Article

An AsCas12f-based compact genome-editing tool
derived by deep mutational scanning and structural
analysis
Graphical abstract

Authors
Tomohiro Hino, Satoshi N. Omura,Ryoya Nakagawa, ..., Tsukasa Ohmori,Atsushi Hoshino, Osamu Nureki

Correspondence
tohmori@jichi.ac.jp (T.O.),a-hoshi@koto.kpu-m.ac.jp (A.H.),
nureki@bs.s.u-tokyo.ac.jp (O.N.)

In brief
A high-throughput approach, combining
deep mutational scanning and structureinformed design, generated miniature
AsCas12f variants with enhanced activity,
enabling efficient knock-in/knock-out
activities and transcriptional activation inmice using an all-in-one AAV vector.

HighlightsdCryo-EM structures of compact AsCas12f in complex with
sgRNA and target DNAd

AsCas12f engineering by structural analysis and deep
mutational scanning methodsdActivities of enAsCas12f variants, SpCas9, and AsCas12a
variants are comparabledenAsCas12f effectors have been harnessed as a compact
transcriptional activation tool

Hino et al., 2023, Cell 186, 4920–4935
October 26, 2023 ª 2023 The Authors. Published by Elsevier Inc.
https://doi.org/10.1016/j.cell.2023.08.031ll

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ArticleAn AsCas12f-based compact genome-editing tool
derived by deep mutational scanningand structural analysis
Tomohiro Hino,1,18 Satoshi N. Omura,2,18 Ryoya Nakagawa,2,18 Tomoki Togashi,3,4,18 Satoru N. Takeda,2Takafumi Hiramoto,3 Satoshi Tasaka,1 Hisato Hirano,2 Takeshi Tokuyama,5 Hideki Uosaki,5 Soh Ishiguro,6Madina Kagieva,6 Hiroyuki Yamano,7 Yuki Ozaki,7 Daisuke Motooka,7,8 Hideto Mori,9,10,11 Yuhei Kirita,12 Yoshiaki Kise,2,13
Yuzuru Itoh,2 Satoaki Matoba,1 Hiroyuki Aburatani,14 Nozomu Yachie,6,11,15 Tautvydas Karvelis,16 Virginijus Siksnys,16
Tsukasa Ohmori,3,17,* Atsushi Hoshino,1,* and Osamu Nureki2,19,*
1Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine,

Kyoto 602-8566, Japan
2Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
3Department of Biochemistry, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan
4Department of Clinical Laboratory Science, Division of Health Sciences, Graduate School of Medical Science, Kanazawa University,
Ishikawa 920-0942, Japan
5Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi 329-0498, Japan
6School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver,BC V6S 0L4, Canada
7Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
8Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI),
Osaka University, Suita, Osaka 565-0871, Japan
9Institute for Advanced Biosciences, Keio University, Yamagata 997-0035, Japan
10Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa 252-0882, Japan
11Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Suita, Osaka, 565-0871, Japan
12Department of Nephrology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
13Curreio, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
14Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan
15Synthetic Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan
16Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius, Lithuania
17Center for Gene Therapy Research, Jichi Medical University, Tochigi 329-0498, Japan
18These authors contributed equally
19Lead contact*Correspondence: tohmori@jichi.ac.jp (T.O.), a-hoshi@koto.kpu-m.ac.jp (A.H.), nureki@bs.s.u-tokyo.ac.jp (O.N.)
https://doi.org/10.1016/j.cell.2023.08.031

SUMMARY

SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively
large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The
type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been
harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutationalscanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells
comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed
that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their
DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.
INTRODUCTIONCRISPR-Cas (clustered regularly interspaced short palindromic
repeats and CRISPR-associated proteins) systems provide

adaptive immunity against mobile genetic elements in bacteria
and archaea and are divided into two classes (classes 1 and 2)
and six types (types I–VI).1,2 Cas9 (type II) from Streptococcus
pyogenes (SpCas9) associates with dual RNA guides (CRISPR

4920 Cell 186, 4920–4935, October 26, 2023 ª 2023 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

ArticleRNA [crRNA] and trans-activating crRNA [tracrRNA] or their artificially connected single-guide RNA [sgRNA]) and cleaves double-stranded DNA (dsDNA) targets flanked by an NGG (where
N is any nucleotide) protospacer adjacent motif (PAM), using
its HNH and RuvC nuclease domains.3,4 By contrast, among
the diverse type V Cas12 enzymes, Cas12a from Acidaminococcus sp. (AsCas12a) binds a crRNA and cleaves dsDNA targets
with TTTV (where V is A, G, or C) PAMs, using a single RuvC
nuclease domain.5 As SpCas9 and AsCas12a exhibit robust
nuclease activities in eukaryotic cells, they are widely used asversatile genome engineering tools.5,6 However, neither SpCas9(1,368 amino acids) nor AsCas12a (1,307 amino acids) can be
efficiently packaged into a single adeno-associated virus (AAV)
vector due to their large gene sizes, which hampers their clinicalapplications to in vivo gene therapy.
Recent studies demonstrated that type V-F Cas12 effectors
are exceptionally compact (400–700 amino acids) RNAguided DNA endonucleases.7,8 Cas12f associates with dual
crRNA:tracrRNA guides and cleaves target DNAs with T-rich
PAMs. Previous structural studies of Cas12f from an uncultured archaeon (UnCas12f), with 529 amino acids, revealed
that UnCas12f functions as a dimer to compensate for its
small size.9,10 However, UnCas12f cleaves DNA targets
only under low salt conditions in vitro and lacks activity in human cells, which limits its application as a genome-editing
tool. The minimal Cas12f from Acidibacillus sulfuroxidans(AsCas12f), which consists of only 422 amino acids, cancleave DNA targets with a TTR (where R is A or G) PAM underphysiological conditions in vitro.8 In addition, AsCas12f exhibits low but detectable genome-editing activity in humancells.11 Therefore, AsCas12f shows great promise as a miniature genome-editing tool that can be packaged into a single
AAV vector.
In this study, we present a comprehensive effort to improve
the AsCas12f system for genome editing. By combining structural analysis and deep mutational scanning (DMS) methods,
we revealed the molecular basis and identified a detailed landscape of amino acid substitutions that greatly augment thenuclease activity of AsCas12f. The synergistic effects of thesemutations, coupled with guide RNA engineering, remarkably
enhanced the genome-editing efficiency of AsCas12f in humancells to levels comparable with those of both SpCas9 and engineered AsCas12a effectors. The compact size of AsCas12f offers an attractive feature for AAV-deliverable sgRNA and partner
genes, such as base editors and epigenome modifiers. Therefore, our newly engineered AsCas12f systems could be a promising genome-editing platform.
RESULTS
Cryo-EM structure of the AsCas12f–sgRNA–target DNA
ternary complex
To understand the molecular mechanism of AsCas12f, we used
cryoelectron microscopy (cryo-EM) to analyze the complex
structure of AsCas12f with a 222-nucleotide (nt) sgRNA (natural
29-nt crRNA and 169-nt tracrRNA sequences connected by a
GAAA tetraloop) and a 38-nt dsDNA with phosphorothioate
modifications of the DNA backbone around the cleavage sitell
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with a TTA PAM and obtained a reconstruction at an overall resolution of 3.1 Å (Figures 1A–1C and S1A–S1D; Table 1). The
structure revealed that two AsCas12f molecules (AsCas12f.1
and AsCas12f.2) assemble with one sgRNA molecule to form
an asymmetric homodimer, as previously observed in UnCas12f.9,10 The Cas12f dimer adopts a bilobed architecture
comprising a recognition (REC) lobe and a nuclease (NUC)
lobe, with the guide RNA–target DNA heteroduplex bound tothe central channel between the two lobes. The REC lobe consists of the wedge (WED) and REC domains of both AsCas12f.1
and AsCas12f.2 (WED.1/WED.2/REC.1/REC.2), whereas the
NUC lobe includes the RuvC and target nucleic-acid-binding(TNB) domains of both AsCas12f.1 and AsCas12f.2 (RuvC.1/
RuvC.2/TNB.1/TNB.2).
The sgRNA consists of the 20-nt guide segment (G1 to C20)
and 202-nt sgRNA scaffold (A(!202) to C(!1)) (Figures 1D and
1E). The sgRNA scaffold adopts an unexpected architecturewith two pseudoknots (PK 1 and 2) and five stems (stems
1–5), which were not predicted from its primary sequence.
Stem 1 contains a 6-base pair (bp) duplex (U(!192):A(!175)
to G(!187):C(!180)) and PK 1 comprises a 4-bp duplex(U(!185):A(!2) to A(!182):U(!5)). Stem 2 includes a 16-bp
duplex (G(!171):U(!125) to U(!150):A(!145)) with two
non-canonical base pairs (G(!171):U(!125) and G(!161):
U(!135)), and stem 3 consists of three base pairs (G(!118):
C(!109) to C(!116):G(!111)). PK 2 contains two base pairs(G(!122):C(!88) and G(!121):C(!87)) and connects stems 2
and 3. Stem 4 comprises a 16-bp duplex with two coaxiallystacked duplexes (U(!102):A(!80) to C(!96):G(!86)). Notably,C(!103), located between stems 3 and 4, is flipped out and inserted into stem 1 and PK 1 to form a base pair with G(!181),
thus generating a continuous helix consisting of stem 1 andPK 1 (Figures 1D and 1E). The 50 region (A(!202) to A(!194))and stem 5 (U(!70) to U(!15)) are disordered in the structure,suggesting their flexibility (Figures 1D and 1E).
AsCas12f is over 100 residues shorter than UnCas12f,
whereas its cognate sgRNA is about 40 nucleotides longer
than that of UnCas12f. A structural comparison of AsCas12f
with UnCas12f revealed that, although the domain configurations are similar, AsCas12f lacks the zinc-finger (ZF) domain inserted between the WED and REC domains, which is observedin UnCas12f (Figure S2A). Instead, in the AsCas12f structure,
the ZF domain is replaced with PK 2 and stem 3, which are notpresent in UnCas12f (Figure S2A). These findings account forthe miniaturization of AsCas12f, which is compensated by its
longer sgRNA.
Miniature AsCas12f scaffold associated with thelarge sgRNA
AsCas12f.1 and AsCas12f.2 interact with each other mainly
through hydrophobic interactions in the REC.1/REC.2 andRuvC.1/RuvC.2 interfaces, thereby facilitating the dimerizationof AsCas12f, as observed in UnCas12f (Figures S2B–S2E).9,10
REC.1 and REC.2 form a symmetrical interface by facing eachother in opposite directions with W43, F48, and I116 playing central roles, whereas RuvC.1 and RuvC.2 form an asymmetricalinterface with each protomer adopting a distinct conformation(Figures S2D and S2E). Notably, the a1 helix and a1-a2 loop of
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A

B

C

D

E

(legend on next page)4922 Cell 186, 4920–4935, October 26, 2023

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ArticleRuvC.1 interact with the helices a1 and a2 of RuvC.2 (Figure S2E). In addition to the protein-protein interactions, the lower
and upper regions of stem 2 extensively interact with AsCas12f.1
and AsCas12f.2, respectively, suggesting that the protein-nucleic-acid interactions also contribute to AsCas12f dimerization(Figures S2F and S2G).
The assembly of the sgRNA scaffold with AsCas12f isfacilitated by both base-specific and non-specific interactions(Figures 2A–E). The continuous helix consisting of stem 1 andPK 1 is primarily recognized and accommodated within thegroove formed by the WED.1 and RuvC.1 domains (Figure 2C).
Stem 2 passes through the gap between AsCas12f.1 and AsCas12f.2 and extensively interacts with both protomers, likely reinforcing their dimerization, as described above (Figures S2F
and S2G). PK 2 is recognized by the WED.1 and REC.1 domains
through sugar-phosphate backbone interactions and further stabilized by the coordination of metal ions (Figure 2D). By contrast,
stems 3 and 4 are exposed to the solvent and have minimal interactions with AsCas12f.
The guide RNA–target DNA heteroduplex is accommodated
within the positively charged central channel and recognizedby AsCas12f through base non-specific interactions (Figures 2Band 2E). The PAM-proximal region of the heteroduplex(G1:dC18 to G12:dC7) is mainly recognized by Cas12f.1,
whereas the PAM-distal region of the heteroduplex (C13:dG6
to G18:dC1) is recognized by AsCas12f.2 (Figure 2E). Notably,
P240.2 in RuvC.2 stacks with the G18:dC1 base pair in the heteroduplex, indicating that 18 nucleotides in the spacer sequencefunction as a guide segment (Figure 2E). A structural comparison
with UnCas12f revealed that the position of RuvC.1 in AsCas12f
is similar to that of RuvC.1 in UnCas12f, which cleaves both thetarget and non-target strands (Figure 2F). These structural observations suggest that AsCas12f.1 is responsible for cleaving
the target DNA, whereas AsCas12f.2 plays a crucial role inrecognizing the PAM-distal region of the heteroduplex.
Engineering of AsCas12f to enhance genome-editing
efficiency in mammalian cells
Previous studies revealed that AsCas12f shows limited
genome-editing activity in human cells.8,11 To expand theutility of this compact protein, we sought to engineer an AsCas12f variant with enhanced activity. We first performed
DMS to determine how all amino acid substitutions impact
the genome-editing efficiency in HEK293T cells. We designed
a plasmid that expresses both EGFP and AsCas12f andcreated an AsCas12f library that encompasses all 20-aminoacid substitutions in the whole sequence (M1–K422) (Figure 3A).
The sgRNA targeting GFP was transduced into HEK293T cells,
and subsequently, the AsCas12f library packaged within a lenti-

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virus was expressed at a multiplicity of infection (MOI) of lessthan 0.2, ensuring that no more than one mutant AsCas12f1
was expressed per cell (Figure 3B).12 Given that the full-length
sgRNA exhibited suboptimal genome-editing efficiency, a stem
5-deleted variant (sgRNA_DS5), which exhibited enhanced
genome editing in human cells, was used for the screening process. We selected lentivirus-infected cells by sorting GFP-positive cells on day 2 post-infection and cultured them for 5 days
further. On day 7 post-infection, we separately extracted
mRNAs from GFP-positive and GFP-negative cells and performed the deep sequencing analysis to identify mutations (Figure 3B). The editing efficiency of each mutation was defined as
the ratio of GFP-negative read-count to total read-count andnormalized by the value of wild-type (WT) AsCas12f (Figure 3C).
DMS experiments were performed in duplicate and yielded
similar results (R2 [coefficient of determination] = "0.6), as inour previous study (Figure S3A).13 We identified over 200 singleamino acid substitutions that resulted in a more than 20%
increase in editing efficiency compared with the WT(Figures 3C, 3D, and S3B). Among them, we first focused on
the S188H and D195K mutations, which are located close to
the nucleic acids in our structure and could form additional interactions to reinforce the target DNA binding by AsCas12f,
and adopted them for further experiments. We next developed
a split-GFP reporter system, which is connected by a frameshift
linker containing the Vascular endothelial growth factor A(VEGFA) sequence, and evaluated the genome-editing activityof WT and several mutants by measuring the ratio of frameshifted GFP-positive cells (Figure S3C).14 Consistent with the
DMS analysis, the S188H and D195K mutants exhibited higher
genome-editing activities than WT (Figure 3E). To create variants with higher genome-editing activity than the single
S188H or D195K variants, we combined the S188H or D195K
substitution with other substitutions selected based on the
DMS results and our structural perspective. Although somedouble mutants showed decreased genome-editing activity,
S188H/V232A and D195K/V232A exhibited improved activity(Figures 3E and S3D). To further increase their activities, we
introduced additional mutations that enhanced the activity
when combined with S188H or D195K, resulting in the generation of seven triple mutants. The S188H/V232A/E316M and
D195K/D208R/V232A triple mutants showed further improved
genome-editing activities (Figures 3E and S3E). Lastly, we constructed 29 quadruple mutants by combining the S188H/
V232A/E316M or D195K/D208R/V232A triple mutants withother effective substitutions identified through the DMS
analysis and assessed their genome-editing activities. Mostof these variants showed comparable or lower activities
than the triple mutants, but the F48Y/S188H/V232A/E316M

Figure 1. Cryo-EM structure of the AsCas12f–sgRNA–target DNA ternary complex(A) Domain structure of AsCas12f.
(B and C) Cryo-EM maps (B) and structural models (C) of the AsCas12f–sgRNA–target DNA ternary complex. The zinc and magnesium ions in the TNB domain
and sgRNA scaffold are shown as gray spheres.
(D) Schematic of sgRNA and the target DNA. The disordered regions are enclosed by dashed boxes. TS, target strand; NTS, non-target strand; PK, pseudoknot.
To differentiate between NTS and TS within the manuscript, asterisks are used to denote NTS.
(E) Structure of the sgRNA and target DNA complex. The dotted arrows represent the backbone direction, from 50 to 30 .
See also Figures S1 and S2.

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Table 1. Data collection, processing, model refinement, and validation
Data collection and processing
SampleAsCas12f–sgRNA–target DNA

AsCas12f-YHAM–sgRNA_
DS3–5_v7–target DNA

AsCas12f-HKRA–sgRNA_DS
3–5_v7–target DNA

EMDB ID

EMD-35912

EMD-35926

EMD-35965

PDB ID

8J12

8J1J

8J3R

Microscope

Titan Krios G3i

Detector

Gatan K3 camera

4,398

Magnification

105,000

Voltage (kV)300

Electron exposure (e!/Å2)

50

Defocus range (mm)!0.8 to !1.6

Pixel size (Å)

0.83

Symmetry imposed

C1

Number of movies

4,027

4,154

Initial particle images (no.)

1,304,079

2,118,348

3,835,663

Final particle images (no.)

79,011

146,448

155,198

Map resolution (Å)

3.08

2.91

2.95

FSC threshold

0.143

3DFSC analysis
Global resolution (Å)

3.20

3.32

3.38

Sphericity

0.893

0.843

0.805

!73.8

!53.6

!58.1

6,574

6,588

6,545

Model building and refinement
Map sharpening B factor (Å2)
Model composition
Protein atoms
Nucleic acid atoms

4,121

3,202

2,977

Metal ions

9

4

4

Model-map CC (CCmask/CCbox/
CCpeaks/CCvolume)

0.84/0.84/0.82/0.84

0.85/0.84/0.84/0.85

0.84/0.84/0.82/0.84

Resolution (Å) by model to map
FSC, threshold 0.50 (masked/unmasked)

3.11/3.05

2.89/2.89

2.94/2.94

Average B factor (Å2) (protein/
nucleotides/metal ion)

74.20/91.91/75.44

69.20/79.37/96.22

72.01/93.51/73.69

Bond lengths (Å)

0.003

0.004

0.003

Bond angles (# )

0.445

0.528

0.498

MolProbity score

1.81

1.67

1.60

CaBLAM outliers (%)

1.40

2.29

0.76

Clashscore

6.90

6.59

5.57

Model refinement

RMS deviations

Validation

Rotamer outliers (%)

2.14

1.43

2.46

Cb outliers (%)

0.00

0.00

0.00

EMRinger score

2.67

3.09

3.14

Favored (%)

96.99

96.87

97.99

Allowed (%)

3.01

2.88

2.01

Outliers (%)

0.00

0.25

0.00

Ramachandran plot

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A

B

C

D

F

E

Figure 2. Recognition of the guide RNA and target DNA(A) Recognition sites of the guide RNA scaffold and target DNA.
(B) Electrostatic surface potential of AsCas12f. The sgRNA–target DNA heteroduplex and the stem 1, stem 2, and PK 1 regions of the sgRNA scaffold areaccommodated within the positively charged grooves of the AsCas12f dimer.
(C–E) Recognition of stem 1 and PK 1 (C), stem 3 (D), and the guide RNA–target DNA heteroduplex (E).
(F) Structural comparison of the TS, NTS, and RuvC active site of AsCas12f with those of UnCas12f (Cas12f from an uncultured archaeon) (PDB: 7C7L). The
positions of the RuvC.1 active site relative to the target DNA are similar in both structures, suggesting that AsCas12f also cleaves target DNA by using its RuvC.1
domain.
See also Figure S2.

and I123H/D195K/D208R/V232A quadruple mutants exhibitedfurther enhanced genome-editing activities (Figures 3E andS3F). We designated the F48Y/S188H/V232A/E316M andI123H/D195K/D208R/V232A variants as AsCas12f-YHAM and
AsCas12f-HKRA, respectively.
Optimization of sgRNA for AsCas12f
A recent study reported that deleting stem 5 from the sgRNA enhances the genome-editing activity of AsCas12f.15 Consistent
with this finding, stem 5 is completely disordered in our structure.

Our present structure also revealed that, although PK1, PK2,
stem 1, and stem 2 form extensive interactions with the
AsCas12f protein, stems 3 and 4 are exposed to the solvent
and form few interactions with the protein (Figure 2A). Thus,
we sought to further engineer the sgRNA by truncating these domains. We designed seven sgRNA variants (sgRNA_DS3–5_v1–v7) to eliminate stems 3, 4, and 5 while connecting the originalarchitecture of the remaining regions by different linker sequences (Figure S4A). As expected, all the sgRNA variants exhibited increased genome-editing activities compared with

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sgRNA_DS5, with sgRNA_DS3–5_v7 being the most effective,
albeit by a slight margin (Figure S4B). In our initial DMS experiment, the sgRNA transduced by a multi-copy lentivirus led tohigher genome-editing activities than that by a single-copy lentivirus. Therefore, we hypothesized that the expression level ofsgRNA in cells may influence the activity of AsCas12f. Indeed,
the expression level of sgRNA_DS3–5_v7 was 4.5-fold higher
than that of sgRNA_DS5 in HEK293 cells (Figure S4C), suggesting that shortening the sgRNA sequence led to an increase
in its expression, and consequently enhanced the genome-editing activity. To further elucidate the effects of our sgRNA modification, we transduced the GFP-targeting sgRNA_DS5 andsgRNA_DS3–5_v7 by single-copy lentivirus infection and evaluated the deletion rate of GFP. sgRNA_DS3–5_v7 significantly
improved the GFP deletion efficiencies of both AsCas12f-WT
and AsCas12f-HKRA, compared with sgRNA_DS5 (Figures 3F
and S4D), suggesting that sgRNA_DS3–5_v7 is effective
in situations with a limited expression level, such as in vivo
gene delivery.
Cryo-EM structures of AsCas12f variants with
optimized sgRNA
To gain mechanistic insights into the enhanced DNA cleavage
activity exhibited by our AsCas12f variants, we determinedthe cryo-EM structures of the AsCas12f-YHAM–sgRNA_DS3–
5_v7–target DNA and AsCas12f-HKRA–sgRNA_DS3–5_v7–
target DNA complexes, both at overall resolutions of 2.9 Å
(Figures 4A and S1E–S1L; Table 1). The overall structures of
the AsCas12f-YHAM and AsCas12f-HKRA variants are similar
to that of WT AsCas12f, revealing that the mutations introducedby our DMS experiment do not substantially affect the overall
structures of the complexes.
The sgRNA_DS3–5_v7 consists of the 20-nt guide segment(G1 to C20) and 96-nt sgRNA scaffold (A(!96) to C(!1))(Figures S4E–S4H). As we expected, sgRNA_DS3–5_v7 lacks
the stem 3, 4, and 5 regions while maintaining the stem 1, 2,
and PK 1 regions and extensively interacts with AsCas12f.
Notably, C(!6), which corresponds to the flipped C(!103) inthe WT sgRNA, base pairs with G(!83) and connects PK
1/stem 1 to form a continuous helix (Figures S4I and S4J). The
designed linker region (G(!24) to A(!7)) forms the stem-loop
structure connected by a typical GAAA tetraloop, containing
the G(!24):C(!8) to G(!23):C(!9) base pairs and C(!21):
G(!14) to C(!20): G(!15) base pairs (Figures S4F and S4H). Unexpectedly, G(!12) in the stem loop is flipped out and stacks
with Trp17, akin to the interaction between G(!90) and Trp17
in the WT, contributing to the stable interaction between
sgRNA_DS3–5_v7 and AsCas12f (Figures S4K and S4L). Collectively, the sgRNA_DS3–5_v7 can stably bind to AsCas12f
through both structurally maintained scaffolds and mimicked regions (e.g., C(!6) and G(!12)), explaining why sgRNA_DS3–
5_v7 exhibits efficient genome-editing activity.
In the AsCas12f-YHAM structure, in addition to the hydrophobic interactions observed in WT AsCas12f, Tyr48.1 (F48Y.1),
located at the dimer interface, may form a hydrogen bond withthe main chain of G57.2, likely promoting more stable dimerization (Figure 4B). The side chain of His188.1 (S188H.1) is stabilized by Ile2 and interacts with the backbone phosphate4926 Cell 186, 4920–4935, October 26, 2023

Articlegroup between dT19 and dC18, indicating that the S188H
mutation promotes the unwinding of the target DNA (Figure 4C).
Met316.1 and.2 (E316M.1 and.2) form additional hydrophobic
interactions with Thr239 and Ala241, thereby potentiallyenhancing the overall stability (Figure 4D).
In the AsCas12f-HKRA structure, His123.1 (I123H.1) andLys195.1 (D195K.1) provide hydrophobic and electrostatic interactions with the ribose moiety of A5 and the backbone phosphate groups of A3 and A5, respectively, thus stabilizing theguide RNA–target DNA heteroduplex (Figure 4E). Arg208.1(D208R.1) forms hydrogen-bonding and hydrophobic interactions with dT19 and Ile2, respectively, analogous to His188.1
of AsCas12f-YHAM, thereby facilitating the initial heteroduplex
formation (Figure 4F). Furthermore, Arg208.2 of another subunit(D208R.2) also forms an electrostatic interaction with the phosphate backbone of G(!43) in the sgRNA scaffold to stabilize
the complex structure (Figure 4G).
Despite the presence of the V232A mutation in both the
AsCas12f-YHAM and AsCas12f-HKRA variants, our structures
do not provide clear insight into its contribution to the enhanced
DNA cleavage activities of both variants. These findings suggestthat DMS-based engineering approaches offer significant potential for generating highly active mutants that cannot be predictedfrom structural information alone.
Characterization of AsCas12f variants in human cells
To assess the PAM specificity of enAsCas12f variants toward a
broad range of targets, we developed a self-targeting librarycomposed of both the sgRNA and its target sequence andmeasured insertion–deletion (indel) formations induced by WT
AsCas12f (referred to as AsCas12f for simplicity), AsCas12fYHAM, and AsCas12f-HKRA toward 750 different spacer sequences with 16 NTTN PAMs (Figures 5A and S5A). The deep
sequence analysis demonstrated that the three enzymes
induced indels at the NTTR PAM, but not the NTTY (where Y is
T or C) PAM, indicating that they commonly recognize theNTTR sequences as the PAM (Figure 5B). Our cryo-EM structures revealed that the nucleobases of dT(!3*) and dT(!2*) inthe PAM duplex form hydrophobic interactions with Tyr76.1,
whereas the N6 of dA(!1*) forms a hydrogen bond with
His72.1 (Figure S5B). When dA(!1*) is replaced by dG, the O6of dG(!1*) can form a hydrogen bond with His72.1, providing aclear explanation for the NTTR PAM preference. At the NTTR
PAM, AsCas12f induced indels at 3.0% on average, whereas
AsCas12f-YHAM and AsCas12f-HKRA induced indels at
40.8% and 44.7% on average, respectively (Figure 5B). These
results established that the enAsCas12f variants exhibit
genome-editing activities much higher than that of AsCas12f atvarious target sequences with the NTTR PAM.
Next, we compared the genome-editing efficiencies of
AsCas12f and enAsCas12f variants with those of AsCas12aand AsCas12a variants (UltraCas12a and enAsCas12a),16,17 atfive target sites with the NTTG PAM in HEK293T cells. AsCas12f,
AsCas12f-YHAM, AsCas12f-HKRA, AsCas12a, UltraCas12a,
and enAsCas12a generated indels at these five sites with
8.5%, 22.3%, 14.3%, 9.0%, 11.0%, and 11.1% frequencies on
average, respectively (Figure 5C). These results indicated thatthe enAsCas12f variants, but not AsCas12f, can induce indels

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Figure 3. Engineering AsCas12f variants with deep mutational scanning(A) The design of the deep mutational scanning (DMS) library for AsCas12f.
(B) A schematic of the DMS approach to evaluate the genome-editing efficiency in the context of the GFP gene deletion.
(C) Heatmap illustrating how all single mutations affect the genome-editing activity. Squares are colored by mutational effect according to the scale bars on theright, with blue indicating deleterious mutations. Squares with a diagonal line through them indicate wild-type amino acid.
(D) Close-up view of the selected amino acid substitutions. Squares are colored by mutational effect according to the scale bars on the right, with blue indicating
deleterious mutations. Mutations selected for two quadruple mutants (AsCas12f-YHAM and AsCas12f-HKRA) are highlighted with gray-colored borders.
(E) The genome-editing activities of AsCas12f mutants carrying editing efficiency-enhancing mutations, determined by flow cytometry (n = 4).
(F) Time course of GFP deletion induced by AsCas12f and AsCas12f-HKRA in HEK293T cells expressing d2EGFP (n = 3, mean ± SD). sgRNA_DS5 andsgRNA_DS3–5_v7 were transduced by a single-copy lentivirus infection. See also Figures S3 and S4.

at efficiencies comparable with those of the AsCas12a and AsCas12a variants. We also compared the genome-editing efficiencies with those of SpCas9 at eight target sites, which had

the same spacer sequences with a 50 -TTTG PAM for AsCas12f
and an NGG-30 PAM for SpCas9, in HEK293T cells (Figure 5D).
AsCas12f, AsCas12f-YHAM, AsCas12f-HKRA, and SpCas9

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Figure 4. Cryo-EM structure of AsCas12f variants with sgRNA_DS3–5_v7(A) Structural models of the AsCas12f–sgRNA–target DNA (left), AsCas12f-YHAM–sgRNA_DS3–5_v7–target DNA (center), and AsCas12f-HKRA–sgRNA_DS3–
5_v7–target DNA (right) complexes. Mutated residues are shown as Corey-Pauling-Koltun (CPK) models.
(B–D) Dimer interface (B), target DNA recognition (C), and hydrophobic interactions (D) of AsCas12f-YHAM. Mutated residues are highlighted in red.
(E–G) Recognition of the guide RNA–target DNA heteroduplex (E), target DNA (F), and sgRNA scaffold (G) of AsCas12f-HKRA. Mutated residues are highlighted
in red.
See also Figures S1 and S4.

generated indels at the eight target sites with 1.7%, 14.8%,
16.9%, and 22.9% frequencies on average (Figure 5D). Furthermore, we measured indel formations at three therapeutic targets, PCSK9 and ANGPTL3 for atherosclerosis18 and TTR fortransthyretin amyloidosis,19 in HEK293T cells. We observed
higher indel frequencies for AsCas12f-YHAM and AsCas12fHKRA than those for SpCas9 at two of the three target sites (Figure 5E). Similar results were obtained at other target sites and inother human cell lines, Huh-7 hepatoma cells, and HT-1080 sarcoma cells (Figures S5C–S5F), indicating that both AsCas12f-

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YHAM and AsCas12f-HKRA exhibit genome-editing activities
comparable with those of SpCas9 and AsCas12a variants
regardless of the target sites or cell lines.
To examine the specificities of enAsCas12f variants, we investigated their mismatch tolerance. Both enAsCas12f variants
showed broad tolerance of a single mismatch but negligible
tolerance of double mismatches, except for those at the PAMdistal region (positions 16–20), as observed with Cas12a andother Cas12fs (Figure S6A).15,20,21 We also examined the
genome-wide specificities of AsCas12f-HKRA and SpCas9

Articleusing GUIDE-seq (genome-wide, unbiased identification of double-stranded breaks (DSBs) enabled by sequencing)22–24 at ninetarget sites. AsCas12f-HKRA and SpCas9 had comparable
numbers of off-target sites, and AsCas12f-HKRA tolerated mismatches at the PAM-distal 5-nt region, consistent with our
mismatch experiments (Figures S6B and S6C). These results
demonstrated that enAsCas12f variants with sgRNA_DS3–5_v7
exhibit genome-editing activities and specificities comparablewith those of SpCas9 and AsCas12a, despite their extremelycompact size (Figure S5F).
Therapeutic potential of AsCas12f-mediated genome
editing
To evaluate the potentials of the variants for therapeutic applications, we generated induced pluripotent stem cells (iPSCs) withthe deletion of Duchenne muscular dystrophy (DMD) exon 44(hDMDDEx44), which is the major mutation of DMD, and differentiated them into cardiomyocytes (iPSC-CMs) (Figures S7A–
S7D). Dystrophin deficiency causes myopathy and cardiomyopathy, and the skipping of exon 45 to restore the DMD protein is
now in clinical trials.25,26 We first expressed AsCas12f-HKRA
and paired sgRNAs designed to skip DMD exon 45, using an
all-in-one AAV serotype 6 vector in WT iPSC-CMs, and foundthat AsCas12f-HKRA efficiently reduced the amount of dystrophin protein (Figure 6A). Furthermore, this all-in-one treatment
partially restored the dystrophin protein in hDMDDEx44-iPSCCMs (Figures 6B and 6C), confirming the potential of AsCas12f
variants to treat DMD using AAV gene delivery.
Next, to investigate the therapeutic potential of AsCas12f
in vivo, we applied AsCas12f-HKRA to mouse liver genome editing. We constructed an AAV vector encoding AsCas12f or
AsCas12f-HKRA under the HCRhAAT promoter and a U6 promoter-driven sgRNA targeting the TTR gene for transthyretin
amyloidosis (Figure 6D).27 We injected 7-week-old mice with
3 3 1011 or 1 3 1012 vg of the hepatotropic AAV serotype 8
and evaluated the expression level of plasma transthyretin. AsCas12f failed to modulate the plasma transthyretin level,
whereas AsCas12f-HKRA reduced it in a dose-dependent
manner (Figure 6E). 8 weeks after AAV-based gene delivery,
the target locus was analyzed by targeted amplicon deep
sequencing, which showed that a high editing rate (66.3%)
was achieved by 1 3 1012 vg of AsCas12f-HKRA (Figure 6F).
We then evaluated the in vivo knock-in efficiency of the AsCas12f system to insert the EGFP gene at the mAlb 30 UTR.
The knock-in of genes at the Alb locus is an intensively explored
platform to ectopically produce therapeutic proteins, including
coagulation factors and lysosomal enzymes, from the liver.28,29
We prepared two AAV vectors, one expressing AsCas12f or
AsCas12f-HKRA and the sgRNA targeting the mAlb 30 UTR,
and the other providing a donor template to knock in EGFP at
the DSB only by homology-directed repair (HDR) (Figure 6G).
We intraperitoneally injected these two AAV vectors into
C57BL/6 WT neonatal mice and assessed EGFP expression in
the liver at 4 weeks after the vector injection by immunofluorescence microscopy. We confirmed a significant increase in
EGFP-positive liver cells with the injection of the AAV vector
harboring AsCas12f-HKRA (Figures 6H and 6I). We next replaced the EGFP gene of the donor vector with the coagulationll
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factor IX (F9) cDNA with the Padua mutation and then injected
the vectors into neonatal hemophilia B mice (F9-deficientmice). The knock-in of the F9 gene at the Alb locus using
AsCas12f-HKRA, but not AsCas12f, significantly increased theplasma coagulation factor IX (FIX) activity beyond the therapeutic range (Figure 6J). These data indicate that our engineered
AsCas12f variants with the optimal sgRNA can be harnessed
for in vivo gene therapies (e.g., for hemophilia).
Applications of compact enAsCas12f
Given its small size, the AsCas12f gene can be packaged into an
all-in-one AAV vector along with multiple sgRNAs and/or large
partner genes, enabling its application to genome-editing treatments that were not possible with previous genome-editing
tools. We designed a single AAV vector, encoding AsCas12f(or AsCas12f-HKRA) under a liver-tropic Ttr promoter, a donor
sequence, and a U6 promoter-driven sgRNA, to insert the F9cDNA with the Padua mutation into the Alb 30 UTR locus (Figure 7A).30 We injected the single AAV serotype 8 vector into
neonatal hemophilia B mice and found that both the plasma
FIX activity (FIX:C) and antigen (FIX:Ag) were significantly
increased into the therapeutic range by the AAV vector harboring
AsCas12f-HKRA, but not by that carrying AsCas12f (Figures 7B
and 7C). Consistent with these results, the coagulation time assessed by activated partial thromboplastin time (APTT) and theexpression level of F9 mRNA assessed by quantitative realtime PCR (RT-PCR) were significantly improved (Figures 7D
and 7E). The amylose gel analysis of the mRNA PCR fragments
revealed that cDNA insertion via HDR had occurred, althoughsome insertions mediated by non-homologous end joining(NHEJ) were also observed (Figure 7F).
Finally, to investigate the utility of enAsCas12f for epigenome
editing, we performed a transcriptional activation assay in
Huh-7 cells, which stably express luciferase driven by the minimal cytomegalovirus (CMV) promoter with two HEXA sgRNArecognition sites (Figure 7G). To enhance the transcriptional activity of enAsCas12f, we engineered an sgRNA with the MS2 aptamer inserted into its stem loops.31 We transfected the Huh-7
cells with the plasmids expressing catalytically inactive
AsCas12f-HKRA (dAsCas12f-HKRA) conjugated with VP64 andan MS2-fused activator (MS2-p65-HSF1), as well as engineered
sgRNAs targeting HEXA (Figure 7G). As shown in Figures 7H andS7E, dAsCas12f-HKRA in combination with the MS2 aptamerinserted sgRNA significantly enhanced luciferase expression(Figure 7H). The conjugation of VP64 at the end of dAsCas12fHKRA was most effective for transcriptional activation, although
the direct conjugation of VPR (VP64, p65, and Rta) failed toenhance the transcription by MS2-p65-HSF1 (Figures S7F and
S7G). In addition, we transduced Huh-7 cells with the all-inone AAV serotype 6 vector, encoding dAsCas12f or dAsCas12f-HKRA conjugated with VP64, MS2-p65-HSF1, andsgRNA, and confirmed the significant increase of luciferase
expression in a dose-dependent manner (Figures 7I and S7H).
Lastly, to investigate the utility of enAsCas12f-mediated transcriptional activation in vivo, we administered the AAV vector
to mice (Figure S7I). We observed the significant increase of
luciferase activity in mice treated with dAsCas12f-HKRA, but
not with dAsCas12f (Figures 7J and 7K). These results indicated

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Figure 5. Genome-editing activities of AsCas12f variants in human cell lines(A) A schematic of the self-targeting library screening for large-scale genome-editing efficiency in HEK293T cells.
(B) Indel efficiencies on 750 target sequences in each NTTN PAM. Red, blue, and yellow represent AsCas12f, -YHAM, and -HKRA, respectively. Bars indicate
median indel rates.
(C) Indel efficiencies by AsCas12f, AsCas12f-YHAM, AsCas12f-HKRA, AsCas12a, Ultra, and enAsCas12a at five endogenous target loci in HEK293T cells (n = 3).
(D) Indel efficiencies by AsCas12f, AsCas12f-YHAM, AsCas12f-HKRA, and SpCas9 at eight endogenous target loci in HEK293T cells. The same spacer
sequences with a 50 -TTTG PAM for AsCas12f and an NGG-30 PAM for SpCas9 were used (n = 3).
(E) Indel efficiencies by AsCas12f, AsCas12f-YHAM, AsCas12f-HKRA, and SpCas9 at three therapeutic target loci in HEK293T cells (n = 3).
See also Figures S5 and S6.

that enAsCas12f conjugated with a transcriptional activator canbe harnessed for a transcriptional activation tool deliverable using a single AAV vector.
DISCUSSION
In this study, we determined the cryo-EM structures of the
AsCas12f–sgRNA–target DNA ternary complex and revealed
that two AsCas12f molecules (AsCas12f.1 and AsCas12f.2)
assemble with a single sgRNA to form an asymmetric homo-

4930 Cell 186, 4920–4935, October 26, 2023

dimer, similar to UnCas12f. This observation suggests that thecompact type V-F CRISPR-Cas nucleases commonly function
as dimers.
We applied the DMS technique to CRISPR-Cas effectors andsuccessfully engineered compact AsCas12f variants withenhanced activities. The DMS approach combines exhaustive
protein mutagenesis and functional screening with deep
sequencing, enabling the assessment of the effects of thousands of mutations in a single experiment. One of the typical applications of DMS is the introduction of the library into the yeast

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Figure 6. Applications of AsCas12f-HKRA for therapeutic mouse and human models(A) Western blot analysis of dystrophin protein in wild-type iPSC-CMs (AR21-5) after AAV transduction at a titer of 1 3 105 vg/cell.
(B and C) In hDMDDEx44-iPSC-CMs, AsCas12-HKRA (5 3 105 vg/cell) partially restored dystrophin, which was confirmed by western blotting (B) and immunostaining (C). Scale bar represents 40 mm.
(D) A schematic of the AAV vector used for mouse liver genome editing. NLS, SV40; pA, poly A sequence.
(E) Time course of plasma transthyretin levels (n = 3–4, mean ± SD).
(F) Indel frequency at the Ttr locus in mouse livers (n = 3–4, mean ± SD). Statistical significance between AsCas12f and AsCas12f-HKRA was analyzed by twotailed Student’s t test.
(G) A schematic of the AAV vectors for EGFP or coagulation factor IX (F9) knockin at the Alb 30 UTR. NLS, SV40; pA, poly A sequence.
(H) Immunohistochemistry (IHC) staining of livers from AAV-injected mice at 4 weeks after the vector injection. Endothelial cells were stained with antibodies
specific to CD146 (red). Nuclei were counterstained with DAPI (blue). Scale bars, 200 mm.
(I) The ratio of EGFP-positive cells. Statistical significance between AsCas12f and AsCas12f-HKRA was analyzed by two-tailed Student’s t test.
(J) The increase in plasma factor IX activity (FIX:C). Statistical significance between donor only and donor plus AsCas12f-HKRA was analyzed by two-tailed
Student’s t test.
See also Figure S7.

surface display system and the evaluation of the binding affinityof ligands, including antibodies and viral glycoproteins.32 In thisstudy, to evaluate genome-editing activities, we applied theyeast screening system to mammalian cell-based screening.
We constructed a library that encompasses all 20 single aminoacid substitutions at each position in the whole sequence ofCell 186, 4920–4935, October 26, 2023 4931

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Figure 7. Application of enAsCas12f for knock-in and transcriptional activation by a single AAV vector(A) A schematic representation of the knock-in strategy targeting the Alb locus by a single AAV vector in vivo. Neonatal hemophilia B mice were treated with the
AAV vector (3 3 1011 vg/body). NLS, SV40; pA, poly A sequence.
(B and C) Increase in plasma factor IX activity (FIX:C) (B) and antigen (FIX:Ag) (C) (n = 6–8, mean ± SD).

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ArticleAsCas12f (422 amino acids) and identified over 200 effective mutations at various positions. To efficiently explore the effective
combinations of these mutants, we employed a structural
perspective. In the absence of an experimentally determined
structure, structure prediction models like AlphaFold would bea useful approach for efficiently selecting mutations identified
through DMS.
Various CRISPR-Cas variants have been engineered with theassistance of structural information33,34 and directed evolution
with random mutagenesis.35 However, the former approach
has limited capacities to test the number of mutations, whereas
the latter method carries the risk of introducing unnecessary mutations that do not contribute to activity improvement. By
contrast, by combining comprehensive DMS with rationallystructure-informed design, our approach enables the development of variants with enhanced activity in a more reliable andefficient manner. By applying this powerful approach to otherCas enzymes with different PAM sequences, we can potentiallygenerate efficient genome-editing enzymes capable of targetinga wide range of genes. Moreover, with suitable adaptations to
the evaluation system, this approach can be applied to enzymes
beyond the scope of genome editing.
Limitations of the study
Although our DMS technique discovered numerous effective
substitutions, it is not clear whether the best set of mutations
was selected in the final optimized variants, due to the lack ofa reliable method for selecting the optimal combination of effective mutations. One future solution is to use computationalmodeling to predict effective combinations without compromising protein stability.36 Another is a machine learning-based
approach. By training a machine learning algorithm using a dataset from the DMS containing multiple mutations,37 the resultant
model could predict genome-editing activity values for all
possible mutation combinations.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY

B Lead contact

OPEN ACCESSB Materials availability
B Data and code availabilityd METHOD DETAILSB Protein and RNA preparation for structural analysis
B Electron

microscopy sample preparation and datacollection
B Single-particle cryo-EM data processing
B Model building and validation
B Plasmid generation for cell biological experiments
B Cell culture
B Lentivirus production
B T7 endonuclease assay and amplicon sequencing
B DMS Library construction, FACS, and Illumina
sequencing analysis
B Self-targeting library construction, FACS, and Illumina
sequencing analysis
B GUIDE-Seq
B Plasmid constructs for AAV vector production
B Human iPS cell-derived cardiomyocyte model of DMD
B Confirmation of DMD exon skipping by genomic anddroplet digital PCR
B Western blotting of dystrophin
B Immunofluorescent staining of dystrophin
B Animal experimentation
B Quantitative RT-PCR
B FIX activity and ELISA
B Immunohistochemistry
B Measurement of luciferase activity
B Measurement of luciferase activity by in vivo imaging
in miced QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.cell.
2023.08.031.
ACKNOWLEDGMENTS
Experimental schemes were created in part through BioRender.com. V.S. acknowledges the Research Council of Lithuania for the support of the European
Joint Programme on Rare Diseases project GET-READY (grant number
S-EJPRD-21-1). T.O. was supported by AMED grant numbers JP22fk0410037
and JP22ae0201007. A.H. was supported by AMED grant number
JP19am0401005 and the Japan Foundation for Applied Enzymology. O.N. was(D) Plasma coagulation time assessed by activated partial thromboplastin time (APTT) in C57BL/6 mice, hemophilia B mice, and hemophilia B mice treated with
the AAV vector (n = 4–8, mean ± SD).
(E) The expression of hF9 in liver, measured by quantitative RT-PCR (n = 4–8, mean ± SD). Statistical significance was analyzed by one-way ANOVA with post hoc
Tukey’s multiple comparison test.
(F) Amylose gel analysis of mRNA PCR fragments. When a donor AAV fragment is inserted into the DSB by NHEJ, the duplication of exon 14 results in the 68-bp
longer transcript, compared with those modified by HDR.
(G) A schematic representation of transcriptional activation with the insertion of the MS2 aptamer in the stem loop of the sgRNA of AsCas12f. The plasmid andsgRNA targeting HEXA were transduced into Huh-7 cells stably expressing luciferase under the control of the minimal CMV promoter with HEXA sgRNA-bindingsites. NLS, SV40; pA, poly A sequence.
(H) Increased luciferase activity in the cells transduced with the plasmid vector (n = 3, mean ± SD).
(I) Increased luciferase activity in the cells transduced with the AAV vector (n = 4, mean ± SD).
(J) Representative IVIS imaging system data at 3 weeks after the vector administration.
(K) Quantitative data expressed as photon units. Values are presented as mean ± SD (n = 4). Statistical significance was analyzed by two-tailed Student’s t test.
See also Figure S7.

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supported by AMED grant numbers JP233fa627001 and JP19am0401005, thePlatform Project for Supporting Drug Discovery and Life Science Research (Basisfor Supporting Innovative Drug Discovery and Life Science Research [BINDS])
from AMED under grant number JP23am121002 (support number 3272) andJP23ama121012 (support number 4848, A.H.), and the Cabinet Office, the Government of Japan, and the Public/Private R&D Investment Strategic Expansion
Program (PRISM) grant number JPJ008000.
AUTHOR CONTRIBUTIONS
T.H., S.T., Y.K., and A.H. conducted deep mutational scanning and subsequent data analysis with assistance from S.M. T.H. and S.T. performedin vitro assays to identify enhanced mutants. A.H. performed self-targetinglibrary experiments. H.Y., Y.O., D.M., and H.M. contributed to the data analysis. T.H. performed the GUIDE-seq analysis. S.I., M.K., H.A., and N.Y.
contributed to data collection. T.T. and H.U. conducted the iPSC-derived cardiomyocyte experiments. T.T., T.H., and T.O. conducted AAV preparation andmouse experiments. S.N.O. and R.N. performed biochemical and structural
analyses with assistance from S.N.T., H.H., Y.K., and Y.I. T.K. and V.S.
conceived the project. S.N.O., R.N., H.U., T.O., A.H., and O.N. wrote themanuscript with help from all authors. T.O., A.H., and O.N. supervised theresearch.
DECLARATION OF INTERESTS
T.H., S.N.O., R.N., Y.K., S.M., T.O., A.H., and O.N. have filed a patent application related to this work.
Received: April 21, 2023
Revised: June 27, 2023
Accepted: August 23, 2023
Published: September 29, 2023
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ArticleOPEN ACCESS

STAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCESOURCEIDENTIFIER

Antibodies
mouse anti-dystrophin

Sigma-Aldrich# D8168, RRID:AB_259245

mouse anti-dystrophin antibody

Santa Cruz Biotechnology# sc-73592, RRID:AB_1122390

mouse anti-GAPDHSanta Cruz Biotechnology# sc-32233, RRID:AB_627679

anti-mouse CD146

BioLegend# 134702, RRID:AB_1731991

Anti-GFP rabbit polyclonal antibody

MBL# 598, RRID:AB_591816

Goat anti-Rabbit IgG (H+L) CrossAbsorbed Secondary AlexaFluor 488

Thermo Fisher Scientific# A32731

Goat anti-Rat IgG (H+L) Cross-Absorbed
Secondary AlexaFluor 594

Thermo Fisher Scientific# A11007

Donkey anti-Mouse IgG (H+L) Highly CrossAdsorbed Secondary Antibody, Alexa
Fluor! Plus 488

Thermo Fisher Scientific# A32766

Peroxidase AffiniPure donkey anti-mouse
IgG (H+L)

Jackson ImmunoResearch# 715-035-150

DNA-913F

Bacterial and virus strains
Competent Quick DH5a

TOYOBO

Endura ElectroCompetent Cells

Lucigen60242-2

AAV6 AsCas12f DMD

This paper

N/A

AAV6 AsCas12f-HKRA DMDThis paper

N/A

AAV8 AsCas12f Ttr

This paper

N/A

AAV8 AsCas12f-HKRA Ttr

This paper

N/A

AAV8 AsCas12f Alb

This paper

N/A

AAV8 AsCas12f-HKRA Alb

This paper

N/A

AAV8 Alb-EGFP

This paper

N/A

AAV8 Alb-F9 Padua

This paper

N/A

AAV8 mTTR AsCas12f1 sgRNA_Alb-F9
Padua donor

This paper

N/A

AAV8 mTTR AsCas12f1-HKRA sgRNA_AlbF9 Padua donor

This paper

N/A

AAV6 CMV AsCas12f (D225A)VP64_P2A_MS2-p65-HSF1_sgRNA(HEXA) Stem4

This paper

N/A

AAV6 CMV AsCas12f-HKRA (D225A)VP64_P2A_MS2-p65-HSF1_sgRNA(HEXA) Stem4

This paper

N/A

AAV8 mTTR AsCas12f (D225A)VP64_P2A_MS2-p65-HSF1_sgRNA(HEXA) Stem4

This paper

N/A

AAV8 mTTR AsCas12f-HKRA (D225A)VP64_P2A_MS2-p65-HSF1_sgRNA(HEXA) Stem4

This paper

N/A

AAV8 CMVmini (AsCas12f1-HEXA)Luciferase

This paper

N/A

AsCas12f

Bigelyte et al.8

N/A

AsCas12f variants

This paper

N/A

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ll

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Continued
REAGENT or RESOURCESOURCEIDENTIFIER

DMEM

Sigma-Aldrich# D6429

DMEM

WAKO

044-29765

FBS

Thermo Fisher Scientific#10270-106

RPMI1640

Sigma-Aldrich
Thermo Fisher Scientific# R8758# 11875-093

StemFit AK02N

ReproCell

RCAK02N

B-27 supplement (50X), serum free

Thermo Fisher Scientific17504044

B-27 Supplement, minus insulin

Thermo Fisher ScientificA1895601

iMatrix-511 silk

Matrixome

892021

Y-27632

Fujifilm/Wako Pure Chemical

257-00614

CHIR99021

Cayman13122

C59, Wnt Antagonist (WntC59)

Abcam

ab142216

human EGF

Thermo Fisher Scientific#13247-051

ITS-X Supplement

Thermo Fisher Scientific#51500-056

GlutaMAX

Thermo Fisher Scientific#35050-061

penicillin/streptomycin

Thermo Fisher Scientific# 15140122

Fugene HD

Promega

E2311

Lipofectamine 3000

Thermo Fisher Scientific#3000015

Cellmatrix type I-C

Nitta Gelatin# 637-00773

TRIzolThermo Fisher Scientific# 15596018

Direct-zol RNA MiniPrep

Zymo Research Corporation# R2052

PrimeScript II Reverse Transcriptase

Takara Bio# 2690A

SimplePrep reagent for DNA

Takara Bio# 9180

ExTaq DNA polymerase

Takara Bio# RR006A

PrimeSTAR HS (Premix)

Takara Bio# R040A

KOD One PCR Master Mix

TOYOBOKMM-101

T7 endonuclease

Nippon Gene

# 313-08801

NEBuilder HiFi DNA Assembly Master Mix

NEB

E2621XDNA Ligation Kit

Takara Bio# 6023

PrimeScript RT Master Mix

Takara Bio# RR036A

KAPA SYBR FAST

Kapa Biosystems# 7959630001

SE Cell Line 4D-Nucleofecto X Kit L

Lonza# V4XC-1024

Tissue-Tek optimal cutting temperaturecompound

Sakura Fintek Japan# 45833

VECTASHIELD Mounting Medium
with DAPI

Vector Laboratories#H-1500

Alt-R" S.p. Cas9 Nuclease V3 Alt-R" S.p.
Cas9 Nuclease V3

Integrated DNA Technologies

1081058

Alt-R" CRISPR-Cas9 tracrRNAIntegrated DNA Technologies

1072533

4–15% Mini-PROTEAN" TGX! Precast
Protein Gels

Bio-Rad#4561083

10x Tris/Glycine/SDS

Bio-Rad#1610732

Trans-Blot Turbo Midi 0.2 mm PVDF
Transfer Packs

Bio-Rad#1704157

PVDF Blocking Reagent for Can Get Signal

TOYOBONYPBR01

Can Get Signal" Immunoreaction Enhancer
Solution

TOYOBO

NKB-101

Tris Buffered Saline with Tween"20 (TBS-T)
Tablets, pH 7.6

Takara Bio

T9142(Continued on next page)

Cell 186, 4920–4935.e1–e11, October 26, 2023 e2llArticleOPEN ACCESS

Continued
REAGENT or RESOURCESOURCEIDENTIFIER

Immobilon Western Chemiluminescent
HRP Substrate

Merck

WBKLS0500

cOmplete! EDTA-free protease inhibitor
cocktailRoche

11836170001

phosphatase inhibitor cocktail 2

Sigma-Aldrich

P5726

phosphatase inhibitor cocktail 3

Sigma-Aldrich

P0044

Droplet Generation Oil for Probes

Bio-Rad#1863005

ddPCR Droplet Reader Oil

Bio-Rad#1863004

ddPCR! Buffer Control for Probes

Bio-Rad#1863052

ddPCR! Supermix for Probes (No dUTP)

Bio-Rad#1863023

Thrombocheck APTT

Sysmex#15350Thrombocheck Factor IX

Sysmex#15450Luciferase Assay System

Promega

E1501

Passive lysis buffer 5x

Promega

E194A

TaqMan Gene Expression Assays_ F9

Thermo Fisher Scientific

Hs01592597_m1
Mm99999915_g1

TaqMan Gene Expression Assays_Gapdh

Thermo Fisher Scientificanti-human FIX antibody

CEDARLANE

CL20039AP

anti-human FIX antibody conjugated withhorseradish peroxidase

Affinity Biologicals

GAFIX-APHRP

D-Luciferin sodium salt

OZ Biosciences#LN10000

ABTS Microwell Peroxidase Substrate

Seracare

5120-0032

Critical commercial assays
NucleoSpin Plasmid EasyPure

Takara Bio# U0727

NucleoBond Xtra Midi Plus EF

Takara Bio# U0420

NucleoSpin Gel and PCR Clean-Up

Takara Bio# U0609

NucleoSpin Tissue

Takara Bio# U0952

Prealbumin ELISA Kit

Aviva Systems Biology#OKIA00111

AsCas12f–sgRNA–DNA complex
coordinates

This paper

PDB: 8J12

AsCas12f-YHAM–sgRNA_DS3–5_v7–DNA
complex coordinates

This paper

PDB: 8J1J

AsCas12f-HKRA–sgRNA_3–5_v7–DNA
complex coordinates

This paper

PDB: 8J3R

Deposited data

AsCas12f–sgRNA–DNA complex EM map

This paper

EMDB: EMD-35912

AsCas12f-YHAM–sgRNA_DS3–5_v7–DNA
EM mapThis paper

EMDB: EMD-35926

AsCas12f-HKRA–sgRNA_3–5_v7–DNA
EM mapThis paper

EMDB:EMD-35965

Data sets from DMS, self-targeting library,and GUIDE-seq

This paper

BioProject ID: PRJNA937472

Experimental models: Cell lines
E. coli Mach1

Thermo Fisher Scientific

C862003

E. coli Rosetta 2 (DE3)

Novagen

71397

HEK293JCRB Cell Bank#JCRB9068

HT1080JCRB Cell Bank#JCRB9113

AAVpro 293T

Takara Bio#632273

Huh-7

RIKEN BRC

#RCB1942

TLR3

JCRB Cell Bank

IFO50380(Continued on next page)e3 Cell 186, 4920–4935.e1–e11, October 26, 2023

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ArticleOPEN ACCESS

Continued
REAGENT or RESOURCESOURCEIDENTIFIER

Lenti-X 293T

Clontech

Cat# 632180

Human iPS cells

PMID: 33749676

AR21-5DMD Knock-out human iPS cells

This paper

DMD #2-16, 33, 61, 84

Experimental models: Organisms/strains
Mouse: C57BL/6J wild type

SLC Japan, Inc.

N/A

Mouse: B6.129P2-F9tm1Dws

The Jackson Laboratory

RRID:IMSR_JAX:004303

Sequences used for structural analysis, see
Table S1This paper

Table S1Spacer and designed full sgRNAsequences, see Table S2This paper

Table S2

Primers for qPCR and NGS, see Table S3This paper

Table S3This paper

https://benchling.com/s/
seq-NjTfYidGYdXGiBCkyxBQ?
m=slm-vlGrnO4tH1mikHG33rGB

Oligonucleotides

Recombinant DNA
pE-mH6-AsCas12f

lentiCRISPR v2

AddgeneCat# 52961

lentiGuide-RFP670

This paper

N/A

pBS sgRNA (AsCas12f)

This paper

N/A

pBS sgRNA (SpCas9)

This paper

N/A

pBS gRNA (AsCas12a)This paper

N/A

pMD2.G

AddgeneCat# 12259

psPAX2AddgeneCat# 12260

Cbhv5 AsCas12f DMD

This paper

N/A

Cbhv5 AsCas12f-HKRA DMDThis paper

N/A

pAAV HCRhAAT AsCas12f Ttr

This paper

N/A

pAAV HCRhAAT AsCas12f-HKRA Ttr

This paper

N/A

pAAV HCRhAAT AsCas12f Alb

This paper

N/A

pAAV HCRhAAT AsCas12f-HKRA Alb

This paper

N/A

pAAV Alb-EGFP

This paper

N/A

pAAV Alb-F9 Padua

This paper

N/A

pRC8

This paper

N/A

pRC6

Takara Bio

Cat# 6651

pHelper

Takara Bio

Cat# 6651

pLenti CMV AsCas12f EF1a EGFP

This paper

N/A

pLenti CMV SplitGFP-VEGFAThis paper

N/A

pLenti CMV SplitGFP-TTR

This paper

N/A

pLenti CMV d2EGFPThis paper

N/A

pLenti U6 sgRNA EF1a RFP670

This paper

N/A

pcDNA4TO mCherry

This paper

N/A

pcDNA4TO AsCas12f

This paper

N/A

pcDNA4TO SpCas9This paper

N/A

pcDNA4TO AsCas12aThis paper

N/A

pLenti CMV AsCas12f PGK mCherry

This paper

N/A

pLenti CMV SpCas9 PGK mCherry

This paper

N/A

pLenti CMV AsCas12a PGK mCherry

This paper

N/A

pAAV mTTR AsCas12f1 sgRNA_Alb-F9
Padua donor

This paper

N/A(Continued on next page)

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REAGENT or RESOURCESOURCEIDENTIFIERpAAV mTTR AsCas12f1-HKRA sgRNA_AlbF9 Padua donor

This paper

N/A

pSI545 CMV AsCas12f1-HKRA(D225A) VP64

This paper

N/A

pcDNA3 MS2-p65-HSF1

This paper

N/A

pcDNA3 miniCMV (AsCas12f1-HEXA)Luciferase

This paper

N/A

pBS sgRNA for AsCas12f1_HEXA

This paper

N/A

pBS sgRNA for AsCas12f1_HEXA
MS2 stem2

This paper

N/A

pBS sgRNA for AsCas12f1_HEXA
MS2 stem3This paper

N/A

pBS sgRNA for AsCas12f1_HEXA
MS2 stem4

This paper

N/A

pBS sgRNA for AsCas12f1_HEXA MS2
stem2+3This paper

N/A

pBS sgRNA for AsCas12f1_HEXA MS2
stem2+4This paper

N/A

pBS sgRNA for AsCas12f1_HEXA MS2
stem3+4This paper

N/A

pBS sgRNA for AsCas12f1_HEXA MS2
stem2+3+4

This paper

N/A

pAAV CMV AsCas12f (D225A)VP64_P2A_MS2-p65-HSF1_sgRNA(HEXA) Stem4

This paper

N/A

pAAV CMV AsCas12f-HKRA (D225A)VP64_P2A_MS2-p65-HSF1_sgRNA(HEXA) Stem4

This paper

N/A

pSI545 CMV VP64-AsCas12f1HKRA (D225A)

This paper

N/A

pSI545 CMV VP64-AsCas12f1-HKRA(D225A)-VP64

This paper

N/A

pSI545 CMV AsCas12f1-HKRA(D225A)-VPR

This paper

N/A

pSI545 CMV AsCas12f1-HKRA (D225A)

This paper

N/A

pSI545 CMV AsCas12f1 (D225A)-VP64

This paper

N/A

pAAV mTTR AsCas12f (D225A)VP64_P2A_MS2-p65-HSF1_sgRNA(HEXA) Stem4

This paper

N/A

pAAV mTTR AsCas12f-HKRA (D225A)VP64_P2A_MS2-p65-HSF1_sgRNA(HEXA) Stem4

This paper

N/A

pAAV CMVmini (AsCas12f1-HEXA)Luciferase

This paper

N/A

EPU software

Thermo Fisher Scientifichttps://www.thermofisher.com/jp/ja/home/about-us/events/industrial/smart-epu.html

cryoSPARC v3.3.2 software

Punjani et al.38

https://cryosparc.com/updates

AlphaFold2

Jumper et al.39

https://alphafold.ebi.ac.uk/download

COOT

Emsley and Cowtan40

https://www2.mrc-lmb.cam.ac.uk/
personal/pemsley/coot/

PHENIX

Afonine et al.41

https://www.phenix-online.org/

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REAGENT or RESOURCE
MolProbity

SOURCEIDENTIFIER
42

Chen et al.

https://www.phenix-online.org/
documentation/reference/
molprobity_tool.html

CueMol

N/A

http://www.cuemol.org

ImageJ

NIH

https://imagej.nih.gov/ij/

FlowJoThree Star

https://www.flowjo.com/

Prism9GraphPad

https://www.graphpad.com/
scientific-software/prism/

RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Osamu
Nureki (nureki@bs.s.u-tokyo.ac.jp).
Materials availability
All unique/stable reagents generated in this study are available from the lead contact with a completed Materials Transfer Agreement.
Data and code availabilityd The atomic models have been deposited in the Protein Data Bank under the accession codes 8J12 (AsCas12f–sgRNA–DNA
complex), 8J1J (AsCas12f-YHAM–sgRNA_DS3–5_v7–DNA) and 8J3R (AsCas12f-HKRA–sgRNA_DS3–5_v7–DNA). The
cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-35912(AsCas12f–sgRNA–DNA complex), EMD-35926 (AsCas12f-YHAM–sgRNA_DS3–5_v7–DNA) and EMD-35965 (AsCas12fHKRA–sgRNA_DS3–5_v7–DNA).
d Data sets from DMS, self-targeting library, and GUIDE-seq have been deposited with the National Center for Biotechnology
Information Sequence Read Archive under BioProject ID PRJNA937472.
d Microscopy data reported in this paper will be shared by the lead contact upon request.
d The data of unprocessed image files have been deposited in the Mendeley Data repository (https://doi.org/10.17632/
dn9h5k4tpf.1).
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
METHOD DETAILS
Protein and RNA preparation for structural analysis
The N-terminally His6-tagged wild-type AsCas12f, AsCas12f-YHAM, and AsCas12f-HKRA proteins were expressed in Escherichia
coli Rosetta2 (DE3). Transformed E. coli cells were cultured at 37# C until the OD600 reached 0.8, and protein expression was theninduced by the addition of 0.1 mM isopropyl b-D-thiogalactopyranoside (Nacalai Tesque). E. coli cells were further cultured at
20# C overnight and harvested by centrifugation. The cells were then resuspended in buffer A (20 mM HEPES-NaOH, pH 7.6,
20 mM imidazole, and 1 M NaCl), lysed by sonication, and centrifuged. The supernatant was mixed with 3 ml Ni-NTA Superflow resin(QIAGEN), and the mixture was loaded into an Econo-Column (Bio-Rad). Proteins were eluted with buffer B (20 mM HEPES-NaOH,
pH 7.6, 0.3 M imidazole, 0.5 M NaCl) and then loaded onto a 5-ml HiTrap Heparin HP column (GE Healthcare) equilibrated with buffer
C (20 mM HEPES-NaOH, pH 7.6, and 0.5 M NaCl). The proteins were eluted with a linear gradient of 0.5–2 M NaCl and further purified
by chromatography on a HiLoad 16/600 Superdex 200 column (GE Healthcare) equilibrated in buffer D (20 mM HEPES-NaOH, pH
7.6, 0.5 M NaCl). The purified proteins were stored at !80# C until use. The wild-type sgRNA and sgRNA_DS3–5_v7 were transcribedin vitro with T7 RNA polymerase and purified by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis.
Electron microscopy sample preparation and data collection
The AsCas12f–sgRNA–target DNA ternary complex was reconstituted by mixing purified AsCas12f, the 222-nt sgRNA, the 38-nt
target DNA, and the 38-nucleotide non-target DNA at a molar ratio of 1:1:1:1. Each DNA strand has phosphorothioate modifications
within the phosphate backbone around the cleavage site to inhibit DNA hydrolysis (Table S1). The AsCas12f-YHAM–sgRNA_DS3–
5_v7–target DNA and AsCas12f-HKRA–sgRNA_DS3–5_v7–target DNA ternary complexes were reconstituted in the same way. The
ternary complexes were purified by size-exclusion chromatography on a Superdex 200 Increase 10/300 column (GE Healthcare)

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Articleequilibrated with buffer E (20 mM HEPES-NaOH, pH 7.6, 50 mM NaCl, 10 mM MgCl2, and 10 mM ZnCl2). The purified complex solution (A260 nm = 10 for wild type and A260 nm = 4 for mutants) was then applied to Au 300-mesh R1.2/1.3 grids (Quantifoil) thatwere glow-discharged after adding 3 ml of amylamine in a Vitrobot Mark IV (FEI) at 4# C, with a waiting time of 10 sec and a blotting
time of 4 sec under 100% humidity conditions. The grids were plunge-frozen in liquid ethane and cooled to the temperature of liquid
nitrogen.
Micrographs for all datasets were collected with a Titan Krios G3i microscope (Thermo Fisher Scientific) running at 300 kV andequipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting
mode (The University of Tokyo, Japan). Datasets of the AsCas12f–sgRNA–target DNA ternary complex were collected with a total
dose of approximately 50 electrons per Å2 per 48 frames by the standard mode, and datasets of the AsCas12f-YHAM–
sgRNA_DS3–5_v7–target DNA and AsCas12f-HKRA–sgRNA_DS3–5_v7–target DNA ternary complexes were collected with a total
dose of approximately 50 electrons per Å2 per 64 frames by the CDS mode, using the EPU software (Thermo Fisher Scientific). The
dose-fractionated movies were subjected to beam-induced motion correction and dose weighting using Patch Motion Correction,and the contrast transfer function (CTF) parameters were estimated using Patch-based CTF estimation in cryoSPARC v3.3.2.
Single-particle cryo-EM data processing
Data were processed using cryoSPARC v3.3.2.38 For the AsCas12f–sgRNA–target DNA ternary complex, 1,304,079 particles were
initially selected from the 4,027 motion-corrected and dose-weighted micrographs using Blob picker and Topaz, and extracted at a
pixel size of 3.32 Å. These particles were subjected to several rounds of 2D classification to curate particle sets. The particles were
further curated by heterogeneous refinement, using a map derived from ab initio reconstruction as the template. The selected
150,071 particles were then re-extracted at a pixel size of 1.33 Å and subjected to 3D variability analysis. The resulting maps withdifferent conformations were used for subsequent heterogeneous refinement. The selected particles after heterogeneous refinement
were refined using non-uniform refinement with optimization of the CTF value,43 yielding a map at 3.08 Å, according to the Fouriershell correlation (FSC) criterion of 0.143.44
The datasets for the AsCas12f-YHAM–sgRNA_DS3–5_v7–target DNA and AsCas12f-HKRA–sgRNA_DS3–5_v7–target DNA
ternary complexes were processed using cryoSPARC, in a similar manner to that used for the wild-type AsCas12f–sgRNA–target
DNA ternary complex. For data processing details, see Figure S1.
Model building and validation
The model was built using the predicted model of the AsCas12f protein created by AlphaFold239 (the AlphaFold model) as the reference, followed by manual model building with COOT40 against the density map sharpened using DeepEMhancer. The model was
refined using phenix.real_space_refine ver. 1.20.141 with secondary structure, base pair, and metal coordination restraints. The metal
coordination restraints were generated using ReadySet, as implemented in PHENIX. Structure validation was performed using MolProbity.42 In the model of the wild-type AsCas12f–sgRNA–target DNA ternary complex, residues 56–65, 268–284, 326–330, 380–384,
and 422 of AsCas12f.2, nucleotides (!202) to (!194), (!70) to (!14), and 19 to 20 of the sgRNA, nucleotides (!8) to (!1) and 29 to 30of the TS, and nucleotides (!12*) to (!11*) and 2* to 26* of the NTS were not included in the final model due to their poor resolutions in
the density map. In the model of the AsCas12f-YHAM–sgRNA_DS3–5_v7–target DNA ternary complex, residues 59–64, 266–284,
327–330, 380–384, and 422 of AsCas12f.2, nucleotides (!96) to (!95) and 19 to 20 of the sgRNA, nucleotides (!8) to (!1) and 29
to 30 of the TS, and nucleotides (!12*) to (!11*) and 2* to 26* of the NTS were not included in the final model due to their poor resolutions in the density map. In the model of the AsCas12f-HKRA–sgRNA_DS3–5_v7–target DNA ternary complex, residues 59–64,
266–284, 327–330, 380–384, and 422 of AsCas12f.2, nucleotides (!96) to (!95) and 19 to 20 of the sgRNA, nucleotides (!8) to 2
and 26 to 30 of the TS, and nucleotides (!12*) to (!8*) and 2* to 26* of the NTS were not included in the final model due to theirpoor resolutions in the density map.
Plasmid generation for cell biological experiments
Using a plasmid encoding both AsCas12f and sgRNA (addgene #171614), AsCas12f and its mutants were cloned into pcDNA4TO(Thermo Fisher Scientific) using a NEBuilder HiFi DNA Assembly Kit (New England Biolabs). The sgRNAs were separately cloned intopBS or lentiGuide (addgene #52963). Other Cas enzymes, including AsCas12a, UnCas12f, SpCas9 and their mutants, were mutagenized and cloned into pcDNA4TO (Thermo Fisher Scientific) with the following plasmids: addgene #114091, #176269, and #98290,
respectively. The engineered sgRNA was designed by extracting stem sequences. Both the 50 and 30 side fragments were cloned into
pBS. The reporter for genome editing was constructed with GFP1-10, GFP11 and the linker containing the target sequence. Thissequence was cloned into pLenti CMV (addgene #17448).
Cell culture
HEK293T cells (Clontech) were cultured at 37 # C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, WAKO), containing
10% fetal bovine serum (FBS, Thermo Fisher Scientific) and penicillin/streptomycin (100 U/ml, Thermo Fisher Scientific). HEK293
cells (JCRB Cell Bank), HT1080 cells (JCRB Cell Bank), and AAVpro 293T cells (Takara Bio) were cultured in DMEM (Sigma-Aldrich),supplemented with 10% FBS (Thermo Fisher Scientific) and GlutaMAX (Thermo Fisher Scientific). Huh-7 cells (RIKEN BRC) were
cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS and 2 mM L-glutamine. Murine immortalized liver cells (TLR3

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cells, JCRB Cell Bank) were maintained in DMEM containing 2% FBS, 5 ng/ml of human EGF, and ITS-X Supplement (Thermo Fisher
Scientific). All cell lines tested negative for mycoplasma contamination.
Lentivirus production
Ten-centimeter plates of 70% confluent HEK293T (Clontech) cells were transfected with 9 mg of the plasmid library, 6 mg of the
psPAX2 vector and 3 mg pMD2.G, using Fugene HD (Promega) according to the manufacturer’s instructions. Supernatants werecollected after 48 h, centrifuged at 3,000 rpm for 10 min at 4 # C, filtered through a 0.45 mm low protein-binding filter (SFCA), andthen frozen at –80 # C.
T7 endonuclease assay and amplicon sequencing
One day before transduction, the cells (5 3 104 cells/well) were seeded into 48 well plates coated with collagen type I (Cellmatrix typeI-C, Nitta Gelatin). The plasmids (200 ng) were incubated together with Lipofectamine 3000 (Thermo Fisher Scientific) according to
the manufacturer’s recommendations, and then directly added to the cell culture. At 72 h after the transduction, the cells were lysedwith the SimplePrep reagent for DNA (Takara Bio). The supernatants were directly used for polymerase chain reaction (PCR). DNA
fragments were amplified with ExTaq DNA polymerase (Takara Bio). Purified PCR products were denatured and re-annealed using athermal cycler, and then treated with T7 endonuclease (Nippon Gene). DNA fragments were analyzed by a microchip electrophoresis
system (MCE-202 MultiNA; Shimadzu). The target gene sequences and primer sequences used in the analysis are described inTables S2 and S3, respectively. When indicated, PCR amplicons were subjected to 150 bp pair-end read sequencing using theIllumina MiSeq at The University of Tokyo, or to 300 bp pair-end read sequencing using the Illumina MiSeq at Bioengineering Lab.
Co., Ltd. (Kanagawa, Japan). The frequencies of the mutations were assessed by CRISPResso2.45
DMS Library construction, FACS, and Illumina sequencing analysis
The AsCas12f sequence was divided into 6 units, and saturation mutagenesis was introduced separately in these 6 units. Pooled
oligonucleotides with degenerate NNK codons were synthesized by Integrated DNA Technologies, Inc. Synthesized oligos wereextended by overlap PCR and cloned into pLenti CMV EF1-GFP, modified from the plasmid (addgene #17448) using an NEBuilder
HiFi DNA Assembly Kit (New England Biolabs). An 80 ml ligation reaction was performed using 10 mg of the gel-purified inserts and5 mg of the vector, and was then purified with a Gel and PCR Clean-up kit (Takara Bio) and eluted by 20 ml of distilled water. Next,
200 mg ("4 ml) of the purified reaction was transformed into 50 ml of electrocompetent cells (Lucigen) and expanded according to
the manufacturer’s protocol with 1500 V electroporation by an ECM 399 system (BTX). A 1,000-fold dilution of the full transformation
was plated to estimate the scale of the mutant library. The six libraries were packaged into the lentivirus separately to avoid swapping
contamination, and then transduced into HEK293T cells. At 48 h after transduction, the GFP-expressing library cells were sorted. The
six libraries were pooled and further cultured for 5 days, and then cells were sorted on a MA900 cell sorter (Sony). Dead cells, doublets, and debris were excluded by first gating on the main population by forward and side scatter. GFP-positive and -negative cells
were collected. The total numbers of collected cells were about 10 million cells for each group. Total RNA was extracted fromcollected cells with TRIzol (Life Technologies) and Direct-zol RNA MiniPrep (Zymo Research Corporation), according to the manufacturers’ protocols. First-strand complementary DNA (cDNA) was synthesized with PrimeScript II Reverse Transcriptase (TakaraBio) primed with a gene-specific oligonucleotide. After cDNA synthesis, each library was amplified with specific primers. Following
a second round of PCR, primers with added adapters for annealing to the Illumina flow cell and sequencing primers, together withbarcodes for experiment identification, were used. The PCR products were sequenced on an Illumina NovaSeq 6000 using a 2 3 150
nucleotide paired-end protocol, in the Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University. Data were analyzed by comparing the read counts within each group normalized relative to the WT sequence read-count.
Editing rates for all individual mutations were calculated as the ratio of GFP-negative reads to the total (GFP-positive and -negative)
reads.
Self-targeting library construction, FACS, and Illumina sequencing analysis
The custom pooled oligonucleotides containing pairs of sgRNAs, corresponding target sequences and barcodes were synthesized
by Twist Bioscience. The library includes NTTN 16 PAM sequences and 750 random target sequences, adapted from a previous paper,46 per PAM sequence. Synthesized pooled oligonucleotides were extended by overlap PCR and cloned into lentiGuide EF1-RFP(adapted from addgene #52963), packaged into lentivirus, and transduced into HEK293T cells in a multiple copy infection manner,using the same protocol as for the DMS library. These library cells were infected with lentivirus carrying AsCas12f or enAsCas12f and
PGK-mCherry, and sorted with mCherry 2 days after infection. The cells were maintained for 7 days and then harvested to extract
DNA. This library contains 12,000 different sequences and for more than 500-fold coverage, at least 6 million cells were maintained ineach step. Genomic DNA was extracted by the salt precipitation method, as previously reported.47 From one confluent 100 mm dish,"15 million cells were harvested in a 15 ml conical tube, and 6 ml of NK Lysis Buffer (50 mM Tris, pH 8.0, 50 mM EDTA, 1% SDS) and
30 ml of 20 mg/ml Proteinase K (QIAGEN 19131) were added and the mixture was incubated at 55# C overnight. The next day, 30 ml of
10 mg/ml RNase A (QIAGEN 19101) was added to the lysed sample, which was then inverted 25 times and incubated at 37# C for
30 min. Samples were then cooled on ice. To precipitate the proteins, 2 ml of pre-chilled 7.5 M ammonium acetate (Sigma-Aldrich
A1542) was added and the samples were vortexed at high speed for 20 s or until a white, turbid solution was obtained. After

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Articlecentrifugation at 8,000 3 g for 10 min, a tight pellet was visible in each tube and the supernatant was carefully decanted into a new
15 ml conical tube. After 6 ml 100% isopropanol was added, the tube was briefly vortexed and centrifuged at 8,000 3 g for 10 min.
Genomic DNA was visible as a small white pellet in each tube. The supernatant was discarded, and 6 ml of freshly prepared 70%
ethanol was added. The tube was vortexed briefly, and then centrifuged at 8,000 3 g for 1 min. The supernatant ethanol was removed
completely, and the pellet was air-dried for 10–30 min. After the pellet turned slightly translucent, 500 ml of TE buffer was added andthe tube was incubated at 65# C for 1 h and at room temperature overnight to fully resuspend the DNA. After gDNA extraction, 100 mg
portions were subjected to PCR to attach the adapters and barcodes. The PCR products were purified with a Gel and PCR Clean-up
kit (Takara Bio) and sequenced on an Illumina NovaSeq 6000, using a 2 3 150 nucleotide paired-end protocol, in the Department of
Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University.
GUIDE-Seq
GUIDE-seq experiments were performed essentially in accordance with the protocols described previously. 23 Briefly, 2 x 105
HEK293T cells were transfected with 360 ng enAsCas12f- or SpCas9-encoding plasmid, 360 ng sgRNA expression plasmid,and 50 pmol end-protected double-strand oligodeoxynucleotide (dsODN) GUIDE-seq tag that encodes an internal NdeI restriction site, on a 4D-Nucleofector with the SE Cell Line Nucleofector Kit and the CM-137 program, according to the manufacturer’s
instructions (Lonza). Genomic DNA was isolated with a NucleoSpin Tissue Kit (Takara Bio) and sheared to an average size of
500-bp with 6 cycles of 30 s sonication in mode H of a Bioruptor UCD-250 (Cosmo Bio Co., Ltd.). Restriction enzyme assays
with NdeI and T7E1 were performed at the target loci to determine tag integration efficiencies and modification percentages,
respectively. High-throughput sequencing libraries were generated in accordance with the protocols and subsequently
sequenced with about 10 million reads per sample, using an Illumina Novaseq 6000 system in a 150-bp paired-end mode.
The obtained sequence data were analyzed using the published pipeline (https://github.com/tsailabSJ/guideseq) with thefollowing modifications: demultiplexing with bcl2fastq (v2.20.0.422), human reference genome (hg19), window size of 80-bp,
and configurable PAM sequence.
Plasmid constructs for AAV vector production
A DNA fragment encoding a promoter, the AsCas12f cDNA, the SV40 polyadenylation signal, and an sgRNA sequence driven by
the U6 promoter was introduced between inverted terminal repeats into the pAAV plasmid. We employed the cytomegalovirus(CMV) promoter and the liver-tropic promoter [HCRhAAT promoter (an enhancer element of the hepatic control region of the
Apo E/C1 gene and the human anti-trypsin promoter) or murine transthyretin (Ttr) promoter] used for the cell transduction
and the mouse liver transduction in vivo, respectively. The guide RNA sequences for AsCas12f used in this study are shownin Table S2. We also created a pAAV donor plasmid containing a P2A self-cleaving peptide sequence-conjugated EGFP
sequence or human FIX with the Padua mutation, possessing homologous arms at the target site for inserting the target
gene only by HDR. For DMD experiments, AsCas12f and sgRNA sequences were inserted in the AgeI-EcoRI sites and XhoINotI sites of the Cbhv5 plasmid, respectively. The AAV genes were packaged by triple plasmid transfection of AAVpro293T cells
to produce the AAV vector (helper-free system), as described previously.48 The titers of recombinant AAV vectors were determined by quantitative PCR.49
Human iPS cell-derived cardiomyocyte model of DMD
To model the cardiomyocytes of Duchenne muscular dystrophy, the parental hiPS cell line (AR21-5) and four DMD exon 44 deleted
cell lines (2-16, 33, 61, 84) were differentiated toward cardiomyocytes using the previously reported method.50 Briefly, 125,000 undifferentiated hiPS cells were plated in the wells of a 6-well plate, cultured in AK02N for 4 days, and then induced to cardiomyocytesby serial treatments with CHIR99021 (6 mM, days 0–2) and Wnt-C59 (2 mM, days 2–4), in RPMI 1640 supplemented with B27 minus
insulin and GlutaMAX. Cells were further cultured in RPMI 1640+B27-Insulin for another 3 days, and purified by puromycin selectionin RPMI 1640 with B27 for 3 to 8 days. At day 11 or 15, cardiomyocytes were replated in iMatrix and 0.1% gelatin coated dishes forgenome editing experiments. One day after replating, the AAVs encoding AsCas12f or enAsCas12f-HKRA targeting DMD exon 45
were transduced to iPSC-derived cardiomyocytes at 1 x 104 vg/cell to 5 x 105 vg/cell. Two separate AAVs encoding split
SpCas9-N and -C were used as controls.
Confirmation of DMD exon skipping by genomic and droplet digital PCR
DNA and proteins were isolated at 6 to 13 days after transduction, and subsequently subjected to PCR and western blotting toconfirm exon skipping. The deletion of DMD exon 45 was detected by genomic PCR (Fwd, CTTGTGGGGACAAGAAATCG; Rev,GCTCAAGTTCCCCTTCAAGA) and droplet digital PCR (HEX-conjugated dHsaCP2506429 for DMD exon 26 as a control; FAM-conjugated dHsaCNS992188911 for DMD exon 45; Bio-Rad). Quantitative digital droplet PCR was performed using the QX100 Droplet
Digital PCR system (Bio-Rad), according to the manufacturer’s instructions. The plate was heat-sealed with a foil seal and amplified
on the C1000 Touch thermal cycler (Bio-Rad) using the following protocol: 95# C for 10 min, 40 cycles of 94# C for 30 s and 53# C for
1 min, and 98# C for 10 min. Quantification was determined based on the number of positive droplets in each reaction, using the
QuantaSoft Analysis software (Bio-Rad).

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Western blotting of dystrophin
For western blotting of the dystrophin protein, iPSC-CMs were lysed in Laemmli SDS sample buffer supplemented with DTT(100 mM), cOmplete! EDTA-free protease inhibitor cocktail (11836170001, Roche), phosphatase inhibitor cocktail 2 (P5726,
Sigma-Aldrich) and phosphatase inhibitor cocktail 3 (P0044, Sigma-Aldrich). After heating at 95# C for 5 min, samples were resolved
on 4–15% polyacrylamide gels (Mini Protean TGX gels, Bio-Rad) and transferred to 0.2 mm PVDF membranes with a trans-blot turbo
transfer system (Bio-Rad). The blot was blocked using PVDF blocking reagent (NYPBR01, TOYOBO). Primary and secondary antibodies were each diluted in Can Get Signal Solution (NKB-101, TOYOBO). The blot was incubated with mouse anti-dystrophin antibody (1:1,000, D8168, Sigma-Aldrich), mouse anti-dystrophin antibody (1:1,000, sc-73592, Santa Cruz) or mouse anti-GAPDH antibody (1:1,000, sc-32233, Santa Cruz) at room temperature for 1 h, and then with peroxidase AffiniPure donkey anti-mouse IgG (H+L)(1:2,000, Jackson ImmunoResearch) at room temperature for 30 min. The protein band was visualized using Immobilon Western
Chemiluminescent HRP Substrate (WBKLS0500, Merck), according to the manufacturer’s instructions.
Immunofluorescent staining of dystrophin
We also replated cardiomyocytes for an immunofluorescent analysis to confirm the restoration of dystrophin in cardiomyocytes.
Immunofluorescence staining of DMD was performed as described previously.51 Cells were fixed with ice-cold acetone for 10 minutes at !20# C, and then blocked with 10% donkey serum (S30-100ML, Sigma-Aldrich) / TBS-T (T9141, Takara Bio) for 1 h atroom temperature. Primary and secondary antibodies were added to cells in blocking buffer for 2 h and 1 h, respectively. Nuclei
were counterstained using Hoechst 33342 (62249, Thermo Fisher Scientific). Antibodies used in this article were dystrophin(MANDYS8, D8168, Sigma-Aldrich, 1:800 dilution) and Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody,
Alexa Fluor! Plus 488 (A32766, Thermo Fisher Scientific, 1:500 dilution). All imaging was performed using an Olympus FV1000 laser
scanning confocal microscope.
Animal experimentation
All animal experimental procedures were approved by The Institutional Animal Care and Concern Committee of Jichi Medical University (permission number: 20051-8), and animal care was conducted in accordance with the committee’s guidelines and ARRIVE
guidelines. Coagulation factor IX (FIX)-deficient mice (B6.129P2-F9tm1Dws) were obtained from The Jackson Laboratory (Sacramento, CA). C57BL/6 mice were purchased from SLC Japan (Shizuoka, Japan). To obtain plasma samples, mice were anesthetizedwith isoflurane (1–3%), and the blood sample was drawn from the jugular vein using a 29G micro-syringe (TERUMO) containing 1/10
(volume/volume) sodium citrate. Platelet-poor plasma was obtained by centrifugation and then frozen and stored at !80# C until analysis. The AAV vector was administered intravenously through the jugular vein (100–150 mL) and intraperitoneally (10 mL) in adult andneonatal mice, respectively.
Quantitative RT-PCR
Total RNA was isolated from cells with an RNeasy Mini Kit (QIAGEN). The RNA samples were reverse-transcribed using a PrimeScript
RT Reagent Kit (Takara Bio). Quantitative real-time PCR was performed using TaqMan Gene Expression Assays [(F9:Hs01592597_m1; Gapdh: Mm99999915_g1 (Thermo Fisher Scientific)] and THUNDERBIRD Probe qPCR Mix (TOYOBO) in a
QuantStudio 12K Flex Real-Time PCR system (Thermo Fisher Scientific). Reactions were analyzed in duplicate, and F9 expression
levels were normalized to Gapdh mRNA levels.
FIX activity and ELISA
Human FIX activity (FIX:C) was measured with a one-stage clotting-time assay and an automated coagulation analyzer (Sysmex CS1600). Plasma coagulation time was measured as activated partial thromboplastin time (APTT), using an automated coagulation
analyzer (Sysmex CA-500). The FIX antigen (FIX:Ag) was measured as follows: microtiter plates were coated with an anti-human
FIX antibody (CEDARLANE). After blocking with 5% casein, diluted plasma samples were incubated for 1 h at 37# C. The antigen binding was detected with the anti-human FIX antibody conjugated with horseradish peroxidase (Affinity Biologicals) and ABTS microwell
peroxidase substrate (Seracare). Plasma transthyretin was measured with a Prealbumin ELISA Kit (Aviva Systems, San Diego, CA),according to the manufacturer’s recommendations.
Immunohistochemistry
Anesthetized mice were perfused with phosphate buffered saline (PBS). The isolated tissues were fixed with 4% paraformaldehyde,
incubated with PBS containing sucrose, and then frozen in optimal cutting temperature compound (Sakura Fintek Japan, Tokyo,
Japan). The tissue sections were blocked with 5% donkey serum and then incubated overnight at 4# C with an anti-GFP antibody(MBL Co., Aichi, Japan) and an anti-CD146 antibody. The sections were incubated with anti-rabbit IgG conjugated with
AlexaFluor 488 (Thermo Fisher Scientific) and anti-rat IgG conjugated with AlexaFluor 594 (Thermo Fisher Scientific) for 2 h at
4# C. The sections were mounted with VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA).
Immunofluorescence staining was observed and photographed using an all-in-one microscope (BZ-X700, Keyence, Tokyo, Japan).
The EGFP-positive cells were quantified with the BZ-X 700 imaging software (Keyence).

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ArticleMeasurement of luciferase activity
The luciferase gene, driven by the minimal cytomegalovirus promoter conjugated with two HEXA sgRNA-binding sequences, wascloned into the pcDNA3 vector (Thermo Fisher Scientific). The linearized plasmid was transduced into Huh-7 cells, using Lipofectamine 3000 (Thermo Fisher Scientific). To select the transfected cell clones, G418 (Nacalai Tesque) was added to the culture medium.
For the luciferase activity measurements, the cells were lysed with 100 ml of lysis reagent (Promega), and then aliquots (10 mL) wereadded into the wells of a 96-well plate. The 96-well plate was placed in a luminometer (Centro LB 960, BERTHOLD Technologies), and50 mL of the Luciferase Assay Reagent (Promega) was injected into each well, using the automatic injector.
Measurement of luciferase activity by in vivo imaging in mice
The anesthetized mice were injected intraperitoneally with the luciferin substrate (3 mg/body). We injected two AAV serotype 8 vectors, one encoding dAsCas12f or dAsCas12f-HKRA conjugated with VP64, MS2-p65-HSF1, and sgRNA, and the other encoding
luciferase with a HEXA sgRNA binding site. Photons derived from luciferase activity were measured by an IVIS" Imaging System
and Living Image software (Xenogen Corp., Alameda, CA). Quantitative data were expressed as photon units (photons/second).
QUANTIFICATION AND STATISTICAL ANALYSIS
All data are expressed as mean ± SD. No statistical methods were used to predetermine sample size. Sample size was based on
experimental feasibility and sample availability. Samples were processed in random order. Statistical analyses were performed using
GraphPad Prism 10 (Graph Pad Software, San Diego, CA). All data are presented as the mean ± standard deviation (SD). Statisticalsignificance was analyzed by two-tailed Student’s t test, one-way ANOVA with post hoc Tukey’s multiple comparison test, or twoway ANOVA with post post hoc Sidak’s multiple comparison test. P<0.05 was considered as statistically significant.

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