← 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_52313973_F | Primer3: not found at this Tm | N.d. | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313973_R | Primer3: not found at this Tm | N.d. | 1.00 |
| mm4_exon_Trpv1_chr11_73254819_F | TCGTCGGCAGCGTCGGGTGGATGAGGTGAACTGG | 60.0 | 0.40 |
| mm4_exon_Trpv1_chr11_73254819_R | GTCTCGTGGGCTCGGCATCCAGCACACCTCACCTC | 60.3 | 0.40 |
| mm4_exon_Gm11532/Sp6_chr11_97022541_F | TCGTCGGCAGCGTCGAAGGCGCCAAAGAGGAGG | 60.7 | 0.34 |
| mm4_exon_Gm11532/Sp6_chr11_97022541_R | GTCTCGTGGGCTCGGACATCCATGGGGCTCAGTTG | 60.0 | 0.34 |
| mm4_intergenic_Tmem247|Atp6v1e2_chr17_86931006_F | TCGTCGGCAGCGTCTCTTAGGGAGGAGCAGAGCC | 60.3 | 0.30 |
| mm4_intergenic_Tmem247|Atp6v1e2_chr17_86931006_R | GTCTCGTGGGCTCGGTGCAGTTACGAAGACGCACT | 59.9 | 0.30 |
| mm4_intergenic_Gm25403|March1_chr8_65550437_F | Primer3: not found at this Tm | N.d. | 0.26 |
| mm4_intergenic_Gm25403|March1_chr8_65550437_R | Primer3: not found at this Tm | N.d. | 0.26 |
| mm4_intergenic_H19/H19_1/Mir675/H19_2|Igf2_chr7_142603291_F | TCGTCGGCAGCGTCGTAAAAGAGGGACCAGGGGC | 60.0 | 0.25 |
| mm4_intergenic_H19/H19_1/Mir675/H19_2|Igf2_chr7_142603291_R | GTCTCGTGGGCTCGGAACTCACTTCCTGGCATGGG | 59.9 | 0.25 |
| mm3_intergenic_Srfbp1|Gm27026_chr18_52465936_F | TCGTCGGCAGCGTCGAGACAGGGTCCACGCGG | 62.4 | 0.21 |
| mm3_intergenic_Srfbp1|Gm27026_chr18_52465936_R | GTCTCGTGGGCTCGGTGATTGTGCAATGCGTGACC | 59.7 | 0.21 |
| mm3_intron_G930045G22Rik_chr6_50849885_F | Primer3: not found at this Tm | N.d. | 0.17 |
| mm3_intron_G930045G22Rik_chr6_50849885_R | Primer3: not found at this Tm | N.d. | 0.17 |
| mm4_exon_Kremen2/9530082P21Rik_chr17_23746038_F | TCGTCGGCAGCGTCCCTTGGCGGAATTCCCCAA | 60.3 | 0.13 |
| mm4_exon_Kremen2/9530082P21Rik_chr17_23746038_R | GTCTCGTGGGCTCGGTGGGAGATCAGAGGACTCGG | 60.1 | 0.13 |
| mm4_intron_Dock5_chr14_67896496_F | Primer3: not found at this Tm | N.d. | 0.13 |
| mm4_intron_Dock5_chr14_67896496_R | Primer3: not found at this Tm | N.d. | 0.13 |
| mm4_intergenic_Gm13530|Lmx1b_chr2_33549018_F | Primer3: not found at this Tm | N.d. | 0.12 |
| mm4_intergenic_Gm13530|Lmx1b_chr2_33549018_R | Primer3: not found at this Tm | N.d. | 0.12 |
| mm4_intergenic_1700010I14Rik|6530411M01Rik_chr17_9016915_F | TCGTCGGCAGCGTCCTCATGGGTCAGGATGCTCC | 59.8 | 0.12 |
| mm4_intergenic_1700010I14Rik|6530411M01Rik_chr17_9016915_R | GTCTCGTGGGCTCGGGCAGACTCAGTGGACCTGTC | 60.0 | 0.12 |
| mm4_exon_Lphn1_chr8_83930064_F | Primer3: not found at this Tm | N.d. | 0.12 |
| mm4_exon_Lphn1_chr8_83930064_R | Primer3: not found at this Tm | N.d. | 0.12 |
| mm4_exon_Champ1_chr8_13879395_F | TCGTCGGCAGCGTCGCTTCGGGCTCATCTGAGTC | 60.5 | 0.11 |
| mm4_exon_Champ1_chr8_13879395_R | GTCTCGTGGGCTCGGGGGAGAAGCAGGAAGGACTG | 59.7 | 0.11 |
| mm4_intron_Tspan18_chr2_93334022_F | TCGTCGGCAGCGTCACCCCTTCACTCTCGGTACA | 59.8 | 0.10 |
| mm4_intron_Tspan18_chr2_93334022_R | GTCTCGTGGGCTCGGGGTCAGGTGCAGCCATTCT | 60.0 | 0.10 |
| mm4_intron_Abcb9_chr5_124081294_F | Primer3: not found at this Tm | N.d. | 0.09 |
| mm4_intron_Abcb9_chr5_124081294_R | Primer3: not found at this Tm | N.d. | 0.09 |
| mm4_exon_Slc39a13_chr2_91069265_F | TCGTCGGCAGCGTCCAAGAGGTGGGGCTGAAGAG | 60.0 | 0.09 |
| mm4_exon_Slc39a13_chr2_91069265_R | GTCTCGTGGGCTCGGGCTCATAAGGGACTACAGTCAGT | 59.2 | 0.09 |
| mm4_intergenic_Epas1|Tmem247_chr17_86833567_F | Primer3: not found at this Tm | N.d. | 0.09 |
| mm4_intergenic_Epas1|Tmem247_chr17_86833567_R | Primer3: not found at this Tm | N.d. | 0.09 |
| mm4_exon_Sox1/Gm25239_chr8_12395375_F | Primer3: not found at this Tm | N.d. | 0.09 |
| mm4_exon_Sox1/Gm25239_chr8_12395375_R | Primer3: not found at this Tm | N.d. | 0.09 |
| mm4_intron_Msh3_chr13_92252148_F | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intron_Msh3_chr13_92252148_R | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intergenic_Gm20682|3300002A11Rik_chr12_99315003_F | TCGTCGGCAGCGTCGGCAGATCTAGAACTTGTTGGC | 59.3 | 0.08 |
| mm4_intergenic_Gm20682|3300002A11Rik_chr12_99315003_R | GTCTCGTGGGCTCGGAAGCCCACACAGAGTGTTGT | 59.7 | 0.08 |
| mm4_intron_Ccser1_chr6_61898381_F | TCGTCGGCAGCGTCACAACTTGTGTCAGCCCACA | 60.0 | 0.08 |
| mm4_intron_Ccser1_chr6_61898381_R | GTCTCGTGGGCTCGGCATCCTCAGTAGCTTGGGCA | 59.4 | 0.08 |
| mm4_intergenic_1700031F10Rik|Eefsec_chr6_88241879_F | TCGTCGGCAGCGTCCCTGGTAGGTGAACCTCAGC | 59.7 | 0.08 |
| mm4_intergenic_1700031F10Rik|Eefsec_chr6_88241879_R | GTCTCGTGGGCTCGGGGCTACTGTCCCCAAAGAGG | 59.7 | 0.08 |
| mm3_intron_Ptprt_chr2_161709090_F | TCGTCGGCAGCGTCTGAAAGACCACATGGCCCTG | 60.2 | 0.07 |
| mm3_intron_Ptprt_chr2_161709090_R | GTCTCGTGGGCTCGGTGCTTCTAGGAGGGCTTTGC | 60.0 | 0.07 |
| mm4_intron_Prkg2_chr5_99036891_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intron_Prkg2_chr5_99036891_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intron_Creb3l2_chr6_37354307_F | TCGTCGGCAGCGTCCTGGTCAGCGGGAGAACTAC | 59.8 | 0.06 |
| mm4_intron_Creb3l2_chr6_37354307_R | GTCTCGTGGGCTCGGTTGAAAGCCCTGGAATGCCC | 60.9 | 0.06 |
| mm4_intergenic_Gm14835|4933401B06Rik_chrX_107431738_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Gm14835|4933401B06Rik_chrX_107431738_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Vmn1r-ps52|Nlrp4b_chr7_10681573_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Vmn1r-ps52|Nlrp4b_chr7_10681573_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Gm22597|Gm24514_chr18_55586551_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Gm22597|Gm24514_chr18_55586551_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intron_Immp2l_chr12_41043537_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intron_Immp2l_chr12_41043537_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_MDGA2/Mdga2|Mdga2_chr12_66821103_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_MDGA2/Mdga2|Mdga2_chr12_66821103_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_exon_Gm13898_chr6_17710825_F | TCGTCGGCAGCGTCCCCTTAACCTCACCCGTGTT | 59.6 | 0.04 |
| mm4_exon_Gm13898_chr6_17710825_R | GTCTCGTGGGCTCGGGGCCTGCTTTGGAGAACTCT | 59.9 | 0.04 |
| mm4_intron_Kif7_chr7_79715056_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Kif7_chr7_79715056_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Arhgdig|Rgs11_chr17_26202849_F | TCGTCGGCAGCGTCCACACTGCAGGCTATCCCAA | 60.0 | 0.03 |
| mm4_intergenic_Arhgdig|Rgs11_chr17_26202849_R | GTCTCGTGGGCTCGGCCCGCTAAGGTTAACCGTGT | 60.0 | 0.03 |
| mm4_intergenic_Gm14221|Gm14222_chr2_160581214_F | TCGTCGGCAGCGTCGGAAGAGAGCTGCTTCCCAG | 60.1 | 0.03 |
| mm4_intergenic_Gm14221|Gm14222_chr2_160581214_R | GTCTCGTGGGCTCGGAGCTTCCAGGCGAGAGAATG | 59.8 | 0.03 |
| mm4_intergenic_4933405O20Rik|Nell1_chr7_50618928_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_4933405O20Rik|Nell1_chr7_50618928_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_exon_Gm17501_chr3_145650792_F | TCGTCGGCAGCGTCAACGAGTGCAGAGTTCTCCC | 59.6 | 0.03 |
| mm4_exon_Gm17501_chr3_145650792_R | GTCTCGTGGGCTCGGGTCTCCTCCGCTTCTGCTTT | 60.0 | 0.03 |
| mm4_intergenic_Rpl37a|Igfbp2_chr1_72774413_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_Rpl37a|Igfbp2_chr1_72774413_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intron_Wbscr17_chr5_131174162_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Wbscr17_chr5_131174162_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_exon_Slc22a23_chr13_34344652_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_exon_Slc22a23_chr13_34344652_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Rps6kc1_chr1_190789157_F | TCGTCGGCAGCGTCGGCAAAGACACCAAGCCAAG | 59.9 | 0.02 |
| mm4_intron_Rps6kc1_chr1_190789157_R | GTCTCGTGGGCTCGGAATACTGCGTGGTCTGGAGC | 60.1 | 0.02 |
| mm4_intron_Espn_chr4_152129768_F | TCGTCGGCAGCGTCATCAGGGACAGCAAAGAGGG | 59.3 | 0.02 |
| mm4_intron_Espn_chr4_152129768_R | GTCTCGTGGGCTCGGCCAGCAAGATGGCCTCTCTT | 59.7 | 0.02 |
| mm3_intergenic_Col13a1|Gm5424_chr10_62033921_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm3_intergenic_Col13a1|Gm5424_chr10_62033921_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm2_intergenic_4930428D18Rik|Gm5793_chrX_76481468_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm2_intergenic_4930428D18Rik|Gm5793_chrX_76481468_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Ywhag_chr5_135934153_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Ywhag_chr5_135934153_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Atp5g2|Mir688_chr15_102670898_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Atp5g2|Mir688_chr15_102670898_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm20033|Klf3_chr5_64803115_F | TCGTCGGCAGCGTCGAGGAGGCCAGGACAGCTA | 60.3 | 0.00 |
| mm4_intergenic_Gm20033|Klf3_chr5_64803115_R | GTCTCGTGGGCTCGGCTCTCCACCCTGGGCCTC | 61.0 | 0.00 |
| mm4_intergenic_Asb1|Twist2_chr1_91630354_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Asb1|Twist2_chr1_91630354_R | Primer3: not found at this Tm | N.d. | 0.00 |
These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.
| mm4_exon_Trpv1_chr11_73254819 |
GGGTGGATGAGGTGAACTGGACTACCTGGAACACCAACGTGGGCATCATCAACGAGGACCCAGGCAACTGTGAGGGCGTC AAGCGCACCCTGAGCTTCTCCCTGCGGTCAGGCCGAGGTGAGGTGTGCTGGATG |
| mm4_exon_Gm11532/Sp6_chr11_97022541 |
GAAGGCGCCAAAGAGGAGGCGGCGGCGGCGGCCCAAGGCGAGGGCAAGGCCGGTGGCGTGGTGGAACCACCCGGGGGCAA AGGCAAACGTGAGGCCGAAGGCAGCTCGGCGTCCTCCAACTGAGCCCCATGGATGT |
| mm4_intergenic_Tmem247|Atp6v1e2_chr17_86931006 |
TCTTAGGGAGGAGCAGAGCCTCTGAAGGTCTCCCCAAGGTTGCCTGAGCCTCCAGACTTCCTGCAGTGTGCTCACAGCCT GACACCATGGAGGGCTTGAGTGCGTCTTCGTAACTGCA |
| mm4_intergenic_H19/H19_1/Mir675/H19_2|Igf2_chr7_142603291 |
GTAAAAGAGGGACCAGGGGCCTAGCACCCTAGCTGGGGCTGTAGTCTTAGGCAAAGCCACATGGTTGCCTGAGCTCCTGT GGGCTCCAGGGACCCAGAGATGCCTGCTGCCCACTGGCCCATGCCAGGAAGTGAGTT |
| mm3_intergenic_Srfbp1|Gm27026_chr18_52465936 | GAGACAGGGTCCACGCGGGTCGGTCCCCGGTCTCAGGCGACCGTGGGGAGGTCACGCATTGCACAATCA |
| mm4_exon_Kremen2/9530082P21Rik_chr17_23746038 |
CCTTGGCGGAATTCCCCAAGCGGTGCCCCCCACCTGGAGGGGAGGGGGAGGGGAGCAGGCCCAGGCAAGCGTGGGGGGAA ATTCCCCGCCCCCTCCCCCTGAGCCCGACTCCTCCGAGTCCTCTGATCTCCCA |
| mm4_intergenic_1700010I14Rik|6530411M01Rik_chr17_9016915 |
CTCATGGGTCAGGATGCTCCGTGCCTGTGGAGCACCTGTGGTGCCTATGGAGCACTGGTGCTGGGAACCCCCTGGATGTG CTTCCTCTCTGTTGCCTGAGCCTGCTTCATCCCAGACAGGTCCACTGAGTCTGC |
| mm4_exon_Champ1_chr8_13879395 |
GCTTCGGGCTCATCTGAGTCACCGAAGGTAGCGTCAGACATATGGAAGCCTGTCTTGTCTATCGATGCTGAGCCTAGGAA GTCCACACTGTTTCCTGAGCCCACCAAAGCAGTCCTTCCTGCTTCTCCC |
| mm4_intron_Tspan18_chr2_93334022 |
ACCCCTTCACTCTCGGTACAGCCCCGGCCAACACACCGGCTCTGGGAGTCCCACCTGCCGCCTTACTGTCGCCTGAGCCC CCGAGATCGCTGGGCGCAGAATGGCTGCACCTGACC |
| mm4_exon_Slc39a13_chr2_91069265 |
CAAGAGGTGGGGCTGAAGAGAAGAGATGCCTCACAGTTGCCAGAGCCAGAGGCTGTGGTAACTAACTTATGGCTAATTAA TTTGGTGCTCTAGATTGTTAACTGACTGTAGTCCCTTATGAGC |
| mm4_intergenic_Gm20682|3300002A11Rik_chr12_99315003 |
GGCAGATCTAGAACTTGTTGGCATTTGAGAACTGAGATACATTCAAGAATGGAAAGAAGACACAGCCCGGGCTCAGACAA CTGCGGGGGATTTGAGATGTCATTTTGTCACAACACTCTGTGTGGGCTT |
| mm4_intron_Ccser1_chr6_61898381 |
ACAACTTGTGTCAGCCCACATGAATTATGATGCTTCTATATATTCTGCTCTCCAAAATCCCTCCCGTGCATGCAGGTGCT CAGGCAGCCGTGTGGAGGCTGCCCAAGCTACTGAGGATG |
| mm4_intergenic_1700031F10Rik|Eefsec_chr6_88241879 |
CCTGGTAGGTGAACCTCAGCAAATCCCCAATTTTCAAGCTTTGGGGGCTCAGGAAACCGTCAGGTGGTGATTGGGCCAGT CAGCTGGCCAGCGTAAAAAAAAAACTCCCTAAATCCTCTTTGGGGACAGTAGCC |
| mm3_intron_Ptprt_chr2_161709090 |
TGAAAGACCACATGGCCCTGGTGAGCTGGTTATAATTCTTGCTGTAAATGTGAGCCCAGGCTCAGGCAACTGTGCAGCAA GCAAAGCCCTCCTAGAAGCA |
| mm4_intron_Creb3l2_chr6_37354307 |
CTGGTCAGCGGGAGAACTACTTTCTGGGCTGAAAAGAGATGGTCCTTAGTCCTCACGGTTCTCTGAGCCCCCAGGGCATT CCAGGGCTTTCAA |
| mm4_exon_Gm13898_chr6_17710825 |
CCCTTAACCTCACCCGTGTTACATCCGCACTCTTACCTGAGCCCTCGCTGCCGCTTCCACCTGTGATGGGGCGATTGCAG CTGCTGCACTTGGGTGCAAAGAGTTCTCCAAAGCAGGCC |
| mm4_intergenic_Arhgdig|Rgs11_chr17_26202849 |
CACACTGCAGGCTATCCCAAGTCACATCTTGCGAGGGCTCAGGCATCTCTGTGGTTCGAGAACCCGTGTATCTCATGGGT CTCGAGGCTCACACGGTTAACCTTAGCGGG |
| mm4_intergenic_Gm14221|Gm14222_chr2_160581214 |
GGAAGAGAGCTGCTTCCCAGTGATCCTGGCTTCCAGTGACTCCAGCGGGCTCAGCCAAGTGTGAGGGATTTCTACAACAC CCTTGGGGGAGGGCTGCCACCAGCTGCCATTCTCTCGCCTGGAAGCT |
| mm4_exon_Gm17501_chr3_145650792 |
AACGAGTGCAGAGTTCTCCCGGAGCCTGACGTCCACTTCCTCACGGATGCCGGAGGCCGGGTCAACCCTCCTGGCCTCTC TCCCTGGACTGGCGGCGGTGGAAACTGAGCAGCCAAAGCAGAAGCGGAGGAGAC |
| mm4_intron_Rps6kc1_chr1_190789157 |
GGCAAAGACACCAAGCCAAGCACGCTCACGGGCTCAGGGAACCGGACGGACACTGGGGGCAGAGTTAGAGCCACCTTCAC ACTTGCAGGGCCAGGGGTGCTGCTCCAGACCACGCAGTATT |
| mm4_intron_Espn_chr4_152129768 |
ATCAGGGACAGCAAAGAGGGCCAGGGAGGGCAGGAGGGGGAGAAGAGGGGTGGAGCTCGCCTGCCCTTGTGACCCTGGCT CAGGCCACTGTGGGGCCCCACAAAGGTGGTCCTCTAAGAGAGGCCATCTTGCTGG |
| mm4_intergenic_Gm20033|Klf3_chr5_64803115 |
GAGGAGGCCAGGACAGCTAGGGCGGGGCCGCGGGCTCAGGGCGCCGGGCGGAGCGCTCAGGCGGGGTGGAGGCCCAGGGT GGAGAG |