Supplementary Table 1: Small molecules that are reported ...10.1186/s13059-017-1164... ·...
36
Supplementary Table 1: Small molecules that are reported to increase HDR efficiency. Type Small molecule Concentration Time Cell line Fold change Reference Function NHEJ inhibitors SCR7 0.01~1μM 24-48h A549 1.8-3.1 Byrne SM, et al, 2015 SCR7 targets the DNA binding domain of DNA ligase IV, reducing its affinity for DSBs and inhibiting its function 0.01~1μM 24-48h MelJuSo (melanoma) 10.9-19.1 1μM 24-48h DC2.4 (dendritic cell line) 9.3-14.1 1mM Inject together with Cas9 and sgRNA Mouse zygote 1.0-4.0 1μM 12-72h HEK293 5 Maruyama T, et al, 2015 1μM 0-72h HEK293A 1.2 Pinder J, et al, 2015 20-80μM 0-20h Rabbit embryo NS Song J, et al, 2016 1μM 16h-64h HEK293 2 Robert F, et al, 2015 NU7441 2μM 16h-64h HEK293 2 Pharmacological inhibitors of DNA- PKcs KU- 0060648 250nM 16h-64h HEK293 2 HR activator RS-1 10μM 0-72h HEK293A 3-6 Pinder J, et al, 2015 Stabilize association of RAD51 with DNA 10μM 0-72h U2OS 1.5-3 7.5μM, 15μM 0-20h Rabbit embryo 2-5 Song J, et al, 2016 1
Transcript of Supplementary Table 1: Small molecules that are reported ...10.1186/s13059-017-1164... ·...
Supplementary Table 1: Small molecules that are reported to increase HDR efficiency.
Type Small molecule Concentration Time Cell line Fold
change Reference Function
NHEJ inhibitors
SCR7
0.01~1μM 24-48h A549 1.8-3.1
Byrne SM, et al, 2015 SCR7 targets the DNA binding domain of DNA ligase IV, reducing its affinity for DSBs and inhibiting its function
0.01~1μM 24-48h MelJuSo (melanoma) 10.9-19.1
1μM 24-48h DC2.4 (dendritic cell line) 9.3-14.1
1mM Inject together with Cas9 and
sgRNA Mouse zygote 1.0-4.0
1μM 12-72h HEK293 5 Maruyama T, et al, 2015 1μM 0-72h HEK293A 1.2 Pinder J, et al, 2015
20-80μM 0-20h Rabbit embryo NS Song J, et al, 2016 1μM 16h-64h HEK293 2
Robert F, et al, 2015 NU7441 2μM 16h-64h HEK293 2 Pharmacological inhibitors of DNA-PKcs
KU-0060648 250nM 16h-64h HEK293 2
HR activator RS-1
10μM 0-72h HEK293A 3-6 Pinder J, et al, 2015 Stabilize association of
RAD51 with DNA 10μM 0-72h U2OS 1.5-3 7.5μM, 15μM 0-20h Rabbit embryo 2-5 Song J, et al, 2016
1
Table 1 (Continued)
Type Small molecule Concentration Time Cell line Fold
change Reference Function
Cell cycle inhibitors
Lovastatin (Lov) 40μM 0-17h
HEK293T NS
Lin S, et al, 2014
Blocks at early G1 and partially at G2/M phase Fibroblast 0.8%
Mimosine (Mim) 200μM 0-17h
HEK293T NS
Arrest cells at the G1-S border prior to onset of DNA replication
Fibroblast 0.7% Aphidicolin
(Aph) 2μg/ml 0-17h, HEK293T 1.1-1.5
25h-42h Fibroblast 0.6%-1.3% Thymidine
(Thy) 5mM 0-17h, HEK293T NS
25h-42h Fibroblast 0.6% Hydroxyurea
(HU) 2mM 0-17h HEK293T NS Fibroblast 0.6%
Nocodazole (Noc) 200ng/ml
0-17h HEK293T 1.5-3
Arrest at G2/M phase 0-17h Fibroblast NS
0-16h, Aph 3h Human ESC 2%
Others
L755507 5μM 0-72h HEK293A NS Pinder J, et al, 2015 A β3-adrenergic receptor agonist L755507 5μM 0-24h Mouse ESC 3
Yu C, et al, 2015
Brefeldin A 0.1μM 0-24h Mouse ESC 2
An NHEJ inhibitor of intracellular protein transport from the ER to the Golgi apparatus
NS: not significant.
2
a
Supplementary Figure 1: The HDR efficiency of different homology arm length in three 293T reporter lines. Single 293T-puro-wpre cell clones 1# (a), 2# (b) and 3# (c) were isolated by sorting. The HDR-mediated knockin efficiencies were determined by FACS at day 7 after co-transfection with Cas9/sgRNA1 and the donor plasmids with various homology arm length. The data presented are average of three technical replicates.
293T-puro-wpre Reporter line 1
293T-puro-wpre Reporter line 2
293T-puro-wpre Reporter line 3
b c
3
Supplementary Figure 2: The cleavage efficiency of sgCTNNB1 and sgPRDM14 in iPSCs. To confirm the target locus cleavage activity of sgCTNNB1 and sgPRDM14, we nucleofected iPSCs of each sgRNA together with Cas9 plasmid, and carried out T7E1 assay using total genomic DNA. CTNNB1-F1 and CTNNB1-R1 primers, PRDM14-F2 and PRDM14-R2 primers were used for PCR. The cleavage frequency was calculated from the proportion of cut bands intensity to total bands intensity.
500bp
sgCTNNB1T7E1 assay
－ ＋ － ＋
sgPRDM14
700bp
1000bp
400bp
Cleavage (%) 0 060 30
T7E1
4
G
T
G
A
C
G
G
Sg
s
5
Rep
GTGGCCTGGCAC
TGTTAACGTCTAT
GGCCAGGATGCC
ACCTCATGGATG
CTTTTACTCTGCC
GGAACATTGGAA
GCTGTTTTAAACA
Supplementagray shadow. PAM is indicatline and right sequences in lhomology arm
Primer CTNN
51bp (HA50bp
placed sequenc
CTGAGTAATTACT
TGTCTGCTTCTCT
CTTGGGTATGGA
GGGCTGCCTCCA
CTACAGAACTTCA
ATGTTCTCAGATT
ATTAATAGCAGC
ary Figure 3aThe stop codoted in bold andhomology armleft and right a
ms. The primers
NB1-F1
p)
RecoPAM
ce in left arm
304bp (HA
Cut s
TTGGCTAAAGAAA
TCCTCTCTCTTTT
ACCCCATGATGGA
GGTGACAGCAAT
AGAAAGACTTGG
TTCTGGTTGTTAT
CTTTCTCTCTTTA
: Design of gon is indicatedd the predicted
m with a blue lirms are separas used for knoc
ognition site of
Replaced
93b
A300bp)
site
AGCTGGAGGTTG
TGCCTTCCTTCTT
AACAGAGATGGG
TCAGCTGGCCTG
GTTGGTAGGGTGG
TGTGATCATGTGT
ATACAGCTGTATT
gene editing ad in red. The red cut site is indne. The precisately indicatedckin pattern an
sgCTNNB1
d sequence in r
308
bp (HA100bp)
GAAGAGGCTAGA
TGCCTATTTTGTT
GTGGCCACCCAC
GGTTTGATACTGA
GGAGTGGTTTAG
TGGAAGTTATTAA
TGTCTGAACTTGC
at the human ecognition sitedicated by a rese length of ho in purple and
nalysis by PCR
87bp (HAright arm
bp (HA300bp
)
AAAGCGTTGTTTT
TGACACCCTGACT
CCCTGGTGCTGAC
ACCTGTAAATCAT
GGCTATTTGTAAA
ACTTTAATGTTTTT
CATTGTGATTGGC
CTNNB1 exoe of sgCTNNBed triangle. Thmology arm ispink. Sequenc
R are indicated
Lef
P
100bp)
Stop Codon
p) TCTGACAAGTTTG
TCTTCTAGATCCT
CTATCCAGTTGAT
TCCTTTAGGTAAG
ATCTGCCACAAAA
TTGCCACAGCTT
CCTGTAGAGTTG
n15. The exon1 is in bold an
he left homolog indicated undces in grey indby black arrow
ft homology
Primer CTNNB
GCTGCTGAACTTT
TAGCTATCGTTCT
TGGGCTGCCAGA
GAAGTTTTAAAAA
AACAGGTATATAC
TTTGCAACTTAATA
GCTGAG
n sequence ofd underlined bgy arm is indicer a thick arrowicate genomic
ws.
y arm
B1-R1
Right ho
Ex
145bp (HA
TGGATGCCCTAA
TTTTCACTCTGGT
ATCTGGGGCATG
AGCCAGTTTGGG
CTTTGAAAGGAGA
ACTCAAATGAGTA
CTNNB1 geneby a green linecated with an ow. Replaced DNA outside o
140bp (HA
41bp (HAomology arm
xon15
A150bp)
CCTCAG
TGGATAT
GCCCAGG
GTAAAATA
ATGTCTT
AACATTT
e is in e, The orange
of
A150bp)
A50bp) m
5
S
G
T
G
A
G
G
G
T
G
Suppleme
GTGGCCTGGCAC
TGTTAACGTCTAT
GGCCAGGATGCC
ACCTCATGGATG
GGTGGTGGATCC
GGGTCAGGGCAC
GCTTCCATCAGT
TCCCTTACTGTTA
GACCGCTGCGGA
Primer CTN
entary Figu
CTGAGTAATTACT
TGTCTGCTTCTCT
CTTGGGTATGGA
GGGCTGCCTCCtG
CATGGTGAGCAA
CCGGCAATCCAA
TACCTGCCCTACC
AACTACCGCTAC
ACTGGTGCAGGT
NB1-F1
PAM
ure 3b: Pr
TTGGCTAAAGAAA
TCCTCTCTCTTTT
ACCCCATGATGGA
GGcGAtAGCAATC
AGGGCGAGGAGG
AATGATGGTTATG
CCTGACGGGATG
ACCTACGAGGGA
TCGAAGAAGACTT
Destroyed rec
CTNNB
recisely ed
AGCTGGAGGTTG
TGCCTTCCTTCTT
AACAGAGATGGG
CAGCTGGCCTGG
GATAACATGGCCT
GAGGAGTTAAACC
GTCGCCTTTCCAG
AAGCCACATCAA
TACCCCAACGAC
ognition site of
B1 with sense m
308
dited CTNN
GAAGAGGCTAGA
TGCCTATTTTGTT
GTGGCCACCCAC
GTTTGATACTGAC
TCTCTCCCAGCG
CTGAAGTCCACC
GGCCGCCATGGT
AAGGAGAGGCCC
CAAAACCATCATC
f sgPRDM14
mutations
m
8bp (HA300bp
87bp (HA1
NB1 locus
AAAGCGTTGTTTT
TGACACCCTGACT
CCCTGGTGCTGAC
CCTGGGTGGTGG
GACACATGAGTTA
CAAGGGTGACCTC
TAGATGGCTCCGG
AGGTGAAGGGG
CAGTACCTTTAAG
mNeonGreen
p)
00bp)
s.
TCTGACAAGTTTG
TCTTCTAGATCCT
CTATCCAGTTGAT
GTGGTTCTGGTGG
ACACATCTTTGGC
CCAGTTCTCCCC
GATACCAAGTCC
ACTGGTTTCCCT
GTGGAGTTACACC
Left hom
GS
n
GCTGCTGAACTTT
TAGCTATCGTTCT
TGGGCTGCCAGA
GTGGTGGTTCCG
CTCCATCAACGGT
CCTGGATTCTGGT
CATCGCACAATGC
TGCTGACGGTCCT
CACTGGAAATGG
ology arm
S Linker 4
TGGATGCCCTAAC
TTTTCACTCTGGT
ATCTGGGGCATG
GGCGGCGGCGGC
TGTGGACTTTGA
TCCCTCATATCGG
CAGTTTGAAGATG
TGTGATGACCAA
GCAAGCGCTACCG
Exon15
140bp (HA
41bp (HA50bp
CCTCAG
TGGATAT
GCCCAGG
CTCTGGT
ACATGGT
GGTATG
GGTGCC
ACTCGCT
GGAGCA
A150bp)
p)
6
C
A
T
A
A
T
T
T
A
G
C
CTGCGCGGACCA
AACTTCAAGGAG
TTGACTGGTATTC
AATCCTGGTTGC
ACCTGTCAGCTC
TGACAATTCCGT
TCAATCCAGCGG
TGATAAGATACAT
ATAAACAAGTTAA
GATCCGGCTGCC
CCGTCAGGCGTC
ACCTACACCTTTG
GTGGCAAAAGGCC
CTTAACTATGTTG
CTGTCTCTTTATGA
CCTTTCCGGGACT
GGTGTTGTCGGG
GACCTTCCTTCCC
TTGATGAGTTTG
ACAACAACAATTG
CTCGCGCGTTTC
CAGCGGGTGTTG
GCCAAGCCAATG
CTTTACCGATGT
GCTCCTTTTACGC
AGGAGTTGTGGC
TTTCGCTTTCCCC
GGAAGCTGACGT
CGCGGCCTGCTG
GACAAACCACAA
GCATTCATTTTAT
CGGTGATGACGGT
GGCGGGTGTCGG
GGCGGCTAACTAT
GATGGGCATGGA
CTATGTGGATACG
CCCGTTGTCAGG
CCTCCCTATTGCC
TCCTTTCCATGGC
GCCGGCTCTGCG
ACTAGAATGCAGT
TGTTTCAGGTTCA
TGAAAACCTCTG
GGGCGCAGCCAT
TCTGAAGAACCA
ACGAGCTGTACA
GCTGCTTTAATG
GCAACGTGGCGTG
CACGGCGGAACT
CTGCTCGCCTGT
GGCCTCTTCCGCG
TGAAAAAAATGCT
AGGGGGAGGTGT
ACACATGCAGCT
TGAGTAAATCATC
Stop Codon
AGCCGATGTACGT
AAGTAAGTTTAAA
CCTTTGTATCATG
GGTGTGCACTGT
TCATCGCCGCCT
TGTTGCCACCTGG
GTCTTCGCCTTCG
TTTATTTGTGAAA
TGGGAGGTTTTTT
TCCCGGAGACGG
CCTTTAGGTAAGA
TGTTCCGTAAGA
ACGCGTCGACAAT
GCTATTGCTTCC
TGTTTGCTGACGC
TGCCTTGCCCGCT
GATTCTGCGCGG
GCCCTCGACGAG
ATTTGTGATGCTA
TAAAGCAAGTAAA
GTCACAGCTTGTC
AAGTTTTAAAAAG
Right hom
CGGAGCTCAAGC
TCAACCTCTGGA
CGTATGGCTTTC
CAACCCCCACTG
TGCTGGACAGGG
GGACGTCCTTCTG
GTCGGATCTCCC
ATTGCTTTATTTG
AACCTCTACAAA
CTGTAAGCGGAT
GCCAGTTTGGGTA
Wpre
mology arm
51b
CACTCCAAGACC
ATTACAAAATTTGT
CATTTTCTCCTCCT
GGTTGGGGCATTG
GGCTCGGCTGTT
GCTACGTCCCTTC
CTTTGGGCGGATC
TAACCATTATAAG
ATGTGGTATGGCT
TGCCGGGAGCAG
AAAATACTTTTAC
pl
m
bp (HA50bp)
CGAGCTC
TGAAAGA
TTGTATA
GCCACC
TGGGCAC
CGGCCC
CCAGACA
GCTGCA
TGATTAT
GACAAGC
CTCTGCC
oyA
7
T
T
A
S
g
a
S
TACAGAACTTCAG
TGTTCTCAGATTT
ATTAATAGCAGC
Supplementamarked in red
green line. Th
arm is indicate
Sequences in
black arrows.
304bp (H
GAAAGACTTGGT
TCTGGTTGTTATG
CTTTCTCTCTTTA
ary Figure 3b. Silent mutati
e left homolog
ed under a thic
grey indicate g
HA300bp)
93bp
TTGGTAGGGTGG
GTGATCATGTGTG
ATACAGCTGTATT
: Precisely edons are introd
gy arm is indic
k arrow. The G
genomic DNA
p (HA100bp)
GGAGTGGTTTAGG
GGAAGTTATTAAC
TGTCTGAACTTGC
dited CTNNB1uced to destro
ated with an o
GS linker seque
outside of hom
GCTATTTGTAAAT
CTTTAATGTTTTT
CATTGTGATTGG
1 locus. The e
oy the recogni
orange line and
ence is in yello
mology arms. T
P
TCTGCCACAAAAA
TTGCCACAGCTTT
CCTGTAGAGTTG
exon of CTNNB
tion site of sgC
d the right HA
ow and mNeon
The primers us
Primer CTNNB
ACAGGTATATACT
TTGCAACTTAATA
GCTGAG
B1 gene is in g
CTNNB1, whic
A with a blue lin
nGreen-Wpre-p
ed for knockin
B1-R1
145bp (HA1TTTGAAAGGAGA
ACTCAAATGAGTA
gray shadow. T
ch is in bold an
ne. The precise
ployA sequenc
pattern analys
50bp) ATGTCTTGGAACA
AACATTTGCTGTT
The stop codo
nd underlined
e length of hom
ce in green colo
sis are indicate
ATTGGAA
TTTAAAC
on is
by a
mology
or.
ed by
8
Supplementary Figure 3c: Backbone sequence cut off from pD-mNeonGreen-sg (CTNNB1).
ggtgacagcaatcagccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaa
aaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgg
gttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagc
aactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactg
cggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacga
gcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttc
tgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacgggg
agtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaag
gatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgct
tgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagtta
ggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggc
gcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaagg
cggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgat
gctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgccttt
gagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagc
tggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcgg
ataacaatttcacacagctgattgctgtcacc
Partial recognition site of sgCTNNB1
Partial recognition site of sgCTNNB1
Donor plasmid backbone sequence
9
Supplementary Figure 3c: Backbone sequence cut off from pD-mNeonGreen-sg (CTNNB1). The sequence of donor plasmid
backbone is in lower case and indicated by a red line. The residual partial sgCTNNB1 recognition sequence after linearization by
CRISPR is in bold and indicated by green lines.
10
Supplementary Figure 3d: Knockin pattern analysis at the CTNNB1 locus. pD-mNeonGreen-sg-HA50-50bp (CTNNB1) Left homology arm Replaced sequence in left arm Replaced sequence in right arm Right homology arm Wild type TGCCAGATCTGGGGC::::::GGCTGCCTCCa------------------------------------------------------------------------ggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::GGTAAAATACTTTTA
Left homology arm CTNNB1 with sense mutations GS-mNeonGreen-Wpre-ployA Right homology arm Expected TGCCAGATCTGGGGC::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG::::::CAGCCATGAGTAAATCATCC:::::GGTAAAATACTTTTA Cl1-19 TGCCAGATCTGGGGC::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG::::::CAGCCATGAGTAAATCATCC:::::GGTAAAATACTTTTA HDR Cl20-21 TGCCAGATCTGGGGC::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG::::::CAGCCATGAGtaaatcatcc:::::::::::::::::::ggtaaaatac
-----------------------------cctggtttgatcatgacctgTAAATCATCC:::::GGTAAAATACTTTTA 3-NHEJ 71bp ins Clone22 TGCCAGATCTGGGGC::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG:::::CAGCCATGAGtaaatcatcc::::::::::::::::::::ggt------- -----gacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::GGTAAAATACTTTTA 3-NHEJ 80bp ins Clone23 TGCCAGATCTGGGGC::::::GGCTGCCTCCagtggagtattttacccaaactgg::::cctgggcatgccccagatcctccagggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::GGTAAAATACTTTTA 5,3-NHEJ mNeonGreen reverse insertion Clone24 TGCCAGATCTGGGGC::::::GGCTGCCTCCaggacagcaatcagccaggtg:::::tcacacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::GGTAAAATACTTTTA 5,3-NHEJ Backbone forward insertion Clone25 TGCCAGATCTGGGGC::::::GGCTGCCTCCagtggtgacagcaatcagccag::::tcacacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::GGTAAAATACTTTTA 5,3-NHEJ Backbone forward insertion Clone26 TGCCAGATCTGGGGC::::::GGCTGCCT----cagcaatcagccaggtggcac::: ::tcacacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::GGTAAAATACTTTTA 5,3-NHEJ Backbone forward insertion Cl27-28 TGCCAGATCTGGGGC::::::GGCTGCCTCCagggtgacagcaatcagctcctg::agtgccacctggctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::GGTAAAATACTTTTA 5,3-NHEJ Backbone reverse insertion Clone29 TGCCAGATCTGGGGC::::::GGCTGCCT-------------------------tcagctcctg::::agtgccacctggctgattgctgt----ggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::GGTAAAATACTTTTA 5,3-NHEJ Backbone reverse insertion Clone30 TGCCAGATCTGGGGC:::::-----------------ggtgacagcaatcagctcctg:::ccccgaaaagtgccacctggctgatt----------------------------gctggcctggtttgatactgacctgTAAATCATCC:::::GGTAAAATACTTTTA 5,3-NHEJ Backbone reverse insertion
pD-mNeonGreen-sg-HA100-100bp (CTNNB1) Left homology arm Replaced sequence in left arm Replaced sequence in right arm Right homology arm Wild type GAGATGGGTGGCCAC::::::GGCTGCCTCCa-----------------------------------------------------------------------ggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::TGGTTGGTAGGGTGG
Left homology arm CTNNB1 with sense mutations GS-mNeonGreen-Wpre-ployA Right homology arm Expected GAGATGGGTGGCCAC::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG::::CAGCCATGAGTAAATCATCC:::::TGGTTGGTAGGGTGG Cl1-16 GAGATGGGTGGCCAC::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG::::CAGCCATGAGTAAATCATCC:::::TGGTTGGTAGGGTGG HDR Clone17 GAGATGGGTGGCCAC::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG::::CAGCCATGAGtaaatcatcc::::::::::::::::::::::tggttggt--
---------------------------tggcctggtttgatactgacctgTAAATCATCC::::TGGTTGGTAGGGTGG 3-NHEJ 114bp ins Clone18 GAGATGGGTGGCCAC::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG::::--------------------------------------------
--------------caatcagctggcctggtttgatactgacctgTAAATCATCC::::TGGTTGGTAGGGTGG 3-NHEJ 493bp del; 31bp ins Clone19 GAGATGGGTGGCCAC:::::----------------tggagggtggccac::::ggctgcctcctggcgatagcaatcagctggcctggtttgatactgacctgggtggtggtg::cagccatgagTAAATCATCC:::::TGGTTGGTAGGGTGG 5-NHEJ 41bp del; 131bp ins Clone20 GAGATGGGTGGCCAC::::::GGCTGCCTCCatggagggtggccac::::ggctgcctcctggcgatagcaatcagctggcctggtttgatactgacctgggtggtggtg::cagccatgagtaaatcatcc::::::::::::::::::::::tggttggtagcctccag
ggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::TGGTTGGTAGGGTGG 5-NHEJ 91bp ins; 3-NHEJ 139bp ins Clone21 GAGATGGGTGGCCAC::::::GGCTGCCTCCaggtgacagcaatcagccaggtg:::::cacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::TGGTTGGTAGGGTGG 5,3-NHEJ Backbone forward insertion Cl22-23 GAGATGGGTGGCCAC::::::GGCTGCCTCCagggtgacagcaatcagccaggtg:::cacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::TGGTTGGTAGGGTGG 5,3-NHEJ Backbone forward insertion Clone24 GAGATGGGTGGCCAC::::::GGCTGCCTCC-ggtgacagcaatcagccaggtg:::::cacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::TGGTTGGTAGGGTGG 5,3-NHEJ Backbone forward insertion Cl25-26 GAGATGGGTGGCCAC::::::GGCTGCCTCC-------agcaatcagccaggtg:::: :::cacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::TGGTTGGTAGGGTGG 5,3-NHEJ Backbone forward insertion Clone27 GAGATGGGTGGCCAC::::::GGCTGCCT-------gacagcaatcagccaggtg:::::cacaggagctgattgctgtca-----------------------tcagctggcctggtttgatactgacctgTAAATCATCC:::::TGGTTGGTAGGGTGG 5,3-NHEJ Backbone forward insertion Clone28 GAGATGGGTGGCCAC::::::GGCTGCCTCCa---gacagcaatcagccaggtg:::::cacaggagctgattgctg----------------------------------gcctggtttgatactgacctgTAAATCATCC:::::TGGTTGGTAGGGTGG 5,3-NHEJ Backbone forward insertion Clone29 GAGATGGGTGGCCAC::::::GGCTGCCTCCag-----------cagccaggtg:::::cacaggagctgattgctgtcaccagctgctgatt
-gtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::TGGTTGGTAGGGTGG 5,3-NHEJ Backbone forward insertion Clone30 GAGATGGGTGGCCAC::::::GGCTGCCTCCagggtgacagcaatcagctcctgtgt::::cacctggctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::TGGTTGGTAGGGTGG 5,3-NHEJ Backbone reverse insertion
pD-mNeonGreen-sg-HA150-150bp (CTNNB1)
11
Left homology arm Replaced sequence in left arm Replaced sequence in right arm Right homology arm Wild type ACTCTGGTGGATATG::::::GGCTGCCTCCa------------------------------------------------------------------------ggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::AGGTATATACTTTGA
Left homology arm CTNNB1 with sense mutations GS-mNeonGreen-Wpre-ployA Right homology arm Expected ACTCTGGTGGATATG::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG::::CAGCCATGAGTAAATCATCC:::::AGGTATATACTTTGA Cl1-24 ACTCTGGTGGATATG::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG:::::CAGCCATGAGTAAATCATCC::::AGGTATATACTTTGA HDR Clone25 ACTCTGGTGGATATG::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG:::::CAGCCATGAGtaaatcatcc:::::::::::::::::::aggtatatac
---------------------------------tggtttgatactgacctgTAAATCATCC:::::AGGTATATACTTTGA 3-NHEJ 163bp ins Clone26 ACTCTGGTGGATATG::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG:::::CAGCCATGAGtaaatcatcc:::::::::---------------
---------------------agctggcctggtttgatactgacctgTAAATCATCC:::::AGGTATATACTTTGA 3-NHEJ 156bp ins Clone27 ACTCTGGTGGATATG::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG:::::-------------------------------------------
-gtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::AGGTATATACTTTGA 3-NHEJ 339bp del, 38bp ins Clone28 ACTCTGGTGGATATG::::::GGCTGCCTCCagcctggaggtggatatg::::::ggctgcctcctggcgatagcaatcagctggcctggtttgatactgacctg
GGTGGTGGTG:::::CAGCCATGAGTAAATCATCC::::AGGTATATACTTTGA 5-NHEJ 148bp ins Clone29 ACTCTGGTGGATATG::::::GGCTGCCTCCagctgaaggtggatatg::::::ggctgcctcctggcgatagcaatcagctggcctggtttgatactgacctgggtggtggtg::cagccatgagtaaatcatcc::::--------------- ggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::AGGTATATACTTTGA 5-NHEJ 147bp ins; 3-NHEJ 165bp ins Clone30 ACTCTGGTGGATATG::::::GGCTGCCTCCaggtgacagcaatcagccaggtg::::cacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::AGGTATATACTTTGA 5,3-NHEJ Backbone forward insertion Clone31 ACTCTGGTGGATATG::::::GGCTGCCTCC-ggtgacagcaatcagccaggtg::::cacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::AGGTATATACTTTGA 5,3-NHEJ Backbone forward insertion Cl32-35 ACTCTGGTGGATATG::::::GGCTGCCTC-------cagcaatcagccaggtg:::: :::cacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::AGGTATATACTTTGA 5,3-NHEJ Backbone forward insertion Clone36 ACTCTGGTGGATATG::::::GGCTGCCTCCagggtgacagcaatcagccaggt::::cacaggagctgattgctgtca-------------gcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::AGGTATATACTTTGA 5,3-NHEJ Backbone forward insertion pD-mNeonGreen-sg-HA300-300bp (CTNNB1)
Left homology arm Replaced sequence in left arm Replaced sequence in right arm Right homology arm Wild type TTGAAGAGGCTAGAA::::::GGCTGCCTCCa----------------------------------------------------------------------ggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::ATAGCAGCCTTTCTC
Left homology arm CTNNB1 with sense mutations GS-mNeonGreen-Wpre-ployA Right homology arm Expected TTGAAGAGGCTAGAA::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG::::CAGCCATGAGTAAATCATCC:::::ATAGCAGCCTTTCTC Cl1-33 TTGAAGAGGCTAGAA::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG::::CAGCCATGAGTAAATCATCC:::::ATAGCAGCCTTTCTC HDR Clone34 TTGAAGAGGCTAGAA::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG::::CAGCCATGAGtaaatcatcc::::::::::::::::::::atagcagcctcca
---------gcaatcagctggcctggtttgatactgacctgTAAATCATCC::::ATAGCAGCCTTTCTC 3-NHEJ 339bp ins Clone35 TTGAAGAGGCTAGAA::::::GGCTGCCTCCaggtgacagcaatcagccaggtg:::::cacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::ATAGCAGCCTTTCTC 5,3-NHEJ Backbone forward insertion Clone36 TTGAAGAGGCTAGAA:::::::GGCTGC-------ggtgacagcaatcagccaggtg::: :::cacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::ATAGCAGCCTTTCTC 5,3-NHEJ Backbone forward insertion Clone37 TTGAAGAGGCTAGAA::::::GGCTGCCT----------cagcaatcagccaggtg::: :: ::cacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::ATAGCAGCCTTTCTC 5,3-NHEJ Backbone forward insertion Clone38 TTGAAGAGGCTAGAA::::::GGCTGCCT----------cagcaatcagccaggtg:: ::::cacaggagctgattgctgtcacctggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC::::ATAGCAGCCTTTCTC 5,3-NHEJ Backbone forward insertion Clone39 TTGAAGAGGCTAGAA::::::GGCTGCCTCCaggc-------------ccaggtg:::: :: :: cacaggagctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::ATAGCAGCCTTTCTC 5,3-NHEJ Backbone forward insertion Clone40 TTGAAGAGGCTAGAA::::::GGCTGCCTCCagggtgacagcaatcagctcctgtg::::gccacctggctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::ATAGCAGCCTTTCTC 5,3-NHEJ Backbone reverse insertion Clone41 TTGAAGAGGCTAGAA::::::GGCTGCCTCCagaggtgacagcaatcagctcctgtg::gccacctggctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::ATAGCAGCCTTTCTC 5,3-NHEJ Backbone reverse insertion Cl42-43 TTGAAGAGGCTAGAA::::::GGCT----------ggtgacagcaatcagctcctgtgtg:: :: ::gccacctggctgattgctgtcaccggtgacagcaatcagctggcctggtttgatactgacctgTAAATCATCC:::::ATAGCAGCCTTTCTC 5,3-NHEJ Backbone reverse insertion
Supplementary Figure 3d: Knockin pattern analysis at the CTNNB1 locus. The stop codon is indicated in red. The left homology arm is highlighted in yellow shadow and right homology arm in blue shadow. Replaced sequences at upstream and
12
downstream of DSB are separately indicated in purple and pink lowercase. The inserted sequence is in red lowercase. Sequences in grey indicate genomic DNA outside of homology arms.
13
G
T
G
A
C
G
G
S3stag
Repl
GTGGCCTGGCAC
TGTTAACGTCTAT
GGCCAGGATGCC
ACCTCATGGATG
CTTTTACTCTGCC
GGAACATTGGAA
GCTGTTTTAAACA
Supplementa300bp-RR1-0sgCTNNB1 is triangle. The learm is indicategenomic DNA
Primer CTNN
laced sequence
CTGAGTAATTACT
TGTCTGCTTCTCT
CTTGGGTATGGA
GGGCTGCCTCCA
CTACAGAACTTCA
ATGTTCTCAGATT
ATTAATAGCAGC
ary Figure 4abp). The exonin bold and uneft homology a
ed under a thicoutside of hom
NB1-F1
343bp (H
RecPAM
Cut se in left arm
TTGGCTAAAGAAA
TCCTCTCTCTTTT
ACCCCATGATGGA
GGTGACAGCAAT
AGAAAGACTTGG
TTCTGGTTGTTAT
CTTTCTCTCTTTA
: Improved den sequence of Cnderlined by aarm is indicatek arrow. Repla
mology arms. T
HA300bp)
cognition site o
site
AGCTGGAGGTTG
TGCCTTCCTTCTT
AACAGAGATGGG
TCAGCTGGCCTG
GTTGGTAGGGTGG
TGTGATCATGTGT
ATACAGCTGTATT
esign of genoCTNNB1 is in
a green line. Thed with an oranaced sequenceThe primers us
30
of sgCTNNB1
GAAGAGGCTAGA
TGCCTATTTTGTT
GTGGCCACCCAC
GGTTTGATACTGA
GGAGTGGTTTAG
TGGAAGTTATTAA
TGTCTGAACTTGC
ome editing agray shadow.he PAM is indnge line and th
e at upstream osed for knockin
8bp (HA300bAAAGCGTTGTTTT
TGACACCCTGACT
CCCTGGTGCTGAC
ACCTGTAAATCAT
GGCTATTTGTAAA
ACTTTAATGTTTTT
CATTGTGATTGGC
at human CTN The stop codicated in bold he right arm wof DSB is indicn pattern analy
Pr
Lef
p)
Stop Codon
Right homo
TCTGACAAGTTTG
TCTTCTAGATCCT
CTATCCAGTTGAT
TCCTTTAGGTAAG
ATCTGCCACAAAA
TTGCCACAGCTT
CCTGTAGAGTTG
NNB1 exon15 on is indicatedand the expec
with a blue linecated in purple.sis are indicate
imer CTNNB1
ft homology
ology arm
GCTGCTGAACTTT
TAGCTATCGTTCT
TGGGCTGCCAGA
GAAGTTTTAAAAA
AACAGGTATATAC
TTTGCAACTTAATA
GCTGAG
(pD-mNeonGd in red. The rected cut site ise. The precise . Sequences ined by black arr
1-R1
y arm TGGATGCCCTAAC
TTTTCACTCTGGT
ATCTGGGGCATG
AGCCAGTTTGGG
CTTTGAAAGGAGA
ACTCAAATGAGTA
Green-sg-HA3ecognition site
s indicated by alength of homon grey indicate rows.
Exon15
CCTCAG
TGGATAT
GCCCAGG
GTAAAATA
ATGTCTT
AACATTT
300-e of a green ology
14
S3 G
T
G
A
G
G
G
T
G
Suppleme300bp-RR
GTGGCCTGGCAC
TGTTAACGTCTAT
GGCCAGGATGCC
ACCTCATGGATG
GGTGGTGGATCC
GGGTCAGGGCAC
GCTTCCATCAGT
TCCCTTACTGTTA
GACCGCTGCGGA
Primer CTN
entary FiguR1-0bp
CTGAGTAATTACT
TGTCTGCTTCTCT
CTTGGGTATGGA
GGGCTGCCTCCtG
CATGGTGAGCAA
CCGGCAATCCAA
TACCTGCCCTACC
AACTACCGCTAC
ACTGGTGCAGGT
NNB1-F1
PAM
ure 4b: Pr
TTGGCTAAAGAAA
TCCTCTCTCTTTT
ACCCCATGATGGA
GGcGAtAGCAATC
AGGGCGAGGAGG
AATGATGGTTATG
CCTGACGGGATG
ACCTACGAGGGA
TCGAAGAAGACTT
Destroyed rec
CTNNB1 w
recisely ed
AGCTGGAGGTTG
TGCCTTCCTTCTT
AACAGAGATGGG
CAGCTGGCCTGG
GATAACATGGCCT
GAGGAGTTAAACC
GTCGCCTTTCCAG
AAGCCACATCAA
TACCCCAACGAC
30
cognition site o
with sense muta
dited CTNN
GAAGAGGCTAGA
TGCCTATTTTGTT
GTGGCCACCCAC
GTTTGATACTGAC
TCTCTCCCAGCG
CTGAAGTCCACC
GGCCGCCATGGT
AAGGAGAGGCCC
CAAAACCATCATC
08bp (HA300b
of sgCTNNB1
ations
m
NB1 locus
AAAGCGTTGTTTT
TGACACCCTGACT
CCCTGGTGCTGAC
CCTGGGTGGTGG
GACACATGAGTTA
CAAGGGTGACCTC
TAGATGGCTCCGG
AGGTGAAGGGG
CAGTACCTTTAAG
bp)
mNeonGreen
s with pD-m
TCTGACAAGTTTG
TCTTCTAGATCCT
CTATCCAGTTGAT
GTGGTTCTGGTGG
ACACATCTTTGGC
CCAGTTCTCCCC
GATACCAAGTCC
ACTGGTTTCCCT
GTGGAGTTACACC
Left hom
G
n
mNeonGre
GCTGCTGAACTTT
TAGCTATCGTTCT
TGGGCTGCCAGA
GTGGTGGTTCCG
CTCCATCAACGGT
CCTGGATTCTGGT
CATCGCACAATGC
TGCTGACGGTCCT
CACTGGAAATGG
ology arm
S Linker
E
een-sg-HA
TGGATGCCCTAAC
TTTTCACTCTGGT
ATCTGGGGCATG
GGCGGCGGCGGC
TGTGGACTTTGA
TCCCTCATATCGG
CAGTTTGAAGATG
TGTGATGACCAA
GCAAGCGCTACCG
Exon15
A300-
CCTCAG
TGGATAT
GCCCAGG
CTCTGGT
ACATGGT
GGTATG
GGTGCC
ACTCGCT
GGAGCA
15
CTGCGCGGACCACCTACACCTTTGCCAAGCCAATGGCGGCTAACTATCTGAAGAACCAGCCGATGTACGTGTTCCGTAAGACGGAGCTCAAGCACTCCAAGACCGAGCTC
AACTTCAAGGAGTGGCAAAAGGCCTTTACCGATGTGATGGGCATGGACGAGCTGTACAAGTAAGTTTAAACGCGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGA
TTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATA
AATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACC
ACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCAC
TGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCC
TCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCGACGAGTCGGATCTCCCTTTGGGCGGATCCAGACA
TGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCA
ATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTAT
GATCCGGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGC
CCGTCAGGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGAGGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCCTTTAGGTAAGAA
Stop Codon
Wpre
ployA
Right homology arm
16
G
A
G
T
Ss
c
GTTTTAAAAAGCC
AGGTATATACTTT
GCAACTTAATACT
TGAG
Supplementasequence of Cintroduced to dbold. The left homology armcolor. Sequencby black arrow
CAGTTTGGGTAA
TGAAAGGAGATG
TCAAATGAGTAA
ary Figure 4bCTNNB1 is in gdestroy the rehomology arm
m are indicated ces in grey ind
ws.
AAATACTTTTACTC
GTCTTGGAACATT
CATTTGCTGTTTT
: Precisely edgray shadow. Tcognition site
m is indicated wunder thick arr
dicate genomic
CTGCCTACAGAA
TGGAATGTTCTCA
TAAACATTAATAG
dited CTNNB1The stop codoof sgCTNNB1
with an orangerows. The GS
c DNA outside
343
ACTTCAGAAAGAC
AGATTTCTGGTTG
GCAGCCTTTCTCT
1 locus with pon is indicated
, which is in be line and rightlinker sequencof homology a
3bp (HA300bp
CTTGGTTGGTAGG
GTTATGTGATCAT
TCTTTATACAGCT
pD-mNeonGrein red. Silent
bold and undert homology arce is in yellow
arms. The prim
p)
GGTGGGAGTGGT
TGTGTGGAAGTTA
TGTATTGTCTGAA
een-sg-HA300mutations (indrlined by a grem with a blue and mNeonGrers used for kn
TTTAGGCTATTTG
ATTAACTTTAATG
ACTTGCATTGTGA
0-300bp-RR1-dicated in loweeen line. The P
line. The precreen-Wpre-ploynockin pattern
GTAAATCTGCCAC
GTTTTTTGCCACA
ATTGGCCTGTAG
-0bp. The exoercase) were PAM is indicatecise lengths of yA sequence ianalysis are in
Primer CTNN
CAAAAAC
AGCTTTT
AGTTGC
on
ed in
n green ndicated
NB1-R1
17
pD-mNeonGreen-sg-HA300-300bp-RR1-0bp (CTNNB1) Left homology arm Replaced sequence in left arm Right homology arm Wild type TTGAAGAGGCTAGAA::::::GGCTGCCTCCa-------------------------------------------------------------------------------------------------------------------------------GGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCC:::::ATAGCAGCCTTTCTC
Left homology arm CTNNB1 with sense mutations GS-mNeonGreen-Wpre-ployA Right homology arm Expected TTGAAGAGGCTAGAA::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG:::::CAGCCATGAGGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCC:::::ATAGCAGCCTTTCTC Cl1-37 TTGAAGAGGCTAGAA::::::GGCTGCCTCCtGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG:::::CAGCCATGAGGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCC:::::ATAGCAGCCTTTCTC HDR Clone38 TTGAAGAGGCTAGAA::::::GGCTGCCTCCagtggaggctagaa:::::ggctgcctcc
tGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG:::::CAGCCATGAGGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCC:::::ATAGCAGCCTTTCTC 3-NHEJ 312bp ins
Clone39 TTGAAGAGGCTAGAA::::::----------------tggaggctagaa::::::ggctgcctcc tGGcGAtAGCAATCAGCTGGCCTGGTTTGATACTGACCTGGGTGGTGGTG:::::CAGCCATGAGGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCC:::::ATAGCAGCCTTTCTC
3-NHEJ 13bp del, 310bp ins Clone40 TTGAAGAGGCTAGAA::::::-----------------------------::::::---------
---------------------------------------------------------------------------------------------------------:::CAGCCATGAGGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCC:::::ATAGCAGCCTTTCTC
3-NHEJ 176bp del Clone41 TTGAAGAGGCTAGAA::::::----------------------agcaatcagccaggtggcacttttcggggaaatgtgcgc::::::::: :::::::: ::::::: :::::::::: :::::::acacaggagctgattgctgtcaccGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCC:::::ATAGCAGCCTTTCTC
5,3-NHEJ Backbone reverse insertion Clone42 TTGAAGAGGCTAGAA::::::GGCTGCCTCCag-----------------------------------------------------------------------------------------:::acacaggagctgattgctgtcaccGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCC:::::ATAGCAGCCTTTCTC
5,3-NHEJ Backbone reverse insertion
Supplementary Figure 4c: Knockin pattern analysis at the CTNNB1 locus edited by pD-mNeonGreen-sg-HA300-300bp-RR1-0bp (CTNNB1). The stop codon is indicated in red. The left homology arm is indicated in yellow shadow and right homology arm in blue shadow. Replaced sequence in the left arm is indicated in purple lowercase. The inserted sequence is in red lowercase. Sequences in grey indicate genomic DNA outside of homology arms.
18
sgRNA1
PuroPuroEF1EF1 GFPGFP WpreWpreHA HA
PuroPuroEF1EF1 GFPGFP WpreWpre
PuroPuroEF1EF1 GFPGFP WpreWpre
PuroPuroEF1EF1 GFPGFP WpreWpre
PuroPuroEF1EF1 GFPGFP WpreWpreStuffer200bp
Stuffer200bp
Stuffer50bp
Stuffer200bp
sgRNA1
PuroPuroEF1EF1 GFPGFP WpreWpreHA HA
Reporter system with replaced sequence on one side
Lentivirus transductionPuromycin selection
Cas9+sgRNA1+mCherry donortransfection
293T cells 293T-reporter cells HDR-mediated KI
Reporter system with replaced sequence on both sides
a
b
c
mCherry HDR reporter system
*****
*** ***
RS0-0bp
RS0-200bp
RS200-0bp
RS0-0bp
RS50-50bp
RS200-200bp
293T-reporter cells
293T-reporter cells
pD-mCherry-sg PuroPuro mCherry WpreWpresgRNA1 sgRNA1
pD-mCherry-sg PuroPuro mCherry WpreWpresgRNA1 sgRNA1
19
Supplementary Figure 5: The effects of replaced sequence length adjacent to DSB site (sgRNA1 cleavage site) on HDR efficiency. (a) Schematic outline of the HDR reporter systems. 293T cells transduced with lentiviral vectors harboring different lengths of replaced sequences in (b) and (c) were used as reporter cell lines. (b-c) The HDR-mediated knockin efficiency was determined by FACS at day 7 after co-transfection with Cas9/sgRNA1 and the donor plasmid pD-mCherry-sg-HA300-300bp. Significance was calculated using Student’s paired t-test: **P≤0.01; ***P≤0.001.
20
S
C
G
T
A
G
G
C
G
G
1
6
4
Suppleme
CCAGCCTGCAAT
GGAGTCCTTTCG
TGGTTGTCCAGC
AGAAGCCCTTCA
GTGGGAAAATCT
GGCAGAGGTGG
CATGCCTGTGGT
GACAGAACCAGA
GGGTTATAGAAGPrim
60bp (HA150
Primer PRDM
60bp (HA50b
46bp (HA50bp
entary Figu
TCTGCTTTTTTAC
GGATTAAGGCTAC
CTCGTGGAAGTTG
AAATGCAAGTACT
TTCTCAGATCAAG
GTGGATCACTCA
TTCCAGCCACTCA
ACCCTGTCTCAAA
GTCCCTGCAGTTGmer PRDM14-R
148b
0bp)
M14-F1
p)
p)
Rig
ure 6a: De
CAAACTGGACCTC
CTGATGATGACCA
GACTTTTTCTTGT
TGTGGTAAATCTT
GAAACATTCTACT
AAGTCCAGGAGTT
AGGAGGTCGAGG
AACAAAACAAAAC
GGC
Prim
R1
bp (HA150bp)
ght homolo
esign of ge
CCAGATAATCTAG
AGGAGTGCTCTA
TGTTCTGTATTTC
TTTGCATCCCATG
TCCCACATGAAGT
TCGAGACCAGCC
GTGGCAGGATGG
CAAAAAAAATGAG
Replac
mer PRDM14-
317
)
ogy arm
R
enome edi
GCCATTGAAGAAC
ATGGCCTTAAAAC
CTGTTCTCTGCAG
GCTGCCCATGAC
TTTCATGAAGACT
CTGGGCAACATG
GTTTGAGCACAGG
GTAGCCCTCAAG
ced sequence i
-F2
7bp (HA300b
Recognition sit
iting at the
CCACTGTTCTAAC
CCCAAGGTTCGT
GAGGTTCACAGCC
CAGCCATGTCCGG
TACTAGCCCTGC
GGTGAAATCCTGT
GAGACGGAGGTT
GAGTGTGGAGAC
in left arm Cut s
3
p)
111bp (HA10
te of sgPRDM14
e PRDM14
CATCCAGTGCTT
CCATAAGGATGA
CTCCAGCATACT
GCGTTCACACAA
CCAGGCACAATG
TCTCTACCAAAAA
TGCTGTGAGCTG
AATGTAAAAACAA
105bp (HAsite
322bp (HA300
PAM
00bp)
4
4 locus
TCTCAAAACTAGC
AGCAACAGTGAAA
TCCGCACACACAT
AGGAGGATGATGG
ACTCACGCCTGT
AAATACAAAAATC
GAGATCGCCCCAC
AGAGATTCGGAT
A300bp)
0bp)
Primer
Left hom
Ex
CCAGAAGGACACA
ATTTACCCCTTAG
TCAGGCAGCACT
GCTGCTCATGCA
TAATCCCAGCACT
CAGCTGGGGGTG
CTGCTTTTCAACC
TTCTCTCTATTTCPRDM14-R2
mology arm
xon8
ATCAGT
GTTTAAT
TCCGGGG
AGCATCT
TTTGGGA
GGTGGCA
CTGGGT
CTTTTAT
21
Supplementary Figure 6a: Design of genome editing at the human PRDM14 exon8. The exon8 of PRDM14 is indicated in gray shadow. The stop codon is indicated in red. The recognition site of sgPRDM14 is in bold and underlined by a green line. The PAM is indicated in bold and the cut site is indicated by a red triangle. The left homology arm is indicated with an orange line and right homology arm with a blue line. The precise length of homology arm is indicated under a thick arrow. Replaced sequence upstream of DSB is indicated in purple. Sequences in grey indicate genomic DNA outside of homology arms. The two pairs of primers used for knockin pattern analysis by PCR are indicated by black arrows.
22
S C
G
T
A
G
C
G
C
C
G
6
Suppleme
CCAGCCTGCAAT
GGAGTCCTTTCG
TGGTTGTCCAGC
AGAAGCCCTTCA
GTGGGAAAATCT
CCAGGTCCCATG
GGCGAGGGCGA
CAGTGCTTCAGC
CTACAAGACCCG
GGAGTACAACTA
Primer PRDM
60bp (HA50bp
entary Figu
TCTGCTTTTTTAC
GGATTAAGGCTAC
CTCGTGGAAGTTG
AAATGCAAGTACT
TTCTCAGATCAAG
GGTGAGCAAGGG
ATGCCACCTACGG
CCGCTACCCCGA
GCGCCGAGGTGA
ACAACAGCCACAA
148b
M14-F1
p)
ure 6b: Pr
CAAACTGGACCTC
CTGATGATGACCA
GACTTTTTCTTGT
TGTGGTAAATCTT
GAAACATTCTACT
GCGAGGAGCTGT
GCAAGCTGACCC
CCACATGAAGCA
AAGTTCGAGGGC
ACGTCTATATCAT
P
GFP
bp (HA150bp)
recisely ed
CCAGATAATCTAG
AGGAGTGCTCTA
TGTTCTGTATTTC
TTTGCATCCCATG
TCCCACATGAAGT
TTCACCGGGGTGG
CTGAAGTTCATCT
AGCACGACTTCTT
GACACCCTGGTG
TGGCCGACAAGC
rimer PRDM1
Destr
dited PRDM
GCCATTGAAGAAC
ATGGCCTTAAAAC
CTGTTCTCTGCAG
GCTGCCCATGAC
TTTCATGAAGACT
GTGCCCATCCTG
TGCACCACCGGC
TCAAGTCCGCCA
GAACCGCATCGA
CAGAAGAACGGC
14-F2
royed recogniti
M14 locus
CCACTGTTCTAAC
CCCAAGGTTCGT
GAGGTTCACAGCC
CAGCCATGTCCGG
TACCAGTGTACT
GGTCGAGCTGGA
CAAGCTGCCCGTG
ATGCCCGAAGGC
AGCTGAAGGGCA
CATCAAGGTGAAC
105bp (H
ion site of sgPR
s.
CATCCAGTGCTT
CCATAAGGATGA
CTCCAGCATACT
GCGTTCACACAA
TAATTATGCTCTCT
ACGGCGACGTAAA
GCCCTGGCCCAC
TACGTCCAGGAG
ATCGACTTCAAGG
CTTCAAGATCCGC
322bp (HA30
HA100bp)
RDM14
TCTCAAAACTAGC
AGCAACAGTGAAA
TCCGCACACACAT
AGGAGGATGATGG
TTGAAATTGGCT
ACGGCCACAAGT
CCCTCGTGACCA
GCGCACCATCTTC
GAGGACGGCAAC
CCACAACATCGA
2A
Left hom
0bp)
CCAGAAGGACACA
ATTTACCCCTTAG
TCAGGCAGCACT
GCTGCTCATGCA
GGAGATGTTGAG
TTCAGCGTGTCC
ACCCTGACCTACG
CTTCAAGGACGA
CATCCTGGGGCAC
AGGACGGCAGCG
mology arm
Exon8
ATCAGT
GTTTAAT
TCCGGGG
AGCATCT
GAGCAAC
GGCGAG
GGCGTG
ACGGCAA
CAAGCT
GTGCAGC
23
TCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAA
GCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGTTTAAACGCGTCGACAATCAACCTCTGGATTAC
AAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTT
TCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTT
GGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCT
CGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTA
CGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCGACGAGTCGGATCTCCCTTTG
GGCGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAAC
CATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTG
GTATGGCTGATTATGATCCGGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCG
GGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGAGGCCAGGCACAATGACTCACGCCTGTAATCCCAGCACTTTGGGAG
Wpre
PAM
PolyA
24
G
A
A
T
S
ga
GCAGAGGTGGGT
ATGCCTGTGGTT
ACAGAACCAGAC
TCCTTTTATGGGT
Supplementais indicated in bold. The left homology armgreen color. Seanalysis by PC
160bp (HA15
46bp (HA50bTGGATCACTCAA
TCCAGCCACTCAG
CCCTGTCTCAAAA
TTATAGAAGTCC
ary Figure 6bred. The dest
homology armm are indicated
equences in grCR are indicate
50bp)
Primer
bp) AGTCCAGGAGTTC
GGAGGTCGAGGT
ACAAAACAAAACA
CTGCAGTTGGC
: Precisely edtroyed recogni
m is indicated wwith thick arrorey indicate geed by black arr
r PRDM14-R1
Right ho
CGAGACCAGCCT
TGGCAGGATGGT
AAAACAAAACAAA
dited PRDM14tion site of sgP
with an orangews. The riboso
enomic DNA ourows.
omology ar
TGGGCAACATGG
TTTGAGCACAGGA
AAAAAATGAGTAG
4 locus. The ePRDM14 is in e line and rightome-skipping sutside of homo
317bp
rm
GTGAAATCCTGTC
AGACGGAGGTTG
GCCCTCAAGAGT
exon8 of PRDMbold and undet homology arsequence 2A isology arms. Th
p (HA300bp)
111bp (HA10CTCTACCAAAAAA
GCTGTGAGCTGA
TGTGGAGACAATG
M14 is indicateerlined by a grm with a blue s in yellow ande two pairs of
00bp) AATACAAAAATCA
AGATCGCCCCACT
GTAAAAACAAGA
ed in gray shadreen line, and line. The prec
d GFP-Wpre-plprimers used f
AGCTGGGGGTGG
TGCTTTTCAACCT
AGATTCGGATTCT
dow. The stop PAM is indica
cise lengths of loyA sequencefor knockin pat
Primer PRD
GTGGCAC
TGGGTG
TCTCTATT
codon ated in
e in ttern
M14-R2
25
Supplementary Figure 6c: Backbone sequence cut off form pD-GFP-sg (PRDM14)
agggctagtagtcttccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaa
aaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtggg
ttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagca
actcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgc
ggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgag
cgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttct
gcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacgggga
gtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaagg
atctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgctt
gcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagtta
ggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggc
gcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaagg
cggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgat
gctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgccttt
gagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagc
tggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcgg
ataacaatttcacacagaagactactagccct
Partial recognition site of sgPRDM14 Donor plasmid backbone sequence
Partial recognition site of sgPRDM14
26
Supplementary Figure 6c: Backbone sequence cut off from pD-mNeonGreen-sg (PRDM14). The sequence of donor plasmid
backbone is in lowercase and indicated by a red line. The residual partial sgPRDM14 recognition sequence after plasmid linearization
by CRISPR is in bold and indicated by green lines.
27
Supplementary Figure 6d: Knockin pattern analysis at the PRDM14 locus. pD-GFP-sg-HA50-50bp (PRDM14)
Left homology arm Replaced sequence in left arm Right homology arm Wild type TCTGTGGGAAAATCT::::::TGAAGACTACtagccct------------------------------------------------------------------------------------------------------------------------GCCAGGCACAA::::::TTTGGGAGGCAGAGG
Left homology arm 2A-GFP-Wpre-ployA Right homology arm Expected TCTGTGGGAAAATCT::::::TGAAGACTACCAGTGTACTAATTAT::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACAA::::::TTTGGGAGGCAGAGG Cl1-38 TCTGTGGGAAAATCT::::::TGAAGACTACCAGTGTACTAATTAT::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACAA::::::TTTGGGAGGCAGAGG HDR Clone39 TCTGTGGGAAAATCT::::::TGAAGACTACCAGTGTACTAATTAT::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACAt:::::::TTTGGGAGGCAGAGG HDR with 1bp mutation Clone40 TCTGTGGGAAAATCT::::::TGAAGACTACtAGcGTACTAATTAT::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACAA::::::TTTGGGAGGCAGAGG HDR with 2bp mutation Clone41 TCTGTGGGAAAATCT::::::TGAAGACTACCAGTGTACTAATTAT::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::GGGCGCAGCCATGAGgccaggcacaa:::::::::::::tttgg----------ccaggca
---------------:::::::--------------::CAATG 3-NHEJ: 48bp ins; 290bp del Clone42 TCTGTGGGAAAATCT::::::TGAAGACTACtagccctcttgccgggaaaatct:::tgaagactacCAGTGTACTAATTAT::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACAA::::::TTTGGGAGGCAGAGG 5-NHEJ: 73bp ins Clone43 TCTGTGGGAAAATCT::::::TGAAGACTACtagcccttgccgggaaaatct::::::tgaagactacCAGTGTACTAATTAT::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACAA::::::TTTGGGAGGCAGAGG 5-NHEJ: 71bp ins Clone44 TCTGTGGGAAAATCT::::::TGAAGACTACtagccccagattttcagttt----::::::------------------------------------:::::::GGGCGCAGCCATGAGGCCAGGCACAA::::::TTTGGGAGGCAGAGG 5-NHEJ: 20bp ins; 531bp del Clone45 TCTGTGGGAAAATCT::::::TGAAGACTACtagccctcttgccgggaaaatct::::tgaagactaccagtgtactaattat:::::::::::::::::::::::::::::::::::::::::::gggcgcagccatgaggccaggcacaa::::::::::::::::::::::tttgggaggcctggca
GCCAGGCACAA::::::TTTGGGAGGCAGAGG 5, 3-NHEJ: left 73bp ins, right 52bp ins Clone46 TCTGTGGGAAAATCT::::::TGAAGACTACtagccctagaaacattc::::::::::::::tgaagactaccagtgtactaattat:::::::::::::::::::::::::::::::::::::::::::gggcgcagccatgaggccaggcacaa::::::::::::::::::::::tttgggaggcctggcat
GCCAGGCACAA::::::TTTGGGAGGCAGAGG 5, 3-NHEJ: left 67bp ins, right 53bp ins Clone47 TCTGTGGGAAAATCT::::::TGAAGACTAC---------ctagtagtcttcctgt:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::gccacctggaagactactagcc-------AGGCACAA::::::TTTGGGAGGCAGAGG 5,3-NHEJ: Backbone reverse insertion Clone48 TCTGTGGGAAAATCT::::::TGAAGA------------------attaaaaatgaagttt::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::gccacctggaagactactagcc-------AGGCACAA::::::TTTGGGAGGCAGAGG 5,3-NHEJ: Backbone reverse insertion Clone49 TCTGTGGGAAAATCT::::::TGAAGACTACtagccctagggctagtagtct:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::acaaataggggttccgcgcaca------------------------::::::TTTGGGAGGCAGAGG 5,3-NHEJ: Backbone reverse insertion
pD-GFP-sg-HA100-100bp (PRDM14)
Left homology arm Replaced sequence in left arm Right homology arm Wild type ATGTCCGGCGTTCAC::::::TGAAGACTACtagccct------------------------------------------------------------------------------------------------------------------------GCCAGGCACA::::::TGAAATCCTGTCTCT Left homology arm 2A-GFP-Wpre-ployA Right homology arm Expected ATGTCCGGCGTTCAC::::::TGAAGACTACCAGTGTACTAATTAT::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACA::::::TGAAATCCTGTCTCT Clo1-37 ATGTCCGGCGTTCAC::::::TGAAGACTACCAGTGTACTAATTAT::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACA::::::TGAAATCCTGTCTCT HDR Clone38 ATGTCCGGCGTTCAC::::::TGAAGACTACCAGTGTACTAATTAT::::------------------------------------------------------------------------------:::::::::-------------
------------------------::::TGAAATCCTGTCTCT 3-NHEJ: 252bp del Clone39 ATGTCCGGCGTTCAC::::::TGAAGACTACtagccct----------::::-------------------------------------------------------::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACA::::::TGAAATCCTGTCTCT 5-NHEJ: 7bp ins, 656bp del Clone40 ATGTCCGGCGTTCAC::::::TGAAGACTACtagcc------------:::::-------------------------------------------------------::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACA::::::TGAAATCCTGTCTCT 5-NHEJ: 5bp ins, 553bp del Clone41 ATGTCCGGCGTTCAC::::::TGAAGACT---------caca-------::::---------------------------------------------------------::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACA::::::TGAAATCCTGTCTCT 5-NHEJ: 4bp ins, 834bp del Clone42 ATGTCCGGCGTTCAC::::::TGAAGACTACtagccctgcttccaggtggcac:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::ggaagactactagccctctGCCAGGCACA::::::TGAAATCCTGTCTCT 5,3-NHEJ: Backbone forward insertion Cl43-44 ATGTCCGGCGTTCAC::::::TGAAG-----------------gtcagaccccgtag::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::caggaagactactagccctg----------------ACA::::::TGAAATCCTGTCTCT 5,3-NHEJ: Backbone forward insertion
28
pD-GFP-sg-HA150-150bp (PRDM14)
Left homology arm Replaced sequence in left arm Right homology arm Wild type TACTGTGGTAAATCT::::::TGAAGACTACtagccct--------------------------------------------------------------------------------------------------------------------------GCCAGGCACA::::::GCACATGCCTGTGGT Left homology arm 2A-GFP-Wpre-ployA Right homology arm Expected TACTGTGGTAAATCT::::::TGAAGACTACCAGTGTACTAATTAT::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACA::::::GCACATGCCTGTGGT Cl1-34 TACTGTGGTAAATCT::::::TGAAGACTACCAGTGTACTAATTAT::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACA::::::GCACATGCCTGTGGT HDR Cl35-36 TACTGTGGTAAATCT::::::TGAAGACTACCAGTGTACTAATTAT::::::------------------------------------------------------------------------------::::::::-----------------
--------------::::::::::::::::GCACATGCCTGTGGT 3-NHEJ: 479bp del Clone37 TACTGTGGTAAATCT::::::TGAAGACTACt-gcc--tggtaaatct::::::tgaagactacCAGTGTACTAATTAT::::::::::: :::::: :::::::::::::GGGCGCAGCCATGAGGCCAGGCACA::::::GCACATGCCTGTGGT 5-NHEJ: 326bp ins Clone38 TACTGTGGTAAATCT::::::TGAAGACTAC------------------------::::::--------------------------------------------::::::::::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACA::::::GCACATGCCTGTGGT 5-NHEJ: 1191bp del Clone39 TACTGTGGTAAATCT::::::TGAAGACTACtagccctagggctagtagtcttccaggtg::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::atttcacacaggaagac-------AGGCACA::::::GCACATGCCTGTGGT 5,3-NHEJ: Backbone forward insertion
pD-GFP-sg-HA300-300bp (PRDM14)
Left homology arm Replaced sequence in left arm Right homology arm Wild type CCATAAGGATGAGCA::::::TGAAGACTACtagccct------------------------------------------------------------------------------------------------------------------------GCCAGGCACA::::::GAGTAGCCCTCAAGA
Left homology arm 2A-GFP-Wpre-ployA Right homology arm Expected CCATAAGGATGAGCA::::::TGAAGACTACCAGTGTACTAATTATG:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACA::::::GAGTAGCCCTCAAGA Cl1-45 CCATAAGGATGAGCA::::::TGAAGACTACCAGTGTACTAATTATG:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::GGGCGCAGCCATGAGGCCAGGCACA::::::GAGTAGCCCTCAAGA HDR
Supplementary Figure 6d: Knockin pattern analysis at the PRDM14 locus. The left homology arm is indicated in yellow shadow and right HA in blue shadow. The inserted sequence is in red lowercase. Replaced sequence in left arm is indicated in purple. Sequences in grey indicate genomic DNA outside of homology arms.
29
WT CTCCCACATGAAGTTTCATGAAGACTACTAGCCCT↓GCCAGGCACAATGACTCACGCCTGT 0
2# CTCCCACATGAAGTTTCATGAAGACTACTAGCCCTtGCCAGGCACAATGACTCACGCCTGT +1
3# CTCCCACATGAAGTTTCATGAAGACTACTAGCCCTtGCCAGGCACAATGACTCACGCCTGT +1
6# CTCCCACATGAAGTTTCATGAAGACTAC-----------CTGCCAGGCACAATGACTCACGCCTGT -5
9# CTCCCACATGAAGTTTCATGAAGACTACTAGCC-----------AGGCACAATGACTCACGCCTGT -5
10# CTCCCACATGAAGTTTCATGAAGACTACTAGCC-T↓GCCAGGCACAATGACTCACGCCTGT -1
12# CTCCCACATGAAGTTTCATGAAG----------------------T↓GCCAGGCACAATGACTCACGCCTGT -11
Supplementary Figure 7: Presence of indels at the non-target allele of all iPSC clones with heterozygous HDR knockin at the PRDM14 locus. The non-targeted allele was amplified by PCR, followed by Sanger sequencing to identify indels.
30
a
Supplementary Figure 8: The effects of small molecules on HDR in the H1 ES cell line. (a-c) The effects of small molecules on HDR efficiency at the CTNNB1 locus (a) or PRDM14 locus (b) . The human H1 ES cells were treated with RS-1 (10μM), Nu7441 (2μM), SCR7 (1μM), Brefeldin A (0.1μM), L755507 (5μM) or Nocodazole (100ng/ml) at 0-24h after nucleofection and the HDR efficiency was determined by FACS on Day 3.
b
31
Supplementary Fig.9a
32
Supplementary Fig.9b
33
Supplementary Figure 9: Illustration of cellular location of genes of interest by fusion with a fluorescent protein after HDR targeting in iPSCs.(a) Localizaiton of PRDM14 at the nucleus as indicated by a tdTomato reporter. A new donor plasmid was created to generate a fusion gene of PRDM14 and tdTomato, which are separated by a GS linker The cells were stained with DAPI and Phalloidin 488 to show the nucleus and plasma membrane. (b) Localizaiton of CTNNB1 at plasma membrane as indicated by a mNeonGreen reporter. The cells were stained with Hoechst 33258 and CellMask Red to show the nucleus and menbrane. (scale bar = 50 μm).
34
CTNNB1 locus
sgCTNNB1Exon13 Exon14 Exon15
pD-mNeonGreen-sgRNA2HA GS-mNeonGreen-Wpre-pA HA
atttcacacaGCTAGTGGGGTTGATAGGAGNGGNNNNNNN NNNNNNNCCNCTCCTATCAACCCCACTAGCcaggtggcac
sgRNA2 sgRNA2
CTNNB1 locus
sgCTNNB1Exon13 Exon14 Exon15
pD-mNeonGreen-sgRNA3 HA GS-mNeonGreen-Wpre-pA HA
atttcacacaGCCGGGAGCAGGCGTGAGTGNGGNNNNNNN NNNNNNNCCNCACTCACGCCTGCTCCCGGCcaggtggcac
sgRNA3 sgRNA3
CTNNB1 locus
HA GS-mNeonGreen-Wpre-pA HA
Exon13 Exon14 Exon15
pD-mNeonGreen-sgCTNNB1
atttcacacaGCTGATTGCTGTCACCTGGNGGNNNNNNN NNNNNNNCCNCCAGGTGACAGCAATCAGCcaggtggcac
sgCTNNB1 sgCTNNB1
sgCTNNB1
a
b
c
sgRNA for genome: sgCTNNB1sgRNA for donor: sgCTNNB1
sgRNA for genome: sgCTNNB1sgRNA for donor: sgRNA2
sgRNA for genome: sgCTNNB1sgRNA for donor: sgRNA3
35
ns nsd
Supplementary Figure 10: The effects of using one vs. two sgRNAs in the double cut donor system on HDR efficiency. (a-c) Schematic outline of genome editing at the CTNNB1 locus using double cut HDR donors, which will be linearized by Cas9 and sgCTNNB1 (a), sgRNA2 (b), or sgRNA3 (c), respectively. The donors contain a GS-mNeonGreen-Wpre-polyA sequence sandwiched by left homology arm (HA, marked in yellow shadow) and right homology arm (HA, marked in blue shadow). (d) The HDR efficiency was determined by FACS at day3 after co-transfection with Cas9/sgRNA and one of the three pD-mNeonGreen-sg donors (a-c). Statistical significance was calculated using the Student’s paired t-test: ns, not significant.
CTNNB1
36
