Extended Data Table 1a. Reagent list.
Oligonucleotides used for cloning, Sanger sequencing, MiSeq or qRT–PCR analysis.
| Engineering tetracycline-inducible hESCs targeting the endogenous AAVS1 locus | ||
|---|---|---|
| sgRNA | Top oligo for cloning | Bottom oligo for cloning |
| sgRNA1-1 | TCCCGCTTCACGGCACCAGGGGTGA | AAACTCACCCCTGGTGCCGTGAAGC |
| sgRNA1-2 | TCCCGCACTAGCCCCACTCCAACC | AAACGGTTGGAGTGGGGCTAGTGC |
| sgRNA2b | TCCCACCCACCAAATAGAACCCCC | AAACGGGGGTTCTATTTGGTGGGT |
| sgRNA4 | TCCCACTTGCAGGTGGTCCGAGTG | AAACCACTCGGACCACCTGCAAGT |
| In vitro transcription of human sgRNAs | |||
|---|---|---|---|
| sgRNA | Forward | Reverse | T7 Guide Primer |
| sgRNA1-1 | CACCGCTTCACGGCACCAGGGGTGA | AAACTCACCCCTGGTGCCGTGAAGC | TTAATACGACTCACTATAGGGGGCGTTCTCTTTGGTGGGT |
| sgRNA2b | CACCGACCCACCAAATAGAACCCCC | AAACGGGGGTTCTATTTGGTGGGTC | TTAATACGACTCACTATAGGACCCACCAAATAGAACCCCC |
| sgRNA4 | CACCGACTTGCAGGTGGTCCGAGTG | AAACCACTCGGACCACCTGCAAGTC | TTAATACGACTCACTATAGGACTTGCAGGTGGTCCGAGTG |
| In vitro transcription of mouse sgRNAs | |||
|---|---|---|---|
| sgRNA | Forward | Reverse | T7 Guide Primer |
| sgRNA1-1 | CACCGCTTCACGGCATTGGGGCGGT | AAACACCGCCCCAATGCCGTGAAGC | TTAATACGACTCACTATAGGCTTCACGGCATTGGGGCGGT |
| sgRNA1-2 | CACCGGCAAAGTCTCCACGCCAACT | AAACAGTTGGCGTGGAGACTTTGCC | TTAATACGACTCACTATAGGGCAAAGTCTCCACGCCAACT |
| sgRNA2b | CACCGACCCACCAAAGAGAACGCCC | AAACGGGCGTTCTCTTTGGTGGGTC | TTAATACGACTCACTATAGGACCCACCAAAGAGAACGCCC |
| sgRNA4 | CACCGCATCCTAGGTGGTTCGAGTA | AAACTACTCGAACCACCTAGGATGC | TTAATACGACTCACTATAGGCATCCTAGGTGGTTCGAGTA |
| Dmc1 sgRNA | CACCGTAGGAATCTGTACCATTAA | AAACTTAATGGTACAGATTCCTAC | TTAATACGACTCACTATAGGGTAGGAATCTGTACCATTA |
| Universal Reverse Primer for IVT | AAAAGCACCGACTCGGTGCC |
| MiSeq and/or Sanger sequencing analysis of human cells | ||
|---|---|---|
| Site | Forward oligo | Reverse oligo |
| sgRNA1-1 on-target site | CTGTGGGCCCCAGGTT | ATCAGGCTGCCCTGTCAT |
| sgRNA1-2 on-target site | TGGAGGTGATGGGCCAG | ACCAGGGGTGACGGTG |
| sgRNA2b on-target site (used primarily) | AGGGGAGATTGATAACTGGTGT | ACTAGGTTCAGGGATACTCCTTAG |
| sgRNA2b on-target site 1000bp | CGCCCAGCAAAGAACTTCTA | GAGAACCACTGCACCAAAGA |
| sgRNA2b on-target site 800bp | TGCATGAGTCAGTGAACAGG | GAGAACCACTGCACCAAAGA |
| sgRNA2b on-target site 140bp | CATGGGTGAGGGTAGTCTGC | TGGGATATACACAGGCCGAT |
| sgRNA2b adjacent to on-target site1 | GCCTGACTGCTTGGACATTC | GGCTCGAGAAGGATGTGAGT |
| sgRNA2b adjacent to on-target site2 | CAGGTGGTGGTGTGAAAAGG | TCGTAGCTCTCCGTCTTTGG |
| sgRNA2b adjacent to on-target site3 | CAGATGGTCGTTTGGCTGAA | TCTGGGAAGAGGTGGTAAGC |
| sgRNA2b adjacent to on-target site4 | CTTCAGGAGCTTGGCAAATTG | AGGGGAGATTGATAACTGGTGT |
| sgRNA2b adjacent to on-target site5 | ACCCATTCCCTGTTCACTGA | GCCAGGGTCTCTCTTTCTGT |
| sgRNA2b adjacent to on-target site6 | TCTCTCACTCAAGTATCACCCC | AAAGCAAGCTGGGGAGAGTA |
| sgRNA4 on-target site | TGTCCTCCTCTAACTGCTCT | CAGAGGAAAGGACACTGGTC |
| NR_036440 putative off-target site | CCTGCACGAGGGTTTCTG | AAGGAGTCCCAGGACATCAA |
| NM_001159542 putative off-target site | AACCCGGAGAAGTCCCAG | TGTTGTCAGCTTCCTCCAC |
| NR_034180 putative off-target site | GCAGGAGTCCCAGAACATC | GGGTTTCTGCTTTGCATGTC |
| NR_131184 putative off-target site | CCAGTCCCAGGACATCTCAA | ACTTCTGCAGCAAGGGC |
| chr3:+101807899 putative off-target site | TATGTGGGCTGGCATTCATAC | TTTTCACCATGGAGCATTGAGTA |
| chr3:+128394390 putative off-target site | ACGGGGTTTCTCCATGTTAC | CTCTTCATTGTTGGCTGCTT |
| chr19:-9072444 putative off-target site | CCGAGGTATCCAGGACAGAT | ATCCACAGAGGGAGGACTTG |
| MiSeq analysis of mouse cells | ||
|---|---|---|
| Site | Forward oligo | Reverse oligo |
| sgRNA2b on-target site | GAACAGTTTGCCAAGCTGCT | CCCCACCTCTGACAGTTCAA |
| Dmc1 on-target site | GATGGACACGAAGCTCATGAC | CAATAAGCTTGTTGGCTGCCTC |
| qRT-PCR | ||
|---|---|---|
| Gene | Forward oligo | Reverse oligo |
| POU5F1 | AGTGAGAGGCAACCTGGAGA | ACACTCGGACCACATCCTTC |
| GAPDH | GATGACATCAAGAAGGTGGTG | GTCTACATGGCAACTGTGAGG |
| NANOG | CATGAGTGTGGATCCAGCTTG | CCTGAATAAGCAGATCCATGG |
| SOX2 | TGGACAGTTACGCGCACAT | CGAGTAGGACATGCTGTAGGT |
| GATA6 | TCCACTCGTGTCTGCTTTTG | TCCTAGTCCTGGCTTCTGGA |
| SOX17 | CGCACGGAATTTGAACAGTA | GGATCAGGGACCTGTCACAC |
| AFP | ACCATGAAGTGGGTGGAATC | TGGTAGCCAGGTCAGCTAAA |
| PAX6 | TCTAATCGAAGGGCCAAATG | TGTGAGGGCTGTGTCTGTTC |
| PAX6 | CTTTGCTTGGGAAATCCGAG | AGCCAGGTTGCGAAGAACTC |
| NESTIN | GAAACAGCCATAGAGGGCAAA | TGGTTTTCCAGAGTCTTCAGTGA |
| ISL1 | GTGCAAGGACAAGAAGCGAA | TATGTCACTCTGCAAGGCGA |
| NKX2.5 | CCTCAACAGCTCCCTGACTC | AGGCTGCAGGATCACTCATT |
| KRT18 | CTGCTGCACCTTGAGTCAGA | TGGTGGTCTTTTGGATGGTT |
| CDX2 | GGAACCTGTGCGAGTGGAT | TCGATATTTGTCTTTCGTCCT |
| GATA2 | CAGACGAAGGCAACCATTTT | CAGAGGAGAAGAGGGTGCAG |
| GSC | GAGGAGAAAGTGGAGGTCTGGTT | CTCTGATGAGGACCGCTTCTG |
| SOX1 | FW+ REV: Quantitec primers (Qiagen): QT00215299 | |
| TBX6 | AAGTACCAACCCCGCATACA | TAGGCTGTCACGGAGATGAA |
| PBGD | GGAGCCATGTCTGGTAACGG | CCACGCGAATCACTCTCATCT |