Cell Stem Cell
Supplemental Information
CRISPR-Cas9-Mediated Genetic Screening
in Mice with Haploid Embryonic Stem Cells
Carrying a Guide RNA Library
Cuiqing Zhong, Qi Yin, Zhenfei Xie, Meizhu Bai, Rui Dong, Wei Tang, Yu-Hang Xing,
Hongling Zhang, Suming Yang, Ling-Ling Chen, Marisa S. Bartolomei, Anne
Ferguson-Smith, Dangsheng Li, Li Yang, Yuxuan Wu, and Jinsong Li
1. Supplemental Figures and Legends:
Figure S1. Deficiency of Dlk1-Gtl2 Cluster in H19△DMR –AGH Cells and Their SC Pups,
Related to Figure 1 and Table 1. (A) Genotyping analysis of derived H19△DMR –AGH cell
lines. Note that all three lines carry mutant H19-DMR but not WT H19-DMR. (B) Around
80% of oocytes reconstructed with H19△DMR –AGH cells (ICAHCI) (right) cleaved into
two-cell embryos the day after activation, at efficiency similar to that of round spermatid injection (ROSI) (left). Scale bar, 100 µm. (C) Newborn SC pups from ICAHCI using
H19△DMR –AGH-1, 2 and 3 cells. Pups and placentas obtained by C-section from
pseudopregnant mice at E19.5 are shown. Passage numbers are indicated in bracket. Asterisks indicate the growth-retarded SC pups and placentas. (D) Genotyping analysis of SC pups
derived from H19△DMR –AGH cells. Note that all tested SC pups carry both mutant H19-DMR
and WT H19-DMR while the control pup carries only WT H19-DMR. (E) Transcription analysis of imprinted genes (Gtl2 and Dlk1) in important organs of growth-retarded (n=3) and
survived (n=3) SC mice generated from H19△DMR –AGH cells. The transcription level of Gtl2
(right) was higher in lung and heart of growth-retarded SC mice compared with normal SC mice and control newborn mice. The expression values (means ±SD) were normalized to that of Gapdh. (F) Growth-retarded pups harbored severe loss of the IG-DMR methylation imprint. Methylation state of the IG-DMR in a WT pup (top), a normal SC pup (middle) and a retarded SC pup (bottom). Open and filled circles represent unmethylated and methylated CpG sites, respectively. (G) Cobra assay of IG-DMR in retarded and normal pups. (H)
Methylation state in IG-DMR of H19△DMR –AGH cells.
Figure S2. DKO-AG-haESCs Are Efficient Donors for SC mice Generation, Related to
Figure 1, 2 and Table 1. (A) Genotyping analysis of H19△DMR- IG△DMR-AGH cells derived
after removal of IG-DMR in H19△DMR-AGH cells. (B) The sequence of one DKO-AG-haESC
line (represented by H19△DMR- IG△DMR-AGH-4). The sgRNA-targeting sequences are
underlined. The deleted sequence is marked in black. Note that a region of 4.161 kb at
IG-DMR is deleted. (C) Two-cell embryos generated by ICAHCI of H19△DMR- IG△DMR-AGH
cells. Scale bar, 100 µm. (D) Genotyping analysis of SC pups. (E) SC pups from ICAHCI
using IG△DMR –AGH-2. Pups and placentas obtained by C-section from pseudopregnant mice
at E19.5 are shown. Asterisks indicate the growth-retarded SC pups and placentas. (F)
Methylation state of H19-DMR in IG△DMR –AGH cells. (G) Methylation state of H19-DMR in
normal and retarded SC pups derived from IG△DMR –AGH-2. (H) The sequence of one
DKO-AG-haESC line (represented by IG△DMR- H19△DMR-AGH-2). The deleted sequence is
marked in black. Note that a region of 4.042 kb at H19-DMR is deleted. (I) SC pups from
ICAHCI using IG△DMR- H19△DMR-AGH-2 cells. (J) Genotyping analysis of SC pups derived
from IG△DMR- H19△DMR-AGH-2 cells.
Figure S3. H19 and IG DMRs Are Two Barriers to High-Efficiency Generation of SC Mice in AG-haESCs, Related to Figure 2 and Table 1. (A) The sequences of two DKO-AG-haESC lines derived after removing H19-DMR and IG-DMR in WT AG-haESCs
(AGH-OG-3). (B) SC pups from ICAHCI using H19△DMR-IG△DMR-AGH-OG3-1 cells. (C)
Genotyping analysis of SC pups derived from H19△DMR-IG△DMR- AGH-OG3-2 cells. (D)
Transcription analysis of imprinted genes (H19, Igf2, Gtl2 and Dlk1) in DKO-AG-haESCs and normal AG-haESCs. Note that the transcription levels of H19 and Gtl2 in AGH-OG-3 were decreased after removal of H19 and IG DMRs. In contrast, the transcription levels of Igf2 and Dlk1 in AGH-OG-3 were increased after removal of H19 and IG DMRs. The expression values (means ±SD) were normalized to that of Gapdh. The experiments were repeated for three times. (E) Bigwig track of Gtl2 and H19. RNA-seq of WT AG-haESCs and DKO-AG-haESCs reveals a low expression level of Gtl2 and H19 in DKO-AG-haESCs compared with WT AG-haESCs. (F) Methylation of H19-Igf2 and Dlk1-Dio3 cluster regions. The heights of vertical grey lines represent the read depth for CpG sites, with at most 30 reads. The heights of vertical red lines represent methylation levels for methylated CpG sites, ranging from 0 to 1. (G) Methylation profiles of DKO-AG-haESCs, WT AG-haESCs and round spermatids based on imprinted genes (Yamaguchi et al., 2013).
Figure S4. Multiple Gene Knockout in DKO-AG-haESCs, Related to Figure 3 and Table 1. (A) The sequences of Tet1, 2 and 3 in Tet-TKO-DAH-3 cells. Deletions are indicated with (-). Insertions are highlighted with white background. (B) SC pups from ICAHCI using Tet-TKO-DAH-3 cells. Passage number is indicated in bracket. (C) The sequences of p53, p63 and p73 in p53-TKO-DAH-1 cells. Deletions are indicated with (-). Insertions are highlighted with white background. (D) SC pups from ICAHCI using p53-TKO-DAH-1 cells. Passage number is indicated in bracket. (E) DNA sequence of PCR products amplified from p53, p63 and p73 genes of one SC pup generated from p53-TKO-DAH-1 and one SC pup from p53-TKO-DAH-2. Two peaks can be observed in the sequences of the SC pups carrying heterozygous mutations in p53, p63 and p73.
Figure S5. Multiple Gene Knockin in DKO-AG-haESCs, Related to Figure 3 and Table 1. (A) Schematic for exogenous double double-stranded vectors, including Tet1-EGFP, Tet2-mCherry and Tet3-ECFP. (B) Genotyping analysis of DKO-AGH-haESCs carrying Tet1-EGFP knockin. (C) Genotyping analysis of DKO-AGH-haESCs carrying Tet3-ECFP knockin. (D) Genotyping analysis of Tet1&3-KI-DAH-1 cells. (E) The sequences of Tet1-EGFP and Tet3-ECFP in Tet1&3-KI-DAH-1 cells. The border sequences on genome are labeled in black. The sequences of gene encoding EGFP and ECFP are labeled in red. The sequences of left arm (LA) and right arm (RM) for homology recombination are labeled in blue. The sequence of P2A peptide is labeled in yellow. The sequence of restriction enzyme cutting site is labeled in purple. (F) SC pups from ICAHCI using Tet1&3-KI-DAH-1 cells (passage 40) at 3 wks with a foster mother (ICR strain). (G) Genotyping analysis of SC pups from Tet1&3-KI-DAH-1 cells. (H) The sequence of Tet2-mCherry in Tet-TKI-DAH-1 cells. The border sequences on genome are labeled in black. The sequences of gene encoding EGFP and ECFP are labeled in red. The sequences of left arm (LA) and right arm (RM) for homology recombination are labeled in blue. The sequence of P2A peptide is labeled in yellow. The sequence of restriction enzyme cutting site is labeled in purple. (I) Newborn SC pups from Tet-TKI-DAH-2 cells (passage 50). (J) Genotyping of SC pups from Tet-TKI-DAH-1 cells.
Figure S6. Generation of Biallelic Mutant Mice by DKO-AG-haESC Carrying sgRNA Library, Related to Figure 5 and Table 2. (A) Schematic for generation of biallelic mutant mice by injection of haploid cells carrying sgRNAs, which had been transiently transfected with pX330-mCherry plasmids, into mature oocytes, followed by injection of Cas9 mRNA into reconstructed oocytes (strategy of “Lenti-sgRNA+pX330+Cas9 injection”). (B) PCR analysis of sgRNA in haploid cell clones expanded from single cells. All tested clones carry sgRNA. (C) SC pups from DKO-AG-haESCs carrying sgRNA library. (D) PCR analysis of sgRNA in SC pups. (E) Sequencing analysis of biallelic mutant mice generated via the strategy of “Lenti-sgRNA+pX330+Cas9 injection”. Represented by one SC mouse carrying biallelic mutant Slco5a1 gene, indicated by more than two peaks in the sequences of PCR products. (F) Sequence of the targeted Slco5a1 gene in the mouse-tail by TA cloning and sequencing analysis. 18 of 20 tested clones carry frameshift indel mutations. Deletions are indicated with (-). Insertions are highlighted with white background. (G) Summary of TA cloning and sequencing analysis of 4 biallelic mutant mice. Over 70% tested clones carry frameshift indels.
2. Supplemental Tables:
Table S1. Detailed Information for In Vivo Development of ICAHCI Embryos
from Different AG-haESCs, Related to Figure 1, 2 and Table 1
Donor Cell Type
Haploid ES Cell Line
Passage Number
No. of Embryos Transferred
No. of Growth-retarded Pups (% of Transferred Embryos)
No. of Normal Pups (% of Transferred Embryos)
H19-DMR KO AG-haESCs
H19△DMR-AGH-1
p8 375 10 (2.7) 10 (2.7) p9 180 9 (5.0) 13 (7.2)
H19△DMR-AGH-2
p8 210 5 (2.4) 6 (2.9) p9 116 2 (1.7) 4 (3.4) p11 123 3 (2.4) 3 (2.4)
H19△DMR-AGH-3
p8 271 1 (0.4) 29 (10.7) p17 168 9 (5.4) 21 (12.5)
IG-DMR KO AG-haESCs
IG△DMR-AGH-1
p14 84 0 0 p15 100 4 (4) 0 p17 54 4 (7.4) 1 (1.9) p23 81 1 (1.2) 0
IG△DMR-AGH-2
p8 180 3 (1.7) 3 (1.7)
H19△DMR-IG△D
MR-AGH Cells H19△DMR-IG△D
MR-AGH -1 p19 175 1 (0.6) 44 (25.1)
H19△DMR-IG△D
MR-AGH-2 p19 245 2 (0.8) 56 (22.9)
H19△DMR-IG△D
MR-AGH-3 p29 204 0 46 (22.5)
H19△DMR-IG△D
MR-AGH-4 p29 165 1 (0.6) 32 (19.4) p33 150 0 32 (21.3)
IG△DMR-H19△D
MR-AGH Cells IG△DMR-H19△D
MR-AGH-1 p24 120 1 (0.8) 26 (21.7)
IG△DMR-H19△D
MR-AGH-2 p24 180 2 (1.1) 47 (26.1) p28 244 2 (1.3) 32 (22.2)
H19△DMR-IG△D
MR-AGH-OG3 Cells
H19△DMR-IG△D
MR-AGH-OG3-1
p26 118 0 17 (14.4)
H19△DMR-IG△D
MR-AGH-OG3-2
p30 192 1 (0.5) 36 (18.8) p37 200 0 34 (17)
WT AG-haESCs
AGH-OG-3a p12 82 0 1 (1.2) p20 93 4 (4.3) 0
p24 140 2 (1.4) 5 (3.6) p26 64 0 1 (0.9)
AGH-2b p19 114 0 1 (0.9) p20 62 3 (4.8) 0
AGH-3b p9 118 0 1 (1.2)
a: AG-haESCs generated in this study.
b: AG-haESCs generated in our previous study (Yang et al., 2012)
Table S2:Off-Target Analysis in DKO-AG-haESCs, Related to Figure 1 and 2
Targeted Site and
Sequence
Potential Off-target Sites
Sequence Tested Cell Line Indel Mutation
IG-DMR-sgRAN1: chr12:110
765036 CGTACAGAGCTCCATGGCACAGG
chr8:116672129 CGCCCAGAGTTCCATGGCACCAG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr4:71893313 CTTCCAGAGCTCCATGGCAGTAG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr4:126538001 CTGTCAGGGCTCCATGGCACCAG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr17:41076900 AGGTCAGAGGTCCATGGCACAAG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr18:47648088 CTGACACAGCCCCATGGCACTGG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr3:95480895 AGGACAGAGGTCAATGGCACAAG
H19△DMR-IG△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr1:160441184 GGCACAGAGGTC
CATGGCCCAGG H19△DMR-IG△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr3:94252225 TGTACAGCCCTCC
ATGGCTCAAG H19△DMR-IG△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr17:35793498 GGCTCAGAGCTC
CCTGGCACTGG H19△DMR-IG△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr6:90569642 CGTACAGGGCTC
CAAGGGAGAGG H19△DMR-IG△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr11:77992776 CGCACAGAGCAC
CCTGGGACTGG H19△DMR-IG△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N
Targeted Site and
Sequence
Potential Off-target Sites
Sequence Tested Cell Line Indel Mutation
IG-DMR-sgRNA2: chr12:110
769204 CTGCTTAGAGGTACTACGCTAGG
chr18:4314724 ATGCTTACAGGTACTATGCTTGG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr1:92909983 CTGGCTATAGGTTCTACGCTAGG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr13:97194693 AAGCTGAGAGGTACTACGCCTGG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr7:132553861 CTTCTTGAAGGTACTAAGCTCAG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr13:14524990 GTACTTAGAGTTACTATGCTAAG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr13:45267048 CTGATTACAGGTTCTAAGCTTGG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr9:120725277 CTTCTTGGAGTTACTAGGCTAAG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr16:31987622 CTACTTGGAGGTTCTAAGCTAAG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr10:4532468 CTGCTAAAAGATACAACGCTCGG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr17:85478026 CAGCTTACAGGTTCTTCGCTTGG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr7:133963384 GTGCTTTGTGGTATTACGCTGGG
H19△DMR-IG△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
Targeted Site and
Sequence
Potential Off-target Sites
Sequence Tested Cell Line Indel Mutation
H19-DMR-sgRNA1:
chr7:149768813
CATGAACTCAGAAGAGACTGA
GG
chr7:103320976 AAAGAACTCAGAAGAGACTGGAG
IG△DMR-H19△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 Y
chr12:105735036 CAGGAATTCAGAAGAGACTGGGG
IG△DMR-H19△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr7:125049085 ACTGAACACAGAAGAGACTGGAG
IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr3:56173560 CATGAACTCCGG
AGAGACTGAAG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr3:32443165 GATGAACTAGGA
AGAGACTGAGG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr8:126921829 CTTGAAGTCAGG
AGAGACTGTGG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr10:80037252 CCTGCACAGAGA
AGAGACTGGGG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr2:8044072 AAAGAACTGGGA
AGAGACTGAGG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr9:118786319 CGGGAACCCAGA
AGAGACTCTGG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr4:11454132 CACGATCTCGGA
AGAGACTCTGG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr11:48661555 CTTCATCTCAGAT
GAGACTGTGG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N
Targeted Site and
Sequence
Potential Off-target Sites
Sequence Tested Cell Line Indel Mutation
H19-DMR-sgRNA2:
chr7:149764924
AGGTGAGAACCACTGCTGAGT
GG
chrX:160504351 AGTTGAGAACCACTGCTTAGGGG
IG△DMR-H19△DMR-AGH-2 Y IG△DMR-H19△DMR-AGH-OG3-2 Y
chr12:54007189 AGATGATAAGCACTGCTGAGAAG
IG△DMR-H19△DMR-AGH-2 N IG△DMR-H19△DMR-AGH-OG3-2 N
chr1:93014037 AAGTGAGCCCCACTGCTGAGAGG
IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr18:83528678 TGGGCAGAAGCA
CTGCTGAGAAG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr3:127517958 GTGTCAGAAACA
CTGCTGAGAAG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr7:137266149 AGGAGACAGCCA
CTGCTGAGCAG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr18:69559398 AGGTCAGTACCA
ATGCTGAGGGG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chrX:47778863 AGAAGAGAGACA
CTGCTGAGAGG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr2:163460149 AGGTATGACTCA
CTGCTGAGGGG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr7:29681873 AGGTGAGCACCA
CTGCCCAGGGG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N chr7:148268503 ACGTCAGATCCA
CGGCTGAGCGG IG△DMR-H19△DMR-AGH-2 N
IG△DMR-H19△DMR-AGH-OG3-2 N
Mismatchs are highlighted with underlie. PAM is marked in blue. N: non-indel mutation. Y: indel mutation.
Table S3. Detailed Information for In Vivo Development of ICAHCI Embryos
from Gene-modified DKO-AG-haESCs, Related to Figure 3 and Table 1
Donor Cell Type
Haploid ES Cell Line
Passage Number
No. of Embryos Transferred
No. of Growth-retarded Pups (% of Transferred Embryos)
No. of Normal Pups (% of Transferred Embryos)
DKO-AG-haESCs Carrying Tet1, 2 and 3 Mutations
Tet-TKO-DAH-1
p37 131 2 (1.5) 17 (13)
Tet-TKO-DAH-2
p37 54 0 9 (16.7)
Tet-TKO-DAH-3
p36 174 1 (0.6) 27 (15.5)
Tet-TKO-DAH-4
p35 48 1 (2.1) 6 (12.5)
DKO-AG-haESCs Carrying p53, p63 and p73 Mutations
p53-TKO-DAH-1
p42 182 1 (0.5) 34 (18.7) p44 186 0 32 (17.2)
p53-TKO-DAH-2
p41 154 0 23 (14.9) p42 48 1 (2.1) 8 (16.7)
p53-TKO-DAH-3
p46 90 0 14 (15.6)
DKO-AG-haESCs Carrying Tet1 and 3 Knockin
Tet1&3-KI-DAH-1
p40 58 1 (1.7) 10 (17.2)
p47 80 0 11 (13.8)
DKO-AG-haESCs Carrying Tet1, 2 and 3 Knockin
Tet-TKI-DAH-1
p47 48 1 (2.1) 8 (16.7) p49 174 0 32 (19)
Tet-TKI-DAH-2
p47 96 3 (3) 10 (10.4) p50 136 1 (0.7) 22 (16.2)
Tet-TKI-DAH-3
p49 48 0 8 (16.7)
Tet-TKI-DAH-4
p50 102 1 (1.0) 14 (13.7)
Tet-TKI-DAH-5
p51 174 0 39 (22.4)
Tet-TKI-DAH-6
p51 96 0 18 (18.8)
Table S4. Results of TA Cloning and Sequencing Analysis of Biallelic Mutant
Mice Generated by DKO-AG-haESCs Carrying SgRNA Library, Related to
Figure 4, 5 and Table 2
Strategies Mutant Genes
No. of Tested Clones
No. of Clones with 3n+1/2 bp Deletions or Insertions (n≥0) (% of Total Tested Clones)
No. of Clones with 3n bp Deletions or Insertions (n≥0) (% of Total Tested Clones)
No. of Clones with WT Sequence or Replacement (% of Total Tested Clones)
Lenti-sgRNA+Cas9 injection
Mthfd2 10 4 3 3 Eif2ak4 16 7 0 9 4930444A02Rik 11 8 0 3 Cd53 16 8 0 8 Olfr1053 17 12 5 0 Mthfs 14 8 0 6 1700024P16Rik 18 17 0 1 Subtotal 102 64 (62.7) 8 (7.8) 30 (29.4)
Lenti-sgRNA+pX330+Cas9 injection
0610007P14Rik 10 6 0 4 Wdr47 4 3 0 1 Hyal2 30 27 3 0 Xlr3c 17 13 0 4 Slco5a1 20 15 3 2 Subtotal 81 64 (79) 6 (7.4) 11 (13.5)
Lenti-Cas9+lenti-sgRNA
Gphn 13 4 8 1 Gm572 5 5 0 0 Polm 26 24 0 2 Kng1 31 27 1 3 Slc2a12 10 7 0 3 Ddrgk1 9 0 9 0 Ccdc75 7 4 1 2 Trim7 27 21 1 5 Scube1 27 21 2 4 Nmu 9 6 0 3 Zfp202 11 5 3 3 Rbp1 11 7 0 4 Smyd4 12 4 1 7
Sec23ip 13 7 0 6 Mlana 7 2 1 4 D2hgdh 7 7 0 0 F10 11 9 0 2 Prom1 10 8 1 1 Zfp872 12 1 7 4 Sar1b 10 8 0 2 Ankrd22 13 12 0 1 Tead2 12 5 7 0 C1ql4 13 9 0 4 Crlf2 12 4 4 4 Vipas39 9 8 0 1 Pml 11 9 0 2 Subtotal 338 224 (66.3) 46 (13.6) 68 (20.1)
Table S5. Primer Information, Related to Experimental Procedures Primer Name Sequence (5’-3’) Application Reference Gapdh-F CACTCTTCCACCTTCGATGC
Realtime PCR
(Inoue et al., 2002)
Gapdh-R CTCTTGCTCAGTGTCCTTGC Igf2-F CTAAGACTTGGATCCCAGAACC Igf2-R GTTCTTCTCCTTGGGTTCTTTC Gtl2-F TTGCACATTTCCTGTGGGAC Gtl2-R AAGCACCATGAGCCACTAGG Dlk-F ACTTGCGTGGACCTGGAGAA
Realtime PCR (Ogawa et al., 2006) Dlk-R CTGTTGGTTGCGGCTACGAT
H19-F CATGTCTGGGCCTTTGAA Realtime PCR
(Ogawa et al., 2003) H19-R TTGGCTCCAGGATGATGT
H19-DMR-BS-OF GAGTATTTAGGAGGTATAAGAATT Bisulfite sequencing
(Li et al., 2004)
H19-DMR-BS-OR ATCAAAAACTAACATAAACCCCT H19-DMR-BS-IF GTAAGGAGATTATGTTTATTTTTGG H19-DMR-BS-IR CCTCATTAATCCCATAACTAT
IG-DMR-BS-OF TTAAGGTATTTTTTATTGATAAAATAATGTAGTTT
Bisulfite sequencing
(Gu et al., 2011b)
IG-DMR-BS-OR CCTACTCTATAATACCCTATATAATTATACCATAA
IG-DMR -BS-IF TTAGGAGTTAAGGAAAAGAAAGAAATAGTATAGT
IG-DMR -BS-IR TATACACAAAAATATATCTATATAACACCATACAA
H19-DR WT -F AGATGGGGTCATTCTTTTCC H19-DMR WT genotyping
In this study
H19-DMR WT -F ATTGCTCTTAGCTTCTGTTG H19-DMR KO–F1 GCTCCCCTGGATGTTTCACT H19-DMR △
3.8K genotyping H19-DMR KO–R1 ACTCCTCACCGTCCCTTTTC H19-DMR KO –F2 GTGGTTAGTTCTATATGGGG Genotyping of
deleted H19-DMR by CRISPR-Cas9
H19-DMR KO –R2 TCTTACAGTCTGGTCTTGGT
IG-DMR KO–F TGTGCAGCAGCAAAGCTAAG Genotyping of deleted IG-DMR by CRISPR-Cas9
IG-DMR KO–R ATACGATACGGCAACCAACG
Tet1 LA-F TTTGTGTCTATGAACTACCAGTGAG Genotyping of Tet1-EGFP Knockin
Tet1 LA-R CAGGCCCGGGGTTTTCTTC Tet1 RA-F CAACGAGAAGCGCGATCACA Tet1 RA-R TTTTGACTGATCCCAATTTGCCT Tet2 LA-F CACACCCTTCACCAACAGACG
Genotyping of Tet2-mCherry Knockin
Tet2 LA-R ATCTCGAACTCGTGGCCGTT Tet2 RA-F AAGACCACCTACAAGGCCAAG Tet2 RA-R GGTAGGCAAAGTGCTTTTCTAAGAC
Tet3 LA-F TGTTCACTGGTGAAGGCCAG Genotyping of Tet3-ECFP knockin
Tet3 LA-R GAACAGCTCCTCGCCCTTG Tet3 RA-F TGAGCAAAGACCCCAACGAG Tet3 RA-R ATCGACAAACTTTGGGGCGA
Lenti-sgRNA-F GTTACTCGAGCCAAGGTCGG Genotyping of SC pups generated from DKO-AG-haESCs-Carried sgRNA library
In this study
Lenti-sgRNA-R GACTCGGTGCCACTTTTTCA
Cas9 RT-F CTGAGCAAGGACACCTACGA Realtime PCR
In this study
Cas9 RT-R CTCGGTGTTCACTCTCAGGA Realtime PCR Polm check-F TCCGATGGGAAGCCAAAAGC Polm check primer Polm check-R CGTACCGCAACCGCGAAGTA Polm check primer Scube1 check-F CCATAATAATCCACTTCCAT Scube1 check Scube1 check-R CCAACCCCTGTCCACTACCT Scube1 check Slco5a1 check-F GAGAAGTGCGAGTCAGAGTC Slco5a1 check Slco5a1 check-R ATAGGGGGGTGAGATAAAGT Slco5a1 check
3. Supplemental Experimental Procedures
Bisulphite Sequencing
Genomic DNA was extracted from tails or FACS-derived AG-haESCs by pretreating
with proteinase K lysis, followed by phenol-chloroform extraction. Bisulphite
conversion was performed in agarose beads as described (Hajkova et al., 2002) or EZ
DNA methylation Gold kit (ZYMO Research). The PCR products were cloned into
pMD19-T vectors (Takara) and individual clones were sequenced by Invitrogen,
Shanghai. Bisulphite primer information is presented in Table S4.
Quantitative Reverse Transcription PCR
Total RNA was isolated from the cells or organs using Trizol reagent (Invitrogen).
One microgram of total RNA was reverse transcribed using a First Strand cDNA
Synthesis kit (TOYOBO). Real-time quantitative PCR reactions were set up in
triplicate using the SYBR Green Realtime PCR Master Mix (TOYOBO) and run on a
Bio-Rad CFX96. All the gene expression levels were normalized to the internal
standard gene, Gapdh. The primer sequences are listed in Table S4.
Cobra Assay
Approximately 100 ng of the purified PCR products was digested with restriction
enzyme. TaqI (T/CGA) was used to digest bisulfate-treated IG-DMR for its
methylation analysis.
RNA-seq and Gene Expression Analysis
RNA-Seq libraries were prepared from total RNAs under different treatments
according to the manufacturer’s instructions, and then applied for deep sequencing on
Illumina HiSeq 2000 at CAS-MPG Partner Institute for Computational Biology
Omics Core, Shanghai, China. Roughly, 32 and 52 million 1X100 single reads from
two biological replicates of WT AG-haESCs, 48 million 1X100 single reads from
H19△DMR-IG△DMR-AGH-2, 48 million 1X100 single reads from
H19△DMR-IG△DMR-AGH-OG3-2, 31 million 1X100 single reads from
IG△DMR-H19△DMR-AGH-2, and 65 million 1X100 single reads from round spermatid
were individually obtained and used for further analyses as described previously
(Yang et al., 2011). Briefly, sequence reads were uniquely aligned to the mouse
mm10 genome by Tophat2 (version 2.0.9) (Kim et al., 2013) with up to two
mismatches. Normalized gene expression levels were determined in units of RPKM
(Reads Per Kilobase per Million mapped reads). For visualization, Bigwig files were
generated using UCSC bedGraphToBigWig from Bedgraph files, which were
generated using genomeCoverageBed_2.13.3 from Tophat generated Bam files. An
unsupervised hierarchical clustering for all genes or imprinting genes was individually
performed in cluster 3.0 (de Hoon et al., 2004; Eisen et al., 1998).
Reduced Representation Bisulfite Sequencing (RRBS)
RRBS library were generated from MspI-digested genomic DNA and subjected to
sequence with Illumina HiSeq 2000 as previously described, with some modifications
(Gu et al., 2011a). All sequenced reads were mapped against the mouse genome
mm10 with Bismark (version 0.12.2) (Krueger and Andrews, 2011). The methylated
CpG sites were extracted by bismark methylation extractor script. CpG sites covered
by at least 5 reads were chosen for further analyses. Integrative Genomics Viewer
(IGV) (Thorvaldsdottir et al., 2013) was applied to visualize the pattern of
methylation at CpGs. The methylated level was calculated by C/(C+T) for each CpG
site. An unsupervised hierarchical clustering for methylated CpG sites identified in all
six samples was performed by cluster 3.0 (de Hoon et al., 2004; Eisen et al., 1998).
The promoters of imprinting genes with at lease four CpG sites that were covered
with at least five reads were selected for subsequent analyses. Promoters were defined
from -1.5kb to +1.5 kb of RefGene transcription start sites. The methylated levels of
imprinting gene promoters were calculated by C/(C+T) and an unsupervised
hierarchical clustering for methylated CpG sites in imprinting gene promoter regions
was performed by cluster 3.0 (de Hoon et al., 2004de Hoon et al., 2004; Eisen et al.,
1998).
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5. Supplemental Spreadsheet, Related to Figure 4, 5 and Table 2
An independent excel file, including information about all analyzed SC pups derived
from DKO-AG-haESC carrying sgRNA library. 6. Supplemental Movie, Related to Experimental Procedures and ICAHCI
experiments (Figure 1 to 5)