← return to the list of all guides
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_52313693_F | TCGTCGGCAGCGTCCGCATCACAAGGTGACCCTA | 59.7 | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313693_R | GTCTCGTGGGCTCGGCTCCTAATCCCAGGCTCCCT | 60.1 | 1.00 |
| mm4_intergenic_Abca5|Map2k6_chr11_110344773_F | TCGTCGGCAGCGTCACTGTATGTACGCATGCTCACA | 60.0 | 0.30 |
| mm4_intergenic_Abca5|Map2k6_chr11_110344773_R | GTCTCGTGGGCTCGGAGTCCTAGTGTCCAGCCGAT | 60.0 | 0.30 |
| mm4_exon_A830082K12Rik/Nr2f1_chr13_78199277_F | TCGTCGGCAGCGTCGACATAGAGGAAGCGGCGAG | 60.3 | 0.30 |
| mm4_exon_A830082K12Rik/Nr2f1_chr13_78199277_R | GTCTCGTGGGCTCGGCTCCTCCAGCAGCGTCTG | 59.8 | 0.30 |
| mm4_exon_Gpc1_chr1_92831626_F | Primer3: not found at this Tm | N.d. | 0.21 |
| mm4_exon_Gpc1_chr1_92831626_R | Primer3: not found at this Tm | N.d. | 0.21 |
| mm4_exon_Shank1_chr7_44336008_F | Primer3: not found at this Tm | N.d. | 0.20 |
| mm4_exon_Shank1_chr7_44336008_R | Primer3: not found at this Tm | N.d. | 0.20 |
| mm4_exon_Arrb1_chr7_99535649_F | Primer3: not found at this Tm | N.d. | 0.17 |
| mm4_exon_Arrb1_chr7_99535649_R | Primer3: not found at this Tm | N.d. | 0.17 |
| mm3_intergenic_Gm10398|Gm26660_chr14_25317853_F | TCGTCGGCAGCGTCGTAGGCCCTCAGCAGATGTG | 60.1 | 0.16 |
| mm3_intergenic_Gm10398|Gm26660_chr14_25317853_R | GTCTCGTGGGCTCGGGCTGGGGTCCTTAGCTCCT | 60.6 | 0.16 |
| mm3_exon_N4bp1_chr8_86884970_F | TCGTCGGCAGCGTCCCCGAACAGAGCCTCGATG | 60.2 | 0.14 |
| mm3_exon_N4bp1_chr8_86884970_R | GTCTCGTGGGCTCGGCCGGGTGGTGCTGGATGA | 61.7 | 0.14 |
| mm4_intergenic_Mir466f-4|Irx1_chr13_71509814_F | Primer3: not found at this Tm | N.d. | 0.12 |
| mm4_intergenic_Mir466f-4|Irx1_chr13_71509814_R | Primer3: not found at this Tm | N.d. | 0.12 |
| mm3_intron_Nsg1_chr5_38159118_F | TCGTCGGCAGCGTCCACAGCCAGAAGGACTCCG | 60.0 | 0.10 |
| mm3_intron_Nsg1_chr5_38159118_R | GTCTCGTGGGCTCGGGCCTGAGACTCTGATCTGGC | 59.8 | 0.10 |
| mm4_intergenic_Gm14342|Mir124a-3/Gm27032_chr2_180893152_F | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intergenic_Gm14342|Mir124a-3/Gm27032_chr2_180893152_R | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intergenic_Hnrnpa0|1700066J03Rik_chr13_58128751_F | Primer3: not found at this Tm | N.d. | 0.09 |
| mm4_intergenic_Hnrnpa0|1700066J03Rik_chr13_58128751_R | Primer3: not found at this Tm | N.d. | 0.09 |
| mm4_exon_Yars_chr4_129189950_F | TCGTCGGCAGCGTCGGACTAGCGTGACAGTTCCC | 60.1 | 0.09 |
| mm4_exon_Yars_chr4_129189950_R | GTCTCGTGGGCTCGGGATGCAGCTTCTCCTCAGGG | 60.1 | 0.09 |
| mm4_exon_Rnf38_chr4_44233771_F | TCGTCGGCAGCGTCCGACCGAGGCTGGAAGTG | 60.1 | 0.08 |
| mm4_exon_Rnf38_chr4_44233771_R | GTCTCGTGGGCTCGGGAGTCGAGTCGCAGTCCG | 59.9 | 0.08 |
| mm4_exon_9130019P16Rik/Wipf3_chr6_54429792_F | TCGTCGGCAGCGTCCAGAAGCAACGCCAGGCA | 60.9 | 0.08 |
| mm4_exon_9130019P16Rik/Wipf3_chr6_54429792_R | GTCTCGTGGGCTCGGCCGACTGGACTTTAGCTGGC | 60.7 | 0.08 |
| mm4_exon_Sirt1_chr10_63338714_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_exon_Sirt1_chr10_63338714_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Gm7545|Gm10443_chr6_86315575_F | TCGTCGGCAGCGTCAGGAGACCCGGGGTTATTCA | 59.9 | 0.06 |
| mm4_intergenic_Gm7545|Gm10443_chr6_86315575_R | GTCTCGTGGGCTCGGCCCTCTTCTTCTTGGGCAGG | 60.0 | 0.06 |
| mm4_intergenic_Slco2a1|Rab6b_chr9_103111947_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Slco2a1|Rab6b_chr9_103111947_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intron_Gatad2a_chr8_69973751_F | TCGTCGGCAGCGTCCGCGCAGGGACTCTTATTCT | 59.8 | 0.05 |
| mm4_intron_Gatad2a_chr8_69973751_R | GTCTCGTGGGCTCGGACAGCACGTGGACGAGTG | 59.9 | 0.05 |
| mm4_exon_Nkx2-4_chr2_147084125_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_exon_Nkx2-4_chr2_147084125_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intron_Nudc_chr4_133545755_F | TCGTCGGCAGCGTCCGTTGGAGCGAGACTTCACT | 60.0 | 0.05 |
| mm4_intron_Nudc_chr4_133545755_R | GTCTCGTGGGCTCGGGAGGTAAACGACGTGGCCC | 60.7 | 0.05 |
| mm4_exon_Agap2_chr10_127079476_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_exon_Agap2_chr10_127079476_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_1700030C10Rik|3110053B16Rik_chr12_20815748_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_1700030C10Rik|3110053B16Rik_chr12_20815748_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Fgfr1_chr8_25519435_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Fgfr1_chr8_25519435_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm3_exon_Nrip1_chr16_76372996_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm3_exon_Nrip1_chr16_76372996_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_Lrrc8b|Lrrc8c_chr5_105519299_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_Lrrc8b|Lrrc8c_chr5_105519299_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_exon_Six5_chr7_19094689_F | TCGTCGGCAGCGTCCCAAACATGGCTACCTCGC | 58.9 | 0.03 |
| mm4_exon_Six5_chr7_19094689_R | GTCTCGTGGGCTCGGCAGAGTCTGCAGAAGCTGGC | 61.0 | 0.03 |
| mm4_exon_Ctbp2/Fgfr2_chr7_133123150_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_exon_Ctbp2/Fgfr2_chr7_133123150_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_exon_Ugcg_chr4_59189274_F | TCGTCGGCAGCGTCCAGAGAGCACGGACCAGAAC | 60.3 | 0.01 |
| mm4_exon_Ugcg_chr4_59189274_R | GTCTCGTGGGCTCGGGGCTCCCGTGGTCCAGAC | 62.0 | 0.01 |
| mm4_exon_Stox2_chr8_47289066_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Stox2_chr8_47289066_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Lpin2_chr17_71184023_F | TCGTCGGCAGCGTCGCGAGGTGCAGTTATTCCCT | 60.1 | 0.01 |
| mm4_exon_Lpin2_chr17_71184023_R | GTCTCGTGGGCTCGGACATCTAAACCAGGTCCGCG | 60.1 | 0.01 |
| mm4_exon_Lysmd3_chr13_81657801_F | TCGTCGGCAGCGTCGCCTACTCTACGCGATGACG | 60.3 | 0.01 |
| mm4_exon_Lysmd3_chr13_81657801_R | GTCTCGTGGGCTCGGCCGACTCTAGGTTCTCCGGT | 60.3 | 0.01 |
| mm4_exon_Zcchc11_chr4_108460081_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Zcchc11_chr4_108460081_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Adra1b_chr11_43776276_F | TCGTCGGCAGCGTCAGAGAGTCCTTCCGCGACT | 60.0 | 0.01 |
| mm4_exon_Adra1b_chr11_43776276_R | GTCTCGTGGGCTCGGTTCATGCGTATCCTGGGGTG | 59.8 | 0.01 |
| mm4_exon_Gm10762/Zc3h6_chr2_128967675_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Gm10762/Zc3h6_chr2_128967675_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Efcab2_chr1_178420539_F | TCGTCGGCAGCGTCACCCAGCCTAAGTCACATGC | 60.0 | 0.00 |
| mm4_intron_Efcab2_chr1_178420539_R | GTCTCGTGGGCTCGGACTCTGACAGAAAATAGTGTCTACT | 57.2 | 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_52313693 |
CGCATCACAAGGTGACCCTAGCTCCCCACTGCCATCTCCGCGGTCGCCGTCGACACGGCGCTGGGGCTACCCGGCGCCTG CCTTGTCGCCTTAGCTCCTCTTCTCAGCCAAGATCCCAGGGAGCCTGGGATTAGGAG |
| mm4_intergenic_Abca5|Map2k6_chr11_110344773 |
ACTGTATGTACGCATGCTCACATTTTNNNNNNNNNNNNNNNNNNNNNNNAGGTGGTGGTGTGTGAAGCAGGGTTTCCTGC TGCAGCATCGGCTGGACACTAGGACT |
| mm4_exon_A830082K12Rik/Nr2f1_chr13_78199277 | GACATAGAGGAAGCGGCGAGGGCGGCGGCGGCCGCGGAGGAGGAGGTCGCGGCTGCAGACGCTGCTGGAGGAG |
| mm3_intergenic_Gm10398|Gm26660_chr14_25317853 |
GTAGGCCCTCAGCAGATGTGAGAGCGATTATTATAATCTCTCCGCCCCCAGATGGCATGGTCCGGGTTGCCCCCTCCTCC GCCTGCCAGGCCCCTCTCCGCGGTCCCCGGGGACAGGAGCTAAGGACCCCAGC |
| mm3_exon_N4bp1_chr8_86884970 |
CCCGAACAGAGCCTCGATGCGCCCGCGGCTCCGCTCCAGCAGCGCCGCCTTCTCCGCGGGCGCCGTGAACTCATCCAGCA CCACCCGG |
| mm3_intron_Nsg1_chr5_38159118 |
CACAGCCAGAAGGACTCCGCACACCCACCCGCTGTCTCCGCGGTCGCTGTCCCCGCCTTATCTCCCCAGAGAGGTAGCAG GGACGCGGCGATCTAGCCAGATCAGAGTCTCAGGC |
| mm4_exon_Yars_chr4_129189950 |
GGACTAGCGTGACAGTTCCCGGCACGCGGAGACGACGGCGAACGAGGAAAGGGCGCGGGTGCCGGCTGAGCGCGGGAAAC AGAGACAGCGGAGCCATGGGTGATGCTCCAAGCCCTGAGGAGAAGCTGCATC |
| mm4_exon_Rnf38_chr4_44233771 |
CGACCGAGGCTGGAAGTGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTTC CGCTCATGGCGGGGCCGCGGGCAGCGGCTCGGACTGCGACTCGACTC |
| mm4_exon_9130019P16Rik/Wipf3_chr6_54429792 |
CAGAAGCAACGCCAGGCACTGTCTGCAGCCCAGCCGGGATCCTCGANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNGAGGCGCCAGCTAAAGTCCAGTCGG |
| mm4_intergenic_Gm7545|Gm10443_chr6_86315575 |
AGGAGACCCGGGGTTATTCAGCCTTCTCCGAGGGAGCCGTCAACTGTACTCACCCCTCCCTCAAGGCAGGCAGGTCCAAG CCCTGAGAGGGAGCATGGCGGAAGCAGCCCTCGCCTGCCCAAGAAGAAGAGGG |
| mm4_intron_Gatad2a_chr8_69973751 |
CGCGCAGGGACTCTTATTCTGACACGGGATCCTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAATTTAACGCCGCCGCC GGGCTCGGACCCCCGACCCTCCCGTGGACTCTCACCCCACGCCACTCGTCCACGTGCTGT |
| mm4_intron_Nudc_chr4_133545755 |
CGTTGGAGCGAGACTTCACTCGCGGGCTGGGCAGACCGGAAGCTGGGACGCGGGGACCCGGGGACCCGCGCGGCCGGGGT GGGGGAGGTCTCGGCCGACCGCGGAGAAGGGCTGCGGCGCGGGCCACGTCGTTTACCTC |
| mm4_exon_Six5_chr7_19094689 |
CCAAACATGGCTACCTCGCCTGCGGAGCCGANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACCGAGGAGCA GGAGGAGGAAGCGCGCCAGCTTCTGCAGACTCTG |
| mm4_exon_Ugcg_chr4_59189274 |
CAGAGAGCACGGACCAGAACGCAGAGCCGCGGCGAGCGAAAGGGANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNCCAAGGGTCTGGACCACGGGAGCC |
| mm4_exon_Lpin2_chr17_71184023 |
GCGAGGTGCAGTTATTCCCTCAGGCCGTTGGCTTGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCCTGCAGGCCGCGG ACCTGGTTTAGATGT |
| mm4_exon_Lysmd3_chr13_81657801 |
GCCTACTCTACGCGATGACGCACCGCAGGTTTGCGGACGTGGCGGCGGCTGTCGGTGCGGAANNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNAGAAGCGGACCCAGACACCGGAGAACCTAGAGTCGG |
| mm4_exon_Adra1b_chr11_43776276 |
AGAGAGTCCTTCCGCGACTGCGACCTCTCCAGCGAGCCGCCGCGGGTCCACGGCCGGTAGGTGTAAGCGCAGCCTCCTAG ANNNNNNNNNNNNNNNNNNNNNNNNNCACCGCGGCACTGGCACCCCAGGATACGCATGAA |
| mm4_intron_Efcab2_chr1_178420539 |
ACCCAGCCTAAGTCACATGCCTACTCTATTGTCGTCAGCGACCTGGGAGAGGGCATATCTACTTTATTTGTCTTCTCTAG TGAGAAGTAGACACTATTTTCTGTCAGAGT |