Xiao Wang, Chengfeng Ding, Wenxia Yu, Ying Wang, Siting He ...€¦ · Xiong, Jia Wei, Jifang Li,...

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


#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


#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11

2

1

0

3

rA1-dCas12a-BE/mouse offspring tailsF


Embryo #5

Embryo #6

Embryo #4


Embryo #8

Embryo #9

Embryo #7


Embryo #10

Embryo #11


80

40

080

40

080

40

0

Embryo #2

Embryo #3

Embryo #1


Embryo #5

Embryo #6

Embryo #4


Embryo #7

Embryo #8


80

40

080

40

080

40

0

Embryo #2

Embryo #3

Embryo #1

80

40

0

Embryo #3


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






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








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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


#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


#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

Xiao Wang, Chengfeng Ding, Wenxia Yu, Ying Wang, Siting He ...€¦ · Xiong, Jia Wei, Jifang Li,...

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