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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_52313708_F | TCGTCGGCAGCGTCCGCATCACAAGGTGACCCTA | 59.7 | 1.00 |
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313708_R | GTCTCGTGGGCTCGGCTCCTAATCCCAGGCTCCCT | 60.1 | 1.00 |
mm3_exon_Bcor_chrX_12080258_F | Primer3: not found at this Tm | N.d. | 0.46 |
mm3_exon_Bcor_chrX_12080258_R | Primer3: not found at this Tm | N.d. | 0.46 |
mm4_intergenic_Gm24987|Socs6_chr18_88658673_F | Primer3: not found at this Tm | N.d. | 0.33 |
mm4_intergenic_Gm24987|Socs6_chr18_88658673_R | Primer3: not found at this Tm | N.d. | 0.33 |
mm4_intron_Gm16338_chr5_124197316_F | Primer3: not found at this Tm | N.d. | 0.32 |
mm4_intron_Gm16338_chr5_124197316_R | Primer3: not found at this Tm | N.d. | 0.32 |
mm4_intron_1500017E21Rik_chr19_36703153_F | Primer3: not found at this Tm | N.d. | 0.30 |
mm4_intron_1500017E21Rik_chr19_36703153_R | Primer3: not found at this Tm | N.d. | 0.30 |
mm4_intron_Map2k4_chr11_65782604_F | Primer3: not found at this Tm | N.d. | 0.28 |
mm4_intron_Map2k4_chr11_65782604_R | Primer3: not found at this Tm | N.d. | 0.28 |
mm4_intergenic_Gprc5b|Gpr139_chr7_119038882_F | Primer3: not found at this Tm | N.d. | 0.28 |
mm4_intergenic_Gprc5b|Gpr139_chr7_119038882_R | Primer3: not found at this Tm | N.d. | 0.28 |
mm4_intron_Nbea_chr3_55650860_F | TCGTCGGCAGCGTCCACTGCTGAGCATGGAAGGA | 60.0 | 0.25 |
mm4_intron_Nbea_chr3_55650860_R | GTCTCGTGGGCTCGGAAATGCGGTGTAGGAGGACG | 60.1 | 0.25 |
mm4_intergenic_Mier2|Theg_chr10_79557648_F | Primer3: not found at this Tm | N.d. | 0.16 |
mm4_intergenic_Mier2|Theg_chr10_79557648_R | Primer3: not found at this Tm | N.d. | 0.16 |
mm4_exon_P2ry2_chr7_100996864_F | Primer3: not found at this Tm | N.d. | 0.11 |
mm4_exon_P2ry2_chr7_100996864_R | Primer3: not found at this Tm | N.d. | 0.11 |
mm4_intergenic_Gm25062|Gm15487_chr6_37753403_F | Primer3: not found at this Tm | N.d. | 0.09 |
mm4_intergenic_Gm25062|Gm15487_chr6_37753403_R | Primer3: not found at this Tm | N.d. | 0.09 |
mm4_intergenic_B230206L02Rik|Tob1_chr11_94172607_F | Primer3: not found at this Tm | N.d. | 0.08 |
mm4_intergenic_B230206L02Rik|Tob1_chr11_94172607_R | Primer3: not found at this Tm | N.d. | 0.08 |
mm4_intron_Tab1_chr15_80134800_F | TCGTCGGCAGCGTCTGAAAGCTGCTAGTCTGGGC | 60.0 | 0.07 |
mm4_intron_Tab1_chr15_80134800_R | GTCTCGTGGGCTCGGTGTCCAGGGTTCCCTATGGA | 59.5 | 0.07 |
mm4_intron_Poln_chr5_34094787_F | Primer3: not found at this Tm | N.d. | 0.07 |
mm4_intron_Poln_chr5_34094787_R | Primer3: not found at this Tm | N.d. | 0.07 |
mm4_intron_Gdf11_chr10_128885744_F | Primer3: not found at this Tm | N.d. | 0.06 |
mm4_intron_Gdf11_chr10_128885744_R | Primer3: not found at this Tm | N.d. | 0.06 |
mm4_intergenic_Ddx47|Gprc5a_chr6_135050980_F | Primer3: not found at this Tm | N.d. | 0.05 |
mm4_intergenic_Ddx47|Gprc5a_chr6_135050980_R | Primer3: not found at this Tm | N.d. | 0.05 |
mm4_intergenic_Gm23216|Gm23974_chr1_45708908_F | Primer3: not found at this Tm | N.d. | 0.05 |
mm4_intergenic_Gm23216|Gm23974_chr1_45708908_R | Primer3: not found at this Tm | N.d. | 0.05 |
mm4_exon_Hoxa2_chr6_52164430_F | TCGTCGGCAGCGTCCATCCAGGGATACTCAGGCG | 59.6 | 0.04 |
mm4_exon_Hoxa2_chr6_52164430_R | GTCTCGTGGGCTCGGAGACCATTCCCAGCCTGAAC | 59.6 | 0.04 |
mm4_intron_Umodl1_chr17_30973557_F | Primer3: not found at this Tm | N.d. | 0.04 |
mm4_intron_Umodl1_chr17_30973557_R | Primer3: not found at this Tm | N.d. | 0.04 |
mm4_intergenic_Tln2|mmu-mir-7241_chr9_67602103_F | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intergenic_Tln2|mmu-mir-7241_chr9_67602103_R | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intergenic_Gm26766|Gm16136_chr15_37141685_F | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intergenic_Gm26766|Gm16136_chr15_37141685_R | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intron_Gxylt2_chr6_100803041_F | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intron_Gxylt2_chr6_100803041_R | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intergenic_Hlcs|Ripply3_chr16_94321371_F | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intergenic_Hlcs|Ripply3_chr16_94321371_R | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_exon_Nkx6-1_chr5_101663678_F | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_exon_Nkx6-1_chr5_101663678_R | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_intergenic_Tmem132e|Gm24612_chr11_82584278_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Tmem132e|Gm24612_chr11_82584278_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_RP24-282D16.5|Mns1_chr9_72431780_F | TCGTCGGCAGCGTCAGGATCTCCTTTGCCCATGT | 58.6 | 0.00 |
mm4_intergenic_RP24-282D16.5|Mns1_chr9_72431780_R | GTCTCGTGGGCTCGGTCCGGCTCTCACAGACATTG | 59.7 | 0.00 |
mm4_intergenic_Ncald|Gm3267_chr15_37491593_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Ncald|Gm3267_chr15_37491593_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Herpud2|Sept7_chr9_25229127_F | TCGTCGGCAGCGTCGGATCTCCTTTGCCCAGGTT | 59.6 | 0.00 |
mm4_intergenic_Herpud2|Sept7_chr9_25229127_R | GTCTCGTGGGCTCGGACAGCCATTGCAGTGCATTG | 60.0 | 0.00 |
mm4_intergenic_Gm23834|Zbtb40_chr4_137048618_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Gm23834|Zbtb40_chr4_137048618_R | Primer3: not found at this Tm | N.d. | 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_52313708 |
CGCATCACAAGGTGACCCTAGCTCCCCACTGCCATCTCCGCGGTCGCCGTCGACACGGCGCTGGGGCTACCCGGCGCCTG CCTTGTCGCCTTAGCTCCTCTTCTCAGCCAAGATCCCAGGGAGCCTGGGATTAGGAG |
mm4_intron_Nbea_chr3_55650860 |
CACTGCTGAGCATGGAAGGAGCGCAGGTATGCTGTAGATGGCCCTTGACACAGTGCTGCGGCTAGTTGCATCTCCATCTA CAATTCTCATTTACAAAGTACTTCAATTTAACCATCCGTCCTCCTACACCGCATTT |
mm4_intron_Tab1_chr15_80134800 |
TGAAAGCTGCTAGTCTGGGCCTCTTGGGAAGATGAAAGGGCAGCAGGGGAAGAGGAGCGTGAGCCCCAGCACCGTGGCTA AGGCATAGTCAGCGTGGACTTCCATAGGGAACCCTGGACA |
mm4_exon_Hoxa2_chr6_52164430 |
CATCCAGGGATACTCAGGCGGCTGCAGGGCGCCGGCAGGCACCGGGCTGCCGCGACTGCCCGCGGGGCTCGACTTGGGGC GGCCGCCAACGCCAGCGCCGTGGCGAGGGTGACTGCCCGGGTTCAGGCTGGGAATGGTCT |
mm4_intergenic_RP24-282D16.5|Mns1_chr9_72431780 |
AGGATCTCCTTTGCCCATGTTTAGTTGATTTTCCTCCGCGAGGCACAGAATCGCCCAGTGCCCGTCCAGCTCGCGCTTGC CCCAGCGCTGTCTCTATGGAGCTCAATGTACTGCAATGTCTGTGAGAGCCGGA |
mm4_intergenic_Herpud2|Sept7_chr9_25229127 |
GGATCTCCTTTGCCCAGGTTTAGCTGATTTTCCTCCATGAGGCAAAGAGTCGCCCAGTGCCCATCTGGCTCTCACTTGCC CCAGCGCTGTCTCTATGGAATTCAATGCACTGCAATGGCTGT |