R ES E A RC H

RESEARCH ARTICLE SUMMARY◥GENOME EDITING

Targeted nucleotide editing using
hybrid prokaryotic and vertebrate
adaptive immune systems
Keiji Nishida, Takayuki Arazoe, Nozomu Yachie, Satomi Banno, Mika Kakimoto,
Mayura Tabata, Masao Mochizuki, Aya Miyabe, Michihiro Araki, Kiyotaka Y. Hara,
Zenpei Shimatani, Akihiko Kondo*
INTRODUCTION: To combat invading patho-

a powerful genome editing tool, because it canbe programmed to cleave specific DNA sequenceby providing custom gRNAs.
RATIONALE: Although the precise mechanism
by which AID specifically mutates the immunoglobulin locus remains elusive, targeting of AID
activity is facilitated by the formation of a singlestranded DNA region, such as a transcriptional
RNA/DNA hybrid (R-loop). The CRISPR/Cas system can be engineered to be nuclease-inactive.
The nuclease-inactive form is capable of unfolding the DNA double strand in a protospacer
adjacent motif (PAM) sequence-dependent manner so that the gRNA binds to complementarytarget DNA strand and forms an R-loop. The
nuclease-deficient CRISPR/Cas system may serve
as a suitable DNA-targeting module for AID tocatalyze site-specific mutagenesis.
RESULTS: To determine whether AID activity

can be specifically targeted by the CRISPR/Cas
system, we combined dCas9 (a nuclease-deficientmutant of Cas9) from Streptococcus pyogenes
and an AID ortholog, PmCDA1 from sea lamprey,
to form a synthetic complex (Target-AID) by either
engineering a fusion between the two proteins or
attaching a SH3 (Src 3 homology) domain to the
C terminus of dCas9 and a SHL (SH3 interaction
ligand) to the C terminus of PmCDA1. Both of

CONCLUSION: By expanding the genome edit-ing potential of the CRISPR/Cas9 system bydeaminase-mediated hypermutation, TargetAID demonstrated a very narrow range oftargeted nucleotide substitution without theuse of template DNA. Nickase Cas9 and uracil
DNA glycosylase inhibitor protein can be used
to boost the targeting efficiency. The reduced
cytotoxicity will be beneficial for use in cells
that are sensitive to artificial nucleases. Use ofother types of nucleotide-modifying enzymes
and/or other CRISPR-related systems withdifferent PAM requirements will expand our
genome-editing repertoire and capacity.

▪

The list of author affiliations is available in the full article online.
*Corresponding author. Email: akondo@kobe-u.ac.jp
Cite this article as K. Nishida et al., Science 353, aaf8729(2016). DOI: 10.1126/science.aaf8729

A crippled CRISPR/Cas targets AID. In vertebrate adaptive immunity, cytosine deaminase (AID or PmCDA1) induces somatic hypermutation at
single-stranded DNA regions formed during transcription. The bacterial CRISPR/Cas9 immunity system recognizes and cleaves invasive DNA in a gRNAdependent manner. AID and nuclease-deficient CRISPR/Cas9 are engineered to form a hybrid complex (Target-AID) that performs programmablecytosine mutations in a range of a few bases surrounding the –18 position upstream of PAM sequence of the noncomplementary DNA strand.
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gens, cells develop an adaptive immune responseby changing their own genetic information. In
vertebrates, the generation of genetic variation(somatic hypermutation) is an essential process
for diversification and affinity maturation of
antibodies that function to◥
ON OUR WEBSITE
detect and sequester various foreign biomolecules.
Read the full article
The activation-induced cytiat http://dx.doi.
org/10.1126/
dine deaminase (AID) carscience.aaf8729
ries out hypermutation by..................................................
modifying deoxycytidine
bases in the variable region of the immunoglobulin locus that produces antibody. AIDgenerated deoxyuridine in DNA is mutagenic
as it can be miss-recognized as deoxythymine,
resulting in C to T mutations. CRISPR (clusteredregularly interspaced short palindromic repeats)/
Cas (CRISPR-associated) is a prokaryotic adaptive
immune system that records and degrades invasive foreign DNA or RNA. The CRISPR/Cas system cleaves and incorporates foreign DNA/RNA
segments into the genomic region called the
CRISPR array. The CRISPR array is transcribedto produce crispr-RNA that serves as guide RNA(gRNA) for recognition of the complementary
foreign DNA/RNA in a ribonucleoprotein complex with Cas proteins, which degrade the target.
The CRISPR/Cas system has been repurposed asthese complexes performed highly efficient sitedirected mutagenesis. The mutational spectrum
was analyzed in yeast and demonstrated thatpoint mutations were dominantly induced at
cytosines within the range of three to five bases
surrounding the –18 position upstream of the
PAM sequence on the noncomplementary strand
to gRNA. The toxicity associated with the nucleasebased CRISPR/Cas9 system was greatly reducedin the Target-AID complexes. Combination of
PmCDA1 with the nickase Cas9(D10A) mutant,which retains cleavage activity for noncomplementary single-stranded DNA, was more efficient in yeast but also induced deletions as wellas point mutations in mammalian cells. Addition of the uracil DNA glycosylase inhibitor protein, which blocks the initial step of the uracil
base excision repair pathway, suppressed collateral deletions and further improved targeting efficiency. Potential off-target effects wereassessed by whole-genome sequencing of yeast
as well as deep sequencing of mammalian cells
for regions that contain mismatched target sequences. These results showed that off-target
effects were comparable to those of conventional
CRISPR/Cas systems, with a reduced risk ofindel formation.

R ES E A RC H

RESEARCH ARTICLE◥

GENOME EDITING

Targeted nucleotide editing using
hybrid prokaryotic and vertebrate
adaptive immune systems
Keiji Nishida,1 Takayuki Arazoe,1 Nozomu Yachie,2,3,4 Satomi Banno,1 Mika Kakimoto,1
Mayura Tabata,1 Masao Mochizuki,1 Aya Miyabe,1 Michihiro Araki,1 Kiyotaka Y. Hara,5Zenpei Shimatani,1 Akihiko Kondo1,6*T

he activation-induced cytidine deaminase(AID) is responsible for targeted hypermutation (1) by modifying the deoxycytidineof the variable region of the immunoglobulin
locus (2, 3). Although the precise mechanism
by which AID specifically targets the immunoglobulin locus remains elusive, targeting of AID
is facilitated by the formation of a single-stranded
DNA region, such as a transcriptional RNA/DNA
hybrid (R-loop) (4, 5). AID generates deoxyuridine
in DNA, which is further processed by uracil DNA
glycosylase for class-switch recombination (6) as
well as repair (7). CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPRassociated) is a prokaryotic adaptive immune
system (8). The CRISPR/Cas system records andcleaves invasive foreign DNA. A ribonucleoprotein
complex of Cas nuclease and guide-RNA (gRNA)binds to complementary target DNA sequences,
forms an R-loop, and then cleaves the DNA. The
CRISPR/Cas system has been developed as a ge1

Graduate School of Science, Technology and Innovation,
Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo
657-8501, Japan. 2Synthetic Biology Division, ResearchCenter for Advanced Science and Technology, the Universityof Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
3
Institute for Advanced Bioscience, Keio University,
Tsuruoka, Yamagata, Japan. 4Precursory Research for
Embryonic Science and Technology (PRESTO), Japan
Science and Technology Agency (JST), Tokyo, Japan.
5
Department of Environmental Sciences, Graduate School of
Nutritional and Environmental Sciences, University of
Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
6
Department of Chemical Science and Engineering, Graduate
School of Engineering, Kobe University, 1-1 Rokkodai-cho,Nada-ku, Kobe, Hyogo 657-8501, Japan.
*Corresponding author. Email: akondo@kobe-u.ac.jp

SCIENCE sciencemag.org

nome editing tool (9, 10). Here, by using hybrid
vertebrate and bacterial immune systems, we
provide genome engineering tools that enabletargeted nucleotide substitution without integration or the use of template DNA.
Cytidine deamination is targeted by
CRISPR/Cas system
To determine whether the single-stranded DNA
region formed by the CRISPR/Cas system also
serves as a preferable substrate for AID, we created a synthetic complex (Target-AID) of dCas9(a nuclease-deficient mutant of Cas9) (11) from
Streptococcus pyogenes and an AID ortholog,
PmCDA1 from sea lamprey by either a fusion protein or attaching a SH3 (Src 3 homology) domain(12) to the C terminus of dCas9 and a SHL (SH3interaction ligand) to the C terminus of PmCDA1(Fig. 1A). The protein complex was expressed in
Saccharomyces cerevisiae, which lacks an endogenous AID-related system. Chimeric gRNAs (11)were designed (13) to target a genomic negative
selectable marker gene, CAN1, whose loss of function results in resistance to the drug canavanine.
The rate of occurrence of the canavanine-resistantmutant was counted as an on-target mutation.
To assess the potential genome-wide, nonspecificmutator phenotype (off-target mutation), anothernegatively selectable marker, LYP1, was monitored by resistance to S-aminoethyl-L-cysteine. Although the expression of PmCDA1 increased thebackground mutation frequency, the specificmutation frequency of CAN1 increased by morethan 1000-fold over PmCDA1 alone when dCas9SH3 and SHL-PmCDA1 were expressed simulta-

DNA nicking promotes
deamination-mediated mutagenesis
Because nucleotide deamination also occurs
spontaneously, cells are capable of correcting
most deaminated bases, and only a small portion of deamination normally results in mutagenesis (7). To exploit the full potential of
Target-AID, we sought ways to bypass the nucleotide repair pathway without compromisingoverall genome integrity. Because the repair
pathway requires base excision and templatedependent polymerization of nucleotides, wehypothesized that the introduction of a singlestrand break (nick) in the DNA close to the deaminated site may perturb the repair system
and result in a greater rate of mutagenesis. The
Cas9 protein contains two nuclease domains,and each cleaves the opposite strand of DNA
independently (11). Substituting aspartic acidwith alanine in position 10 and histidine withalanine at position 840 prevents cleavage of thenoncomplementary and complementary strandsof DNA, respectively, to produce the Cas9 nickases
nCas9(D10A) and nCas9(H840A) (11). Both nickases
alone demonstrated a modest increase in thespecific mutation rate, whereas the fusion protein of nCas9(D10A) and PmCDA1 consistently
demonstrated an almost saturating mutation
frequency (Fig. 2A), indicating that introducing
nick on the opposite strand of deamination sitefacilitates mutagenesis, either by promoting therepair of the nicked, nonedited strand using theedited strand as a template or by providing better accessibility for PmCDA1. The mutational
spectrum of nCas9(D10A)-PmCDA1 was similar
to that of dCas9-PmCDA1 and distinct from thoseof Cas9 nickases or the complete nuclease alone(fig. S1). nCas9(D10A) has been shown to induce
C-T substitution on the noncomplementary strandof target sites in cancer cell lines that overexpress
endogenous cytidine deaminases, suggesting thatnCas9-induced base substitution is comediatedby the deaminase activity (14). This is consistent
with our observation that the single-stranded

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The generation of genetic variation (somatic hypermutation) is an essential process for theadaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide
substitution of DNA using hybrid vertebrate and bacterial immune systems components.
Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic
repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog
PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly
efficient target-specific mutagenesis. Specific point mutation was induced primarily atcytidines within the target range of five bases. The toxicity associated with the nuclease-based
CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A)
and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel)in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formationand improved the efficiency.

neously (Fig. 1B, 4). Specific mutation was observed even without synthetic attachment ofdCas9 and PmCDA1 (Fig. 1B, 3), suggesting thatthe single-stranded DNA formed by dCas9-gRNA
is a preferred substrate for PmCDA1. The fusion
protein of dCas9 and PmCDA1 was also effective,
especially when a large peptide linker (100 aminoacids) was used (Fig. 1B, 6). Most mutations were
found at g782, which is located at the –16 position
upstream of the protospacer adjacent motif (PAM)
sequence (11), except for PmCDA1 or dCas9 alone(Fig. 1C and table S1), which indicates that thesteric effect of the protein complex affects theefficiency but not the location of the mutation.
This also suggests that a limited region of DNA is
made accessible for PmCDA1 by dCas9-gRNA.
Most mutations were found as point mutations
at a G and C, which suggests that deamination
occurred at the noncomplementary strand of the
C (table S1). The fusion protein with the extended
linker version (Fig. 1B, 6) was used in the following studies because of its simplicity.

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DNA formation by dCas9-gRNA is sufficient
to recruit PmCDA1 (Fig. 1B, 3) and that the
mutation is induced at C on the noncomplementary strand, which is facilitated by nickase
activity of nCas9(D10A). The combination ofnCas9(H840A) and PmCDA1 reduced mutation
frequency, suggesting that nicking on the same

strand of the deamination site may prevent the
mutagenic effect, presumably by facilitating theexcision of deaminated base. It also appearedthat Target-AID, using nCas9(D10A) and PmCDA1,
was much less toxic than the full CRISPR/Cas9
nuclease, which causes growth defects or cell
death in yeast (Fig. 2B). The low efficiency of fullCas9 nuclease in yeast may be attributed to its
toxicity. This facilitates emergence of “escaper
cells” that have lost the system (15, 16).
Mutation spectrum induced by Target-AID
To detect the presence of associated mutations,deep sequencing of the target-flanking region

Fig. 2. Effect of nickase activity on Target-AID.
(A) Forms of fusion protein of Cas9 nuclease mutants[Cas9, dCas9, nCas9(D10A), or nCas9(H840A)] andPmCDA1 are indicated on the left. The schematic illustration in the middle depicts the process of DNA
modification by the fusion proteins. Ladder indicatestarget DNA. Red arrowhead indicates DNA-strand
break introduction. Yellow star indicates cytosine deamination introduction. Yeast cells expressing eachprotein, with gRNA targeting a single site (791 to 772)
in CAN1, were analyzed for canavanine resistance, andthe mutation frequencies are plotted. Seven biologicalreplicates are shown in different colors. Box bars indicate the 95% confidence interval for a sample meanby t test statistical analysis. (B) Yeast cells expressing Cas9, dCas9 dCas9-PmCDA1 or nCas9(D10A)PmCDA1, and gRNA targeting CAN1 were measured
for cell viability. Viable cells were counted and plottedbased on colony formation at each time point. Relative values (1 at 0 hour) of the mean of the biologicaltriplicate are plotted. Error bars indicate SD. (C) Mutation spectra of canavanine-resistant cells produced
by Cas9, dCas9-PmCDA1, or nCas9-PmCDA1, analyzed by deep sequencing at the targeted genomicsite (32681 to 32700 at chromosome V or 786 to 767
in Can1). Frequencies of SNVs and deletions or insertions at each nucleotide position were plotted fromthe –31 to the 9-bp position relative to target PAM
sequence. The nucleotide sequence is shown withthe target sequence (box) and the PAM sequence(inverted red).

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Fig. 1. Complex formation of dCas9-gRNA and
PmCDA1 induces targeted mutation. (A) Schematics of the complex forms of dCas9, gRNA, thetarget DNA, and PmCDA1 examined in this study.
(1) PmCDA1 alone. (2) dCas9-gRNA alone. (3)PmCDA1 and dCas9-gRNA without direct attachment. (4) PmCDA1-SHL attached to dCas9-SH3gRNA via SH3 and SHL interaction (SH3 linker).
(5) PmCDA1 fused to dCas9 with short linker (17amino acids; NLS plus 2x glycine-serine peptide
linker). (6) PmCDA1 fused to dCas9 with long linker(100 amino acids). (B) Yeast cells expressing eachprotein complex numbered as in (A), with gRNAtargeting a single site (786 to 767) in CAN1, wereanalyzed by a colony formation assay. S-aminoethylL-cysteine and canavanine select for the cells withthe lyp1 mutation (nonspecific mutation) and thecan1 mutation (on-target mutation), respectively.
Mutation frequency per five generations is plotted.
Eight biological replicates are shown in different
colors. Box bars indicate 95% confidence interval
for a sample mean by t test statistical analysis.
(C) Partial nucleotide sequence of CAN1 with the target site (box) and the PAM sequences (inverted) are indicated with open reading frame nucleotide
number. Representative sequence of the canavanine-resistant mutants induced by Target-AID is aligned. Mutation site and mutated nucleotide arehighlighted by yellow and red, respectively. A mutation list can be found in table S1.

RE S E ARCH | R E S E A R C H A R T I C L E

was performed for canavanine-selected cells mutated at the Can1 locus by Cas9, dCas9-PmCDA1,
or nCas9-PmCDA1 (Fig. 2C). Cas9 induced deletions and insertions at the –1 to –5 positions.
dCas9-PmCDA and nCas9-PmCDA induced apoint mutation predominantly at the –16 position
with a frequency of 94.7 and 96.1%, respectively.
The second most frequent single-nucleotide variants (SNVs) were at the –22 position for dCas9PmCDA and the –10 position for nCas9-PmCDA,
with a frequency of 1.7 and 4.3%, respectively.
Outside of the –22 to –10 position, no SNVs over
1% were detected for dCas9-PmCDA1, and one
SNV at the +24 position for nCas9-PmCDA1 wasfound (1.2%). Insertions and deletions (indels)
were rarely observed for both dCas9-PmCDA
and nCas9-PmCDA with the highest frequencyof 0.18 and 0.24%, respectively, at the –16 position.
The high efficiency of Target-AID using
nCas9(D10A) and PmCDA1 allowed us to obtainmutants without canavanine-selection and to
perform nonbiased sequencing analyses, whichshows that the mutations were focused at cytosine bases located 15 to 19 bases upstream of
the PAM sequence on the noncomplementarystrand to gRNA, while the length of the gRNAtarget sequence (i.e., the 5′ end position of gRNA)
did not affect mutation location (fig. S2). C to G
and C to T mutations of the noncomplementarystrand were observed with nearly equal frequency
SCIENCE sciencemag.org

target sites in Ade1 gene were selected for editing by Target-AID based on
the criteria that the cytosine mutations in the –19 to –16 position may introduce a stop codon, resulting in a red colony phenotype. Red colony frequency (mean ± SD of biological triplicate) for each target site is indicatedon the left. Sequences of identified mutations are aligned with the numberof clones over the number of total sequenced clones for each colony color(red or none). Reference wild-type sequences with translated amino acidsequences are shown with the target site (box) and the PAM sequence(inverted). The bottom target sequence is complementary to the sequence
shown.

(9 of 18 and 8 of 18, respectively), whereas C to A
was rare (1 of 18).
To obtain a comprehensive mutational spectrum for nCas9-PmCDA1, three intergenic target
sites that were C-rich were selected (Fig. 3A). At
the three targets, a peak of SNV frequency wasconsistently observed at the –18 position (41 to51%), with lower frequencies at the –17 and –19
positions (18 to 40%). The SNV frequencies decreased at the –16 position (4.5 to 6.5%) and –20
position (1.3 to 6.3%) and further decreased in
the surrounding area. Frequencies of more than
1% were rarely observed outside the –13 to –21
positions. This suggests that the mutational efficiency is highly dependent on the relative position within the target sequence. It also implies
that processive deamination of poly-C by PmCDA1
is limited, if it occurs at all. On the other hand,changed nucleotides showed positional bias within
the poly-C region (Fig. 3A, insets). C to T mutations were more common at the 5′ side of thepoly-C region, whereas the C to G mutation
ratio increased on the 3′ side. In Saccharomyces
cerevisiae, the translesion DNA polymerase eta
prefers to insert C opposite an abasic lesion,
resulting in a G insertion at an abasic site (17),whereas a T insertion at an abasic site is common in most other organisms (18). The 3′ sideof the poly-C region is more likely to be subjectedto error-prone repair by the translesion polymer-

ase eta, whereas mutations on the 5′ side areleft as the deaminated base (i.e., uracil), whichis then recognized as T in DNA synthesis.
To assess the general effectiveness of TargetAID, four sites were systematically selected fromthe Ade1 gene [918 base pairs (bp)] to introduce
a stop codon, following the criteria that C to G
or C to T mutations should be introduced at –16
to –19 positions upstream of the PAM sequence(Fig. 3, B and C). The four targets resulted in an
approximately 47, 29, 17, and 16% rate of ade1
gene disruption (red colony phenotype), respectively (Fig. 3C). Sequencing analysis of both red
and white colonies showed that the genetic mutation frequency was even greater because not all
mutations resulted in gene disruption. All sequenced mutations were localized at the –16 to–18 position (Fig. 3C). There seems to be no strong
context preference as reported previously for
PmCDA1 (19).
Multiplex and biallelic editing
by Target-AID
Multiplex editing was performed for the ADE1
and CAN1 genes by simultaneously expressing
ADE1 and CAN1 targeting gRNAs. The independent mutation frequencies for ADE1 and CAN1
were ~54 and 51%, respectively, and doublephenotypic mutants occurred ~31% of the time
(fig. S3A). Sequencing analysis revealed that all

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Fig. 3. Mutable position and target design for nCas9(D10A)-PmCDA1.
(A) Mutation spectra of the targeted poly-C–containing sequences. Threeintergenic target sequences that contain poly-C at 5′ half were edited bynCas9(D10A)-PmCDA1 and analyzed by deep sequencing. Frequencies of
SNV at each position from –31- to 9-bp position relative to target PAM sequence
were plotted. The upper right insets indicate the ratio of nucleotide changes
at positions from –20 to –13 as C to T (yellow), C to G (red), and C to A (blue).
Two biological replicates (Rep1 and Rep2) are shown. (B) Mutational position and frequency by Target-AID. Blue bars indicate average mutation frequency of A at each base position in the 20-base target sequence. (C) FourR ES E A RC H | R E S EA R C H A R T I C LE

lyzed by their recessive phenotype of red colony and canavanine resistance, respectively.
Biallelic mutants were obtained more frequently
than expected if independent allelic mutagenesis
was assumed (fig. S4A), whereas sequencing
analysis verified heteroallelic mutant from phenotypically wild-type cells (fig. S4B). These imply
that microhomology-mediated repair or gene conversion is induced during deamination-mediatedmutagenesis, which results in a higher rate of
biallelic mutagenesis (20).
gRNA mismatch tolerance of Target-AID

Whole-genome sequencing ofTarget-AID–treated yeast

Fig. 4. Target-AID in mammalian cells. (A) Schematics of the mammalian experiment. The mammalianmodifier vector expressing gRNA and the modifier protein is depicted on the top. Linker plus PmCDA1 isinserted before 2A peptide. UGI is also inserted between PmCDA1 and 2A peptide. Experimental flow is
depicted at the bottom. CHO cells were transfected using each modifier vector and harvested after 3 days
for transient assay or transferred to G418-containing media for selection of the transformed cells.
Pulse incubations at 25°C are optionally introduced. After G418 selection, cells were analyzed by deep
sequencing. For the HPRT gene target, cells were further tested for 6-TG resistance. The resistant cells
were counted and then analyzed by Sanger sequencing. (B) 6-TG–resistant (HPRT null) frequencies
induced by each modifier plasmid are shown. Cells were spread onto media with (+) or without (–) 6-TG
in serial dilution, and resistant colonies were counted at optimal colony density for each sample.
Experiment was repeated (Ex.1 and Ex.2) and combined to give average mutation frequency [Ave. mut(%)]. (C) Mutation alignment of the targeted HPRT gene locus. About 0.4-kb fragment around the target
site of 6-TG resistant cells were PCR-amplified and subcloned for sequencing. Frequencies of the same
mutant sequence were indicated as clone count over total reads of the same vector construct.
Mutations are shown in red. Large deletion or insertion is indicated by an asterisk. (D) Mutation spectra
obtained by transient transfection. Cells were transfected with a vector expressing Cas9 or nCas9(D10A)PmCDA1-UGI and gRNA targeting HPRT. Whole-cell culture was subjected to deep sequencing without
any selection or enrichment. Total indel frequency (Indel) within the indicated region (33 nucleotides surrounding the target site) and SNV frequencies above 0.1% at each nucleotide position are shown andhighlighted as indicated at the top right. Target sequence is shadowed and PAM sequence is inverted.

clones (10 of 10) contained mutations at bothsites, whereas phenotypic alterations were linked
to specific types of mutations (fig. S3B).
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Biallelic editing was performed by using yeast
diploid strain YPH501 (fig. S4). Occurrence of
biallelic disruption of Ade1 and Can1 were ana-

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To further assess nonspecific mutator effectsof the deaminase in this system, whole genome
sequencing was performed for each of threeclones expressing dCas9, nCas9, Cas9, PmCDA1,
dCas9-PmCDA1, and nCas9-PmCDA1 (tables S2and S3). Deaminase-expressing strains (PmCDA1,
dCas9-PmCDA1, and nCas9-PmCDA1) were foundto contain seven unique SNVs and two structuralvariants in a total of nine strains, whereas one
SNV and three structural variants were foundin a total of nine no-deaminase strains (dCas9,
nCas9, and Cas9). A slight increase in SNVs inthe deaminase-expressing strains may be attributed to the mutator effect of the deaminase.
Overexpression of deaminases are mutagenic, and
PmCDA1 expression in a yeast strain lacking
uracil DNA glycosylase (ung1-) has been shownto induce genome-wide mutation (20). On theother hand, vertebrate cells that possess AID
family proteins do not suffer from AID-related
mutagenicity at wild-type expression levels. Compared with DNA double-strand breaks, DNA deamination is a more frequent spontaneous DNA
lesion and much easier to repair precisely. Only
intensive deamination or a compromised repair
system may cause mutation. Cas9 expression did
not show obvious off-target or nonspecific mutagenic effects in yeast, possibly because random
DNA double-strand breaks may lead to cell deathin yeast and thus be underrepresented.
DNA nicking plus deamination induces
deletion in mammalian cells
To investigate the feasibility of Target-AID in mammalian cell, Chinese hamster ovary (CHO) cell wastransfected using a vector expressing Cas9, nCas9(D10A), nCas9(D10A)-PmCDA, dCas9-PmCDA,
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Off-targeting effect was assessed by introducing
single mismatches at each base position of thegRNA sequence (fig. S5). Mismatches proximal to
PAM sequence (3, 6, or 9 bases away) were nottolerated, whereas mismatches at 12 or 15 baseposition showed a reduced but detectable mutation rate (27- or 35-fold reduction, respectively).
Mismatch at the 18 base position, where deamination is expected to occur on the opposite
strand, was not tolerated. Mismatches at morethan 20 base positions showed no additional effect. These results are consistent with Cas9 specificity and thus will be improved by Cas9 protein
engineering (21).

RE S E ARCH | R E S E A R C H A R T I C L E

or nCas9(H840A)-PmCDA, with a gRNA targeting the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene. As HPRT converts apurine analog 6-thioguanine (6TG) into a toxic
derivative, homozygous disruption of the HPRT
gene confers 6TG resistance to the cell. Aftertransfection, equal numbers of cells were spreadand grown to form colonies in the presence orabsence of 6TG to calculate mutation frequency(Fig. 4A). nCas9(D10A)-PmCDA1 induced a higher
number of resistant colonies than dCas9-PmCDA1(Fig. 4B). Sequencing analysis showed base substitutions for clones from dCas9-PmCDA1, as expected (Fig. 4C). However, nCas9(D10A)-PmCDA1
induced short deletions with a central focus on
the 5′ distal side of the target sequence, where
deamination was expected to occur. This is likely
due to a combination of nick and deamination thatcannot be repaired properly in mammalian cell.
Suppression of deletion by uracil DNA
glycosylase inhibitor
To assess and bypass the repair pathway, we
introduced uracil DNA glycosylase inhibitor
protein (UGI) (22), which blocks removal ofuracil in DNA and the subsequent repair pathway. UGI was expressed as a fusion protein with
d/nCas9-PmCDA1 to demonstrate improved mutation frequency (Fig. 4B), as well as a substantial
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mismatched bases are highlighted in gray and light blue, respectively, with
nucleotide position (–22 to –11) relative to PAM sequence on the top. Thereis no mismatch in the position from –12 to –1 of the all off-target sites
examined. Total indel frequency within the 43 bases (20 bases of target
region plus upstream 10 bases and downstream 13 bases, including PAM)
is shown in the second column from the left. SNV frequencies above 0.1%
at each nucleotide position (–22 to –11) are separately shown with eachmutant nucleotides. Mutation frequencies are highlighted as indicated at
the bottom right.

change in the mutation spectra, showing a majority of C to T substitutions instead of deletions (Fig. 4C, nCas9(D10A)-PmCDA1-UGI). Pulse
incubations at 25°C after transfection improved
the mutation frequency to 59.9 and 6.71% fornCas9-PmCDA1 and dCas9-PmCDA1, respectively (PmCDA1 is originally from sea lamprey,
which is presumably adapted to low temperatures).
Transient transfection was tested by harvesting thecell culture 3 days after transfection without
any marker selection. Deep sequencing showedthat nCas9-PmCDA1-UGI induced 8.4, 11, and5.1% of C to T base substitutions at –19, –20 and–21 positions, respectively, whereas Cas9 induced
20.8% indels in the same condition (Fig. 4D).
Off-target assessment in mammalian cells
General effectiveness and potential off-target
effect was assessed by using three target loci—
MGAT1 (23), Efemp1 (24), and EMX1 (25)—that
were previously tested as CRISPR/Cas9 targets
in CHO cells or in human cells, in addition to
HPRT locus. After G418 selection for transformants, cells were harvested and subjected to deep
sequencing. Potential off-target genomic regions
were selected using CCtop (http://crispr.cos.uniheidelberg.de/index.html) (26) (tables S6 to S9),and the top six off-target sites were analyzed(Fig. 5 and figs. S6 to S9). Compared with nCas9-

PmCDA1, which induced indels as well as SNVs,
nCas9-PmCDA1-UGI effectively induced C to T
mutations within the –15 to –21 region of the fourtarget sites with a lower frequency of indels.
dCas9-PmCDA1-UGI showed fewer indels anda moderate frequency of C to T mutations. There
were possible off-target mutations detected
at lower than 1.5% for Target-AID throughout
the analyzed off-target sites (Fig. 5 and figs. S5
to S8).
Conclusion
We demonstrated targeted nucleotide substitution
without the use of template DNA by expanding
the potential of the CRISPR/Cas9 system. The
Target-AID performed a very narrow range oftargeted nucleotide substitution within a few
bases. Its reduced cytotoxicity will be beneficial, especially for use in bacteria and other cell
types that are sensitive to artificial nucleases(16). A combination of nickase Cas9(D10A) andPmCDA1 was highly efficient in yeast, whereas
it induced deletions as well as point mutations
in mammalian cells, suggesting that the mutation spectrum is affected by the difference of therepair pathway. The rat apolipoprotein B mRNA
editing enzyme, catalytic polypeptide (rAPOBEC1),
another cytidine deaminase, has also been reported to serve as a programmable base editor

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Fig. 5. Deep-sequencing analysis of target and off-target mutations
induced by Cas9 and a series of Target-AID in CHO cells. Using theindicated modifier vector (left column) expressing gRNA targeting HPRT1,
EMX1, Efemp1, or MGAT1, transformed cell culture was analyzed 7 days after
transfection. The on-target site (yellow) and the top six off-target sites determined by CCtop (http://crispr.cos.uni-heidelberg.de/index.html) as listed
in tables S5 to S8 were analyzed by deep sequencing. Full sets of off-target
analysis are shown in figs. S5 to S8, whereas the most highly mutated offtarget sites (green) for each target site are shown here. Target sequence andR ES E A RC H | R E S EA R C H A R T I C LE

Materials and methods
DNA manipulation
Plasmids available from Addgene are listed intable S4. The human-optimized Streptococcus
pyogenes Cas9 gene-containing plasmid p415GalL-Cas9-CYC1t and the chimeric gRNA–containing
plasmid p426-SNR52p-gRNA.CAN1.Y-SUP4t (13)were kindly provided by Church laboratory andobtained from Addgene (Cambridge, MA, USA).
Nuclease-deficient Cas9 D10A and H840A mutations were introduced using a PCR-based method. The target gRNA sequences were replacedusing a PCR method. A glycine-serine peptide
linker (GGGGS), a SH3 domain, and a 3xFlag tag(DYKDHDGDYKDHDIDYKDDDDK) were addedat the C terminus after the SV40 nuclear localization signal (NLS) (PKKKRKV) in Cas9. The
DNA coding sequences for PmCDA1, the SH3
domain, and the 3xFlag tag were synthesized(Eurofin Genomics, Tokyo, JPN). The DNA fragments were assembled and modified using standard methods. The nucleotide sequence for theentire coding region is in fig. S10.
For CHO transfection plasmid, human codonoptimized PmCDA1 was synthesized on pcDNA3.1
backbone (Genscript, NJ, USA). Human-optimized
Cas9 coding sequence was inserted downstreamof CMV promoter, followed by SV40 NLS, deadSH3 domain, 3xFlag, PmCDA1, and UGI by PCR
sewing and Gibson assembly method. Neomycinresistant gene was linked via 2A peptide (EGRGSLLTCGDVEENPGP) at the end of the coding region.
The nucleotide sequence for the entire coding
sequence is in fig. S11.
Targeted mutagenesis
Saccharomyces cerevisiae BY4741 (MATa his3D0
leu2D0 met15D0 ura3D0) (Open Biosystems,
Huntsville, AL, USA) was used in this study. Yeast
culture and transformation were performed
aaf8729-6

according to standard methods. For the mutational analysis, the yeast cells were grown at
30°C in synthetic complete media with anindicated carbon source and an appropriate
auxotrophic compound complemented by theplasmids. The cells were first inoculated in a
2% glucose medium and grown to saturation,
diluted 16-fold into a 2% raffinose medium,and grown to saturation again. The cells werethen diluted 16-fold or 32-fold into 2% raffinose
medium plus 0.2 or 0.02% galactose medium forinduction. To calculate the mutation rate per
generation, cell culture was diluted 32-fold every
24 hours. After the indicated time, the cell culture was sampled and serially diluted 10-fold inwater and spotted or spread onto a synthetic
medium plate supplemented with the appropriate dropout mix and the appropriate drug. For
CAN1 mutational analysis, arginine was omitted
and 60 mg/l canavanine was added. For LYP1
mutational analysis, lysine was omitted and100 mg/l of S-aminoethyl-L-cysteine was added.
The mutation frequency of ADE1 was counted bycolony color because the loss of function of ade1
results in the accumulation of red pigment whensubjected to an adenine-limited condition. The
plates were incubated at 28°C for 2 to 3 days andthe colonies were counted. Sample size was determined based on countable colony number in asingle spot (up to 40) or dish (up to 400). The t
test statistical analysis was done using Excel
software (Microsoft, WA, USA). The plate images
were acquired using an Image Quant LAS 4000
(GE Healthcare Japan, Tokyo, Japan) or a Cybershot DSC-WX100 digital camera (Sony, Tokyo,
Japan) and were processed using Adobe CS2
software. For the sequencing analysis, the averagesized colonies were randomly picked and directly
amplified by PCR and were analyzed by Sanger
sequencing using a 3130xL Genetic Analyzer(Applied Biosystems, CA, USA) according to the
manufacturer’s instructions.
Viability test
Yeast cells were precultured in synthetic complete media with 2% raffinose minus leucine anduracil for overnight, then diluted by 32-fold andsupplemented with 0.02% galactose to induce
protein expression. Cells were harvested at eachtime point. Ten-fold serial dilution of harvested
culture was spotted onto synthetic complete media
minus leucine and uracil and observed for colony
formation for 2 days.
Mutation spectra analysis
Yeast strains harboring galactose-inducible modifier gene plasmid (Cas9, nCas9, dCas9, dCas9-CDA1,
nCas9-CDA1, or CDA1) containing LEU2 marker
and gRNA-expressing plasmid containing Ura3
marker were precultured in 5 ml SC–Leu–Ura+
Ade media containing 2% galactose overnightat 30°C with rotation. Each cell sample waswashed once with SC–Leu–Ura+Ade media containing 2% raffinose, resuspended in the sameraffinose media at 0.1 OD600 nm, and incubatedovernight at 30°C with rotation. For the modifier
induction, cells were resuspended into SC–Leu–

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Ura+Ade media containing 2% galactose at 0.1
OD600 nm and incubated overnight at 30°C
with rotation. Each sample was then spread on
SC–Leu–Ura–Arg+Ade and SC–Leu–Ura–Arg+Ade
containing 60 mg/ml canavanine plates. From eachplate, colonies were scraped and pooled withsterile water and genomic DNA was extractedusing YeaStar Genomic DNA Kit (Zymo Research).
Target genomic locus was amplified by PCR withprimers containing common sequence ends andreamplified by common Illumina adapter primers
containing index tags for sample multiplexing.
PCR bands were size-selected using E-Gel SizeSelect
2% Agarose Gel (Thermo Fisher Scientific) andquantified by KAPA Library Quantification Kits
for Illumina (KAPA Biosystems). Sequencing libraries were multiplexed at equal molar ratio
and analyzed by Illumina MiSeq Reagent Kit V2(250×250 bp paired-end sequencing). Sequencereads were aligned to the primer sequences andthe target genomic regions using BLAST+ program (short-read option; E-value cutoff of 1e–3)
to demultiplex the read samples and to identify
mutations and indels. Fractions of mutations, baseinsertions and deletions were separately calculated
for every base position of target genomic regionand subtracted by those of the PmCDA1 dataset
derived from corresponding screening condition
to normalize sequencing errors.
Whole-genome sequencing
Yeast strains harboring each expression construct(dCas9, nCas9, Cas9, PmCDA1, dCas9-PmCDA1,
and nCas9-PmCDA1) with gRNA targeting Can1
were preincubated in 2% raffinose medium overnight, diluted by 32-fold into 0.02% galactose
medium for induction and grown overnight. Cells
were spread onto canavanine-containing platemedium to isolate single colonies. Each three
independent colonies were inoculated into YPDA
medium containing 300 ug/ml canavanine. Overnight culture was collected and genomic DNA wasextracted using Wizard Genomic DNA purification Kit (Promega, WI, USA) following manufactures’ instructions. Genomic DNA was thenfragmented by sonication using Bioruptor UCD200 TS Sonication System (Diagenote, NJ, USA)to obtain fragments with size distribution centering at 500 to 900 bp. Genomic DNA library was
prepared by using NEBNext Ultra DNA Library
Prep Kit for Illumina (New England Biolabs, MA,
USA) and labeled by Dual Index Primers Set 1.
Size selection of the library was done using
Agencourt AMPure XP (Beckman Coulter, CA,
USA) to obtain tagged fragments with length
ranging from 600 to 800 bp. Size distribution
was evaluated by the Agilent 2100 Bioanalyzer
system (Agilent Technologies, CA, USA). DNA
was quantified using Qubit HS dsDNA HS Assay
Kit and fluorometer (Thermo Fisher Scientific,
MA, USA). Sequencing was performed using MiSeq
sequencing system (Illumina, CA, USA) and MiSeq
Reagent Kit v3 to obtain 2 × 300 bp read length,
expecting approximately 30-fold coverage for the
genome size. The sequence reads were paired andoverlapping reads within a read pair were merged
then trimmed based on a quality limit of 0.001
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(BE) by fusing it to the N terminus of dCas9 (27).
Consistent with our study, the use of UGI fusion
and D10A nickase increased efficiency in humancells. There are also different features as well.
Using Target-AID, three to five bases surrounding the –18 position upstream of the PAM sequence are substantially subjected to mutation,
whereas five bases surrounding the –15 position
upstream of PAM (or position 4 to 8 from theend distal to the PAM of a 20-base protospacer)
are the major target for BE. This may be attributed to the difference of the enzymatic characteristics (PmCDA1 versus rAPOBEC1) or thedifferent attachments to Cas9 (C terminus versus
N terminus). Possible RNA-editing activity is also
to be assessed, because APOBEC proteins haveRNA-editing activity physiologically (2). Additionally, PmCDA1 exhibited temperature sensitivity that can be exploited to control its activity.
Although direct comparison will be needed, these
two systems may complement each other to extend the repertoire of possible editing sites.
Using other CRISPR-related systems or othermodifier enzymes, such as adenosine deaminase, will also broaden the editing capacity andfurther enrich the genome editing toolbox.

RE S E ARCH | R E S E A R C H A R T I C L E

CHO cell experiments
CHO-K1 adherent cells (ECACC 85051005) wereused in this study. Cells were cultured in Ham’s
F12 medium (Life Technologies, Carlsbad, CA,
USA) supplemented with 10% fetal bovine
serum (Biosera, Nuaille, France) and 100 mg/mL
Penicilline-Streptomycin (Life Technologies,
Carlsbad, CA, USA) at 37°C with a humidified
5% CO2 atmosphere. For the transfection, cells
were plated at 0.5×105 cells per well of 24-wellplates and cultured for 1 day. The cells were transfected with 1.5 mg plasmid and 2 mL Lipofectamine
2000 (Life Technologies, Carlsbad, CA, USA)per well according to manufacturer’s instructions. Five hours after transfection, the medium
was changed to Ham’s F12 medium containing
0.125 mg/mL G418 (InvivoGen, San Diego, CA,USA), and the cells were incubated for 7 days. For
pulse incubation at 25°C, the cells were firsttransfected and incubated at 37°C for 1 day. Cells
were then transferred to 25°C and incubatedfor 1 day, followed by 37°C incubation for 2 days.
This process was repeated twice. To calculate themutation frequency of the HPRT gene, the cells
were released from the well using trypsin-EDTA(Life Technologies, Carlsbad, CA, USA) and seriallydiluted and spread to obtain countable colonydensity onto Ham’s F12 medium containing G418
or G418 plus 5 g/mL 6-TG (Tokyo Chemical Industry, Tokyo, Japan). After 7 days, the numberof resistant colonies was counted. The mutation
frequency was calculated as the rate of 6TG reSCIENCE sciencemag.org

sistant colonies over G418 resistant colonies. For
sequencing analysis, cells were trypsinized andpelleted by centrifugation. Genomic DNA was extracted from the pellets using the NucleoSpin Tissue
XS kit (Macherey-Nagel, Düren, Germany) according
to the manufacturer’s instructions. PCR fragments
including HPRT target site was amplified from thegenomic DNA using a forward primer GGCTACATAGAGGGATCCTGTGTCA and a reverse primer
ACAGTAGCTCTTCAGTCTGATAAAA. PCR products
were cloned into E.coli vector and analyzed bySanger sequencing.
Deep sequencing of target and off-target
region of CHO cells
Target region-containing fragment (~1.5 kb)
was first PCR-amplified using 1st primer pairs
from the extracted genomic DNA. The second
nested PCR was performed to obtain adapteradded amplicon (~0.3 kb) by using the first PCR
products as template and 2nd primer pairs containing adapter sequences. The primes used inthis study are listed in table S5. The amplicon
was labeled using NEBNext Multiplex Oligos forIllumina (Index Primers Set 1 and Dual Index
Primers Set 1) (New England Biolabs, MA, USA).
Deep sequencing was performed using MiSeq
sequencing system (Illumina, CA, USA) to obtainpaired 300 bp read length and more than
100,000 reads per sample on average.
Data analysis was done using CLC Genomics
Workbench 7.0 (CLC bio, Aarhus, Denmark). The
sequence reads were paired and trimmed basedon a quality limit of 0.05 with a maximum of
two ambiguities and then overlapping reads
within a read pair were merged. Reads were
mapped to each reference sequence obtained
from CHOgenome.org (www.chogenome.org/)by the following setting (Masking mode = nomasking, Mismatch cost = 2, Insertion cost = 3,
Deletion cost = 3, Length fraction = 0.2, Similarity fraction = 0.5, Global alignment = No, Autodetect paired distances = Yes, Nonspecific match
handling = ignore). The samples containing lowquality reads (Efem1 on-target, Efemp1 off-target
5, MGAT1 off-target 1 and EMX1 off-target 4)
were reanalyzed by the following setting instead.
The sequence reads were paired and overlapping reads within a read pair were merged thentrimmed based on a quality limit of 0.01 with amaximum of 2 ambiguities. Reads were mappedwith the following settings (Masking mode =no masking, Mismatch cost = 2, Insertion cost = 3,
Deletion cost = 3, Length fraction = 0.6, Similarity fraction = 0.9, Global alignment = No,
Auto-detect paired distances = Yes, Nonspecificmatch handling = ignore). The variant calling
was performed with the following settings (Ignore positions with coverage = 1,000,000, Ignore
broken pairs = No, Ignore Nonspecific matches =Region length 50, Minimum coverage =10, Minimum count = 2, Minimum frequency = 0.1%, Basequality filter = No, Read detection filter = Yes,
Relative read direction filter = 1%, Significance =1%, Read position filter = 1%, Remove pyro-error
variants = No). Output file was rearranged using
Excel (Microsoft, WA, USA).

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Downloaded from https://www.science.org on January 25, 2024with a maximum of 2 ambiguities. Reads were
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Ministry of Economy, Trade and Industry (METI), Japan, by JSPS
KAKENHI grant numbers 26119710 and 16K14654, and by
Cross-ministerial Strategic Innovation Promotion Program (SIP);‘Technologies for creating next-generation agriculture, forestry
and fisheries.’ Patents have been filed related to this work.
SUPPLEMENTARY MATERIALSACKN OWLED GMEN TS

We thank M. Yajima for helpful discussion. This work was supported
by a Special Coordination Fund for Promoting Science and Technology,
Creation of Innovative Centers for Advanced Interdisciplinary Research
Areas (Innovative Bioproduction Kobe) from the Ministry of Education,
Culture, Sports and Technology (MEXT) of Japan. This work was also
partly supported by the commission for Development of Artificial Gene
Synthesis Technology for Creating Innovative Biomaterial from thewww.sciencemag.org/content/353/6305/aaf8729/suppl/DC1
Materials and Methods
Figs. S1 to S11
Tables S1 to S9References
13 April 2016; accepted 25 July 2016
Published online 4 August 201610.1126/science.aaf8729

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