J. Cell Sci. 128: doi:10.1242/jcs.173534: Supplementary information
hI CA 1L
mI CA 1L
rI CA 1L
1 M DS FGQ PR PED N QSV VRR MQ KK YWK T KQ V FIKA TGKK EDEH LV AS DAE LD AKL EVF HS V QET CTE L LKII
1 M DS SEQ LR AED N QSV VSR MQ KN YWR T KQ V FIKA TGKK EDEH VV AS DAE LD AKL EVF HS I QET CNE L VKIV
1 M DS FEH LR PED S QSV VSR MQ KK YWK T KQ V FIKA TGKK EDEH VV AS DAE LD AKL EVF HS I QET CNE L VKAV
hI CA 1L
mI CA 1L
rI CA 1L
71 E KY QLR LN VIS E EEN ELG LF LK FQA E RD A TQAG KMMD ATGK AL CS SAK QR LAL CTP LS R LKQ EVA T FSQR
71 E KY QLR LN VIS E EEN ELG LF LK FQA E RD S TQAG EMMD AAGK AL CS SAK QR LAL CTP LS R LKQ EVA T FSQR
71 E KY QLR LN VIS E EEN ELG LF LT FQA E RD A TQAG KMMG AAGK AL CS SAK QR LAL CTP LS R LKQ EVA T FSQR
hI CA 1L
mI CA 1L
rI CA 1L
141 A VS DTL MT INR M EQA RTE YR GA LLW M KD V SQEL DPDT LKQM EK FR KVQ MQ VRN SKA SF D KLK MDV C QKVD
141 A IS DTL VT INR M ERA RTE YR GA LLW M KD A SQEL DPDT FKQM EK FR KVQ NQ VRN SKD SF D KLK KDV C QKVD
141 A IS DTS VT INR M ERA RTE YR GA LLW M KD A SQEL DPDT FKQV EK FR KVQ IQ VRN SKD CF D KLK MDV C QKVD
hI CA 1L
mI CA 1L
rI CA 1L
211 L LG ASR CN MLS H SLT TYQ RT LL GFW K KT A RMMS QIHE ACIG FH PY DFV AL KQL QDT PS K ISE DNK D EQI211 L LG ASR CN MLS H SLT TYQ RT LL GFW E KT A QMMT QIQE ACAG FH PY DFL AL KRL QDT PG N LTA DCT E GQTE
211 L LG ASR CN MLS H SLA TYQ RT LL GFW E KT A QMMS QIQE ACAG FH PY DFM AL KQL QDT PG N LTA EDK E EQTE
hI CA 1L
mI CA 1L
rI CA 1L
280 G GF LTE QL NKL V LSD EEA SF ES EQA N KD H NEKH SQMR EFGA PQ FS NSE NV AKD LPV DS L EGE DFE K EFSF
281 G SC LTT DL NKV A LSE EEE -- -- --- - -- - ---- ---- ---E ER FE REP AV ARA LPR DS L EGD DFE K EFSF
281 G SC LST NL NKV A LSE E-- -- -- --- - -- - ---- ---- ---E ER FE REP AV ARA LPR DS L EGD DFE K EFSF
hI CA 1L
mI CA 1L
rI CA 1L
350 L NN LLS SG SSS T SEF TQE CQ TA FGS P SA S LTSQ EPSM GSEP LA HS SRF LP SQL FDL GF H VAG AFN N WVSQ
329 L NS LLS PT SSS A SEF TQE CQ PA CGS P CT G LTSQ EPSV GPGS LT SS SQF LP SQL FDL GL H ADG AFN T PNNG
327 L SS LLS PT SSR A SEC TQE CQ PA CGS P S- - LMCQ EPSL GPGP LT SS SQF LP SRL FDL GL H ADG AFN S WASQ
hI CA 1L
mI CA 1L
rI CA 1L
420 E ES ELC LS HTD N QPV PSQ SP KK LTR S PN N GNQD MSAW FNLF AD LD PLS NP DAI GHS DD E LLN A
399 N QD --- -- --- - --- --- -- -- --- - -- - ---- MSAW FNLF AD LG PLS NP DAI GHS DD E LLN A
395 E GS EH- -- -SD T L-- --- -- -- --- - -- - --PV PSQH PKKL KY LG PLS NP DAI GHS DD E LLN A
Figure S1. Evolution conservation of ICA1L BAR domain. Aligment of human, mouse and rat ICA1L was
done by Clustal Omega and presented by Boxshade. Red box shows BAR domains of ICA1L and the borders
of which (aa15-242) were determined by SMART.
Journal of Cell Science • Supplementary information
Figure S1
J. Cell Sci. 128: doi:10.1242/jcs.173534: Supplementary information
Figure S2
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ WT
#1
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ WT
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ +4bp
ATCG
5’ AAGCGACG-----------------------GGTTGCATCCGATGCAGAACTGGAC 3’ -23bp
#2 5’ AAGCGACGGGAAAGAAGGAGGACGA----------GCATCCGATGGAGGACTGGAC 3’ -10bp,m2
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ WT
5’
5’
#4 5’
5’
AAGCGACGGGAAAGAAGGAGGACGA----------GCATCCGATGCAGAACTGGAC
AAGCGACGGGAAAGAAGGAGGACG---ATGTGGTTGCATCCGATGCAGAACTGGAC
AAGCGACGGGAAAGAAGGAGG------ATGTGGTTGCATCCGATGCAGAACTGGAC
AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC
3’
3’
3’
3’
-10bp
-3bp
-6bp
WT
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ WT
#5 5’ AAGCGACGGGAAAGAAGGAGG------ATGTGGTTGCATCCGATGCAGAACTGGAC 3’ -6bp
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ +1bp
C
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ WT
#7
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ +1bp
AAGCGACGGGAAAGAAGGAGGACGA----------------GATGCAGAACTGGAC
AAGCGACGGGAAAGAGGGAGGACGA----------------GATGCAGAACTGGAC
AAGCGACGGGAAAGAAGGAGGACGAGGATGTGGTTGCATCCGATGCAGAACTGGAC
AAGCGACGGGAAAGAAGGAGGAAGAGGATGTGGTTGCATCCGATGCAGAACTGGAC
3’
3’
3’
3’
-16bp
-16bp,m1
m1
+1,m2
5’
5’
#9
5’
5’
A
AAGCGACGGGAAAGAAGGAGGACGAGC-TGTGGTTGCATCCGATGCAGAACTGGAC
AAGCGACGGGAAAGAAGGAGGACG-G-ATGTGGTTGCATCCGATGCAGAACTGGAC
AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC
AAGCGACGGGAAAGAAGGAGGACGAGGATGTGGTTGCATCCGATGCAGAACTGGAC
3’
3’
3’
3’
-1bp
-2bp
WT
+1bp,m1
Founders
5’
5’
#8
5’
5’
C
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ +1bp
A
5’
5’
#20
5’
5’
AAGCGACGGGAAAGAAGGAGG------ATGTGGTTGCATCCGATGCAGAACTGGAC
AAGCGACGGGAAAGAAGGAGGACG----------------------AGAACTGGAC
AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC
AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC
3’
3’
3’
3’
-6bp
-22bp
WT
+1bp
C
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ WT
#21
5’ AAGCGACGGGAAAGAAGGAG---------------------GATGCAGAACTGGAC 3’ -21bp
#23
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ WT
5’ AAGCGACGGGAAAGAAGGAGGACGA----------GCATCCGATGCAGAACTGGAC 3’ -10bp
#27
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ WT
5’ AAGCGACGGGAAAGAAGGAGGACGAGCATGTGGTTGCATCCGATGCAGAACTGGAC 3’ +1bp
C
#29 5’ AAGCGACGGGAAAGAAGGAGG------ATGTGGTTGCATCCGATGCAGAACTGGAC 3’ -6bp
Journal of Cell Science • Supplementary information
T
J. Cell Sci. 128: doi:10.1242/jcs.173534: Supplementary information
Figure S2. Sequences of founders. Fragments containing the targets were PCR amplified from genomic
DNA of founders and cloned into pEGFP-C3 vector and sequenced by Sanger sequencing. Red sequence is
PAM motif and blue letters are point mutations. Mosaic rate was about 50% as previously reported.
Journal of Cell Science • Supplementary information
Interestingly, most insertions happened at sites between the third and fourth base pair before the PAM motif.
J. Cell Sci. 128: doi:10.1242/jcs.173534: Supplementary information
Table S1. Mutation rate for Ica1l
Set
Gene
1(L)
2(S)
3(S)
Ica1l
Ica1l
Ica1l
Cas9/sg
RNA (ng/ul)
100/20
100/20
100/20
Injected
zygotes
NA
NA
NA
Transferred
embryos (recipients)
20(1)
20(1)
20(1)
Newborns
(Dead)
9(0)
12(0)
10(0)
Mutant mice/total mice
7/9
2/12
3/10
Cas9 mRNA, sgRNA mixture (100/20 ng/μl) targeting Ica1l were injected into cytoplasm of one-cell embryos. 60
embryos were transferred into three foster mothers, 20 for each. Two injection parameters were tested-large volume (L)
Journal of Cell Science • Supplementary information
and relatively small volume (S). Injecting of a higher volume resulted in a larger mutation rate.
J. Cell Sci. 128: doi:10.1242/jcs.173534: Supplementary information
Table S2. Off-targeting analysis
Site name
Sequence
Target_Ica1l
GGGAAAGAAGGAGGACGAGCATGTGG
Indel mutation
frequency
(Mutant/Toal)
/
OT1_Ica1l
aGGAAAGAAGGAGGAgGAGgATGAGG
0/12
chr3: 54462626-54462651
OT2_Ica1l
aGGAAAGAAGGAGGAgGAGCAgGAGG
0/12
chr5: 66674025-66674050
OT3_Ica1l
aGGAAAGAAGGAGGACGAGaAgGAGG
0/12
chr2:115930409-115930434
OT4_Ica1l
GGGAcAGAAGGAGGACaAGCtTGAGG
0/12
chr8: 64784497-64784522
OT5_Ica1l
GGGAAAGAgGGAGaACGAGaATGAGG
0/12
chr13: 109584464-109584489
OT6_Ica1l
GGGAAAGAAGGAGGAaGAGgAgGAGG
0/12
chr10: 126570239-126570264
OT7_Ica1l
GGGAAAGAAGGAGGAaGAGgAgGAGG
0/12
chr2: 164720035-164720060
OT8_Ica1l
GGGAAAGAAGGAGGAgGAGgAgGAGG
0/12
chr3: 63845705-63845730
OT9_Ica1l
GGGAAAGAAGGAGGAgGAGgAgGAGG
0/12
chr5: 106430543-106430568
OT10_Ica1l
GGGAAAGAAGGAGGAgGAGgAgGAGG
0/12
chr12: 15928347-15928372
OT11_Ica1l
GGGAAAGAAGGAGGAgGAGgAgGAGG
0/12
chr13: 22343504-22343529
OT12_Ica1l
GGGAAAGAAGGAGGAgGAGgAgGAGG
0/12
chr13: 94702039-94702064
OT13_Ica1l
GGGAAAGAAGGAGGAgGAGgAgGAGG
0/12
chr17: 30181927-30181952
chr1: 60028103-60028128
Mismatches from the on-target sequence are lower-case and highlighted by red. PAM motifs are underlined. Indel mutation
frequencies in 12 founders were calculated by Sanger sequencing. Coordinates in mm10 in which sites are located are
shown. OT, off-targeting. No one base pair or two base pairs mismatches lying at left region of PAM motif were identified.
13 three base pairs mismatches were identified and no off-targeting happened in these mismatch sites.
Journal of Cell Science • Supplementary information
Coordinate (mm10)
J. Cell Sci. 128: doi:10.1242/jcs.173534: Supplementary information
Table S3. Primers for off-targeting analysis
Gene target
Direction
Sequence 5’-3’
Forward
ACGCGTCGACcagcactgtatgtgatcttctcaatatcctcagt
Reverse
ATAAGAATGCGGCCGCctgggcattaatagctaacggtaaat
Forward
ACGCGTCGACgcattcagtaactcagcgctaacatcacagtac
Reverse
ATAAGAATGCGGCCGCgccagaagagctaggaagggcagtatgaagtcat
Forward
ACGCGTCGACcatgcctgtaactccagggtataaggaggcaa
Reverse
ATAAGAATGCGGCCGCtgagcaagagtctcagtgatttgccctagactta
Forward
ACGCGTCGACcttagataagtccatttaagcaagattaagccat
Reverse
ATAAGAATGCGGCCGCgatctgccagttacctctaccagttaccca
Forward
ACGCGTCGACaccttgattatacaatgactttgaagatagtcta
Reverse
ATAAGAATGCGGCCGCggtaagtcattatttgatgaggatacacct
Forward
ACGCGTCGACtcagatcagtccccatgcccaggaatagct
Reverse
ATAAGAATGCGGCCGCtcttgaagctacctgattggtgaaggcacatt
Forward
ACGCGTCGACctagaccaagcatattctaaccaccgcactgggcat
Reverse
ATAAGAATGCGGCCGCgtgaatagacatgcatctgtaggcatgtattcatgct
Forward
ACGCGTCGACacttagccacatgtggtgactcatagttcataatctc
Reverse
ATAAGAATGCGGCCGCaactctgcattggcagaacttttaccactaagccat
Forward
ACGCGTCGACctgaagcatggtatcccaaaatagtagagg
Reverse
ATAAGAATGCGGCCGCtccagttctaggaaagccacgggctgactacat
Forward
ACGCGTCGACatacatgtatgagcaaatgtatgtacgctcacat
Reverse
ATAAGAATGCGGCCGCctagtcactctgtgtggacggcttaaaccttcattg
Forward
ACGCGTCGACaagatattcagggcttactgtagtagattagttgtgctc
Reverse
ATAAGAATGCGGCCGCactgctagtagactagcattgctcctcgagttcca
Forward
ACGCGTCGACaggaactggagcatgcgctatcagtccacct
Reverse
ATAAGAATGCGGCCGCagcgaccgtaattatttgatagtttctgcaattatg
Forward
ACGCGTCGACacagtgtcatccatcctgtatacagtgacatga
Reverse
ATAAGAATGCGGCCGCagtttcctcaggcaccaggcatgcatgtga
OT1_Ica1l
OT2_Ica1l
OT3_Ica1l
OT4_Ica1l
OT5_Ica1l
OT6_Ica1l
OT7_Ica1l
OT8_Ica1l
OT10_Ica1l
OT11_Ica1l
OT12_Ica1l
OT13_Ica1l
Fragments containing the potential off-targeting sites were PCR amplified from founders by the indicated primers
and cloned into pEGFP-C3 vector and sequenced. Boldface letters in forward and reverse primers are restriction
enzyme SalI, NotI cloning site respectively.
Journal of Cell Science • Supplementary information
OT9_Ica1l