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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


chr4:126538001 CTGTCAGGGCTCCATGGCACCAG


chr17:41076900 AGGTCAGAGGTCCATGGCACAAG


chr18:47648088 CTGACACAGCCCCATGGCACTGG


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



IG-DMR-sgRNA2: chr12:110

769204 CTGCTTAGAGGTACTACGCTAGG

chr18:4314724 ATGCTTACAGGTACTATGCTTGG


chr1:92909983 CTGGCTATAGGTTCTACGCTAGG


chr13:97194693 AAGCTGAGAGGTACTACGCCTGG


chr7:132553861 CTTCTTGAAGGTACTAAGCTCAG


chr13:14524990 GTACTTAGAGTTACTATGCTAAG


chr13:45267048 CTGATTACAGGTTCTAAGCTTGG


chr9:120725277 CTTCTTGGAGTTACTAGGCTAAG


chr16:31987622 CTACTTGGAGGTTCTAAGCTAAG


chr10:4532468 CTGCTAAAAGATACAACGCTCGG


chr17:85478026 CAGCTTACAGGTTCTTCGCTTGG


chr7:133963384 GTGCTTTGTGGTATTACGCTGGG


Targeted Site and

Sequence



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


Targeted Site and

Sequence



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


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)

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