so very well in the second experiment (C). (NC; No loxP, PC; positive
control DNA in Cre recombinase (NEB Catlog# M0298S)).

Generation of non‑responsive TATAloxP sequences with T > C mutations. To establish the second
system, we introduced T > C mutations into the TATAloxP sequences to make them insensitive to Cre-induced
recombination. We generated several TATAloxP mutants in which a T base in the 13-bp repeat sequence(Fig. 1C) was replaced by C. These DNAs with different TATAloxP sequences at the 5′ or 3′ side (approximately
3500 bp-vector) were linearized and incubated with Cre recombinase. In this in vitro assay, Cre-induced recombination results in linear DNA of approximately 7000 bp when Cre recombinase can recognize the TATAloxP
sites (Fig. 5A). As shown in Fig. 5B,C, the T > C mutation at the 13th base resulted in reduced recombination inboth mutant/mutant and mutant/wild-type incubations.
We next investigated whether the TATAloxP sequences containing the T > C mutations are resistant to Cremediated recombination in mammalian cells. We generated an expression vector containing a polyA signal
flanked by two TATAloxP sites and a downstream EGFP cassette under the EF1α promoter in which one of the
TATAloxP sites has the T > C mutations. If the mutated TATAloxP site is resistant to Cre-mediated recombination,
EGFP is not expressed even in the presence of Cre recombinase (Fig. 6A). We expressed the TATAloxP (wild-type
or mutant)-EGFP constructs in 293 T cells together with Cre-R32V, a mutant Cre recombinase with improved­fidelity10 (Fig. 6B). However, in contrast to the results of the in vitro experiments, the T > C mutation at 13 basedid not prevent Cre-R32V-induced EGFP expression in either or both 13-bp arms (Fig. 6C,D). Thus, the T > C
mutation at the 13th base was not sufficient to render the TATAloxP sequence insensitive to Cre recombinase
in mammalian cells. Because a previous report showed that double mutations in both 13-bp arms, particularly
at the 7th, 8th, 11th, 12th, and 13th bases efficiently disrupted the loxP s­ tructure11, we subsequently examinedthe effect of double T > C mutations at the 11th and/or 13th bases on their responsiveness to Cre recombinase.
Among the different combinations, we found that double T > C mutations at the 11th and 13th bases became
resistant to Cre-induced EGFP expression in both arms (Fig. 6E,F,G). In this way, we generated a TATAloxP
mutant which does not respond to Cre but will resume the responsiveness to Create by nCAS9-CDA-induced
base editing in mammalian cells.
Inefficient repair of TATAloxP mutations in the chain reaction system. Finally, we examined
whether we could establish the cellular chain reaction system using the TATAloxp mutant. We transduced wildtype and nonresponsive TATAloxP-EGFP, Cre-R32V, and nCas9-CDA into 293 T cells with or without the second sgRNA. The second sgRNA was designed to correct the T > C mutations in the nonresponsive TATAloxPand convert it to a responsive form (Fig. 7A,B). Consistent with previous results, Cre-R32V induced recombination of only the wild-type TATAloxP but not the mutant TATAloxP to induce EGFP expression in 293 T cells(Fig. 7C). Unfortunately, coexpression of the second sgRNA failed to restore EGFP expression, indicating that
the TATAloxP mutant with double T > C mutations at bases 11 and 13 is resistant to nCas9 DNA-mediated baseediting. Thus, this TATAloxP system in its current form is not suitable for cellular chain reactions.

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A

Mutation

EGFP/TATAloxP cassetteEF1α promoter

TATAloxP

PolyA

TATAloxP

Cre recombinase
EGFP

EF1α promoter

C

WT

293T

B

PolyA

TATAloxP
TATAloxP

EGFP

+

EGFP
13thsingle T>C mutant

No loxP

13thdouble T>C mutant

No loxP

Transfection
Piggybac-EGFP/TATAloxP cassette+pLG2 transposase
EGFP/TATAloxP cassette expressing 293T

D

WTTransfection
PMYs-Cre (R32V)

G
WT

Imaging

E

F

EGFP

293T

11thdouble

70.8 %

11thsingle
36.3 %

Transfection
Piggybac-EGFP/TATAloxP cassette+
PMYs-Cre (R32V)

11thdouble
11thsingle/13thsingle

Imaging or FACS analysis

50.0 %

33.3 %

11thsingle/13thsingle
31.7 %

11thdouble/13thsingle

11thdouble/13thsingle
19.4 %

11thsingle/13thdouble

11thdouble/13thdouble

11thsingle/13thdouble

11thdouble/13thdouble

12.1 %

7.63 %No LoxP

EGFP

Figure 6.  Screening of non-responsive TATAloxP with T > C mutations in mammalian cells. (A) Schematic
representation of the EGFP cassette with mutated TATAloxP and polyA sequences under the EF1α promoter.
(B) Experimental scheme used in (C,D). 293 T cells were transduced with the EGFP/TATAloxP cassette, andthe mutant EGFP-expressing 293 T cells were subsequently transfected with Cre-R32V. (C,D) Fluorescence
images of 293 T cells expressing EGFP/ various TATAloxP with the indicated T > C mutations and Cre-R32V.
(E) Experimental scheme as in (F,G). 293 T cells were transfected with EGFP/TATAloxP cassette and Cre-R32V.
(F,G) Fluorescence images (F) and FACS plots (G) of 293 T cells expressing EGFP/various TATAloxP with theindicated T > C mutations and Cre-R32V. WT; wild type.

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www.nature.com/scientificreports/Amutant TATAloxP-targeting sgRNA
Mutation

EGFP/TATAloxP cassetteEF1α promoter

TATAloxP

TATAloxP

PolyA

sgRNA 1D
EGFP

Barcoding
Activate
New sgRNA

nCas9-CDA
Nick
EF1α promoter

TATAloxP

TATAloxP

PolyA

EGFP

Cre recombinase
TATAloxP
EF1α promoter

TATAloxP

EGFP

DNA

PolyA

+

B

Non Homologous End Joining
with Random Indel

EGFP/TATAloxP cassette expressing 293TDNA
Retroviral infection
PMYs-Cre (R32V)

sgRNA 2

Transfection
pLV-nCas9-CDA-U6-sgRNA

BarcodingC

WT

No loxP

Activate
New sgRNA
DNA

EGFP

11thdouble/13thdouble
11thdouble/13thdouble +TATAloxP-targeting sgRNA

EGFPNon Homologous End Joining
with Random IndelDNA

sgRNA 3Figure 7.  Cellular chain reaction systems through sequential repair of loxP mutations. (A) Schematic
representation of the chain reaction by repair of TATAloxP mutations to express EGFP in 293 T cells. (B)Experimental scheme used in (C). 293 T cells were transduced with the EGFP/different TATAloxP cassette with
the indicated T > C mutations, then infected with Cre-R32V, and then transfected with nCas9-CDA and sgRNAtargeting the T > C mutation. (C) Fluorescence images of 293 T cells expressing EGFP/TATAloxP cassette, CreR32V, and nCas9-CDA along with the indicated sgRNAs. When the EGFP/TATAloxP cassette was used with
T > C mutations at the 11th and 13th bases in both arms, no EGFP expression was detected, even in the presenceof the TATAloxP-targeting sgRNA (bottom right). (D) Schematic representation of barcoding method.

Discussion

To develop a system that enables sequential sgRNA activation in mammalian cells, we designed two chain
reaction systems using the nCas9 CDA base editor. As a proof of concept, we successfully established a chain
reaction by repairing sgRNA scaffold mutations in 293 T cells. In our system, there is no theoretical limit to thenumber of sequential reactions. Moreover, our system allows the activation of different sgRNAs at each stageof the chain reaction. Thus, our chain reaction system provides a useful platform for recording and controlling
molecular events in living cells. For example, in combination with the cell cycle-dependent CRISPR-Cas9 activation ­system12, our chain reaction system can be used to develop a counter for cell division. Previously, several
methods to monitor cell division have been reported. Those using BrdU or H2B-EGFP dilution were used to distinguish cells that divided more than 4–5 times from those divided ­less13,14. Another method using Ki67-Cre wasused to mark cells that divided at least ­once15. In contrast to these methods that only record a few cell divisions,
our system has the potential to serially count each cell division. With such systems, we will be able to know how
many times a stem cell divides to be a terminally differentiated cell. In addition, it will be interesting to combine

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the chain reaction system with the DNA barcoding technique. It is known that nCas9 can cause random indelby adding a nick in two ­places16. This random indel can be used as a “barcode”. Therefore, combined with thecell division counting system described above, it is theoretically possible to introduce a barcode to each daughter
cell at each cell division (Fig. 7D). Such systems will enable us to draw the exact cell lineage trees during tissue
development, which is impossible with the current DNA barcoding systems.
However, further optimization will be required to develop more sophisticated chain reaction systems in mammalian cells. Although we achieved the chain reaction using the sgRNA scaffold mutation system, the activity of
the repaired sgRNA was much weaker than that of the control sgRNA. We could not even restore the reactivityof mutant TATAloxP by nCas9-CDA-mediated base editing in mammalian cells. To achieve more robust chain
reactions, it is necessary to increase the mutation repair rate at each step. In order to improve the repair efficiencyof mutant sgRNAs, further screening is required for finding mutants that lose their function after only a single
mutation or adjusting the sequence of the stem loop region of sgRNAs which is changeable without loss of function with checking repairing rate. Our study also showed that the results obtained in yeast or in vitro are not thesame as those obtained in mammalian cells. It has been shown that physiological RNA structure depends not
only on sequence but also on RNA modifications and binding ­proteins17. DNA methylation has also been shown
to affect the efficiency of Cre-mediated ­recombination18. These in vivo-specific regulatory mechanisms could
explain the discrepancy between the in vitro and in vivo results. Thus, our results strongly suggest that in vivo
chain reactions need to be optimized in appropriate cellular contexts.
In summary, we have developed a cellular chain reaction system using nCas9 CDA-mediated base editing of
sgRNA scaffold mutations. Our system, in combination with tissue-specific or time-dependent regulation, may
have broad utility for future biomedical research.

Methods

Plasmids. Plasmids and primers used for plasmid construction are summarized in supplemental Table 1.

Mutations were introduced by PCR with KOD FX NEO (TOYOBO), and mutants were cloned into vectors with
Gibson assembly using Gibson Assembly Master Mix (NEB). The TATAloxP sequence was generated using
gBlocks (IDT). For plasmid construction of pNMA001, the gRNA coding region was constructed by PCR withprimers (5′-GTT​TTA​GAG​CTA​GAA​ATA​GCA​AGT​TAA​AAT​AAG​GCT​AGT​CCG​TTA​TCA​ACT​TGA​AAA​AGT​
GGC​ACC​GAG​TCG​GTG​CTT​TTT​TGT​TCA​CTG​CCG​TAT​ATA​GGC​AG-3′ and 5′-AAA​CTT​CTC​CGC​AGT​GAA​AGA​TAA​ATG​ATC​GTC​AAT​TAC​GAA​GAC​TGA​ACG​TTT​TAG​AGC​TAG​AAA​TAGC-3′). The amplified
product and the p426-SNR52p-gRNA.CAN1. Y-SUP4t (Addgene#43,803) backbone fragment amplified by
PCR using primers (5′- GAT​CAT​TTA​TCT​TTC​ACT​GCGG-3′ and 5′-TGT​TTT​TAT​GTC​TTC​GAG​TCA​TGT​
AATTA-3′) were subsequently assembled by Gibson Assembly. For plasmid construction of pLV-U6p-gRNACMVp-nCas9-PmCDA1-UGI-2A-mCherry, the human U6 promoter and gRNA coding region were amplified by PCR from plasmid pSI-236 using primers (5′-GGG​ACA​GCA​GAG​ATC​CAG​TTA​TCG​ATG​AGG​GCC​
TAT​TTC​CCA​TG-3′) and (5′-GTA​ATT​GAT​TAC​TAT​TAA​TAA​CTA​GTA​AAA​AAG​CAC​CGA​CTC​ GGT-3′).
The amplified product and the pLV-CS-086 backbone fragment digested with SpeI and ClaI were subsequently
assembled using Gibson Assembly.

Cell culture. 293 T cells (CRL-11268, ATCC, Manassas, VA, USA) were cultured in DMEM (Wako) containing 10% fetal bovine serum (FBS) (Biowest) at 37 °C and 5% CO2. 293 T was authenticated by short tandem
repeat analyses and tested for mycoplasma contamination in our laboratory. For selection, 1 μg/ml puromycin(Thermo Fisher Scientific), 5 μg/ml blasticidin S (Thermo Fisher Scientific), and 1 mg/ml zeosin (InvivoGen)
were used.
Transient transfection. Plasmids were transfected using the calcium phosphate co-precipitation m­ ethod19.
A mixture of 10–20 μg plasmid (10–20 μl), 2.5 M CaCl2 (50 μl), and filtered water (430–440 μl) to 2 × HeBS
(500 μl) was added to 293 T cells in a 10-cm dish (2.0 × ­106 cells/dish) for 18 h. To integrate the piggyback vector, 15 μg of piggyback vector and 5 μg of pLG2 (PB200PA-1, SBI) were co-transfected with the above plasmids.
Viral transduction. Lentiviruses and retroviruses were prepared by transient transfection in 293 T cells
using the calcium phosphate method as described above. A mixture of plasmids (3 μg VSVG and 10 μg PAX2 forlentiviruses, 3 μg RD114 and 10 μg M57 for retroviruses along with 12 μg viral vectors), 2.5 M CaCl2 (50 μl), andfiltered water (425 μl) to 2 × HeBS (500 μl) was added to 293 T cells in a 10-cm dish (2.0 × ­106 cells/dish). After
24 h, the medium was removed by aspiration and 10 ml of fresh DMEM(Wako)/10% FBS (biowest) was added.
The virus-containing medium was collected 48 h after transfection.
In vitro Cre recombinase reaction assay.

One μg of the mutant TATAloxP sequences in the zero-blant
vector (Thermo Fisher Scientific) were incubated with 1.5 U/ml BamHI, 1.0 U/ml NotI or 1.0 U/ml MluI to
linearize them. The linear DNAs (20 ng each) were mixed with 10 × Cre Recombinase Reaction Buffer, Cre
Recombinase and H2O and incubated at 37 °C for 30 min and then at 70 °C for 10 min.

Live cell imaging. Live cell imaging was performed using the Invitrogen EVOS FL Auto 2 Imaging System (Thermo Fisher Scientific). Phase contrast images were acquired using a 10 × phase contrast objective andcamera (with a resolution of 1388 × 1040 pixels). EGFP images were acquired using the EVOS™ LED cube, GFP.

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Flowcytometry analysis. The 293 T cells were resuspended in PBS containing 2% FBS. Cells were thenanalyzed using a FACS-Verse (BD Biosciences).
Canavanine assay.

Saccharomyces cerevisiae strain BY4741 (MATa his3Δ0leu2Δ0 met15Δ0 ura3Δ0) wasused for CAN1 mutagenesis analysis of the CRISPR system. Parent strains BY4741 were grown before transformation in YPAD and then propagated in the appropriate synthetic complete media (SC) without the auxotrophic
compound supplemented with the plasmids. BY4741 were spread onto the YPAD plate on day 1. On day 3,
BY4741 single colonies were harvested and cultured in 5 ml YPAD at 30℃ overnight. On day 4, transformationof plasmids containing a galactose-inducible modifier gene plasmid (Cas9, nCas9-CDA) with LEU2 marker
and a gRNA-expressing plasmid with Ura3 marker (250 μg each per transformation) was performed using a
Frozen EZ Yeast Transformation Kit (ZYMO RESEARCH). After transformation, BY4741 were spread on SCLeu-Ura + Ade plates for selection. On day 6, single colonies were taken from each plate and cultured in 5 ml
SC-Leu-Ura-Ade at 30℃. On day 8, the culture medium was changed to SRaffi-Leu-Ura + Ade. On the 10thday, the culture medium was replaced with SGall-Leu-Ura + Ade. On day 11, BY4741 were spread on SC-LeuUra + Ade + canavanine plates or SC-Leu-Ura + Ade plates.

Data availability

Information and inquiries about reagents can be directed to the main contact person, Yosuke Tanaka (ytims@
ims.u-tokyo.ac.jp).
Received: 2 February 2021; Accepted: 19 November 2021

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Acknowledgements

We thank the Center for Flow Cytometry at the Institute of Medical Science, University of Tokyo, for its help. This
project was supported by "Grant-in-Aid for Challenging Exploratory Research" from JSPS to Y.T. (19K22380)
and "Grant-in-Aid for Challenging Exploratory Research" from JSPS to Tsu.F. (20K21613).

Author contributions

T.F. and Y.T. planned most of the experiments. TF., K.A. and A.T performed most of the experiments. N.M.
performed the canavanine assay. S.A., S.I., H.M., and M.S. helped plan the experiments. T.F., S.G., Y.T, and T.K.
wrote the manuscript. Y.T., T.K. Y.N., and S.G. supervised and coordinated the project.

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Competing interests

The authors declare no competing interests.

Additional information

Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​021-​02986-6.
Correspondence and requests for materials should be addressed to Y.T., S.G. or T.K.
Reprints and permissions information is available at www.nature.com/reprints.
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