Cell Reports, Volume 31
Supplemental Information
Cas12a Base Editors Induce
Efficient and Specific Editing
with Low DNA Damage Response
Xiao Wang, Chengfeng Ding, Wenxia Yu, Ying Wang, Siting He, Bei Yang, Yi-ChunXiong, Jia Wei, Jifang Li, Jiayi Liang, Zongyang Lu, Wei Zhu, Jing Wu, ZhiZhou, Xingxu Huang, Zhen Liu, Li Yang, and Jia Chen
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Figure S1. Indels and base substitutions induced by rA1-dCas12a-BE and Cas9. Related to Figure 1.
(A) Immunoblots of DDR proteins and their phosphorylated forms triggered by Cas9-mediated gene editing or
BE2, BE3 and rA1-dCas12a-BE-mediated base editing. The numbers represent individual protein contents
relative to that triggered by Cas9 (setting as 100).
(B) VEGFA target sequences and the indel frequencies induced by Cas9, BE2, BE3 and rA1-dCas12a-BE.
(C) C-to-T editing frequencies induced by Cas9 and indicated BEs.
Means ± SD are from three independent experiments in (B and C). NT, non-transfected. Figure S1
AC-
to-T
edi
ting
frequ
ency
(%)
B
C
0
60
40
20
rA1-dCas12a-BENT
VEGFATTTGC1TC3TC5AAGAC10C11C12AC14AATC18C19AGGC23
TC2AAGAC7 C8 C9 AC11AATC15C16AGGC20CGGCas12a target siteCas9 target site
BE3BE2Cas9
Inde
l fre
quen
cy (%
)0.1
1
rA1-dCas12a-BENT
BE3BE2Cas9
100
10
Cas9rA1-dCas12a-BE
NTUV BE2 BE3
H2AX15 kDa
p-p5350 kDa
p5350 kDa
p-ATM270 kDa
ATM270 kDa
p-ATR270 kDa
ATR270 kDa
Actin
U2OS/Rplicate 2
100 18 49 20 0
100 44 79 54 0
100 49 102 34 12
100 61 83 73 46
100 78 100 74 71
100 73 97 73 80
100 64 94 73 82
100 111 101 121 147
50 kDa
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1
Figure S2. Comparison of BE3 and rA1-dCas12a-BE. Related to Figure 2. 2
(A) The target site sequences and editing windows of BE3 and rA1-dCas12a-BE are shown. 3
(B-D) C-to-T editing frequencies (B), fractions of cytosine substitutions (C) and indel frequencies (D) 4 induced by indicated BEs. 5
(E) Statistical analysis of normalized C-to-T editing frequencies shown in (B), setting the ones induced 6 by BE3 as 100%. 7
(F) Statistical analysis of C-to-T fractions of base editing products shown in (C). 8
Figure S2
AC-to-T editing frequency (%)
40200
BE3-C6rA1-dCas12a-BE-C8
Indel frequency (%)
BE3 rA1-dCas12a
-BE
Fraction of cytosine
substitutions
rA1-dCas12a-BE editing window
BE3 editing windowPAM60
TATTAC6AGAAATACTCTGAATGGCas9 target site
TTTCAGTATTAC8AGAAATACTCTGAATCas12a target site
BE3-C5rA1-dCas12a-BE-C9Cas12a target site TTTGAGGATATGC9AGGTTCTCGTGTCT
Cas9 target site TATGC5AGGTTCTCGTGTCTAGGG
BE3-C5rA1-dCas12a-BE-C9Cas12a target site TTTCCTGCAGATC9GAGGAGACTGGCAG
Cas9 target site AGATC5GAGGAGACTGGCAGAGGG
BE3-C4rA1-dCas12a-BE-C9Cas12a target site TTTAAGGTGGGTC9ATGATAACTGGGTA
Cas9 target site GGTC4ATGATAACTGGGTACGTGG
BE3-C7rA1-dCas12a-BE-C12Cas12a target site TTTGACAGGAAGTTGC12GGGTGGAGGTG
Cas9 target site AAGTTGC7 GGGTGGAGGTGCTGGG
BE3-C7rA1-dCas12a-BE-C9Cas12a target site TTTGGGCAGAAGC9AGGTAGTGATGGCC
Cas9 target site CAGAAGC7AGGTAGTGATGGCCGG
BE3-C5rA1-dCas12a-BE-C9Cas12a target site TTTAAAGCAGGAC9AGAAGGAGCAAGCT
Cas9 target site AGGAC5AGAAGGAGCAAGCTGTGG
210BLM(Bloom syndrome)
C-to-T
C-to-G
C-to-A
BMPR2(Primary pulmonary hypertension)
CHD2(Epileptic encephalopathy)
PAFAH1B1(Lissencephaly 1)
PDE6C(Cone dystrophy 4)
PMS2(Lynch syndrome)
SPAST(Spastic paraplegia 4)
C D
NTrA1-dCas12a-BE
BE3
E
Nor
mal
ized
inde
l fre
quen
cy (%
)
100
80
60
40
20
0
C-to
-T fr
actio
n (%
)
100
90
80
70
60
0
Nor
mal
ized
edi
ting
frequ
ency
(%)
0
120
100
80
60
40
20
120
BE3 rA1-dCas12a-BE
F G
BE3 BE3rA1-dCas12a-BE
rA1-dCas12a-BE
P = 0.001 P = 1 10-14P = 2 10-11
B
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(G) Statistical analysis of normalized indel frequencies shown in (D), setting the ones induced by BE3 9 as 100%. 10
Means ± SD were from three independent experiments in (B and D). n = 21 samples from three 11 independent experiments in (E-G). p value, one-tailed Student’s t test. The median and interquartile 12 range (IQR) are shown. 13 14
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15
Figure S3. Editing window and indel formation induced by engineered hA3A-dCas12a-BEs. 16 Related to Figure 3 and Figure 4. 17
(A) The C-to-T editing frequencies induced by hA3AC101S-dCas12a-BE and hA3AC106S-dCas12a-BE. 18
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(B) The major editing windows and editing efficiencies of the BEs with either Y130F/Y132D or both 19 Y130F and Y132D mutation in hA3A. Positions are counted with the base proximal to the PAM setting 20 as position 1. 21
(C) Comparison of indel frequencies induced by indicated BEs at different genomic target sites. Means 22 ± SD were from three independent experiments. 23
(D) Statistical analysis of normalized indel frequencies induced by indicated BEs in (C), setting the 24 ones induced by hA3AW104A-dCas12a-BE-op as 100%. 25
(E) Statistical analysis of normalized indel frequencies induced by indicated BEs in (C), setting the 26 ones induced by hA3AW98Y/W104A-dCas12a-BE-op as 100%. 27
(F) Statistical analysis of normalized indel frequencies induced by indicated BEs in (C), setting the 28 ones induced by hA3AW104A/P134Y-dCas12a-BE-op as 100%. 29
n = 18 samples from three independent experiments in (D-F). p value, one-tailed Student’s t test. The 30 median and interquartile range (IQR) are shown. 31
32
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Figure S4. DNA damage responses, indels and base substitutions induced by BEACONs and Cas9-BEs. Related to Figure 5 and Figure 6.
(A) Immunoblots of DDR proteins and their phosphorylated forms triggered by Cas9-mediated gene editing, Cas9-BE or BEACON-mediated base editing. The numbers represent individual protein contents relative to that triggered by Cas9 (setting as 100).
(B) Quantification of the relative protein contents normalized with actin in (A) and Figure 5H. Means ±
SD are from two independent experiments.
B
Rela
tive
ratio
to A
ctin
(%) 120
80
040
12080
040
H2AX p-p53
p53
p-ATM
ATM
p-ATR
ATR
BE3
BEACON1
YE1-BE3BE2Cas9
BEACON2NT
E RADAR: RNA-editing Analysis-pipeline to Decode All-twelve-types ofRNA-editing
A
Cas9BE2
Actin
BEACON2
NT
p-ATM270 kDa
ATM270 kDa
p-ATR270 kDa
ATR270 kDa
p-p5350 kDa
p5350 kDa
BE3 BEACON1
YE1_BE3
H2AX15 kDa100 11 26 11 22 16 0
100 34 72 67 22 37 16
100 57 81 72 30 36 15
100 53 98 54 35 40 34
100 72 91 106 51 42 23
100 62 88 61 58 45 19
100 76 95 92 77 39 7
100 94 101 95 93 91 94
U2OS/Replicate 2
50 kDa
C
Cell N
umbe
r (10
4)
Time (h)24 48 72 96
0
30
20
10
NTCas9BE2AncBE4MaxBEACON2
D
DAPIEGFP H2AX Merged
Cas9
BE2
AncBE4Max
BEACON2
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(C) Immunofluorescence staining of gH2AX in U2OS cells transfected with Cas9, BE2, AncBE4Max and BEACON2. The EGFP (green), DAPI (blue) and Alexa Fluor 594 (red) indicates positive staining
for transfected cells, nucleus and gH2AX, respectively. Scale bar: 10 µm.
(D) Cell growth curve illustrates the effects triggered by Cas9 and indicated BEs on cellular proliferation. Means ± SD are from three independent experiments.
(E) Schematic diagrams illustrate the RNA-editing Analysis-pipeline to Decode All twelve-types of RNA-editing events (RADAR).
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Figure S5. On-target editing and indel frequencies induced by BEACONs and rA1-dCas12a-BE in mouse embryos and offspring. Related to Figure 7.
(A) C-to-T editing frequencies induced by rA1-dCas12a-BE at the on-target site in mouse embryos.
(B) C-to-T editing frequencies induced by rA1-dCas12a-BE at the on-target site in tails of mouse offspring.
Figure S5
C-to
-T e
ditin
g fre
quen
cy (%
)
BEACON1/mouse embryos
C6 C11 C15C17C14HSD17B3-ON
C-to
-T e
ditin
g fre
quen
cy (%
) 80
40
080
40
080
40
0
Embryo #2
Embryo #3
Embryo #1
rA1-dCas12a-BE/mouse embryos
C-to
-T e
ditin
g fre
quen
cy (%
)
C-to
-T e
ditin
g fre
quen
cy (%
)
BEACON1/mouse offspring
rA1-dCas12a-BE/mouse offspring A B
C D
BEACON2/mouse embryosE
C-to
-T e
ditin
g fre
quen
cy (%
)
rA1-dCas12a-BE/mouse embryos
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11
BEACON1/mouse embryos
#1 #2 #3 #4 #5 #6 #7 #8
Inde
l fre
quen
cy (%
)
2
1
0
3
#1 #2 #3 #4 #5 #6 #7 #8
2
1
0
3
BEACON1/mouse offspring tails
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 #15 #16 #17 #18 #19 #20 #21 #22 #23 #24
BEACON1/mouse offspring #7 tissues
Hear
t
Live
r
Sple
en
Lung
KIdn
ey
Brai
n
Test
is
BEACON2/mouse embryos
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11
2
1
0
3
rA1-dCas12a-BE/mouse offspring tailsF
C6 C11 C15C17C14HSD17B3-ON
Embryo #5
Embryo #6
Embryo #4
C6 C11 C15C17C14HSD17B3-ON
Embryo #8
Embryo #9
Embryo #7
C6 C11 C15C17C14HSD17B3-ON
Embryo #10
Embryo #11
C6 C11 C15C17C14HSD17B3-ON
80
40
080
40
080
40
0
Embryo #2
Embryo #3
Embryo #1
C6 C11 C15C17C14HSD17B3-ON
Embryo #5
Embryo #6
Embryo #4
C6 C11 C15C17C14HSD17B3-ON
Embryo #7
Embryo #8
C6 C11 C15C17C14HSD17B3-ON
80
40
080
40
080
40
0
Embryo #2
Embryo #3
Embryo #1
80
40
0
Embryo #3
C6 C11 C15C17C14HSD17B3-ON
Embryo #6
Embryo #7
Embryo #5
Embryo #8
80
40
080
40
080
40
0
Embryo #2
Embryo #3
Embryo #1
80
40
0
Embryo #4
Embryo #7
Embryo #8
Embryo #6
Embryo #9
Embryo #12
Embryo #13
Embryo #11
Embryo #14
Embryo #17
Embryo #18
Embryo #16
Embryo #19
Embryo #22
Embryo #23
Embryo #21
Embryo #2480
40
0
Embryo #5 Embryo #10 Embryo #15 Embryo #20
C6 C11 C15C17C14HSD17B3-ON
C6 C11 C15C17C14HSD17B3-ON
C6 C11 C15C17C14HSD17B3-ON
C6 C11 C15C17C14HSD17B3-ON
C6 C11 C15C17C14HSD17B3-ON
80
40
0
Embryo #1 Embryo #3 Embryo #5 Embryo #7 Embryo #9 Embryo #11 Embryo #13
80
40
0
Embryo #2 Embryo #4 Embryo #6 Embryo #8 Embryo #10 Embryo #12 Embryo #14
C6 C11 C15C17C14HSD17B3-ON
C6 C11 C15C17C14HSD17B3-ON
C6 C11 C15C17C14HSD17B3-ON
C6 C11 C15C17C14HSD17B3-ON
C6 C11 C15C17C14HSD17B3-ON
C6 C11 C15C17C14HSD17B3-ON
C6 C11 C15C17C14HSD17B3-ON
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(C) C-to-T editing frequencies induced by BEACON1 at the on-target site in mouse embryos.
(D) C-to-T editing frequencies induced by BEACON1 at the on-target site in tails of mouse offspring.
(E) C-to-T editing frequencies induced by BEACON2 at the on-target site in mouse embryos.
(F) On-target indel frequencies induced by BEACONs and rA1-dCas12a-BE in mouse embryos and offspring.
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Figure S6. Editing frequencies induced by BEACON1 at predicted OT sites in mouse offspring. Related to Figure 7.
C-to-T editing frequencies induced by BEACON1 at predicted OT sites in the tails of mouse offspring #7 and # 11 and in different tissues of mouse offspring #7.
Figure S6
C-to
-T e
ditin
g fre
quen
cy (%
)80
40
0
80
40
0
80
40
0
80
40
0
80
40
0
80
40
0
80
40
0
80
40
0
80
40
0
Offspring #7 - tail
Offspring #11 - tail
Offspring #7 - heart
Offspring #7 - liver
Offspring #7 - spleen
Offspring #7 - lung
Offspring #7 - kidney
Offspring #7 - brain
Offspring #7 - testis
BEACON1/mouse offsprings
C6 C15 C17
HSD17B3-OT-f
C6 C11 C14
HSD17B3-OT-g
C11 C15 C17C14
HSD17B3-OT-e
C6 C11 C15 C17
HSD17B3-OT-d
C6 C15 C17
HSD17B3-OT-c
C6 C11 C17C14
HSD17B3-OT-b
C6 C11 C15 C17C14
HSD17B3-OT-a
C6 C11 C14
HSD17B3-OT-h
C6 C11 C14
HSD17B3-OT-i
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Figure S7. In vivo editing mediated by BE3, AncBE4max and BEACON2 in mouse embryos at overlapped editing sties. Related to Figure 7.
(A) Genotyping of mouse embryos treated with indicated BEs.
(B-D) SCN1A target sequence and C-to-T editing frequency induced by BE3 (B), AncBE4max (C) or BEACON2 (D) in mouse embryos at blastocyst stage.
Figure S7
Mutant ratio
Target gene
No. of indel-containing blastocysts/total blastocysts (%)
No. of examined blastocystsBase editor
No. of base-edited blastocysts/total blastocysts (%)
Scn1A 10/16 (62.5%)11BE3 16/16 (100%)
AncBE4max 8 14/15 (93.3%) 1/15 (6.7%)Scn1A
Scn1A BEACON2 14 8/15 (53.3%) 0/15 (0%)
A
BEACON2/mouse embryos
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10#11#12#13
Inde
l fre
quen
cy (%
) BE3/mouse embryos
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10#11#12#13#14#15#16
20
10
0
30
D
AncBE4max/mouse embryos
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10#11#12#13#14#15
B
100
50
0
Embryo #1
SCN1A TAGC4TC6C7GAGTGTTC15AAGTTGGCC1TAGC5TC7C8GAGTGTTC16AAGTTGG TTTC
BE3/mouse embryos
100
50
0
Embryo #2
100
50
0
Embryo #3
100
50
0
Embryo #4
Embryo #8
Embryo #7
C-to
-T e
ditin
g fre
quen
cy (%
)
E
Embryo #5
Embryo #6
100
50
0
100
50
0
C1TAGC5TC7C8GAGTGTTC16AAGTTGG
AncBE4max/mouse embryos
100
50
0
100
50
0
C1 C5 C8C7 C16
C
F
BEACON2/mouse embryos
#14#15
G
Embryo #9
Embryo #10
Embryo #11
Embryo #12
Embryo #16
Embryo #15
Embryo #13
Embryo #14
C1 C5 C8C7 C16
100
50
0
Embryo #1
100
50
0
Embryo #2
100
50
0
Embryo #3
100
50
0
Embryo #4
Embryo #8
Embryo #7
Embryo #5
Embryo #6
100
50
0
100
50
0
100
50
0
100
50
0
Embryo #9
Embryo #10
Embryo #11
Embryo #12
Embryo #15
Embryo #13
Embryo #14
C-to
-T e
ditin
g fre
quen
cy (%
)
100
50
0
Embryo #1
100
50
0
Embryo #2
100
50
0
Embryo #3
100
50
0
Embryo #4
Embryo #8
Embryo #7
C-to
-T e
ditin
g fre
quen
cy (%
)
Embryo #5
Embryo #6
100
50
0
100
50
0
100
50
0
100
50
0
C4 C7C6 C15
Embryo #9
Embryo #10
Embryo #11
Embryo #12
Embryo #15
Embryo #13
Embryo #14
20
10
0
30
20
10
0
30
C1 C5 C8C7 C16
C1 C5 C8C7 C16 C4 C7C6 C15
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(E-G) On-target indel frequencies induced by BE3 (E), AncBE4max (F) or BEACON2 (G) in mouse embryos.
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Table S1: Comparison of base editors. Related to Figure 1, Figure 5, Figure 6, Figure 7, Figure S1, Figure S5 and Figure S7.
In Vivo
Base editor
Cas protein PAM
Base editing
window Base editing
efficiency DDR
Response RNA off-target
mutation
HSD17B3 SCN1A
efficiency indel efficiency indel
Cas9 Cas9 NGG / / +++++ -
BE2 dCas9 NGG 4 - 8 ~ 1 - 18% + /
BE3 nCas9 NGG 4 - 8 ~13 - 47% +++ ++++
16/16 10/16
YE1-BE3 nCas9 NGG 5 - 7 ~ 4 - 41% + /
AncBE4max nCas9 NGG 4 - 8 ~27 - 70% ++ +
14/15 1/15
BEACON1 dCas12a TTTV 7 - 13 ~38 - 58% ++ + 1/8 0/8
BEACON2 dCas12a TTTV 7 - 12 ~48 - 69% + - 9/14 0/14 8/15 0/15