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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_52313561_F | TCGTCGGCAGCGTCCATTAGCTGCGGGTCTCCTT | 59.8 | 1.00 |
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313561_R | GTCTCGTGGGCTCGGAAGCTGTCATCCTCCTGCTG | 59.7 | 1.00 |
mm4_intergenic_Gm24070|Gm24994_chr12_77830999_F | TCGTCGGCAGCGTCAGCAATTTCAGCTGTCCCC | 58.0 | 0.24 |
mm4_intergenic_Gm24070|Gm24994_chr12_77830999_R | GTCTCGTGGGCTCGGGGCTCATATTTTGCATGGCAC | 58.5 | 0.24 |
mm4_intergenic_Galnt1|Mir187_chr18_24379091_F | Primer3: not found at this Tm | N.d. | 0.22 |
mm4_intergenic_Galnt1|Mir187_chr18_24379091_R | Primer3: not found at this Tm | N.d. | 0.22 |
mm4_intron_Apbb2_chr5_66431136_F | Primer3: not found at this Tm | N.d. | 0.16 |
mm4_intron_Apbb2_chr5_66431136_R | Primer3: not found at this Tm | N.d. | 0.16 |
mm4_intergenic_Tcte2/Mllt4|Mllt4_chr17_13775717_F | TCGTCGGCAGCGTCACAGAGGTCACTGAAGTGGT | 58.2 | 0.14 |
mm4_intergenic_Tcte2/Mllt4|Mllt4_chr17_13775717_R | GTCTCGTGGGCTCGGCCTAAAAGGAAAGCATGAGGGC | 59.5 | 0.14 |
mm4_intergenic_Sod2|Gpr31b_chr17_13020519_F | Primer3: not found at this Tm | N.d. | 0.14 |
mm4_intergenic_Sod2|Gpr31b_chr17_13020519_R | Primer3: not found at this Tm | N.d. | 0.14 |
mm4_intergenic_Pdzrn3|Gm26911_chr6_101191356_F | Primer3: not found at this Tm | N.d. | 0.14 |
mm4_intergenic_Pdzrn3|Gm26911_chr6_101191356_R | Primer3: not found at this Tm | N.d. | 0.14 |
mm4_intergenic_Vps13c|Gm22382_chr9_68097203_F | Primer3: not found at this Tm | N.d. | 0.09 |
mm4_intergenic_Vps13c|Gm22382_chr9_68097203_R | Primer3: not found at this Tm | N.d. | 0.09 |
mm4_intron_Nup214_chr2_31975708_F | TCGTCGGCAGCGTCACACTGGCCCACCACAAAA | 59.9 | 0.09 |
mm4_intron_Nup214_chr2_31975708_R | GTCTCGTGGGCTCGGATTTCCCCATGAGGCCACAC | 60.3 | 0.09 |
mm4_intergenic_Gm8614|Gm23335_chr18_55861326_F | TCGTCGGCAGCGTCTGCTAGCTGACATAAACTGTAGCA | 60.0 | 0.08 |
mm4_intergenic_Gm8614|Gm23335_chr18_55861326_R | GTCTCGTGGGCTCGGTCTATGGTCCAAGCCCAGGG | 60.9 | 0.08 |
mm4_intron_B230216G23Rik_chr6_142413974_F | TCGTCGGCAGCGTCAGAACACAAACCCCGGGTTC | 60.4 | 0.06 |
mm4_intron_B230216G23Rik_chr6_142413974_R | GTCTCGTGGGCTCGGGAGCATAGCTTGGGGAGTCC | 59.8 | 0.06 |
mm4_intergenic_Gm14214|Raly/a_chr2_154784701_F | TCGTCGGCAGCGTCTGCCCTTACTGCACACACAT | 59.8 | 0.05 |
mm4_intergenic_Gm14214|Raly/a_chr2_154784701_R | GTCTCGTGGGCTCGGTGGTCATCCAGCACCGAAAG | 60.3 | 0.05 |
mm4_intergenic_Sh3gl2|Gm12680_chr4_85486336_F | Primer3: not found at this Tm | N.d. | 0.05 |
mm4_intergenic_Sh3gl2|Gm12680_chr4_85486336_R | Primer3: not found at this Tm | N.d. | 0.05 |
mm4_intergenic_Zfp704|Pag1_chr3_9684750_F | Primer3: not found at this Tm | N.d. | 0.03 |
mm4_intergenic_Zfp704|Pag1_chr3_9684750_R | Primer3: not found at this Tm | N.d. | 0.03 |
mm4_intron_Ahdc1_chr4_133037073_F | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intron_Ahdc1_chr4_133037073_R | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intergenic_Gm14261/Sall4|Gm25214_chr2_168879594_F | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_intergenic_Gm14261/Sall4|Gm25214_chr2_168879594_R | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_intron_Slc25a13_chr6_6107405_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Slc25a13_chr6_6107405_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Ccnb2_chr9_70416512_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Ccnb2_chr9_70416512_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Arhgef16_chr4_154279375_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Arhgef16_chr4_154279375_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Fam126a|Klhl7_chr5_24078281_F | TCGTCGGCAGCGTCTCCCATTTGGCAGTGACAGA | 59.2 | 0.00 |
mm4_intergenic_Fam126a|Klhl7_chr5_24078281_R | GTCTCGTGGGCTCGGAGGAAAAGCTCACAGTGGGA | 58.8 | 0.00 |
mm4_intergenic_Cnih3|Ccdc121_chr1_181479034_F | TCGTCGGCAGCGTCGACACCAGGCCTAGTGCTTC | 60.3 | 0.00 |
mm4_intergenic_Cnih3|Ccdc121_chr1_181479034_R | GTCTCGTGGGCTCGGTCCTCTGCCCTATGCTTCCT | 60.0 | 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_52313561 |
CATTAGCTGCGGGTCTCCTTTCATCTTCACTGTGGCAGACGTTTCTATTTATCCACTTGCGTTCGCCGAGTGGCGTCACC AGCGGTACTGTAATGACGATTGCAGCAGGAGGATGACAGCTT |
mm4_intergenic_Gm24070|Gm24994_chr12_77830999 |
AGCAATTTCAGCTGTCCCCCACCCCCACACACAATAAAAACTAATACATATTCTGTGTCTCAAGTGAGTACTACCTTTGG TACTGTTATGAGGATTCAAGATTAAAGAGAATTCTGAATGTGCCATGCAAAATATGAGCC |
mm4_intergenic_Tcte2/Mllt4|Mllt4_chr17_13775717 | ACAGAGGTCACTGAAGTGGTAGAAATTGTAATTACACAACCGCAGGGCCCTCATGCTTTCCTTTTAGG |
mm4_intron_Nup214_chr2_31975708 |
ACACTGGCCCACCACAAAAAAAGCCACCAAGCCCCAGTGGTACAGTCATGACTATTAGGATGATAATATCAAAAAAGATA GATGATTTGTTTCTTTATGAATGGCCAGTGTGGCCTCATGGGGAAAT |
mm4_intergenic_Gm8614|Gm23335_chr18_55861326 |
TGCTAGCTGACATAAACTGTAGCATTAGGCAGATTAAAAAGACAAATGGAAATAGAAGAAGAGAAATAAAAAAGGTCATT ACACTACAGCAGGATCTTGCCCTGGGCTTGGACCATAGA |
mm4_intron_B230216G23Rik_chr6_142413974 |
AGAACACAAACCCCGGGTTCGGACTAGGCAGCCTCTGTTTTTGCTTTTTCCACCCGCTGCATTGTAATAACGATTCTTCT CTCCTCCCTGCACTGCAGCGACTCTCTGAGAAGAGGAACTGGACTCCCCAAGCTATGCTC |
mm4_intergenic_Gm14214|Raly/a_chr2_154784701 |
TGCCCTTACTGCACACACATTCCTTCAGTGAAGGATTTGCATATCGTCTTTACAGCACCCCTGGGTCGGCAGCGTCATGC CCACCCATGTTTATGTAGTCCTTTCGGTGCTGGATGACCA |
mm4_intergenic_Fam126a|Klhl7_chr5_24078281 |
TCCCATTTGGCAGTGACAGAGATGTCTGTGGTCTCTAAGGACACTGGAATCGTTATTACAGTCCCCAGGGCTTTCTCTAA AGCAAGAGGCAAAATTTTCCCACTGTGAGCTTTTCCT |
mm4_intergenic_Cnih3|Ccdc121_chr1_181479034 |
GACACCAGGCCTAGTGCTTCGACGGGGTTATTTTACAAAACCTCATGACAGTCCCGCAGGCAAGCTGTTGTGCAGGTGAG CTGGCAATACACTCCATGGCTGCAGGTGAGGAAGCATAGGGCAGAGGA |