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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 |
|---|---|---|---|
| mm0_exon_Pop5_chr5_115238118_F | Primer3: not found at this Tm | N.d. | 1.00 |
| mm0_exon_Pop5_chr5_115238118_R | Primer3: not found at this Tm | N.d. | 1.00 |
| mm4_exon_Irs2/9530052E02Rik_chr8_11005544_F | TCGTCGGCAGCGTCTCAGAGAGTGGAGAGCGCT | 60.0 | 0.74 |
| mm4_exon_Irs2/9530052E02Rik_chr8_11005544_R | GTCTCGTGGGCTCGGGCGGCCTCATCTTCTTCACT | 60.1 | 0.74 |
| mm4_intron_Sv2c_chr13_95995521_F | TCGTCGGCAGCGTCAAAGAGGGACGGATCTTGCA | 59.0 | 0.27 |
| mm4_intron_Sv2c_chr13_95995521_R | GTCTCGTGGGCTCGGGCATTCCTTGAGCAACAGGC | 60.1 | 0.27 |
| mm4_exon_Zfp831_chr2_174644426_F | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_exon_Zfp831_chr2_174644426_R | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_intergenic_4933406G16Rik|Gm12025_chr11_19342587_F | TCGTCGGCAGCGTCCCAGCATCCCAAACCATCCA | 60.3 | 0.17 |
| mm4_intergenic_4933406G16Rik|Gm12025_chr11_19342587_R | GTCTCGTGGGCTCGGTGCTGGGAGTTAGAGGCTCT | 59.9 | 0.17 |
| mm4_intron_Clcn6_chr4_148026693_F | TCGTCGGCAGCGTCTTCACCTGACCTCACTGCAC | 59.8 | 0.16 |
| mm4_intron_Clcn6_chr4_148026693_R | GTCTCGTGGGCTCGGGGCTATTCTGGGGCAGGATC | 59.9 | 0.16 |
| mm4_intergenic_Krt19|Krt9_chr11_100149379_F | TCGTCGGCAGCGTCGAATCTGGGAGTGTGAGCCC | 60.1 | 0.15 |
| mm4_intergenic_Krt19|Krt9_chr11_100149379_R | GTCTCGTGGGCTCGGGCCCTGGATAGTAGAGTTGGC | 59.9 | 0.15 |
| mm4_intron_Arhgef16_chr4_154290911_F | TCGTCGGCAGCGTCTGTGAACCCCAGAGCTCCTA | 59.8 | 0.14 |
| mm4_intron_Arhgef16_chr4_154290911_R | GTCTCGTGGGCTCGGCTCCCGGTATCCAGCCAAAA | 59.7 | 0.14 |
| mm4_intron_Nav2_chr7_49156301_F | Primer3: not found at this Tm | N.d. | 0.13 |
| mm4_intron_Nav2_chr7_49156301_R | Primer3: not found at this Tm | N.d. | 0.13 |
| mm4_intergenic_Gm24174|n-R5s168_chr6_140193721_F | TCGTCGGCAGCGTCGCCAGGCAGTCAGGAAATCT | 60.0 | 0.12 |
| mm4_intergenic_Gm24174|n-R5s168_chr6_140193721_R | GTCTCGTGGGCTCGGGGGAGTCCAAACCAGGTCAG | 59.9 | 0.12 |
| mm4_intron_Ano7_chr1_93377380_F | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intron_Ano7_chr1_93377380_R | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intron_Nup214_chr2_32022647_F | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intron_Nup214_chr2_32022647_R | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intergenic_Otogl|Ppp1r12a_chr10_107973849_F | TCGTCGGCAGCGTCAGCTCTCTGTACCCAGCACT | 60.2 | 0.09 |
| mm4_intergenic_Otogl|Ppp1r12a_chr10_107973849_R | GTCTCGTGGGCTCGGGGTGAATGGCTTGTGTCCCT | 60.2 | 0.09 |
| mm4_intron_Myo3b_chr2_70260335_F | TCGTCGGCAGCGTCGCTGACACCAAAGTCTTCTGG | 59.1 | 0.09 |
| mm4_intron_Myo3b_chr2_70260335_R | GTCTCGTGGGCTCGGCACCGCACTAACTCTGCTGT | 60.3 | 0.09 |
| mm4_intergenic_Mif|Derl3_chr10_75887464_F | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_Mif|Derl3_chr10_75887464_R | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intron_Vwa8_chr14_79189950_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intron_Vwa8_chr14_79189950_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_4933428G20Rik/Arhgap23|Srcin1_chr11_97502747_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_4933428G20Rik/Arhgap23|Srcin1_chr11_97502747_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intron_Eif4e_chr3_138547020_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intron_Eif4e_chr3_138547020_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Tmcc3|Gm16154_chr10_94592686_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Tmcc3|Gm16154_chr10_94592686_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intron_Rab3gap1_chr1_127871179_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Rab3gap1_chr1_127871179_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Cldn6_chr17_23682175_F | TCGTCGGCAGCGTCATCCTGTCCCAGTCCCAAGA | 59.8 | 0.04 |
| mm4_exon_Cldn6_chr17_23682175_R | GTCTCGTGGGCTCGGAAGTCTGACCAGGAAGGGGT | 60.1 | 0.04 |
| mm4_intron_Psen2_chr1_180239913_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intron_Psen2_chr1_180239913_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_Wdr96|Gsto1_chr19_47840016_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_Wdr96|Gsto1_chr19_47840016_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_Ube2e1|Gm22350_chr14_18338067_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_Ube2e1|Gm22350_chr14_18338067_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Osbp2_chr11_3860486_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Osbp2_chr11_3860486_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_mmu-mir-6393|Zdhhc25_chr15_88487258_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_mmu-mir-6393|Zdhhc25_chr15_88487258_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Brca1_chr11_101525576_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Brca1_chr11_101525576_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Gm12297|Gm12298_chr11_66885447_F | TCGTCGGCAGCGTCAGCCGCAACCTTCTCTGTTT | 60.1 | 0.01 |
| mm4_intergenic_Gm12297|Gm12298_chr11_66885447_R | GTCTCGTGGGCTCGGCAGCCTCTCTGACACCACAC | 60.3 | 0.01 |
| mm4_intergenic_Tshz2/Gm26883|Gm26883_chr2_169772904_F | TCGTCGGCAGCGTCGAGCCCCGGATTAAAAGCCT | 60.1 | 0.01 |
| mm4_intergenic_Tshz2/Gm26883|Gm26883_chr2_169772904_R | GTCTCGTGGGCTCGGTCTGACCCATGAGTGTGCAC | 59.9 | 0.01 |
| mm3_intergenic_Ppp2r5a|Dtl_chr1_191464816_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm3_intergenic_Ppp2r5a|Dtl_chr1_191464816_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Aff3_chr1_38525879_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Aff3_chr1_38525879_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Smn1|Naip2_chr13_100142131_F | TCGTCGGCAGCGTCTCATCCCTGCATGCACAGAG | 60.1 | 0.00 |
| mm4_intergenic_Smn1|Naip2_chr13_100142131_R | GTCTCGTGGGCTCGGTTGGCTGCAAGAGACCTCAG | 59.9 | 0.00 |
| mm4_intron_Vdac1_chr11_52371168_F | TCGTCGGCAGCGTCGGAGTCCCGTTGCATCCTG | 60.4 | 0.00 |
| mm4_intron_Vdac1_chr11_52371168_R | GTCTCGTGGGCTCGGCACCACAAAGAGCTCACAGC | 59.4 | 0.00 |
| mm4_intron_Slc44a1_chr4_53529070_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Slc44a1_chr4_53529070_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Unc5cl|Gm24071_chr17_48601475_F | TCGTCGGCAGCGTCCAGGCAAAGAGGGATGCTGT | 60.3 | 0.00 |
| mm4_intergenic_Unc5cl|Gm24071_chr17_48601475_R | GTCTCGTGGGCTCGGAACTCCACACAGGTCTGTCC | 59.2 | 0.00 |
| mm4_intergenic_Top1mt|Rhpn1_chr15_75681860_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Top1mt|Rhpn1_chr15_75681860_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm6569/Mroh5|Gm7935_chr15_73884687_F | TCGTCGGCAGCGTCAGCTTGGGAGTCACTCAGGA | 60.1 | 0.00 |
| mm4_intergenic_Gm6569/Mroh5|Gm7935_chr15_73884687_R | GTCTCGTGGGCTCGGCCCTCTGGAAGTGAACCCAC | 59.9 | 0.00 |
| mm4_intergenic_Gm25778|Ppapdc1a_chr7_128932489_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm25778|Ppapdc1a_chr7_128932489_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm25769|Gm11487_chr4_73255995_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm25769|Gm11487_chr4_73255995_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Dyrk1a|Kcnj6_chr16_94717683_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Dyrk1a|Kcnj6_chr16_94717683_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Arf2|Crhr1_chr11_104123830_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Arf2|Crhr1_chr11_104123830_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_2900041M22Rik|Tnrc6c_chr11_117637614_F | TCGTCGGCAGCGTCCCCTCCACCTCCAATTTCCC | 60.0 | 0.00 |
| mm4_intergenic_2900041M22Rik|Tnrc6c_chr11_117637614_R | GTCTCGTGGGCTCGGCTGCACTGCACTAGCCTGAT | 60.1 | 0.00 |
These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.
| mm4_exon_Irs2/9530052E02Rik_chr8_11005544 |
TCAGAGAGTGGAGAGCGCTGCCGGCTGTCGCTGCTGGTGCCTGGGGTTCCTGAACCCAGGGATGAAGCAGGACTACTAGC TGAGAGCAGTGAAGAAGATGAGGCCGC |
| mm4_intron_Sv2c_chr13_95995521 |
AAAGAGGGACGGATCTTGCATAATAACCAAGGCCCTTGCCAGCCTCCCCCTGCTGTCGCTGCAGGGCCCTATGGCTGCCT CCTCGGCTGCCTTCCAGCTGCCTGTTGCTCAAGGAATGC |
| mm4_intergenic_4933406G16Rik|Gm12025_chr11_19342587 |
CCAGCATCCCAAACCATCCATCATTTATCCCAAACAAAACTCCTTTCTTGGTTGACTGAGGCTCCTGCTGTCGTCACAGG TACTCTACATTTCCAGAGCCTCTAACTCCCAGCA |
| mm4_intron_Clcn6_chr4_148026693 |
TTCACCTGACCTCACTGCACTTCCGTGGTCACTCAAGGCACCTGCAGCAGCAGCAGGAGAGTAAACTCTGCTAGTCACGC TGGCTGAGAGATCCTGCCCCAGAATAGCC |
| mm4_intergenic_Krt19|Krt9_chr11_100149379 |
GAATCTGGGAGTGTGAGCCCCCAAGAAGAATGGAACAGTGTGTATGAGCCTTGACAGCATAGTGACAAAAATAATAATGA AGGTAACAGTGGTGACAGCAGGTGACCATTGCCAACTCTACTATCCAGGGC |
| mm4_intron_Arhgef16_chr4_154290911 |
TGTGAACCCCAGAGCTCCTAGACACACTGCCCACCCTGCTGTCGGGGCAGGTCCCTACCTTATTTTTGGCTGGATACCGG GAG |
| mm4_intergenic_Gm24174|n-R5s168_chr6_140193721 |
GCCAGGCAGTCAGGAAATCTGTCTGCCAGGGTCCTGTGTGTGTCCCCTTTGCTCCAGCTGTGGCCTCAGATACCATCTCA GTCTGACCTGGTTTGGACTCCC |
| mm4_intergenic_Otogl|Ppp1r12a_chr10_107973849 |
AGCTCTCTGTACCCAGCACTTCTGATCCCCAAGGGAACAAGGTCACTGCTGTGACAGCAGGTTCTGCTATAAAGTATGAG CACAGAGAGAGGACTCTGGGGGTGGAGAATGGAGAATGCAGGGACACAAGCCATTCACC |
| mm4_intron_Myo3b_chr2_70260335 | GCTGACACCAAAGTCTTCTGGTAACAAGGTGCCTACTGAGACAGCAGGAGGCTACAGCAGAGTTAGTGCGGTG |
| mm4_exon_Cldn6_chr17_23682175 | ATCCTGTCCCAGTCCCAAGAGTTCCTGCTGTGGCCGGAGGTAACTCACAGGACAGACGCACCCCTTCCTGGTCAGACTT |
| mm4_intergenic_Gm12297|Gm12298_chr11_66885447 |
AGCCGCAACCTTCTCTGTTTCCGTTAAACCTTTGCTGAATGTGATAGGAGGTCCAGGTAAGGCACCTGCAGCCAGAGCAG GGCTCTGCCCAAGGTGTGGTGTCAGAGAGGCTG |
| mm4_intergenic_Tshz2/Gm26883|Gm26883_chr2_169772904 |
GAGCCCCGGATTAAAAGCCTGTGTTCCTACTGCCGCCGCAGGTTCATTTAAAACCATCTGAACCTCCAGGCACAAGAGCG CTGCCTCGTGAAAGCCCCCCTCATAGTGCACACTCATGGGTCAGA |
| mm4_intergenic_Smn1|Naip2_chr13_100142131 |
TCATCCCTGCATGCACAGAGGATGCTGCCGAGACACTGAGGGAGGTCCTTGCAGCACCAGCTCTAGCCACAGGCACCTTG TGTCACACGCAGTGGCTGGCAGCACTAGCATTCTGAGGTCTCTTGCAGCCAA |
| mm4_intron_Vdac1_chr11_52371168 |
GGAGTCCCGTTGCATCCTGCAGGTTCCAGCTGTGCCAGCAGGCACCTTCACCTGCTGGATTACCTTGCAGGTCCTGCTGT GAGCTCTTTGTGGTG |
| mm4_intergenic_Unc5cl|Gm24071_chr17_48601475 |
CAGGCAAAGAGGGATGCTGTTGTCCCAAATCTCTAAGGGTAGCTGCGGCATCAGCTGGTTCTCTCCTGCAGGAATCCTGA TGTTCAGGACAGACCTGTGTGGAGTT |
| mm4_intergenic_Gm6569/Mroh5|Gm7935_chr15_73884687 |
AGCTTGGGAGTCACTCAGGAGTCTGTCAGGCCTGCTGGAGCCTCAGGTACCTGCCTCAGGTCTGGTTCTGTTAGCCCATG GCTGCGGGCCCAGACCCCTCTCCACAGAAGGAGTGGGTTCACTTCCAGAGGG |
| mm4_intergenic_2900041M22Rik|Tnrc6c_chr11_117637614 |
CCCTCCACCTCCAATTTCCCAGGATTTGGAACACTGGGGCTACAGTGCAGAATCCAGCTTTCCCCGCAGGTTCCTGGGAT GTGAGCTCTGTCATCAGGCTAGTGCAGTGCAG |