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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_52313990_F | TCGTCGGCAGCGTCAACCGCCAGAGAAGATGGTG | 60.0 | 1.00 |
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313990_R | GTCTCGTGGGCTCGGCTGCCTGCTAGTCCATCGAC | 60.2 | 1.00 |
mm3_exon_Dgat1_chr15_76511726_F | TCGTCGGCAGCGTCACTACCACCCTGGACGGAAA | 60.4 | 0.78 |
mm3_exon_Dgat1_chr15_76511726_R | GTCTCGTGGGCTCGGCTCCAAGCGCTGAGAGCC | 60.5 | 0.78 |
mm4_intergenic_Fignl2|Ankrd33_chr15_101077890_F | Primer3: not found at this Tm | N.d. | 0.31 |
mm4_intergenic_Fignl2|Ankrd33_chr15_101077890_R | Primer3: not found at this Tm | N.d. | 0.31 |
mm4_intergenic_Cmtr1/RNF8/Ccdc167|Ccdc167_chr17_29707805_F | TCGTCGGCAGCGTCGTGGCTGCAGAACGGACTTA | 60.3 | 0.29 |
mm4_intergenic_Cmtr1/RNF8/Ccdc167|Ccdc167_chr17_29707805_R | GTCTCGTGGGCTCGGTCAGCAGCCATAACCCATGT | 59.3 | 0.29 |
mm4_intergenic_Gm12417|Sh3gl2_chr4_85186509_F | Primer3: not found at this Tm | N.d. | 0.21 |
mm4_intergenic_Gm12417|Sh3gl2_chr4_85186509_R | Primer3: not found at this Tm | N.d. | 0.21 |
mm4_intergenic_E4f1|Pgp_chr17_24456550_F | Primer3: not found at this Tm | N.d. | 0.19 |
mm4_intergenic_E4f1|Pgp_chr17_24456550_R | Primer3: not found at this Tm | N.d. | 0.19 |
mm4_intergenic_Gm15443|Gm23535_chr1_149423253_F | Primer3: not found at this Tm | N.d. | 0.18 |
mm4_intergenic_Gm15443|Gm23535_chr1_149423253_R | Primer3: not found at this Tm | N.d. | 0.18 |
mm4_intron_Ptafr_chr4_132571464_F | Primer3: not found at this Tm | N.d. | 0.17 |
mm4_intron_Ptafr_chr4_132571464_R | Primer3: not found at this Tm | N.d. | 0.17 |
mm4_intron_Disp1_chr1_183137633_F | Primer3: not found at this Tm | N.d. | 0.14 |
mm4_intron_Disp1_chr1_183137633_R | Primer3: not found at this Tm | N.d. | 0.14 |
mm4_exon_Lin54_chr5_100443223_F | TCGTCGGCAGCGTCACCACACATAACCTCCCAGC | 59.6 | 0.11 |
mm4_exon_Lin54_chr5_100443223_R | GTCTCGTGGGCTCGGTGATGACAGAACTCATACGCAG | 58.2 | 0.11 |
mm4_intergenic_2610028E06Rik|Csf3r_chr4_125967453_F | Primer3: not found at this Tm | N.d. | 0.09 |
mm4_intergenic_2610028E06Rik|Csf3r_chr4_125967453_R | Primer3: not found at this Tm | N.d. | 0.09 |
mm4_intergenic_Gm22983|1700057H15Rik_chr4_124396019_F | TCGTCGGCAGCGTCGGAAGTGGCAGGATTCCACA | 59.9 | 0.08 |
mm4_intergenic_Gm22983|1700057H15Rik_chr4_124396019_R | GTCTCGTGGGCTCGGTCCTGCCACTCAAGTGTGTC | 59.8 | 0.08 |
mm4_intergenic_Qsox1|Cep350_chr1_155813010_F | TCGTCGGCAGCGTCGCGTGTGAGCTGCCAGAG | 61.1 | 0.08 |
mm4_intergenic_Qsox1|Cep350_chr1_155813010_R | GTCTCGTGGGCTCGGCCCTCAACGACCTCGAGTG | 59.7 | 0.08 |
mm3_exon_Bnc2_chr4_84675213_F | Primer3: not found at this Tm | N.d. | 0.07 |
mm3_exon_Bnc2_chr4_84675213_R | Primer3: not found at this Tm | N.d. | 0.07 |
mm4_intron_Faah_chr4_116017361_F | TCGTCGGCAGCGTCACCTGGCGCAGAAAGAGTAC | 60.0 | 0.06 |
mm4_intron_Faah_chr4_116017361_R | GTCTCGTGGGCTCGGCAAGGCTGCGACTTCAACTG | 59.7 | 0.06 |
mm4_intergenic_Gm22235|Epas1_chr17_86753643_F | TCGTCGGCAGCGTCAGCCCGATGTCCCACTCC | 61.0 | 0.06 |
mm4_intergenic_Gm22235|Epas1_chr17_86753643_R | GTCTCGTGGGCTCGGCTCTCGAATGTCCGGCCTC | 59.9 | 0.06 |
mm4_intergenic_Pop7|Gigyf1_chr5_137510742_F | Primer3: not found at this Tm | N.d. | 0.03 |
mm4_intergenic_Pop7|Gigyf1_chr5_137510742_R | Primer3: not found at this Tm | N.d. | 0.03 |
mm4_intergenic_Gm24863|Gm24635_chr13_79796082_F | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_intergenic_Gm24863|Gm24635_chr13_79796082_R | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_intergenic_D430019H16Rik|Bdkrb2_chr12_105515448_F | TCGTCGGCAGCGTCCTTCCTTTGGTGCAGGAGGT | 59.8 | 0.00 |
mm4_intergenic_D430019H16Rik|Bdkrb2_chr12_105515448_R | GTCTCGTGGGCTCGGTCTCCTGTAAGCTGTGGGGA | 59.8 | 0.00 |
mm4_exon_Pax6_chr2_105676442_F | TCGTCGGCAGCGTCCGGCGATGCTTTTGTCCAG | 59.8 | 0.00 |
mm4_exon_Pax6_chr2_105676442_R | GTCTCGTGGGCTCGGCAGTTCTGTGCCGGGAGAAT | 60.0 | 0.00 |
mm4_intergenic_Gm15272|Gm15270_chr10_24535219_F | TCGTCGGCAGCGTCAGGTATAAACAGCAGGCCGG | 59.8 | 0.00 |
mm4_intergenic_Gm15272|Gm15270_chr10_24535219_R | GTCTCGTGGGCTCGGGGAGAGTGACGCAGGCAAA | 60.3 | 0.00 |
mm4_exon_Rpl15_chr14_18270581_F | TCGTCGGCAGCGTCCGGGAATGAGAAGGGCTCTG | 60.1 | 0.00 |
mm4_exon_Rpl15_chr14_18270581_R | GTCTCGTGGGCTCGGTAACCCTGCTGCCAGTTCTG | 59.9 | 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_52313990 |
AACCGCCAGAGAAGATGGTGCCCCACGGTTGCCTGAGCCCGCGAGCCGGCCCTCCGACTTCCCGGGAGCGTGGCGGGGGA GGCCAGGAGGAGGAGCCGGTCGATGGACTAGCAGGCAG |
mm3_exon_Dgat1_chr15_76511726 |
ACTACCACCCTGGACGGAAACCCGCGAGCCGGTCCTCCGACGCCGAGAGCTTCCCGCGCCTCCGCGGTCGCCCATGGCTT CGGCCCGCACCTCGTCGCGGCCCAGTCGTGCTCCGCCGGCTCTCAGCGCTTGGAG |
mm4_intergenic_Cmtr1/RNF8/Ccdc167|Ccdc167_chr17_29707805 |
GTGGCTGCAGAACGGACTTAACCCAAAAGACAGGTCAAAAAGGCAAGAAAAGACCAGAAAATGGACCTTACACAAAGTCC CCAGTTGGAGTGCCGGCTCTCAGGCACATGGGTTATGGCTGCTGA |
mm4_exon_Lin54_chr5_100443223 |
ACCACACATAACCTCCCAGCTCTGAGTCCTCGCGTCTCCAGCGAGCCAGACTTCAGACTTACTGCGTATGAGTTCTGTCA TCA |
mm4_intergenic_Gm22983|1700057H15Rik_chr4_124396019 |
GGAAGTGGCAGGATTCCACAGGAGACCGGACACTCAAGATGGGCACCAGCCTGACCCGCCAGCCAGCTCTCTGACTTCCC CGGGACACACTTGAGTGGCAGGA |
mm4_intergenic_Qsox1|Cep350_chr1_155813010 |
GCGTGTGAGCTGCCAGAGCCGGGGTCCCGGTGCTGGTGGGCGCTCCGCGGTTCCCATCCCCCGGCGAGCCAGACCTCCGA GCTGGGTCCGGAAACGAGTGCCGCCCGGGACCTCAGGCCGCACTCGAGGTCGTTGAGGG |
mm4_intron_Faah_chr4_116017361 |
ACCTGGCGCAGAAAGAGTACGGAATGCATTAGCTCGAGGAGGGGGCGATTCTGTTCCGGGGCTCACATCTGCGCTGCCTG CCAGCCGGCCCTCTGGCGTCCGGGCAGTTGAAGTCGCAGCCTTG |
mm4_intergenic_Gm22235|Epas1_chr17_86753643 |
AGCCCGATGTCCCACTCCCGCGCCCCTCGCGCCCTCCCGCGGGCGGGCCGTCCGAGTTTTTAAAGTGGGCTGACAGCCGA GGAGGCCGGACATTCGAGAG |
mm4_intergenic_D430019H16Rik|Bdkrb2_chr12_105515448 |
CTTCCTTTGGTGCAGGAGGTGGAGGTGACCCCCTAGCCTGCCCTCCCACTTGCCTCTTCGGCAGTTTACCAGATTAGTAG CCATCTCAGACTTCAGGTCAGTCCAGATCCGTCCCCACAGCTTACAGGAGA |
mm4_exon_Pax6_chr2_105676442 |
CGGCGATGCTTTTGTCCAGCCCGGGCCGAGCCAGCGCGCAAAGAGCGCCCAACCAGCCGACTCTCCGCGCCGCCTCCCGC CAGCCCGCGCTCCGGCTTTGTGCAAGATTCTCCCGGCACAGAACTG |
mm4_intergenic_Gm15272|Gm15270_chr10_24535219 |
AGGTATAAACAGCAGGCCGGGCCCAGAAGGCAGGCAGCAAGGAGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNGACTTTTTGCCTGCGTCACTCTCC |
mm4_exon_Rpl15_chr14_18270581 |
CGGGAATGAGAAGGGCTCTGCAGAGGCAGCAAGGCGCAGGGCCGGCTGGGCGGGACTGACTATTCCATACTCCCGGAGGC CGCCAGAACTGGCAGCAGGGTTA |