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BIOTECHNOLOGYEngineered CRISPR-Cas9 nucleasewith expanded targeting space
Hiroshi Nishimasu1*, Xi Shi2,3, Soh Ishiguro4,5,6, Linyi Gao2,7, Seiichi Hirano1,
Sae Okazaki1, Taichi Noda8, Omar O. Abudayyeh2,3,9, Jonathan S. Gootenberg2,3,9,Hideto Mori4,5,6, Seiya Oura8,10, Benjamin Holmes2,3, Mamoru Tanaka4, Motoaki Seki4,
Hisato Hirano1, Hiroyuki Aburatani4, Ryuichiro Ishitani1, Masahito Ikawa8,10,11,Nozomu Yachie1,4,5,6, Feng Zhang2,3,7,9, Osamu Nureki1*1T

he CRISPR RNA-guided endonuclease Cas9
cleaves double-stranded DNA targets complementary to the RNA guide (1) (fig. S1) andhas been harnessed for genome editing ineukaryotic cells (2). However, the widely used
Cas9 from S. pyogenes (SpCas9) strictly recognizes
an NGG sequence (where N is any nucleobase) as
the protospacer adjacent motif (PAM) (3), thereby
restricting the targetable genomic loci. Structureguided directed evolution approaches to address
this limitation yielded several SpCas9 variants
with altered PAM specificities, such as the SpCas9
VQR and VRER variants, which recognize theNGA and NGCG PAMs, respectively (4). In addition, Cas9 and Cas12a (also known as Cpf1)
enzymes with distinct PAM specificities, suchas Staphylococcus aureus Cas9 (SaCas9) (5),Acidaminococcus sp. Cas12a (AsCas12a), andLachnospiraceae bacterium Cas12a (LbCas12a) (6),have extended the targeting range in CRISPRCas–mediated genome editing.
To expand the targeting range of CRISPRCas9, we sought to engineer a SpCas9 variant
with relaxed preferences for the third nucleobaseof the PAM. Previous studies revealed that thesecond and third nucleobases in the NGG PAM
are recognized by Arg1333 and Arg1335 of SpCas9,
respectively (7) (fig. S2). We thus hypothesized that
the PAM constraint can be reduced by eliminating the base-specific interaction between Arg1335
and the third G, and compensating for the loss of
this base-specific interaction by introducing non–base-specific interactions with the PAM duplex.
We first measured the in vitro cleavage activities

Department of Biological Sciences, Graduate School ofScience, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan. 2Broad Institute of MIT and Harvard,Cambridge, MA 02142, USA. 3McGovern Institute for Brain
Research, Cambridge, MA 02139, USA. 4Research Center for
Advanced Science and Technology, The University ofTokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
5Institute for Advanced Biosciences, Keio University, 14-1 Babacho, Tsuruoka, Yamagata 997-0035, Japan. 6Graduate Schoolof Media and Governance, Keio University, 5322 Endo,Fujisawa, Kanagawa 252-0882, Japan. 7Department of
Biological Engineering, Massachusetts Institute ofTechnology, Cambridge, MA 02139, USA. 8Research Institutefor Microbial Diseases, Osaka University, 3-1 Yamadaoka,Suita, Osaka 565-0871, Japan. 9Department of Brain andCognitive Sciences, Massachusetts Institute of Technology,Cambridge, MA 02139, USA. 10Graduate School of
Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka,
Suita, Osaka 565–0871, Japan. 11Institute of Medical Science,
The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku,
Tokyo 108–8639, Japan.
*Corresponding author. Email: nisimasu@bs.s.u-tokyo.ac.jp (H.N.);
nureki@bs.s.u-tokyo.ac.jp (O.N.)

Fig. 1. In vitro cleavage activity. (A) SDS–polyacrylamide gel electrophoresis analysis of wild-type SpCas9, SpCas9-NG, and xCas9.
(B, C, and E) In vitro DNA cleavage activities of wild-type SpCas9 (B), SpCas9-NG (C), and xCas9 (E) toward the TGN PAM targets. Data
are means ± SD (n = 3). (D) PAM preference of SpCas9-NG.

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The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genomeediting tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9)
requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby
restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed
that the loss of the base-specific interaction with the third nucleobase is compensatedby newly introduced non–base-specific interactions, thereby enabling the NG PAM
recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing
NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG andthe activation-induced cytidine deaminase (AID) mediates the C-to-T conversion attarget sites with NG PAMs in human cells.

of purified wild-type SpCas9 and the R1335A(Arg1335 → Ala) mutant toward a target plasmidwith the TGG PAM and confirmed that, whereas
wild-type SpCas9 efficiently cleaves the TGG target,
R1335A has almost no activity (fig. S3, A to C).
We next examined whether the R1335A activity is restored by the substitution of residues surrounding the PAM duplex, and found that thereplacements of Leu1111, Gly1218, Ala1322, and Thr1337
with Arg partially restored the activity of theR1335A mutant (fig. S3, A to C). Furthermore,
the R1335A/L1111R/G1218R/A1322R/T1337R variant (referred to as ARRRR) efficiently cleaved theTGG target (fig. S3, A to C). However, the cleavage
kinetics of ARRRR was slower than that of wildtype SpCas9 (fig. S3, D and E). In the previously
reported VQR (D1135V/R1335Q/T1337R) and VRER
(D1135V/G1218R/R1335E/T1337R) variants, theD1135V mutation provides interactions with thesugar-phosphate backbone of the PAM duplex(8, 9). In addition, molecular modeling suggested
that the E1219F mutation forms hydrophobic interactions with the ribose moiety of the second G,
and that the R1335V mutation stabilizes Arg1333

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Fig. 2. Crystal structure of SpCas9-NG.
(A) Recognition of the PAM duplex.
Arg1333 and the substituted residues
are shown as stick models. (B) Non–base-specific interactions between
the PAM duplex and the substituted
residues. TS, target strand; NTS,
nontarget strand.

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Fig. 3. Gene editing in human cells. (A) Indel formation efficiencies ofwild-type SpCas9, SpCas9-NG, and xCas9 at the 69 endogenous target
sites in HEK293FT cells. Data are means ± SD (n = 3). (B) Summary of theediting efficiencies of SpCas9, SpCas9-NG, and xCas9. Medians and first andNishimasu et al., Science 361, 1259–1262 (2018)

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third quartiles are shown. In (A) and (B), 20% indel frequency is indicatedby dashed lines. (C) Specificities of wild-type SpCas9, SpCas9-NG, andthe enhanced-specificity versions of SpCas9 (ES) and SpCas9-NG (NG-ES).
The off-target cleavages were evaluated by GUIDE-seq.

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Fig. 4. Base editing in human cells.
(A and B) C-to-T conversion efficiencies
at the 20 endogenous target sites(Target-AID and Target-AID-NG) (A)and at the 12 poly-C–containing targetsites (Target-AID, Target-AID-NG, andxCas9-BE4) (B) in HEK293T cells.
The experiments were performedat least twice, and similar results
were obtained.

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TGC targets (fig. S9, D and G); nonetheless, thecleavage kinetics of xCas9 was slower than thatof SpCas9-NG (fig. S9, C, D, F, and G), and thecleavage activity of xCas9 toward the TGA, TGT,and TGC targets was comparable to or lower thanthat of wild-type SpCas9 toward the TGA target(fig. S9, B, D, E, and G). These results demonstratethat SpCas9-NG outperforms xCas9 in recognizing NGH PAMs in vitro.
To clarify the NG PAM recognition mechanism,
we determined the crystal structure of SpCas9NG, in complex with a single guide RNA and its
target DNA containing the TGG PAM, at 2.7 Åresolution (fig. S10A and table S1). The second G
in the PAM (dG2*) forms bidentate hydrogenbonds with Arg1333 (Fig. 2A), which is stabilizedby Val1335 (R1335V). The third G (dG3*) forms ahydrogen bond with Arg1337 (T1337R) (Fig. 2A),
consistent with the preference of SpCas9-NG
for the third G (Fig. 1C). Arg1111 (L1111R), Val1135(D1135V), and Phe1219 (E1219F) interact with thesugar-phosphate backbone of the PAM duplex,
whereas Arg1218 (G1218R) does not directly interact with the DNA backbone (Fig. 2B). A structuralcomparison with the SpCas9 R-loop complex (12)suggested that Arg1322 (A1322R) interacts withthe nontarget DNA strand (fig. S10B).
To assess the activity of SpCas9-NG in mammalian cells, we next measured the formation ofindels (insertions or deletions) induced by wildtype SpCas9, SpCas9-NG, and xCas9 at 69 endogenous target sites with NGN PAMs in humanembryonic kidney (HEK) 293FT cells (table S2).
As expected, wild-type SpCas9 induced indels predominantly at the NGG sites, with some recognition of NGA sites (Fig. 3, A and B). Wild-type
SpCas9 achieved a >20% indel rate at NGG (17 of17 sites) and NGA sites (3 of 19 sites), but not
NGT (0 of 18 sites) or NGC sites (0 of 15 sites)

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(Fig. 3, A and B). In contrast, SpCas9-NG edited
NGA, NGT, and NGG sites, with lower activity atNGC (Fig. 3, A and B), consistent with the in vitrocleavage preference for the third D (D = A, T, or G)
PAM nucleotide (Fig. 1C). SpCas9-NG achieved a>20% indel rate at 13 NGG, 13 NGA, 15 NGT, and 5
NGC sites (Fig. 3, A and B). Relative to SpCas9NG, xCas9 had lower editing efficiency at all of
the NGH sites (Fig. 3, A and B), consistent with
our in vitro cleavage data (Fig. 1E). xCas9 induced
a >20% indel rate at 15 NGG sites, five NGA sites,
and four NGT sites, but none at NGC sites (Fig. 3,A and B).
Using GUIDE-seq (genome-wide, unbiased identification of double-stranded breaks enabled bysequencing) (13), we examined the specificity ofwild-type SpCas9 and SpCas9-NG in human cells
at two previously characterized target sites (EMX1and VEGFA). We found that wild-type SpCas9 andSpCas9-NG have comparable numbers of off-target
sites for the EMX1 target, and that the K848A/
K1003A/R1060A mutations in the high-fidelity
SpCas9 variant [eSpCas9(1.1)] (14) substantiallyreduced the off-target cleavage by SpCas9-NG (Fig.
3C and fig. S11). These results show that SpCas9-NG
has cleavage specificity comparable to that ofwild-type SpCas9, and that its specificity can beenhanced by the high-fidelity mutations. As expected, SpCas9-NG had a different off-target profile with off-target sites harboring NGH PAMs
(fig. S11), further confirming the relaxed PAM
recognition.
The nuclease-inactive version of SpCas9 can beapplied to numerous technologies such as baseediting (15, 16). We examined whether the SpCas9NG D10A nickase fused to the activation-induced
cytidine deaminase (nSpCas9-NG–AID, referred
to as Target-AID-NG) mediates C-to-T conversion
at 32 endogenous target sites with NG PAMs in
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and Phe1219 (E1219F) (fig. S3F). Indeed, the additionof the D1135V, E1219F, and R1335V mutations
enhanced the cleavage activity (fig. S3, D and E).
We designated the R1335V/L1111R/D1135V/G1218R/
E1219F/A1322R/T1337R variant as VRVRFRR.
We next measured the cleavage activities ofVRVRFRR toward the target plasmid with TGN
PAMs. Relative to wild-type SpCas9, VRVRFRR
slowly but more efficiently cleaved the TGA, TGT,and TGC targets (Fig. 1, A to C, and fig. S4). Although VRVRFRR is less active than wild-type
SpCas9 and SaCas9, its cleavage activity wascomparable to those of AsCas12a and LbCas12a(5, 6) (fig. S5). Using an in vitro PAM identification assay (10), we confirmed that whereas wildtype SpCas9 is specific to NGG PAMs, VRVRFRR
preferentially recognizes NG PAMs (Fig. 1D andfig. S6). Although the PAM identification assayrevealed that VRVRFRR slightly recognizes NAN
PAMs (Fig. 1D), in vitro cleavage experiments demonstrated that VRVRFRR is less active toward
TAN PAMs than toward TGN PAMs (fig. S7). Thus,
we concluded that VRVRFRR recognizes a relaxed
PAM, and we refer to this variant as SpCas9-NG, as
it has increased activity on NGH (H = A, T, or C)
PAMs, albeit with reduced relative activity on NGC.
We compared SpCas9-NG with the xCas9 enzyme (a SpCas9 variant with the A262T/R324L/
S409I/E480K/E543D/M694I/E1219V mutations),
which was engineered via directed evolution andrecognizes NG PAMs (11) (fig. S8). We measured
their in vitro cleavage activities toward the target
plasmid with the TGN PAMs. Under our assay
conditions (50 nM Cas9), xCas9 showed almost
no activity toward the TGA, TGT, and TGC targets, and it cleaved the TGG target with lower
efficiency than did SpCas9-NG (Fig. 1, A to C
and E, and fig. S9A). At a higher concentration(200 nM Cas9), xCas9 cleaved the TGA, TGT, andR ES E A RC H | R E PO R T

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RE FERENCES AND NOTES

1. M. Jinek et al., Science 337, 816–821 (2012).
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3. S. H. Sternberg, S. Redding, M. Jinek, E. C. Greene,
J. A. Doudna, Nature 507, 62–67 (2014).
4. B. P. Kleinstiver et al., Nature 523, 481–485 (2015).
5. F. A. Ran et al., Nature 520, 186–191 (2015).
6. B. Zetsche et al., Cell 163, 759–771 (2015).
7. C. Anders, O. Niewoehner, A. Duerst, M. Jinek, Nature 513,569–573 (2014).
8. C. Anders, K. Bargsten, M. Jinek, Mol. Cell 61, 895–902 (2016).
9. S. Hirano, H. Nishimasu, R. Ishitani, O. Nureki, Mol. Cell 61,886–894 (2016).
10. L. Gao et al., Nat. Biotechnol. 35, 789–792 (2017).
11. J. H. Hu et al., Nature 556, 57–63 (2018).
12. F. Jiang et al., Science 351, 867–871 (2016).
13. S. Q. Tsai et al., Nat. Biotechnol. 33, 187–197 (2015).
14. I. M. Slaymaker et al., Science 351, 84–88 (2016).
15. A. C. Komor, Y. B. Kim, M. S. Packer, J. A. Zuris, D. R. Liu,Nature 533, 420–424 (2016).
16. K. Nishida et al., Science 353, aaf8729 (2016).
ACKN OWLED GMEN TS

We thank A. Kurabayashi for assistance with sample preparation,
T. Yamano for Cas12a preparation, B. P. Kleinstiver andJ. K. Joung for assistance with GUIDE-seq, and the beamline
scientists at BL41XU at SPring-8 for assistance with datacollection. Funding: Supported by JST (JPMJPR13L8) and
JSPS (26291010 and 15H01463) (H.N.); JSPS (16J06287)(S.I.); JSPS (17H01394) , AMED (JP18gm5010001), Takeda
Science Foundation, NICHD (P01HD087157 and R01HD088412),
and the Bill & Melinda Gates Foundation (OPP1160866) (M.I.); NEDO(Genome Editing Program), AMED (17g6110007h0002), JST(10814) and JSPS (18H02428) (N.Y.); NIMH (5DP1-MH100706
and 1R01-MH110049), NIDDK (5R01DK097768-03), and the21 September 2018

New York Stem Cell, Simons, Paul G. Allen Family, and Vallee
foundations (F.Z.); AMED (16am0301002h0003) and CSTI, SIP(O.N.). F.Z. is a New York Stem Cell Foundation Robertson
Investigator. Author contributions: H.N. designed the variants,performed in vitro cleavage experiments, and determined thecrystal structure; X.S., L.G., O.O.A., J.S.G., B.H., and F.Z.
performed PAM screens, indel detection, and GUIDE-seq
analyses; S.I., H.M., M.T., M.S., H.A., and N.Y. performedbase-editing analyses; T.N., S.Ou., and M.I. performed indeldetection; S.H. performed crystallization; H.N., S.H., S.Ok., andH.H. prepared the proteins; R.I. assisted with the structural
determination; H.N. wrote the manuscript with help from allauthors; and H.N. and O.N. supervised the research. Competinginterests: H.N. is a scientific adviser for EdiGENE. F.Z. is aco-founder and scientific adviser for Editas Medicine, PairwisePlants, Beam Therapeutics, and Arbor Biotechnologies. F.Z.
serves as a director for Beam Therapeutics and Arbor
Biotechnologies. O.N. is a co-founder, board member, andscientific adviser for EdiGENE. H.N., H.H., and O.N. have filed apatent application related to this work. Data and materials
availability: The atomic coordinates of SpCas9-NG have been
deposited in the Protein Data Bank with PDB code 6AI6. All data
are available in the manuscript or the supplementary material.
SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/361/6408/1259/suppl/DC1Materials and Methods
Figs. S1 to S14
Tables S1 to S3
References (17–24)
4 January 2018; accepted 16 August 2018
Published online 30 August 201810.1126/science.aas9129

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human cells (table S3). nSpCas9–AID (TargetAID) efficiently induced C-to-T conversion atthe NGG sites, with lower activity at NGA sitesand no activity at NGT or NGC sites (Fig. 4Aand figs. S12A and S13). In contrast, TargetAID-NG showed base-editing activity toward all
PAMs assessed, albeit with lower efficiency atNGC sites (Fig. 4A and figs. S12A and S13), consistent with the indel data (Fig. 3A). In addition,
Target-AID-NG showed higher base-editing efficiencies than did the xCas9 D10A nickase fusedto the APOBEC1 cytidine deaminase (xCas9-BE4)
at most of the tested poly-C–containing targetsites (Fig. 4B and figs. S12B and S13). These results demonstrate that the catalytically inactive
version of SpCas9-NG can serve as a useful RNAguided DNA targeting platform.
In this study, we engineered an SpCas9-NG
variant with an increased targeting range (fig. S14).
Nonetheless, the cleavage activity of SpCas9-NG
is lower than that of wild-type SpCas9 at NGG
sites, and SpCas9-NG is less active at NGC sites
relative to NGD sites. Thus, it will be important
to improve the activity of SpCas9-NG by further
molecular engineering. Overall, the rationally designed SpCas9-NG and its high-fidelity variants
can serve as useful genome-editing tools with increased versatility across genomes.