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Articlehttps://doi.org/10.1038/s42003-024-06422-z

Structure and engineering of Brevibacillus
laterosporus Cas9
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Toshihiro Nakane1,10, Ryoya Nakagawa1,10, Soh Ishiguro2, Sae Okazaki3, Hideto Mori4,5,6, Yutaro Shuto
Keitaro Yamashita 3, Nozomu Yachie2,6,7, Hiroshi Nishimasu 3,8,9 & Osamu Nureki 1

1,

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets complementary to
an RNA guide, and is widely used as a powerful genome-editing tool. Here, we report the crystal
structure of Brevibacillus laterosporus Cas9 (BlCas9, also known as BlatCas9), in complex with a guide
RNA and its target DNA at 2.4-Å resolution. The structure reveals that the BlCas9 guide RNA adopts an
unexpected architecture containing a triple-helix, which is speciﬁcally recognized by BlCas9, and that
BlCas9 recognizes a unique N4CNDN protospacer adjacent motif through base-speciﬁc interactions
on both the target and non-target DNA strands. Based on the structure, we rationally engineered a
BlCas9 variant that exhibits enhanced genome- and base-editing activities with an expanded target
scope in human cells. This approach may further improve the performance of the enhanced BlCas9
variant to generate useful genome-editing tools that require only a single C PAM nucleotide and can bepackaged into a single AAV vector for in vivo gene therapy.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats and
CRISPR-associated proteins) systems provide adaptive immunity against
mobile genetic elements in bacteria and archaea1. Cas9 from Streptococcus
pyogenes (SpCas9) associates with dual RNA guides (CRISPR RNA [crRNA]
and trans-activating crRNA [tracrRNA] or their artiﬁcially connected
single-guide RNA [sgRNA]) and cleaves double-stranded DNA (dsDNA)
targets complementary to the RNA guide, using its HNH and RuvC nucleasedomains2,3. Besides the guide RNA–target DNA complementarity, SpCas9
requires an NGG (where N is any nucleotide) protospacer adjacent motif(PAM), located downstream of the target sequence3. Since SpCas9 with its
sgRNA can target endogenous genomic sites in a wide range of cell types andorganisms, it has been widely used for numerous technologies, such as
genome editing, transcriptional regulation, and epigenetic modulation4.
Cas9 orthologs from different microbes function with their cognate guideRNAs, and recognize a variety of PAM sequences5,6. Thus, the use of Cas9
orthologs expands the target range in Cas9-mediated genome engineering.
Structural studies of various Cas9 orthologs, such as SpCas97,8, Staphylococcus aureus Cas9 (SaCas9)9, Francisella novicida Cas9 (FnCas9)10,

Campylobacter jejuni Cas9 (CjCas9)11, Corynebacterium diphtheriae Cas9
(CdCas9)12, Neisseria meningitidis Cas9 (NmCas9)13, and Streptococcus
thermophilus Cas9 (St1Cas9)14, have highlighted the mechanistic conservation of the CRISPR-Cas9 enzymes. Cas9 enzymes commonly adopt abilobed architecture consisting of recognition (REC) and nuclease (NUC)
lobes, with the guide RNA–target DNA heteroduplex accommodated
within the positively charged central channel. The REC lobe mainly consistsof α-helices and recognizes the RNA–DNA heteroduplex and the sgRNA
scaffold, whereas the NUC lobe consists of the RuvC, HNH, Wedge (WED),and PAM-interacting (PI) domains. Cas9 enzymes recognize the PAM
nucleotides by the PI domain, and cleave the target and non-target strands
using the HNH and RuvC domains, respectively. Structural comparisons
between the Cas9 orthologs also revealed the mechanistic diversity among
the CRISPR-Cas9 enzymes8–14. Although they share similar domain organizations, their REC and WED domains are structurally divergent, thereby
recognizing the distinct architectures of their cognate guide RNAs. In
addition, their PI domains adopt a conserved core fold, but recognize different PAM sequences using speciﬁc sets of amino-acid residues.

1

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2School of
Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, BC V6S 0L4, Canada. 3Structural
Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan. 4Institutefor Advanced Biosciences, Keio University, Yamagata 997-0035, Japan. 5Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa 2520882, Japan. 6Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Suita, Osaka 565-0871, Japan. 7Synthetic Biology
Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan. 8Department of Chemistry and Biotechnology,
Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. 9Inamori Research Institute for Science, 620 Suiginyae-mail: nisimasu@g.ecc.u-tokyo.ac.jp;
cho, Shimogyo-ku, Kyoto 600-8411, Japan. 10These authors contributed equally: Toshihiro Nakane, Ryoya Nakagawa.
nureki@bs.s.u-tokyo.ac.jp

Communications Biology | (2024)7:803

1https://doi.org/10.1038/s42003-024-06422-z

Brevibacillus laterosporus Cas9 (BlCas9, also known as BlatCas9)
reportedly recognizes a unique N4CNDD (where D is A, T or G) or
N4CNAA as the PAM, and induces indels in maize15 and mammalian cells16.
Given that most Cas9 orthologs recognize G-rich sequences as the PAM,
BlCas9 can target genomic sites inaccessible by other Cas9 orthologs.
Moreover, BlCas9 consists of 1092 residues, and is 276-residues (~0.8 kb)
smaller than SpCas9 (1368 residues). Thus, as compared with SpCas9,
BlCas9 with its sgRNA can be more efﬁciently packaged into an adenoassociated virus (AAV) vector, making it a potentially valuable asset for invivo therapeutic genome editing. However, the optimal guide length andPAM preference for BlCas9 have not been fully investigated in vitro. In
addition, the PAM recognition mechanism of BlCas9 also remains elusive,
due to the lack of structural information and the limited sequence similarity
between BlCas9 and other structurally characterized Cas9 orthologs.
Here, we performed functional and structural characterizations of
BlCas9. We conﬁrmed that BlCas9 exhibits robust activity with an sgRNA with
an optimal 22-nucleotide (nt) guide and recognizes N4CNDN PAMs with apronounced preference for A at positions 7 and 8. The crystal structure of the
BlCas9–sgRNA–target DNA complex revealed the remarkable diversity in thesgRNA architecture and the PAM recognition mechanism. Furthermore, we
successfully engineered a BlCas9 variant with enhanced cleavage activity andan expanded targeting scope by structure-based rational design.

Results
Biochemical characterization of BlCas9
While the Cas9 orthologs require different guide lengths for efﬁcient DNA
cleavage (20-, 21–23, and 22-nt guides are optimal for SpCas9, SaCas9, andCjCas9, respectively)17,18, the optimal guide length for BlCas9 has not been
fully characterized in vitro. To determine this parameter, we performedin vitro cleavage experiments using puriﬁed BlCas9, sgRNAs with 20–23 nt
guide sequences (sgRNA20–23), which are complementary to three different
targets (Targets 1–3), and their respective plasmid DNA targets with the
23 nt target sequence and a T3CCCAA (Target 1) and T3CCGAA (Targets 2
and 3) PAM (Fig. 1a). BlCas9 with all sgRNAs cleaved the three DNA
targets, and sgRNA22 was superior for all three target sequences (Fig. 1a andSupplementary Fig. 1a). We next performed a PAM identiﬁcation assay,using the puriﬁed BlCas9–sgRNA22 complex and a DNA library containing
the target sequence (Target 1) adjacent to a randomized 8-bp sequence. The
sequence logos of the 8 bp random sequences depleted in this assay showedthat BlCas9 recognizes the N4CNDD PAM, consistent with a previous reportin which a PAM library was cleaved using a 20 nt guide sgRNA15 (Fig. 1b).
However, a detailed 2D proﬁle focused on all 16 possible sequences at the 7th
and 8th positions revealed that BlCas9 does not accommodate all combinations of DD at these positions, and requires an A at either one of them(Fig. 1c). To further examine the PAM preference of BlCas9, we measured
the in vitro cleavage activities of the BlCas9–sgRNA22 complex towardtarget DNAs (Target 1) with 16 different PAMs, in which the fourth toeighth nucleotides in the canonical T3CCCAA PAM were individually
substituted (Fig. 1d and Supplementary Fig. 1b). BlCas9 efﬁciently cleaved
the target plasmids with the T3NCCAA and T3CCNAA PAMs (Fig. 1d andSupplementary Fig. 1b), conﬁrming that it has no preference for the 4th and6th PAM nucleotides. In addition, it only cleaved the T3CCCAA targets, but
not the T3CDCAA targets (Fig. 1d and Supplementary Fig. 1b), indicating
the requirement of the 5th C for the PAM recognition. BlCas9 efﬁciently
cleaved the target plasmids with the T3CCCDA PAMs, but not theT3CCCCA PAM, conﬁrming the requirement of the 7th D for the PAM
recognition (Fig. 1d and Supplementary Fig. 1b). BlCas9 cleaved the target
plasmids with T3CCCAN PAMs, but showed a preference of A > T = G > C
at the 8th position (Fig. 1d and Supplementary Fig. 1b). Together, theseresults indicated that BlCas9 recognizes N4CNDN as the PAM, and prefers
A at both the 7th and 8th positions.
Crystal structure of the BlCas9–sgRNA–DNA complex
To elucidate the PAM recognition mechanism of BlCas9, we attempted todetermine the crystal structure of BlCas9 (1092 residues) in complex with an
Communications Biology | (2024)7:803

ArticlesgRNA and its target DNA, but failed to obtain crystals. Previous studies
revealed that the HNH domain of Cas9 is mobile and dispensable for DNA
recognition7,19,20, suggesting that the HNH domain may hamper crystallization. We thus crystallized a BlCas9-ΔHNH variant, in which the HNH
domain (residues 504–669) is replaced by a GGGSGG linker, as in the caseof CjCas911 (Fig. 2a). After extensive crystallization screening, we determined the crystal structure of BlCas9-ΔHNH in complex with a 110-nt
sgRNA, a 28-nt target DNA strand, and an 8-nt non-target DNA strand
with the T3CCAAA PAM, at 2.4-Å resolution (Fig. 2a–c, and Table 1).
The crystal structure revealed that BlCas9 adopts a bilobed architecture
consisting of the α-helical REC lobe and the NUC lobe, with thesgRNA–target DNA heteroduplex bound within the central channel
between the two lobes, as in the other Cas9 structures7–14 (Fig. 2c andSupplementary Fig. 2). The REC lobe comprises the REC1 (residues
77–229) and REC2 (residues 230–453) domains, whereas the NUC lobe
includes the RuvC (1–44, 454–503, and 670–806), WED (residues 821–920),
and PI (residues 921–1092) domains. The RuvC domain consists of three
separate motifs (RuvC-I–III), with RuvC-I and RuvC-III connected to theREC1 and WED domains via an arginine-rich bridge helix (residues 45–76)
and a phosphate-lock loop (residues 807–820), respectively, as in the otherCas9 orthologs7–14. Consistent with the classiﬁcation of both BlCas9 andCjCas9 in the type II-C category, the overall structure of BlCas9 is moresimilar to that of CjCas911 (PDB: 5X2G, root-mean-square deviation[RMSD] of 2.1 Å for 622 equivalent Cα atoms) than those of other Cas9
orthologs, such as SpCas98 (PDB: 4UN3, RMSD of 3.1 Å for 518 equivalentCα atoms) and SaCas99 (PDB: 5CZZ, RMSD of 3.1 Å for 638 equivalent Cα
atoms) (Supplementary Fig. 2).
The sgRNA guide segment (G1–C20) and the target DNA strand(dG1–dC20) form the RNA–DNA heteroduplex, which is bound within thepositively charged central channel between the REC and NUC lobes(Fig. 2c–e). The target DNA strand (dA(−1)–dT(−8)) and the non-target
DNA strand (dT1*–dA8*) form the PAM duplex, which is bound between
the WED and PI domains (Fig. 2c–e). As in the other Cas9 structures, thephosphate backbone of the sgRNA seed region (C13–C20) is extensively
recognized by the bridge helix and the REC1 domain, while the backbone
phosphate group between dG1 and dA(−1) in the target DNA strand isrecognized by the phosphate-lock loop (Fig. 2c–d). These conserved
structural features indicate that the RNA-guided DNA cleavage mechanismof BlCas9 is similar to those of the other Cas9 orthologs.
Structure and recognition of the sgRNA scaffold
The sgRNA comprises the guide segment (G1–C20), the repeat:antirepeat
duplex (G21•U50–U33:A38), the tetraloop (G34–A37), and the tracrRNAscaffold (A51–U110) (Fig. 3a, b). A57–A60 are disordered, probably due totheir ﬂexibilities. Notably, the present structure revealed that the BlCas9
tracrRNA scaffold contains a triple-helix structure within two stem loops(stem loops 1 and 2) and two stems (stems 1 and 2), which was neither
predicted from its primary sequence nor observed in the other Cas9 orthologs (Fig. 3a, b and Supplementary Figs. 3 and 4). As expected from thenucleotide sequence, the repeat:antirepeat duplex adopts the A-form-like
conformation, which consists of four non-canonical base pairs (G21•U50
and U28•G43–C30•U41) and nine Watson-Crick base pairs(C22:G49–G27:C44 and C31:G40–U33:A38), and is recognized by the bridge
helix and the REC1/WED domains (Fig. 3a–c). In particular, C30•U41 forms
hydrogen bonds with Lys886, indicating the importance of C30•U41 forbase-speciﬁc repeat:antirepeat recognition by BlCas9 (Fig. 3d). Stem loop 1(A52–G66) is formed via four Watson-Crick base pairs (G53:C65–C56:G61)
and a non-canonical base pair (A52•G66), and is recognized by the REC1
domain and the bridge helix (Fig. 3a–c). A62 is ﬂipped out from the stem
loop and forms hydrogen bonds and stacking interactions with Arg69 andArg227, respectively (Fig. 3e). Stem 1 (A67–G71 and C80–U84) and stem 2(G74–A78 and U104–C108) form a triple-helix structure, which isstabilized by two base triples, G71:C80•C103 and U72•A78:U104 (Fig. 3a–c
and Supplementary Fig. 3a, b). U73 hydrogen bonds with the backbone
phosphate of G76, while U79 hydrogen bonds with the main chain of2

Articlehttps://doi.org/10.1038/s42003-024-06422-zaTarget 1

Target 2

Target 3

100

100

100

80

80

80

20-nt

60

40

20-nt

60

40

20-nt

21-nt
20

2

3

4

40

23-nt
0

1

23-nt
60

20

22-nt

23-nt
0

22-nt

21-nt
20

22-nt

0

0

5

0

1

2

Time (min)b

Cleavage (%)

Cleavage (%)

Cleavage (%)

21-nt

3

4

5

0

1

2

Time (min)

3

c

2.0

8th Position

Bits

T
C

0.0

C AA
A

2

3

4

−0.5

G

−0.25
C

0
0.25

GC
T

1

−0.75

A

TT

A
GG

5

T

C

C

7

8

6

0.5
A

C

Positiond5log2 fold depletion
T

1.0

4

Time (min)GT

7th Position

100

2 min

5 min

Cleavage (%)

80

60

40

20

T3CCCAC

T3CCCAG

T3CCCAT

T3CCCCA

T3CCCGA

T3CCCTA

T3CCGAA

T3CCTAA

T3CCAAA

T3CGCAA

T3CTCAA

T3CACAA

T3GCCAA

T3TCCAA

T3ACCAA

T3CCCAA

0

Fig. 1 | In vitro cleavage activity. a In vitro DNA cleavage activities of BlCas9 withthe 20–23 nt guide sgRNAs toward three different target sequences (Targets 1–3).
The linearized plasmid target bearing the T3CCCAA (Target 1) or T3CCGAA(Targets 2 and 3) PAM was incubated with the BlCas9–sgRNA complex at 37 °C for
0.5, 1, 2, and 5 min. The cleavage products were then analyzed by a MultiNA
microchip electrophoresis system. Data are mean ± s.d. (n = 3). b, c Sequence logo(b) and 2D proﬁle (c) of the BlCas9 PAM obtained from the PAM identiﬁcation
assay. d In vitro DNA cleavage activities of BlCas9 with the 22-nt guide sgRNA
toward DNA targets (Target 1) with different PAMs. The linearized plasmid targets
were incubated with the BlCas9–sgRNA complex at 37 °C for 2 and 5 min. Data aremean ± s.d. (n = 3).

Glu1071/Glu1073 in the PI domain (Fig. 3f). Stem loop 2 (A85–U102) isformed via seven Watson-Crick base pairs (A85:U102–U91:A96), and isrecognized by the REC2 and RuvC domains (Fig. 3a–c). In particular, U95 is
ﬂipped out of the stem and forms hydrogen bonds with Glu248 and Lys427
in the REC2 domain (Fig. 3g). Taken together, the BlCas9 sgRNA adopts a
unique conformation for recognition by BlCas9.

and 6 in the N4CNDN PAM. Importantly, the N4 of dC5* and the N7 andO6 of dG(−5) form hydrogen bonds with Asp1022 and Lys1040, respectively (Fig. 4b, c), explaining the observed requirement for the 5th C in the
N4CNDN PAM. While the nucleobase of dA7* in the non-target strand is
not recognized by the protein, the methyl group of dT(−7) in the target
strand forms van der Waals interactions with Thr1025 and Ala1027(Fig. 4b, d), explaining the preference for A at position 7. Similarly, the dA8*
nucleobase in the non-target strand does not contact the protein, whereas
the O4 of dT(−8) forms a hydrogen bond with Lys959, consistent with theobserved preference for A at position 8 (Fig. 4b, d). The single mutations of
Asp1022, Lys1040, and Lys959 abolished or reduced the in vitro DNA

PAM recognition
In the present structure, the PAM duplex is bound between the WED and PI
domains (Fig. 4a). The nucleobases of dT1*–dC4* and dA6* do not directly
contact the protein, consistent with the lack of speciﬁcity for positions 1–4
Communications Biology | (2024)7:803

3https://doi.org/10.1038/s42003-024-06422-z

ArticleFig. 2 | Overall structure of the BlCas9–sgRNA–target DNA complex. a Domain
structure of BlCas9. The HNH nuclease domain was truncated for crystallization.
BH bridge helix, PLL phosphate lock loop. b Diagram of the sgRNA and target DNA
used for crystallization. TS target strand, NTS non-target strand. c Overall structureof BlCas9-ΔHNH in complex with the sgRNA and its target DNA. Disorderedregions are indicated by dotted lines. d, e Surface representations of the
BlCas9–sgRNA–target DNA complex, colored according to the protein domain (d)and electrostatic surface potential (e).

cleavage activities (Fig. 4e and Supplementary Fig. 5a), conﬁrming thefunctional importance of these residues for PAM recognition. By contrast,
the T1025A mutation did not reduce the cleavage activity, but rather relaxed
the PAM preference at the 7th and 8th positions (Fig. 4e, f and Supplementary
Fig. 5a,b), suggesting that the interaction between Thr1025 and dT(−7) is
not crucial for PAM recognition. Taken together, these structural andfunctional analyses revealed that BlCas9 recognizes long, promiscuous
PAM sequences through base-speciﬁc interactions with both the target andnon-target strands. However, the present structure could not fully explain

the tolerance of T/G and the intolerance of C at the 7th position in the
N4CNDN PAM. Therefore, additional structures with different PAM
sequences are required to fully elucidate the PAM recognition by BlCas9.

Communications Biology | (2024)7:803

Molecular engineering
To expand the target range of BlCas9, we sought to engineer a BlCas9 variant
with relaxed PAM preference at the 7th and 8th positions. Previous studies
demonstrated that additional interactions between Cas9 and nucleic acids
augmented the DNA cleavage activity10,18,21. Molecular modeling suggested
4Articlehttps://doi.org/10.1038/s42003-024-06422-z

Table. 1 | Crystallographic data collection, model reﬁnement
and validation
Data collection and processing
SampleBlCas9–sgRNA–target DNA

PDB ID

8X5V

Beamline

SPring-8 BL41XU/SLS X06SA

Wavelength (Å)

1.0Space group

C2

Cell dimensions
a, b, c (Å)

144.8, 99.2, 119.3

β (°)

97.1

Resolution (Å)
Rmeas

*

50–2.0 (2.12–2.0)
0.255 (4.186)

I/σI

19.01 (1.33)

CC(1/2)*0.999 (0.649)

Completeness (%)**

Multiplicity

99.9 (99.8)
28.0 (28.2)

endogenous target sites with N4CNAN/N4CNNA PAMs in humanembryonic kidney (HEK) 293Ta cells. BlCas9 induced indels at 8 out of 21
target sites (at >1% frequencies) with an average frequency of 6.9%, whereas
enBlCas9 induced indels at 12 out of 21 target sites with an average frequency of 10.8% (Fig. 5a). These results demonstrated that, consistent with
our in vitro data, enBlCas9 exhibits higher cleavage activities than BlCas9 atseveral target sites in HEK293T cells.
Finally, we investigated the applicability of BlCas9 to base editing
techniques in mammalian cells. Target-AID, comprising the SpCas9 D10A
nickase mutant fused to the Petromyzon marinus cytosine deaminase 1 anduracil DNA glycosylase inhibitor, mediates C-to-T conversion at target
genomic sites22. We replaced the SpCas9 D10A nickase in Target-AID with
the D8A nickase version of BlCas9 or enBlCas9 to create BlCas9-AID and
enBlCas9-AID, respectively, and then measured C-to-T conversions at 21
target sites (identical to those tested for indel formation) in HEK293Ta cells.
BlCas9-AID induced C-to-T conversions at 7 target sites at >1% frequencies
with an average frequency of 7.3%, whereas enBlCas9-AID induced them at
12 target sites at >1% frequencies with an average frequency of 8.5%
(Fig. 5b). These results indicated that both BlCas9 and enBlCas9 can beutilized for base editing technologies, with enBlCas9 being moreadvantageous.

Reﬁnement
No. reﬂections

107,265

Discussion

Rwork/Rfree

0.1948/0.2333

In this study, we determined the crystal structure of the
BlCas9–sgRNA–target DNA complex, providing high-resolution insights
into its sgRNA architecture and PAM recognition. The BlCas9 sgRNAcontains the conserved repeat:antirepeat duplex, while the tracrRNA scaffold adopts an unpredicted triple-helix structure, which is not observed in
the other Cas9 orthologs. Although the CjCas9 tracrRNA scaffold also
possesses a triple-helix structure, their sequences and architectures aresubstantially different11 (Supplementary Fig. 4). In addition, the triple-helix
structure of CjCas9 is recognized mainly by the bridge helix, whereas that of
BlCas9 is recognized by the RuvC and PI domains (SupplementaryFigs. 2 and 4). These structural differences enable the species-speciﬁc
recognition of their cognate tracrRNA scaffolds. The present structure also
revealed the unique PAM recognition mechanism by BlCas9. Notably,
BlCas9 forms hydrogen bonds with the C:G base pair at position 5 in the
N4CNDN PAM, thereby identifying the characteristic C in the PAM. Whilethe diverse PAM recognition mechanisms of the Cas9 orthologs have beenreported, the mechanism primarily relying on single base-pair recognition is
unique to BlCas9, thereby highlighting the diversity of Cas9-mediated PAM
recognition.
We found that BlCas9 and BlCas9-AID with optimal 22 nt guidesgRNAs can induce indel formation and C-to-T conversion in human cells,suggesting their utility as in vivo genome editing tools. BlCas9 displayed
signiﬁcant variations in indel and C-to-T conversion efﬁciencies among
different target sites with identical PAMs (Fig. 5a, b), indicating that the
genome editing efﬁciencies are substantially affected by the genomic context, as observed previously15. Based on the structural information, we
created the enBlCas9 (E904R/T1025A) variant with improved activity andslightly expanded targeting range. While several Cas9 and Cas12 orthologs
have been reported to exhibit genome-editing activities in mammalian cells,
most Cas9 and Cas12 orthologs require G- and T-rich sequences as their
PAMs, respectively, thereby restricting their targetable genomic loci. In
contrast, enBlCas9 can induce genome- and base-editing at target sites
without G or T, potentially enabling applications in the treatment of genetic
diseases that were previously inaccessible. In addition, since enBlCas9 (1092
residues) is much smaller than SpCas9 (1368 residues), enBlCas9 fused to acompact adenine/cytosine deaminase could be packaged into a single AAV
vector for in vivo therapeutic base-editing23,24. Furthermore, we recently
developed an approach that combines structure-informed design and deep
mutational scanning to engineer variants with enhanced activity in a morereliable and efﬁcient manner22,25. This approach may further boost theperformance of the enBlCas9 variant to generate useful genome-editing
tools that require only a single C PAM nucleotide and can be packaged into a

No. atoms
Protein

7172

Nucleic acid

2983

Others451

B-factors (Å2)
Protein

59.2

Nucleic acid

63.1

Others

59.9

R.m.s. deviations
Bond lengths (Å)

0.008

Bond angles (°)

1.787

Ramachandran plot
Favored (%)

97.28

Allowed (%)

2.72

Outliers (%)

0.00

*

Friedel pairs are treated as different reﬂections.

that Arg904 (E904R) forms a new interaction with the backbone phosphateof dA(−1) in the target strand (Supplementary Fig. 6a). Indeed, the E904R
mutation enhanced the DNA cleavage activity of BlCas9 (Supplementary
Fig. 6b). Thus, we measured the in vitro cleavage activities of the E904R/
T1025A variant towards target plasmids with the T3CCCNA and
T3CCCAN PAMs. The E904R/T1025A variant efﬁciently cleaved all of the
T3CCCNN targets, including T3CCCCA, for which the wild-type BlCas9(referred to as BlCas9 for simplicity) exhibits almost no activity (Fig. 4f and
Supplementary Fig. 5b). We hereafter refer to the E904R/T1025A variant as
the enhanced BlCas9 (enBlCas9). To comprehensively analyze the PAM
speciﬁcity of enBlcas9, we performed the PAM identiﬁcation assay. In
comparison to BlCas9, enBlCas9 showed some preference for the 8th position, but exhibited more relaxed PAM recognition at the 7th and 8th positions(Supplementary Fig. 6c, d). Together, these results demonstrated that ourengineered enBlCas9 improves the cleavage activity and expands the target
range as compared to BlCas9.
BlCas9-mediated genome and base editing in human cells
To assess the activities of BlCas9 and enBlCas9 in mammalian cells, wemeasured indel formation induced by BlCas9 and enBlCas9 at 21
Communications Biology | (2024)7:803

5https://doi.org/10.1038/s42003-024-06422-z

ArticleFig. 3 | Guide RNA architecture and recognition. a Schematic of the sgRNA andtarget DNA. Disordered regions are enclosed in gray boxes. b Structure of the sgRNA
scaffold. The disordered regions are indicated by dotted lines. c Recognition of thesgRNA scaffold by BlCas9. d–g Recognition of the repeat:antirepeat duplex (d), stem
loop 1 (e), stems 1 and 2 (f), and stem loop 2 (g) of the sgRNA scaffold. Hydrogenbonds are depicted with green dashed lines.

single AAV vector. Collectively, our data highlight the structural andmechanistic diversity among the type II CRISPR-Cas9 effector enzymes,
and pave the way for the development of a more compact genome editing
toolbox.

BlCas9 proteins were expressed in Escherichia coli Rosetta2 (DE3) (Novagen). The BlCas9-expressing E. coli Rosetta2 (DE3) cells were cultured at
37 °C in LB medium (containing 20 mg/l kanamycin) until the OD600
reached 0.8, and protein expression was then induced by the addition of
0.1 mM isopropyl-ß-D-thiogalactopyranoside (Nacalai Tesque). The E. coli
cells were further cultured at 20 °C for 18 hr, and harvested by centrifugation
at 5000 g for 10 min. The E. coli cells were resuspended in buffer A (50 mM
Tris-HCl, pH 8.0, 20 mM imidazole, and 500 mM NaCl), lysed by sonication, and then centrifuged at 10,000 g for 10 min. The supernatant was
mixed with 0.3 ml Ni-NTA Superﬂow resin (QIAGEN) equilibrated withbuffer A, and the mixture was loaded into a Poly-Prep Column (Bio-Rad).
The protein was eluted with buffer B (50 mM Tris-HCl, pH 8.0, 300 mM
imidazole, and 500 mM NaCl), and then the concentration of NaCl was

Methods
Sample preparation
The gene encoding full-length BlCas9 (residues 1–1092) was codon optimized, synthesized (Genscript), and cloned between the NdeI and XhoI sitesof the modiﬁed pE-SUMO vector (LifeSensors). The mutations wereintroduced by a PCR-based method, using the vector encoding full-length
BlCas9 as the template, and the sequences were conﬁrmed by DNA
sequencing. For in vitro cleavage experiments, the N-terminally His6-tagged
Communications Biology | (2024)7:803

6

Article

Asn905

Asp1022

NTS

Thr916

BH

Tyr942b

Ile924aAsn921

https://doi.org/10.1038/s42003-024-06422-z

5′

3′

T3*

C4*

C5*

A6*

A7*

A8*

A(−3)

G(−4)

G(−5)

T(−6)

T(−7)

T(−8)

NTS

T2*

5′

A(−2)

PAM

T1*

PLL

A(−1)

WED

3′

TS

5′

PLL

c

WED

Lys959Lys997

Lys992

Ala1027

Thr1025

Lys1040

5′

PI

Arg820TS

Gln818

3′

PI

d
PI

PI
TS
NTS

5′

A1027
dC5*

D1022

TS

T1025
K959

dT(−7)

dA7*
K1040

NTS

dG(−5)5′dT(−8)

5′

dA8*

3′

e 100

f

BlCas9T1025A

enBlCas9

Fig. 4 | PAM recognition. a Binding of the PAM duplex to BlCas9. b Schematics of
the PAM recognition by BlCas9. Hydrogen bonds are depicted by dashed lines. c andd Recognition of the N4CNAN PAM. Nucleotide T(− 7) and residues Thr1025 andAla1027 are depicted by space-ﬁlling models. Hydrogen bonding and electrostaticinteractions are shown as green dashed lines. e In vitro DNA cleavage activities of thewild-type BlCas9 (WT) and PAM recognition mutants. The linearized plasmidCommunications Biology | (2024)7:803

T3CCCAC

T3CCCAG

T3CCCAT

K1040A0
T1025A0
D1022A

20

K959A

20

T3CCCCA40

T3CCCGA

4060

T3CCCTA60

T3CCCAA

Cleavage (%)

80

WT

Cleavage (%)

80

100

3′

targets with the T3CCCAA PAM were incubated with the BlCas9–sgRNA complex
at 37 °C for 2 min. Data are mean ± s.d. (n = 3). f In vitro DNA cleavage activities of
WT BlCas9, the T1025A variant, and enBlCas9 toward DNA targets with different
PAMs. The linearized plasmid targets were incubated at 37 °C for 2 min. Data aremean ± s.d. (n = 3).

7

Articlehttps://doi.org/10.1038/s42003-024-06422-z

a

N4CNAA

N4CNACN4CNAGN4CNAT

N4CNCA

N4CNGA

N4CNTA
Indel frequency (%)

40
30
20

50
40
30
20

N4CNAGN4CNAT

N4CNCA

N4CNGA

PTEN

MECP2

DYRK1A

PTEN

MECP2

DYRK1A

PTEN

MECP2

DYRK1A

PTEN

MECP2

DYRK1A

PTEN

MECP2

DYRK1APTEN

N4CNAC

40

N4CNTA

BlCas9enBlCas9

30

20

10

PTEN

MECP2

DYRK1A

PTEN

MECP2

DYRK1A

PTEN

MECP2

DYRK1A

PTEN

MECP2

DYRK1A

PTEN

MECP2

DYRK1A

PTEN

MECP2

DYRK1A

PTEN

MECP2

0
DYRK1A

BlCas9enBlCas9p = 0.0031
C-to-T conversion efficiency (%)

N4CNAAMECP20
DYRK1A

0
PTEN

10

MECP2

10

b
C-to-T conversion efficiency (%)

p = 0.018

enBlCas950

DYRK1A

Indel frequency (%)

BlCas9

40

30

20

10

0

BlCas9enBlCas9

Fig. 5 | Genome- and base-editing by BlCas9 and enBlCas9. a, b Efﬁciencies of indel formation (a) and C-to-T conversion (b) by BlCas9 (WT) (gray) and enBlCas9 (orange)at endogenous target sites in HEK293Ta cells (n = 3). The p-value was calculated by the Mann–Whitney U-test.

diluted to 300 mM with 50 mM Tris-HCl, pH 8.0. The protein was mixed
with 0.1 ml SP Sepharose High Performance resin (GE Healthcare) equilibrated with buffer C (20 mM Tris-HCl, pH 8.0, and 300 mM NaCl), and the
mixture was loaded into a Poly-Prep Column (Bio-Rad). The protein was
eluted with buffer D (20 mM Tris-HCl, pH 8.0, 1 M NaCl, and 1 mM DTT).
The puriﬁed proteins were stored at –80 °C until use. The 110–113-nt
sgRNAs (containing 20–23 nt guides) were transcribed in vitro with T7
RNA polymerase, and puriﬁed by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis (Supplementary Table 1). RNA bands were
excised from the gel and recovered with an Elutrap System (GE Healthcare).
The sgRNAs were loaded onto a PD-10 desalting column (GE Healthcare),eluted with buffer E (10 mM Tris-HCl, pH 8.0, and 150 mM NaCl), andthen stored at –20 °C until use.
For crystallization, we prepared the BlCas9-ΔHNH variant lacking the
HNH domain (residues 505–670), in which Thr504 (RuvC-II) and Tyr671(RuvC-III) are connected by a GGGSGG linker. The N-terminally His6tagged BlCas9-ΔHNH was expressed in E. coli Rosetta 2 (DE3) and prepared
as described above. The E. coli cells were resuspended in buffer F (50 mM
Tris-HCl, pH 8.0, 20 mM imidazole, and 300 mM NaCl), lysed by sonication, and then centrifuged at 40,000 g for 30 min. The supernatant was
mixed with 4 ml Ni-NTA Superﬂow resin equilibrated with buffer F, and the
mixture was loaded into an Econo-Column (Bio-Rad). The protein was
eluted with buffer G (50 mM Tris-HCl, pH 8.0, 300 mM imidazole, and
300 mM NaCl). The eluted protein was loaded onto a HiTrap SP HP column (GE Healthcare) equilibrated with buffer C. The protein was eluted
with a linear gradient of 0.3–2 M NaCl. To remove the His6-SUMO-tag, theprotein was mixed with TEV protease, and then dialyzed at 4 °C overnight
against buffer H (20 mM Tris-HCl, pH 8.0, 40 mM imidazole, and 500 mM
NaCl). The protein was passed through the Ni-NTA column equilibrated
with buffer H. The protein was further puriﬁed by chromatography on a
Communications Biology | (2024)7:803

HiLoad 16/600 Superdex 200 column (GE Healthcare) equilibrated withbuffer I (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 1 mM DTT).
The 110 nt sgRNA was transcribed in vitro with T7 RNA polymerase,using a partially double-stranded DNA template (Supplementary Table 2).
The transcribed RNA was puriﬁed by 8% denaturing (7 M urea) polyacrylamide gel electrophoresis. The target and non-target DNA strands
were purchased from Sigma-Aldrich (Supplementary Table 2).
Crystallography
The BlCas9-ΔHNH–sgRNA–target DNA complex was reconstituted bymixing the puriﬁed BlCas9-ΔHNH protein, the 110 nt sgRNA, the 28 nt
target DNA strand, and the 8 nt non-target DNA strand (the T3CCAAA
PAM) (molar ratio, 1:1.5:2.3:2.5). The BlCas9-ΔHNH–sgRNA–DNA
complex was puriﬁed by gel ﬁltration chromatography on a Superdex 200
Increase column (GE Healthcare) equilibrated with buffer J (10 mM TrisHCl, pH 8.0, 150 mM NaCl, and 1 mM DTT). The puriﬁed BlCas9ΔHNH–sgRNA–target DNA complex was crystallized at 20 °C, using thehanging-drop vapor diffusion method. Crystals were obtained by mixing
1 µl of complex solution (A260 nm = 25) and 1 µl of reservoir solution(200 mM sodium-acetate (pH 4.5), 15%–20% PEG 500 MME, 200 mM
ammonium sulfate, and 10 mM strontium chloride). X-ray diffraction data
were collected at 100 K on beamlines BL41XU at SPring-8 and X06SA at
SLS. The crystals were cryoprotected in reservoir solution supplemented
with 20% ethylene glycol. X-ray diffraction data were processed using
DIALS26. Finally, 11 datasets were merged using KAMO27 and XSCALE28.
The structure was determined by molecular replacement with Molrep29,
using the coordinates of CjCas9 (PDB:5X2D)11 as the search model. The
model was rebuilt using Buccaneer30, followed by interactive model
rebuilding using COOT31 and structural reﬁnement using phenix.reﬁne32
and Refmac533,34. An AlphaFold2-predicted model using ColabFold35,368

Articlehttps://doi.org/10.1038/s42003-024-06422-z

facilitated the model building of less-ordered regions. Data collection statistics are summarized in Table 1. Structural ﬁgures were prepared using
CueMol (http://www.cuemol.org).
In vitro cleavage assay
The EcoRI-linearized pUC119 plasmid (100 ng, 4.7 nM), containing the
23 nt target sequence and the PAMs (Supplementary Table 1), was incubated at 37 °C for 0.5–5 min with the BlCas9–sgRNA complex (100 nM) in
10 μl of reaction buffer, containing 20 mM HEPES, pH 7.5, 100 mM KCl,
2 mM MgCl2, 1 mM DTT, and 5% glycerol. The reactions were stopped by
the addition of quench buffer, containing EDTA (20 mM ﬁnal concentration) and Proteinase K (40 ng). The reaction products were resolved,
visualized, and quantiﬁed with a MultiNA microchip electrophoresis device(SHIMADZU).
PAM identiﬁcation assay
The PAM identiﬁcation assay was performed as described previously21. The
PAM library (100 ng), containing eight randomized nucleotides downstream of a 22 nt target sequence (Target 1), was incubated at 37 °C with the
puriﬁed BlCas9 (WT and enBlCas9) (100 nM) and the sgRNA22 in 10 µl ofreaction buffer, containing 20 mM HEPES, pH 7.5, 100 mM KCl, 2 mM
MgCl2, 1 mM DTT, and 5% glycerol. The reactions were stopped by theaddition of quench buffer, containing EDTA (20 mM ﬁnal concentration)
and Proteinase K, and then puriﬁed using a Wizard DNA Clean-Up System(Promega). The puriﬁed DNA samples were ampliﬁed for 25 cycles, using
primers containing common adapter sequences. After column puriﬁcation,
each PCR product (~5 ng) was subjected to a second round of PCR for 15
cycles, to add custom Illumina TruSeq adapters and sample indices. The
sequencing libraries were quantiﬁed by qPCR (KAPA Biosystems), and thensubjected to paired-end sequencing on a MiSeq sequencer (Illumina) with
20% PhiX spike-in (Illumina). The sequencing reads were demultiplexed byprimer sequences and sample indices, using NCBI Blast + (version 2.8.1)
with the blastn-short option. For each sequencing sample, the number ofreads for every possible 8 nt PAM sequence pattern (48 = 65,536 patterns in
total) was counted and normalized by the total number of reads in eachsample. For a given PAM sequence, the enrichment score was calculated as
log2-fold enrichment as compared to the untreated sample. PAM sequenceswith enrichment scores of –2.0 or less were used to generate the sequencelogo representation, using WebLogo (version 3.7.1)37. The cumulative distribution and histogram of the read count of each PAM in the unedited
sample conﬁrmed that the plasmid library has sufﬁcient coverage for theindividual PAM sequences.
Genome- and base-editing analyses in human cells
Genome- and base-editing analyses were performed in triplicate, according
to the protocol described previously38. Brieﬂy, HEK293Ta cells weremaintained in DMEM (Sigma) supplemented with 10% (v/v) fetal bovine
serum (FBS) (Thermo Fisher Scientiﬁc) and 1% Penicillin-Streptomycin(Sigma), at 37 °C in a 0.05% CO2 atmosphere. HEK239Ta cells were seeded
at 5 × 103 cells per well in collagen I-coated 96-well plates, 24 h prior totransfection. HEK239Ta cells were transfected with a BlCas9 plasmid or a
BlCas9-derived base-editor plasmid (120 ng) and an sgRNA plasmid(40 ng), using Polyethylenimine Max (Polysciences) (1 mg/ml, 0.5 µl) in
PBS (50 µl) (Supplementary Table 3). The cells were harvested 3 days after
transfection, treated with 50 mM NaOH (100 µl), incubated at 95 °C for
10 min, and then neutralized with 1 M Tris-HCl, pH 8.0 (10 µl). The
obtained genomic DNA was subjected to two rounds of PCR, to prepare thelibrary for high-throughput amplicon sequencing. Genomic regions targeted by sgRNAs were PCR-ampliﬁed to add custom primer-landing
sequences (Supplementary Table 4). The PCR products were puriﬁed by
AMPure XP magnetic beads (Agencourt), and then subjected to a second
round of PCR to attach the custom Illumina TruSeq adapters with sample
indices. After size-selection by agarose gel electrophoresis and column
puriﬁcation, the sequencing libraries were quantiﬁed using a KAPA Library
Quantiﬁcation Kit Illumina (KAPA Biosystems), multiplexed, andCommunications Biology | (2024)7:803

subjected to paired-end sequencing (600 cycles), using a MiSeq sequencer(Illumina) with 20% PhiX spike-in (Illumina). The sequencing reads were
demultiplexed, based on sample indices and primer sequences. Using NCBIBLAST + (version 2.6.0) with the blastn-short option, the sequencing reads
were mapped to the reference sequences to identify indels and substitutionsin the target regions. To remove common PCR errors and somatic mutations, we deleted sequencing reads containing mutations (>1% frequency)
commonly observed in the control samples from the edited samples, andthen normalized the editing frequencies for the target sites by subtracting themutation frequencies of the control samples from those of the edited
samples.
Statistics and reproducibility
In vitro cleavage experiments were performed at least three times. Data areshown as mean ± s.d. (n = 3). Kinetics data were ﬁtted with a one-phase
exponential association curve, using Prism (GraphPad).

Data availability
The atomic coordinates of the BlCas9–sgRNA–target DNA complex havebeen deposited in the Protein Data Bank, with the accession number PDB:
8X5V. The source data behind the graphs in the paper can be found in
Supplementary Data 1–4. Any remaining information can be obtained fromthe corresponding author upon reasonable request.
Received: 15 December 2023; Accepted: 6 June 2024;

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Communications Biology | (2024)7:803

Acknowledgements
We thank the beamline scientists at X06SA of the Swiss Light Source and
BL41XU of SPring-8 for assistance with data collection. We also thank Dr.
Takanori Nakane for assistance with the structure determination. H.N. is
supported by JSPS KAKENHI Grant Numbers 21H05281 and 22H00403,
the Takeda Medical Research Foundation, the Inamori Research Institute for
Science, and JST, CREST Grant Number JPMJCR23B6. O.N. is supported
by AMED Grant Numbers JP223fa627001 and JP19am0401005, the Platform Project for Supporting Drug Discovery and Life Science Research(Basis for Supporting Innovative Drug Discovery and Life Science Research(BINDS)) from AMED under Grant Numbers JP23ama121002 (support
number 3272) and JP23ama121012, and the Cabinet Ofﬁce, Government ofJapan, Public/Private R&D Investment Strategic Expansion Program(PRISM), Grant Number JPJ008000, and Cross-ministerial Strategic Innovation Promotion Program (SIP), “Technologies for Smart Bio-industry andAgriculture” (funding agency: Bio-oriented Technology Research
Advancement Institution).

Author contributions
T.N. performed biochemical experiments and crystallized the complexes,
with assistance from R.N., S.O., Y.S. and H.N.; T.N., K.Y. and H.N.
determined the crystal structures; S.I., H.M. and N.Y. performed cellbiological experiments; R.N. and H.N. wrote the manuscript with assistancefrom S.I. and O.N.; H.N. and O.N. supervised all of the research.

Competing interests
O.N. is a co-founder, board member, and scientiﬁc advisor of Curreio. The
remaining authors declare no competing interests.

Additional information
Supplementary information The online version contains
supplementary material available athttps://doi.org/10.1038/s42003-024-06422-z.
Correspondence and requests for materials should be addressed toHiroshi Nishimasu or Osamu Nureki.
Peer review information Communications Biology thanks the anonymous
reviewers for their contribution to the peer review of this work. Primary
Handling Editors: Dr Ophelia Bu. A peer review ﬁle is available.
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