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Contents:In the table below, Illumina Nextera Handle sequences have been added and highlighted in bold. Primers for the on-target have been added for convenience. The table below is sorted by the CFD off-target score. Sites with very low CFD scores < 0.02 are unlikely to be cleaved, see our study Haeussler et al. 2016, Figure 2.
In the protocol by Matthew Canver, Harvard, two PCRs are run: one PCR to amplify the potential off-target, then a second PCR to extend the handles with Illumina barcodes. Please click here to download the protocol. Alternatively, you can have a look at Fu et al, 2014.
If a primer was not found, the reason is usually that the region around the off-target is too repetitive. To avoid unspecific primers, all repeats are masked for the primer design (not for off-target search). If you think that we should change the parameters here or should use different primer3 settings, please let us know.
| Name | Primer Sequence | Tm | CFD Score |
|---|---|---|---|
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313704_F | TCGTCGGCAGCGTCCGCATCACAAGGTGACCCTA | 59.7 | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313704_R | GTCTCGTGGGCTCGGCTCCTAATCCCAGGCTCCCT | 60.1 | 1.00 |
| mm4_intergenic_Gm5356|Gm26331_chr8_89307978_F | Primer3: not found at this Tm | N.d. | 0.81 |
| mm4_intergenic_Gm5356|Gm26331_chr8_89307978_R | Primer3: not found at this Tm | N.d. | 0.81 |
| mm4_intron_Nin_chr12_70079266_F | TCGTCGGCAGCGTCTACCAGTTTCACCCGCACTG | 60.2 | 0.43 |
| mm4_intron_Nin_chr12_70079266_R | GTCTCGTGGGCTCGGAGGTGATGCTGTGACTGTGG | 59.9 | 0.43 |
| mm4_exon_Lce1h_chr3_92763543_F | Primer3: not found at this Tm | N.d. | 0.19 |
| mm4_exon_Lce1h_chr3_92763543_R | Primer3: not found at this Tm | N.d. | 0.19 |
| mm4_intron_S1pr2_chr9_20975910_F | TCGTCGGCAGCGTCAAATCCCCGGGTGAGTCAAC | 59.9 | 0.13 |
| mm4_intron_S1pr2_chr9_20975910_R | GTCTCGTGGGCTCGGGATGGGGCGTAGGAGTGAAG | 59.8 | 0.13 |
| mm4_exon_Kansl1_chr11_104442190_F | TCGTCGGCAGCGTCGAGCTTCTTCCCGGCCGG | 62.8 | 0.07 |
| mm4_exon_Kansl1_chr11_104442190_R | GTCTCGTGGGCTCGGCATTTTCCCAGTGCCGCC | 59.4 | 0.07 |
| mm3_intron_Ntrk2_chr13_58843469_F | TCGTCGGCAGCGTCTTCCCAGACCCACCATCTCT | 59.8 | 0.05 |
| mm3_intron_Ntrk2_chr13_58843469_R | GTCTCGTGGGCTCGGGAGAACTGAGCACCTCGGAG | 59.8 | 0.05 |
| mm4_intergenic_Brd1|Zbed4_chr15_88751318_F | TCGTCGGCAGCGTCGAAACCACTGAGAACGCGC | 59.7 | 0.03 |
| mm4_intergenic_Brd1|Zbed4_chr15_88751318_R | GTCTCGTGGGCTCGGCTCCTCCGTCAAACTACGCA | 59.7 | 0.03 |
| mm4_intergenic_AI854517|Mir9-3_chr7_79505202_F | TCGTCGGCAGCGTCTCTGTCGGTCCCCTCTGG | 59.9 | 0.03 |
| mm4_intergenic_AI854517|Mir9-3_chr7_79505202_R | GTCTCGTGGGCTCGGGAGAAACGGGCCTCCCATTC | 60.7 | 0.03 |
| mm4_exon_6330416G13Rik_chr4_63584500_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_6330416G13Rik_chr4_63584500_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Elf2_chr3_51340616_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Elf2_chr3_51340616_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Eif4e_chr3_138527732_F | TCGTCGGCAGCGTCGAGAGAGCCGGAGAGGGAG | 60.5 | 0.00 |
| mm4_intron_Eif4e_chr3_138527732_R | GTCTCGTGGGCTCGGTCCTCCGCAGCTCCAGAG | 60.7 | 0.00 |
| mm4_exon_Usp31/1700069B07Rik_chr7_121707087_F | TCGTCGGCAGCGTCGACGCGGCTCATGAAACTG | 59.5 | 0.00 |
| mm4_exon_Usp31/1700069B07Rik_chr7_121707087_R | GTCTCGTGGGCTCGGAAGGAGAAGCGCTCGTTCAG | 60.3 | 0.00 |
These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313704 |
CGCATCACAAGGTGACCCTAGCTCCCCACTGCCATCTCCGCGGTCGCCGTCGACACGGCGCTGGGGCTACCCGGCGCCTG CCTTGTCGCCTTAGCTCCTCTTCTCAGCCAAGATCCCAGGGAGCCTGGGATTAGGAG |
| mm4_intron_Nin_chr12_70079266 |
TACCAGTTTCACCCGCACTGCCGCCAACCCACCCACCAAGCGCCGTGTCTACAACTACCACCTCTACCACAGTCACAGCA TCACCT |
| mm4_intron_S1pr2_chr9_20975910 |
AAATCCCCGGGTGAGTCAACCCATGCTCTAGAGGGGGGCGAGGTCCCAACGCCGTGTAGCCCGCGACAACGCGGACTCCA CGCCACGCGTGCCTTCCTTCACTCCTACGCCCCATC |
| mm4_exon_Kansl1_chr11_104442190 |
GAGCTTCTTCCCGGCCGGGGAGCGCACTTCGGCCGCCCCCCAAGCCGCCGCGGCGAGACGAGTCGGCTTCGCTACGGCGC TTGGTTCTCCCGCCGGCTCAGCGAGCGGTGGCGGTGGCGGCGGCGGCACTGGGAAAATG |
| mm3_intron_Ntrk2_chr13_58843469 |
TTCCCAGACCCACCATCTCTAGGAGCCGTTCTTTTGGGTTCAGCCCTCTGCTTGATATTTATCCTTATGCCCCAGTCGAG CGCCGTGTTGACGGCTAACAGCCAGCATCTCCGAGGTGCTCAGTTCTC |
| mm4_intergenic_Brd1|Zbed4_chr15_88751318 |
GAAACCACTGAGAACGCGCGCTTCTCAAACGCGCAGCAATTCCAACCAACCGCCGTGGCGACTGCGCCTGCGTAGTCTTC GGACCGAACGCCCGACAGGGCAGGTGCGCCAGACCGCGCTTGCGTAGTTTGACGGAGGAG |
| mm4_intergenic_AI854517|Mir9-3_chr7_79505202 |
TCTGTCGGTCCCCTCTGGCTCTCCGCGTGCGCCCGGGGCTGCGGGAGCGGAGTCCGCTCAGGCCCCCGCGCCGGGTCGGC GGCGGCACGTGGGGCCCACAGCGCGGCAGCGGTGCCGGCGAATGGGAGGCCCGTTTCTC |
| mm4_intron_Eif4e_chr3_138527732 |
GAGAGAGCCGGAGAGGGAGCCCGAGGCAGCGGCGCCTCGCCGTCGCCGGCGACCCGCCGCCGGGGGTCCTCGTGCGGGCC GCGCTCTGGAGCTGCGGAGGA |
| mm4_exon_Usp31/1700069B07Rik_chr7_121707087 |
GACGCGGCTCATGAAACTGCCCACCGAGCGCGCCGACGACGGCGACGANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNACTCCGGAACAGCCGCTTGCTGAACGAGCGCTTCTCCTT |