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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_52313569_F | TCGTCGGCAGCGTCTCACTGTGGCAGACGTTTCT | 59.5 | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313569_R | GTCTCGTGGGCTCGGGCCCCATTGCCCTCTTCTTT | 60.6 | 1.00 |
| mm4_intergenic_Gm9630|Gm24080_chr15_14644915_F | Primer3: not found at this Tm | N.d. | 0.62 |
| mm4_intergenic_Gm9630|Gm24080_chr15_14644915_R | Primer3: not found at this Tm | N.d. | 0.62 |
| mm4_exon_Cdyl_chr13_35669789_F | TCGTCGGCAGCGTCCAGAATGCTGGCTGGCTTTC | 59.8 | 0.57 |
| mm4_exon_Cdyl_chr13_35669789_R | GTCTCGTGGGCTCGGGCCTCTGTTCTCTGGTCACC | 60.0 | 0.57 |
| mm4_intergenic_Gm13466|Gm23072_chr2_45870768_F | Primer3: not found at this Tm | N.d. | 0.33 |
| mm4_intergenic_Gm13466|Gm23072_chr2_45870768_R | Primer3: not found at this Tm | N.d. | 0.33 |
| mm4_intergenic_4921515E04Rik|Gm8318_chr15_18101752_F | Primer3: not found at this Tm | N.d. | 0.31 |
| mm4_intergenic_4921515E04Rik|Gm8318_chr15_18101752_R | Primer3: not found at this Tm | N.d. | 0.31 |
| mm4_intergenic_Abcc1|Gm1758_chr16_14484470_F | Primer3: not found at this Tm | N.d. | 0.30 |
| mm4_intergenic_Abcc1|Gm1758_chr16_14484470_R | Primer3: not found at this Tm | N.d. | 0.30 |
| mm4_intergenic_Gm25699|Robo2_chr16_73654447_F | Primer3: not found at this Tm | N.d. | 0.28 |
| mm4_intergenic_Gm25699|Robo2_chr16_73654447_R | Primer3: not found at this Tm | N.d. | 0.28 |
| mm4_intergenic_Gm17091|Gm6455_chr5_10587246_F | Primer3: not found at this Tm | N.d. | 0.23 |
| mm4_intergenic_Gm17091|Gm6455_chr5_10587246_R | Primer3: not found at this Tm | N.d. | 0.23 |
| mm4_intron_Map3k5_chr10_20112143_F | Primer3: not found at this Tm | N.d. | 0.17 |
| mm4_intron_Map3k5_chr10_20112143_R | Primer3: not found at this Tm | N.d. | 0.17 |
| mm4_intergenic_Gm22603|Gria1_chr11_56868270_F | Primer3: not found at this Tm | N.d. | 0.15 |
| mm4_intergenic_Gm22603|Gria1_chr11_56868270_R | Primer3: not found at this Tm | N.d. | 0.15 |
| mm4_intron_Prkd1_chr12_50435396_F | Primer3: not found at this Tm | N.d. | 0.14 |
| mm4_intron_Prkd1_chr12_50435396_R | Primer3: not found at this Tm | N.d. | 0.14 |
| mm4_intergenic_AC154801.1|BC040587_chr16_40442140_F | Primer3: not found at this Tm | N.d. | 0.14 |
| mm4_intergenic_AC154801.1|BC040587_chr16_40442140_R | Primer3: not found at this Tm | N.d. | 0.14 |
| mm4_intron_Pam_chr1_97969943_F | Primer3: not found at this Tm | N.d. | 0.11 |
| mm4_intron_Pam_chr1_97969943_R | Primer3: not found at this Tm | N.d. | 0.11 |
| mm4_intron_Gm12511_chr4_55575464_F | Primer3: not found at this Tm | N.d. | 0.11 |
| mm4_intron_Gm12511_chr4_55575464_R | Primer3: not found at this Tm | N.d. | 0.11 |
| mm4_intergenic_Gm23511|Gm22480_chr3_48965206_F | TCGTCGGCAGCGTCGCGTAGTACAATGGGAGCCA | 59.8 | 0.10 |
| mm4_intergenic_Gm23511|Gm22480_chr3_48965206_R | GTCTCGTGGGCTCGGGCCACGTGTGTTCATTTGCA | 60.2 | 0.10 |
| mm3_intergenic_Rnmtl1|Nxn_chr11_76252645_F | TCGTCGGCAGCGTCACACAATGTGAGCTGAGGGG | 59.9 | 0.10 |
| mm3_intergenic_Rnmtl1|Nxn_chr11_76252645_R | GTCTCGTGGGCTCGGGCTCAGCAGAGTGGTGTGAA | 60.2 | 0.10 |
| mm4_intron_Tmprss6_chr15_78460941_F | TCGTCGGCAGCGTCGAGTGGAAGGAGCTAGCAGC | 60.1 | 0.07 |
| mm4_intron_Tmprss6_chr15_78460941_R | GTCTCGTGGGCTCGGAATGGGGAACTTGCAGCTGA | 59.8 | 0.07 |
| mm4_intergenic_Dnajc12|Gm7075_chr10_63419796_F | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_Dnajc12|Gm7075_chr10_63419796_R | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_Ppm1h|Mirlet7i_chr10_122957164_F | TCGTCGGCAGCGTCTCTTGAGCTGGAAGTGTGCT | 59.2 | 0.07 |
| mm4_intergenic_Ppm1h|Mirlet7i_chr10_122957164_R | GTCTCGTGGGCTCGGCTCCACGCTGACACCTCAG | 60.0 | 0.07 |
| mm4_intron_Dock2_chr11_34756422_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intron_Dock2_chr11_34756422_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Mctp2|Gm10294_chr7_72607904_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Mctp2|Gm10294_chr7_72607904_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Gm25485|Gm12460_chr4_50699800_F | TCGTCGGCAGCGTCAGAGAAAGTGTAGAGTACAGAACAG | 57.7 | 0.05 |
| mm4_intergenic_Gm25485|Gm12460_chr4_50699800_R | GTCTCGTGGGCTCGGTGCCACCCAATATTTAATCCATGT | 58.7 | 0.05 |
| mm4_intergenic_Grik1|Cldn17_chr16_88430472_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Grik1|Cldn17_chr16_88430472_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intron_Prodh_chr16_18083936_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Prodh_chr16_18083936_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Ush2a|Gm25095_chr1_188591650_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Ush2a|Gm25095_chr1_188591650_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Cacng3_chr7_122671836_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Cacng3_chr7_122671836_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Gm4822|Tpm3-rs7_chr14_113086823_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_Gm4822|Tpm3-rs7_chr14_113086823_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_1700023L04Rik|Nrf1_chr6_30028847_F | TCGTCGGCAGCGTCCACCTTCCTTGTGGGACCAT | 59.5 | 0.03 |
| mm4_intergenic_1700023L04Rik|Nrf1_chr6_30028847_R | GTCTCGTGGGCTCGGACTGTGGGCCTAGCTATGGT | 60.3 | 0.03 |
| mm4_intron_Cacna2d3_chr14_29478002_F | TCGTCGGCAGCGTCTGGGTAAAGGCAGAAGCAGA | 58.9 | 0.03 |
| mm4_intron_Cacna2d3_chr14_29478002_R | GTCTCGTGGGCTCGGCAAGGAAGCCCAGGGAGG | 59.3 | 0.03 |
| mm4_intergenic_4922501C03Rik|Tbx18_chr9_87318809_F | TCGTCGGCAGCGTCAGACACATACGGCCACACTG | 60.0 | 0.02 |
| mm4_intergenic_4922501C03Rik|Tbx18_chr9_87318809_R | GTCTCGTGGGCTCGGCTGGCAGAAACCCGATCTGT | 60.0 | 0.02 |
| mm4_intergenic_Gm12367|Gm12368_chr4_35318729_F | TCGTCGGCAGCGTCGCCATAGCTACAGAACACTGA | 57.4 | 0.02 |
| mm4_intergenic_Gm12367|Gm12368_chr4_35318729_R | GTCTCGTGGGCTCGGGATTAGGTAGCTGGCTGGAAA | 57.4 | 0.02 |
| mm3_intergenic_4732491K20Rik|Plg_chr17_12348384_F | TCGTCGGCAGCGTCGACAGACAGGATCCTGAGCC | 59.5 | 0.01 |
| mm3_intergenic_4732491K20Rik|Plg_chr17_12348384_R | GTCTCGTGGGCTCGGCCGAGCTGCTGTTGTAAGGA | 60.0 | 0.01 |
| mm4_intron_Dnah8_chr17_30832975_F | TCGTCGGCAGCGTCCTACCCGTCTGCAACACTGT | 59.9 | 0.01 |
| mm4_intron_Dnah8_chr17_30832975_R | GTCTCGTGGGCTCGGAGTTGGCTATGGGTTATGGCA | 59.4 | 0.01 |
| mm4_intergenic_Mfhas1|Gm22216_chr8_35693542_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Mfhas1|Gm22216_chr8_35693542_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Gm13786|Gm13785_chr2_92250624_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Gm13786|Gm13785_chr2_92250624_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Mtus2_chr5_148100161_F | TCGTCGGCAGCGTCTTCTGACCCTTCTTTCCCCG | 59.3 | 0.00 |
| mm4_intron_Mtus2_chr5_148100161_R | GTCTCGTGGGCTCGGACAGAGAGACCCTGCCTCAA | 60.1 | 0.00 |
| mm4_intron_Acsbg1_chr9_54645123_F | TCGTCGGCAGCGTCCCCAGCAGGTGAGATGACAC | 60.3 | 0.00 |
| mm4_intron_Acsbg1_chr9_54645123_R | GTCTCGTGGGCTCGGACCAGCACTGATTCCAGAGG | 59.3 | 0.00 |
| mm4_intron_Ddx11_chr17_66141645_F | TCGTCGGCAGCGTCCCCTGAGTGCCTTCCAGTTT | 59.8 | 0.00 |
| mm4_intron_Ddx11_chr17_66141645_R | GTCTCGTGGGCTCGGACGGGGAATGACAACTGTTCA | 59.8 | 0.00 |
| mm4_intergenic_Kcnn3|Adar_chr3_89674400_F | TCGTCGGCAGCGTCATTCCATTTGATAGTGCACAGTTT | 57.4 | 0.00 |
| mm4_intergenic_Kcnn3|Adar_chr3_89674400_R | GTCTCGTGGGCTCGGTTGAGGTGGCTCAAAGCTGT | 59.8 | 0.00 |
| mm4_exon_Ephb6_chr6_41614748_F | TCGTCGGCAGCGTCAGGATGGAGTAGGAGGCCAG | 60.1 | 0.00 |
| mm4_exon_Ephb6_chr6_41614748_R | GTCTCGTGGGCTCGGCTTGGCAGAGCTTGTCTCCA | 59.9 | 0.00 |
| mm4_intron_Plcg2_chr8_117600312_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Plcg2_chr8_117600312_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Arntl_chr7_113252038_F | TCGTCGGCAGCGTCAAAGCCAGGAGGATCTGCAG | 59.7 | 0.00 |
| mm4_intron_Arntl_chr7_113252038_R | GTCTCGTGGGCTCGGCCAGTACCCCTTCTGCTGTG | 60.0 | 0.00 |
| mm4_intergenic_Sntb1|Has2_chr15_56624125_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Sntb1|Has2_chr15_56624125_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm9726/mmu-mir-8099-2|Gm26055_chr12_94472387_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm9726/mmu-mir-8099-2|Gm26055_chr12_94472387_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm15742|Xxylt1_chr16_30980229_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm15742|Xxylt1_chr16_30980229_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Myo1d_chr11_80684405_F | TCGTCGGCAGCGTCGGGTCTCCCTTGAAGTCAAAGT | 59.8 | 0.00 |
| mm4_exon_Myo1d_chr11_80684405_R | GTCTCGTGGGCTCGGTGTTGCTGAAGTCAAACTGCG | 59.9 | 0.00 |
| mm4_exon_Gabpb2_chr3_95189939_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Gabpb2_chr3_95189939_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.
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313569 |
TCACTGTGGCAGACGTTTCTATTTATCCACTTGCGTTCGCCGAGTGGCGTCACCAGCGGTACTGTAATGACGATTGCAGC AGGAGGATGACAGCTTAGAAAGAAGAGGGCAATGGGGC |
| mm4_exon_Cdyl_chr13_35669789 |
CAGAATGCTGGCTGGCTTTCGGATTCTGTAAGGACCTGAGAAAGTACCTAAAGTGCCTTGGTTCCCCATCTACTGTAATA AATTCCTCCTGTTGCAATTGTCATTGCAGGTGGTGACCAGAGAACAGAGGC |
| mm4_intergenic_Gm23511|Gm22480_chr3_48965206 |
GCGTAGTACAATGGGAGCCATGACTAACTCACAGAAAAAACTGTAGTGACAACAGCAGCTGGAACGAAGCATCAATATAC TACAGGAGATAATTCCAAAGCTCTACAGCCTTGCAAATGAACACACGTGGC |
| mm3_intergenic_Rnmtl1|Nxn_chr11_76252645 |
ACACAATGTGAGCTGAGGGGGACTGTAATGACTATCACAGCAGGCTGGGTTTTAAAGGCAGCAGGGAAGAATTCACACCA CTCTGCTGAGC |
| mm4_intron_Tmprss6_chr15_78460941 |
GAGTGGAAGGAGCTAGCAGCACCCTGTCCCTAATGCAATCGTCATTTCATTGTATGGATGGGGAGGGGGCAGGGCCCTGA CAATGTCAGCTGCAAGTTCCCCATT |
| mm4_intergenic_Ppm1h|Mirlet7i_chr10_122957164 |
TCTTGAGCTGGAAGTGTGCTTAAAACAGATCCTACAGTCTTAAGCACTGGACTGTAATAAAAGAAATTGTACAAGGTAAA TCTTCTACCAGGAGGCCCAGCTGCAGTAGTTATTACAGAGCCTGAGGTGTCAGCGTGGAG |
| mm4_intergenic_Gm25485|Gm12460_chr4_50699800 |
AGAGAAAGTGTAGAGTACAGAACAGACTGGAATGACTATTGAAGAGGGTAGGTTTCTCTTTTTTTGTAGTAAAATTACCT GTAAATTGACAAAACTATCAGATATAGAAGTCTAACATGGATTAAATATTGGGTGGCA |
| mm4_intergenic_1700023L04Rik|Nrf1_chr6_30028847 |
CACCTTCCTTGTGGGACCATTACTTTCTTGGCTGTACTTTAATGACGATAGCTGTAGGTAAAAGCCAGAGTCGGAGCTAG GAATTGTTTTACCATAGCTAGGCCCACAGT |
| mm4_intron_Cacna2d3_chr14_29478002 |
TGGGTAAAGGCAGAAGCAGAAATGGACATTTGTTTTCCACAGCAAGAAGAAAATGTCTTTTTCCATGACTTTGACAAGGA TGAATACAACCCAGTTCCAATCTTCATTACAGCCCTCCCTGGGCTTCCTTG |
| mm4_intergenic_4922501C03Rik|Tbx18_chr9_87318809 |
AGACACATACGGCCACACTGTCACCCTCTCCTGCCAGCTGCAAACCTCATCACCGTCCCTGTGAAGTACATTTCCACAGA TCGGGTTTCTGCCAG |
| mm4_intergenic_Gm12367|Gm12368_chr4_35318729 |
GCCATAGCTACAGAACACTGAAGTGTAAAGATTTGTTTACCTAGAATTATAATGAGAATTAAATTATAGAGAAAGACTGA AATGAAAATTGCAGGTGGTAATTTTGTCAACCTACTCTTTTTCCAGCCAGCTACCTAATC |
| mm3_intergenic_4732491K20Rik|Plg_chr17_12348384 |
GACAGACAGGATCCTGAGCCGGGCATACGTTCATTCTGCTGCAATCGTCATGAGAGGGAGCCATGCTCTACATGACCAGT CCACCTATCCTTACAACAGCAGCTCGG |
| mm4_intron_Dnah8_chr17_30832975 |
CTACCCGTCTGCAACACTGTGGCATTTACTGTAATGAGAATTACAGGTGGCTACAATCATAGAGAGAAAAATACTATCTT GGTTTTTTTATTTGCCATAACCCATAGCCAACT |
| mm4_intron_Mtus2_chr5_148100161 |
TTCTGACCCTTCTTTCCCCGCCCGCTGCCATGGTCACTTCAGTCCTCTCTAGCAGTCTTTCTACTATTTCCAGATTTTTG TTTTTGATTTTGAGGCAGGGTCTCTCTGT |
| mm4_intron_Acsbg1_chr9_54645123 |
CCCAGCAGGTGAGATGACACTACAGACAGTAATGAGGATAGGAGCCGGCCATTTACACATGACACTCTGGAAGGTTAGAT TCCTCTGGAATCAGTGCTGGT |
| mm4_intron_Ddx11_chr17_66141645 |
CCCTGAGTGCCTTCCAGTTTTCAAATCAGCGTCCCTTAAACTAACCTTCGGCCATCGTCATTACTGTGATCCTATTGGTC ATCTTTGGTACCTTTCTCTGCTTTTGTTAAGCTCTGAACAGTTGTCATTCCCCGT |
| mm4_intergenic_Kcnn3|Adar_chr3_89674400 |
ATTCCATTTGATAGTGCACAGTTTAAATGACATACATGAACTGTAATGTCAATTGGAGGTGGCCTTTCTCTAGTCTGATG TGAAAAACCACTCAAGTGACAGCTTTGAGCCACCTCAA |
| mm4_exon_Ephb6_chr6_41614748 |
AGGATGGAGTAGGAGGCCAGGCAGGGGGCAGCCCCCCAAGGTTACACTGTAATGGCGAAGGCAGGTGGATGGTAGCTGTG GGAGGCTGCCGCTGCCAGCCTGGACACCAGCCTGCTCGTGGAGACAAGCTCTGCCAAG |
| mm4_intron_Arntl_chr7_113252038 |
AAAGCCAGGAGGATCTGCAGAGACCCTCAGGTTCAGTCCCACCATACTGTAATCACTATTCCAGAGGGGATGCTGCCCAG ATTAAAGTAAGTCACAGCAGAAGGGGTACTGG |
| mm4_exon_Myo1d_chr11_80684405 |
GGGTCTCCCTTGAAGTCAAAGTTGATATCCATGTATTTCCCAAACCTGCTGGAATTGTCATTACGATTGGTTTTGGCATT TCCAAAAGCTTCCAAAACGCAGTTTGACTTCAGCAACA |