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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_52313975_F | Primer3: not found at this Tm | N.d. | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313975_R | Primer3: not found at this Tm | N.d. | 1.00 |
| mm3_intergenic_Sohlh1|Kcnt1_chr2_25848497_F | Primer3: not found at this Tm | N.d. | 0.63 |
| mm3_intergenic_Sohlh1|Kcnt1_chr2_25848497_R | Primer3: not found at this Tm | N.d. | 0.63 |
| mm4_intergenic_4930428D18Rik|Gm5793_chrX_76481466_F | Primer3: not found at this Tm | N.d. | 0.52 |
| mm4_intergenic_4930428D18Rik|Gm5793_chrX_76481466_R | Primer3: not found at this Tm | N.d. | 0.52 |
| mm4_intergenic_Cldn23|Gm22030_chr8_35962085_F | TCGTCGGCAGCGTCCTGCAACCGATCCCCGTTAT | 60.1 | 0.34 |
| mm4_intergenic_Cldn23|Gm22030_chr8_35962085_R | GTCTCGTGGGCTCGGAATGCATGCTTTTGGGAGGC | 59.7 | 0.34 |
| mm4_intergenic_Gm773|1600025M17Rik_chrX_56301079_F | TCGTCGGCAGCGTCACAAGGAAGTCAAAGGCGCA | 60.4 | 0.29 |
| mm4_intergenic_Gm773|1600025M17Rik_chrX_56301079_R | GTCTCGTGGGCTCGGACGGAGCTTCTAGGGCTACT | 59.7 | 0.29 |
| mm4_intergenic_Lrrc52|Rxrg_chr1_167561772_F | Primer3: not found at this Tm | N.d. | 0.19 |
| mm4_intergenic_Lrrc52|Rxrg_chr1_167561772_R | Primer3: not found at this Tm | N.d. | 0.19 |
| mm4_intergenic_Cadm1|Nxpe2_chr9_48168039_F | Primer3: not found at this Tm | N.d. | 0.19 |
| mm4_intergenic_Cadm1|Nxpe2_chr9_48168039_R | Primer3: not found at this Tm | N.d. | 0.19 |
| mm4_intergenic_Cblb|Gm23513_chr16_52088764_F | TCGTCGGCAGCGTCTCACACCACAGCTTAGGTGC | 60.2 | 0.11 |
| mm4_intergenic_Cblb|Gm23513_chr16_52088764_R | GTCTCGTGGGCTCGGCAGCACAAACAAACCACCCA | 59.4 | 0.11 |
| mm4_exon_Zcchc14_chr8_121652873_F | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_exon_Zcchc14_chr8_121652873_R | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intergenic_Gltscr1|Zfp541_chr7_16046628_F | TCGTCGGCAGCGTCGAGGAAGGAGCCACTATGCC | 59.8 | 0.10 |
| mm4_intergenic_Gltscr1|Zfp541_chr7_16046628_R | GTCTCGTGGGCTCGGGACAAGGGACCAGGGAGCTA | 60.6 | 0.10 |
| mm4_intron_Mall_chr2_127716573_F | TCGTCGGCAGCGTCAGGAGTGGGTGACATTTGCA | 59.5 | 0.10 |
| mm4_intron_Mall_chr2_127716573_R | GTCTCGTGGGCTCGGCCAGTAGAGGCTTAAGGGGC | 59.5 | 0.10 |
| mm4_intron_Mef2b/2310045N01Rik_chr8_70158702_F | TCGTCGGCAGCGTCTGGGAGCAGGTGATGAAACC | 59.9 | 0.09 |
| mm4_intron_Mef2b/2310045N01Rik_chr8_70158702_R | GTCTCGTGGGCTCGGCAGAAGTGAGAGGCAGGTGG | 60.0 | 0.09 |
| mm4_intergenic_Gm22673|Gm23059_chr12_60309500_F | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intergenic_Gm22673|Gm23059_chr12_60309500_R | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intergenic_2010001E11Rik|G630090E17Rik_chr10_39945806_F | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_2010001E11Rik|G630090E17Rik_chr10_39945806_R | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_Gm26681/Entpd6|Gm26681_chr2_150749616_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Gm26681/Entpd6|Gm26681_chr2_150749616_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Gm12878|Gm12892_chr4_122000829_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Gm12878|Gm12892_chr4_122000829_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intron_Ints7_chr1_191588123_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intron_Ints7_chr1_191588123_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Nmbr|Gm23892_chr10_14861320_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Nmbr|Gm23892_chr10_14861320_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Prkg2_chr5_99036893_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intron_Prkg2_chr5_99036893_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_Stk31|Npy_chr6_49761130_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_Stk31|Npy_chr6_49761130_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_exon_Rpl13_chr8_123102639_F | TCGTCGGCAGCGTCGACCCTGGCCTTGACTCG | 59.7 | 0.03 |
| mm4_exon_Rpl13_chr8_123102639_R | GTCTCGTGGGCTCGGTGCTGCCAATCCTTGTGGAA | 60.1 | 0.03 |
| mm4_intron_Chst8_chr7_34809346_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Chst8_chr7_34809346_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_Smtnl2|Ggt6_chr11_72411717_F | TCGTCGGCAGCGTCGGAGACGTTGAGAGATCCGC | 60.2 | 0.02 |
| mm4_intergenic_Smtnl2|Ggt6_chr11_72411717_R | GTCTCGTGGGCTCGGCTGGGGCTCTAGGGGTCTG | 60.7 | 0.02 |
| mm4_intron_Bbs9_chr9_22543655_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Bbs9_chr9_22543655_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_exon_Exo1_chr1_175901365_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Exo1_chr1_175901365_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Tshz2/A630075F10Rik|Zfp217_chr2_170072271_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Tshz2/A630075F10Rik|Zfp217_chr2_170072271_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Snta1|Cbfa2t2_chr2_154412605_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Snta1|Cbfa2t2_chr2_154412605_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Gm9871|Cntn3_chr6_101920827_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Gm9871|Cntn3_chr6_101920827_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Raver2_chr4_101091089_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Raver2_chr4_101091089_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Prkar2a_chr9_108692894_F | TCGTCGGCAGCGTCGGCGTGTGAGCATTCCAAAT | 59.4 | 0.01 |
| mm4_intron_Prkar2a_chr9_108692894_R | GTCTCGTGGGCTCGGGCACGCTTCATTCAAGGCAG | 60.4 | 0.01 |
| mm4_intron_Sipa1l3_chr7_29360028_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Sipa1l3_chr7_29360028_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Abcc4_chr14_118665667_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Abcc4_chr14_118665667_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_A330074K22Rik_chr8_120222785_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_A330074K22Rik_chr8_120222785_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Gsta4|Gsta1_chr9_78222923_F | TCGTCGGCAGCGTCACAGTCAGTCAATGCTGTTTAGTG | 59.4 | 0.00 |
| mm4_intergenic_Gsta4|Gsta1_chr9_78222923_R | GTCTCGTGGGCTCGGATCCCACGGTGGTGTTTTGT | 60.1 | 0.00 |
| mm4_exon_Tmem88b/Gm13644_chr4_155784358_F | TCGTCGGCAGCGTCAGTTGTTTGTCCTCGTCCGG | 60.2 | 0.00 |
| mm4_exon_Tmem88b/Gm13644_chr4_155784358_R | GTCTCGTGGGCTCGGCTCCTGGTCCGGCGACTA | 60.4 | 0.00 |
| mm4_intron_Itgb1bp1_chr12_21282664_F | TCGTCGGCAGCGTCTAGAACCCACTCTCAGCCCA | 59.8 | 0.00 |
| mm4_intron_Itgb1bp1_chr12_21282664_R | GTCTCGTGGGCTCGGTTGAGAAGGAGACCGGGTGA | 60.1 | 0.00 |
| mm4_intron_G930045G22Rik_chr6_50849883_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_G930045G22Rik_chr6_50849883_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Epha10_chr4_124906504_F | TCGTCGGCAGCGTCCATCGCGGAGGATGGAATCA | 59.9 | 0.00 |
| mm4_intron_Epha10_chr4_124906504_R | GTCTCGTGGGCTCGGCCCTCTCTCCCACCCACTTA | 59.9 | 0.00 |
| mm4_intergenic_Ly6f|9030619P08Rik_chr15_75426607_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Ly6f|9030619P08Rik_chr15_75426607_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Kalrn|Ropn1_chr16_34648532_F | TCGTCGGCAGCGTCTGCCTGTATTCTTCACGCTGA | 59.7 | 0.00 |
| mm4_intergenic_Kalrn|Ropn1_chr16_34648532_R | GTCTCGTGGGCTCGGTGGCAGGTGTGTCTTCTAGG | 59.0 | 0.00 |
| mm4_intergenic_Ino80c|Galnt1_chr18_24145911_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Ino80c|Galnt1_chr18_24145911_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gal|Ppp6r3_chr19_3425262_F | TCGTCGGCAGCGTCAAAGAGAGTCAGGTGCCAGC | 59.9 | 0.00 |
| mm4_intergenic_Gal|Ppp6r3_chr19_3425262_R | GTCTCGTGGGCTCGGACTAGGAGTAGGTGGGCCAC | 60.3 | 0.00 |
| mm4_exon_Fam199x_chrX_137049769_F | TCGTCGGCAGCGTCCAGCAACCTTTGTGCAGAGC | 60.3 | 0.00 |
| mm4_exon_Fam199x_chrX_137049769_R | GTCTCGTGGGCTCGGGATGGGCTACTCTGGCACTC | 59.8 | 0.00 |
| mm4_exon_A830073O21Rik/Fam174b_chr7_73740428_F | TCGTCGGCAGCGTCGTTCTTCCGGGCTCTGCAG | 60.7 | 0.00 |
| mm4_exon_A830073O21Rik/Fam174b_chr7_73740428_R | GTCTCGTGGGCTCGGGAGGTCTGCGTCCCGGAG | 61.8 | 0.00 |
These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.
| mm4_intergenic_Cldn23|Gm22030_chr8_35962085 |
CTGCAACCGATCCCCGTTATGATTTAGGTTAAGTGGAAGCTCTTATTATGTGTTCATTTAATAACTTTGCTGGACTTGAG AGTTCAGGCAACCGTGGCTAATTCATAAAAGGAACAGCCTCCCAAAAGCATGCATT |
| mm4_intergenic_Gm773|1600025M17Rik_chrX_56301079 |
ACAAGGAAGTCAAAGGCGCATAAGCTTAGGCCCCCAGAGAACGGAGCTGCTGGCACTGCCCACCACGGTTGCCTGAACCC AGGAATATCTTCTCCTGTGAGAGGAACAGAAAGAAGTAAGTAGCCCTAGAAGCTCCGT |
| mm4_intergenic_Cblb|Gm23513_chr16_52088764 |
TCACACCACAGCTTAGGTGCCCTCTGAGGCTCAGGCAAACGGGGGCTAGGTTGGGTCATCTTCACAGTAAGCATTGGTTT CCCAGCCCCTTGGGTGGTTTGTTTGTGCTG |
| mm4_intergenic_Gltscr1|Zfp541_chr7_16046628 |
GAGGAAGGAGCCACTATGCCTTCCAATCACCGCGGTCGCCCGAGCCCGCGCCCTCCCGCGGCTCGTCGCCCGGTCCTCCG GCTGGCGGAGGCCCAAGGCCTGGCTGGGTAGCTCCCTGGTCCCTTGTC |
| mm4_intron_Mall_chr2_127716573 |
AGGAGTGGGTGACATTTGCAGGCCTGAGCATGATATATATAAAGAAAAATCCCCAGAGGCTCACGGGCTCAGGAAAACAT GGTGCCCCTTAAGCCTCTACTGG |
| mm4_intron_Mef2b/2310045N01Rik_chr8_70158702 |
TGGGAGCAGGTGATGAAACCGCAGCCCGGGTTGCGTGAGGCCGCGTGTCCCGCGGGCCTGGCTCCCCACCTGCCTCTCAC TTCTG |
| mm4_exon_Rpl13_chr8_123102639 |
GACCCTGGCCTTGACTCGCCGCCCTCCTCTCCCTCGCGGCCTTAGGCAGCCATGGCGCCCAGCCGGAATGGCATGATACT GAAGCCCCACTTCCACAAGGATTGGCAGCA |
| mm4_intergenic_Smtnl2|Ggt6_chr11_72411717 |
GGAGACGTTGAGAGATCCGCAGCTCCTTGGGACGACTGGCGGGCCGCGGCTGGCTGAGCCCGAGGGGCGGCCGGAGGGGG CGGGAGCAGGGCGGGGCAGACCCCTAGAGCCCCAG |
| mm4_intron_Prkar2a_chr9_108692894 |
GGCGTGTGAGCATTCCAAATTGAGCTCCGCGGTTCTCTGAGCCCGAGCGTTCACGGAGAGTGATTTGTCAGATTCTGCCT TGAATGAAGCGTGC |
| mm4_intergenic_Gsta4|Gsta1_chr9_78222923 |
ACAGTCAGTCAATGCTGTTTAGTGACTTCTGGCGGGATCACGGAACCGAGGGGGACTCTGAGGGAAACAGAGAAGGACAG GGAAGAGGAAATGAGGTACAAAACACCACCGTGGGAT |
| mm4_exon_Tmem88b/Gm13644_chr4_155784358 |
AGTTGTTTGTCCTCGTCCGGGTGGCCGTCGGCCAGATCTCCGCTGCTCCTGGGCGGGCGGCGCGGAGCCGGGGTCCTCGC GGGATCAGGCAGCGCAGGGCGTAGTCGCCGGACCAGGAG |
| mm4_intron_Itgb1bp1_chr12_21282664 |
TAGAACCCACTCTCAGCCCACCTTCTAGGCCACCTGCCATGGTGGCCTGAGCCTGAGAGTTTAGTCAGCCTCTCACCCGG TCTCCTTCTCAA |
| mm4_intron_Epha10_chr4_124906504 |
CATCGCGGAGGATGGAATCAAGCTCCAGCTGCTCCTGTGCGATTAGGATTCTCCCGAGTGCCTCGCGGGCTCTGGCCCTC GGGGGATCTTGAACACAAGCAAAGAGGAAAAAGTCAGTAAGTGGGTGGGAGAGAGGG |
| mm4_intergenic_Kalrn|Ropn1_chr16_34648532 |
TGCCTGTATTCTTCACGCTGAAGAAAGAAACCAGTACATCTTTACGTATGCACAACAGACCTCGGTGGCCAGAGCCCAGG AGACATCAAATAGACTTCTCCATCTCCTTTCTTTTTCCTCCTAGAAGACACACCTGCCA |
| mm4_intergenic_Gal|Ppp6r3_chr19_3425262 |
AAAGAGAGTCAGGTGCCAGCCGTGGGGACCCTGGGGCTGCAGTCCTACAGGCGGAGGTCCCACAGGCTCAGGCCACCGTG GCCACTGATGAAAAGCAGGTTGTCAAATCACACAACGTGGCCCACCTACTCCTAGT |
| mm4_exon_Fam199x_chrX_137049769 |
CAGCAACCTTTGTGCAGAGCCCTCCCCTCCTCGGGCGGCTGCGGTGGGCACACTCGGGGGCGGAGGCCACCGCGGCCAGG TTAGCGTGGGGGAAGGGGCGAGTGCCAGAGTAGCCCATC |
| mm4_exon_A830073O21Rik/Fam174b_chr7_73740428 |
GTTCTTCCGGGCTCTGCAGCTTCCTGCCGAGCATCGCGCAGCCGCCTCGGCAGCCGGAGACCCCGGCGCAGGCCTCCCGG GTGCCAGCCTCTGGGGCTCAGGCCTCCGGGGTGCAAGCCTCCGGGACGCAGACCTC |