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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_52314227_F | TCGTCGGCAGCGTCAGAAATCGAGGTGAGCTGCA | 59.3 | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52314227_R | GTCTCGTGGGCTCGGGAAGGGAACTGTGGGTGGAG | 59.9 | 1.00 |
| mm4_intron_Mxi1_chr19_53323439_F | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_intron_Mxi1_chr19_53323439_R | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_intron_0610012H03Rik_chr2_105227344_F | TCGTCGGCAGCGTCCCTCCTCTGGGTACTTTCCG | 59.1 | 0.17 |
| mm4_intron_0610012H03Rik_chr2_105227344_R | GTCTCGTGGGCTCGGCATCCTGGCTATAGCGTGGG | 60.0 | 0.17 |
| mm4_intergenic_Gm2373|5330416C01Rik_chr13_97613658_F | TCGTCGGCAGCGTCATGATGCCTGGAGCCCAAAA | 59.9 | 0.15 |
| mm4_intergenic_Gm2373|5330416C01Rik_chr13_97613658_R | GTCTCGTGGGCTCGGGCATCCAAGTCCATGGCTTC | 59.2 | 0.15 |
| mm4_intron_Col6a2_chr10_76603946_F | TCGTCGGCAGCGTCCCACACGTGTGCCTATGAGA | 59.7 | 0.15 |
| mm4_intron_Col6a2_chr10_76603946_R | GTCTCGTGGGCTCGGTCATGGCTCTGCTACACACC | 59.7 | 0.15 |
| mm3_intron_Stard8_chrX_99061058_F | TCGTCGGCAGCGTCCGGCATCCTGCTTATCCTCC | 60.3 | 0.15 |
| mm3_intron_Stard8_chrX_99061058_R | GTCTCGTGGGCTCGGAACAGATGTGAGGCCATTGC | 58.8 | 0.15 |
| mm4_intergenic_Rfwd2|Gm10530_chr1_159501911_F | TCGTCGGCAGCGTCGCAGGTCTAGAGCTAAGGCC | 59.6 | 0.14 |
| mm4_intergenic_Rfwd2|Gm10530_chr1_159501911_R | GTCTCGTGGGCTCGGGGACTCCCCAAAGCACGAAG | 60.9 | 0.14 |
| mm3_exon_Mdm1_chr10_118159651_F | TCGTCGGCAGCGTCAGTGTGACCGACATGTGCTT | 59.8 | 0.13 |
| mm3_exon_Mdm1_chr10_118159651_R | GTCTCGTGGGCTCGGCATCTTGGGAGGACGCTCTC | 59.8 | 0.13 |
| mm2_intergenic_Gm26435|Snx19_chr9_30427191_F | TCGTCGGCAGCGTCGAGCTTGTTGTGGCTGCTG | 59.7 | 0.13 |
| mm2_intergenic_Gm26435|Snx19_chr9_30427191_R | GTCTCGTGGGCTCGGTACGAACTCCGAAGCAGCAG | 60.1 | 0.13 |
| mm4_intron_Gm14114_chr2_146839264_F | TCGTCGGCAGCGTCCCCCGGTTCCTTCTTTCCAA | 59.8 | 0.12 |
| mm4_intron_Gm14114_chr2_146839264_R | GTCTCGTGGGCTCGGGGTCACAGGAAAACAGTCGC | 59.4 | 0.12 |
| mm3_intron_Nudt9_chr5_104062131_F | TCGTCGGCAGCGTCGGCTGTTTGGTGATGGGGTA | 59.9 | 0.11 |
| mm3_intron_Nudt9_chr5_104062131_R | GTCTCGTGGGCTCGGAACAACACACGCTCACGACA | 60.7 | 0.11 |
| mm4_intergenic_Kl|Stard13_chr5_151022580_F | TCGTCGGCAGCGTCGATCAGGCAGAGCTGGTCTC | 59.8 | 0.10 |
| mm4_intergenic_Kl|Stard13_chr5_151022580_R | GTCTCGTGGGCTCGGCCACACATGGTCATGGAAGA | 57.5 | 0.10 |
| mm4_intron_Map3k2_chr18_32188513_F | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intron_Map3k2_chr18_32188513_R | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intergenic_Rapgef5|Gm24741_chr12_117528937_F | TCGTCGGCAGCGTCACACACATGGTTCTCTCTGG | 57.1 | 0.10 |
| mm4_intergenic_Rapgef5|Gm24741_chr12_117528937_R | GTCTCGTGGGCTCGGTAGACAGACCTGAGCCTGGC | 60.9 | 0.10 |
| mm4_intergenic_Mrps22|PISRT1_chr9_98624746_F | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intergenic_Mrps22|PISRT1_chr9_98624746_R | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intron_Chrna4_chr2_181042035_F | TCGTCGGCAGCGTCGCTCAGCTCTGTTGCCTGTT | 60.8 | 0.07 |
| mm4_intron_Chrna4_chr2_181042035_R | GTCTCGTGGGCTCGGGAGAGACTGCCCGAGAGAGA | 60.1 | 0.07 |
| mm4_intron_Mgmt_chr7_136927938_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intron_Mgmt_chr7_136927938_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Cbln2|Gm5096_chr18_87505211_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Cbln2|Gm5096_chr18_87505211_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Gm13857|Bpgm_chr6_34452529_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Gm13857|Bpgm_chr6_34452529_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intron_Lmx1b_chr2_33567118_F | TCGTCGGCAGCGTCGATAGCAGTGGGTCATGGGG | 59.8 | 0.05 |
| mm4_intron_Lmx1b_chr2_33567118_R | GTCTCGTGGGCTCGGGCGAAGAGCTTTCAAGGCAT | 59.1 | 0.05 |
| mm4_intron_Ssbp3_chr4_106994997_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Ssbp3_chr4_106994997_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm3_exon_Tsnaxip1_chr8_105844366_F | TCGTCGGCAGCGTCTAACATGCTGGGCCCTTCAG | 60.0 | 0.02 |
| mm3_exon_Tsnaxip1_chr8_105844366_R | GTCTCGTGGGCTCGGCCCACGCATCTTCAGTAGGG | 60.1 | 0.02 |
| mm4_intron_Dbn1_chr13_55487771_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Dbn1_chr13_55487771_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Cdh6_chr15_13053940_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Cdh6_chr15_13053940_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Olfr1440|Olfr1441_chr19_12397119_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Olfr1440|Olfr1441_chr19_12397119_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Cntn4_chr6_106001067_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Cntn4_chr6_106001067_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Mylk_chr16_34868160_F | TCGTCGGCAGCGTCGCAACCTCAGCCATCCTCAA | 60.3 | 0.00 |
| mm4_intron_Mylk_chr16_34868160_R | GTCTCGTGGGCTCGGGGCGGGTGATCATGATAGCA | 59.9 | 0.00 |
| mm4_exon_Mpc1/4930506C21Rik_chr17_8297941_F | TCGTCGGCAGCGTCTCTCCCTGCCAAACTGCATC | 60.3 | 0.00 |
| mm4_exon_Mpc1/4930506C21Rik_chr17_8297941_R | GTCTCGTGGGCTCGGTGGGTGTCTGTGCTAGGACT | 60.1 | 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_52314227 |
AGAAATCGAGGTGAGCTGCACCCCAGACTGCGCCACCGGGAACGCCGAGTACCAGCACAGCAAAGGTAGCCACCTTGCCC CTCCGCTCCCCGGTCCGCCCACTCCACCCACAGTTCCCTTC |
| mm4_intron_0610012H03Rik_chr2_105227344 |
CCTCCTCTGGGTACTTTCCGTAAGACACATTTTGGAAATGTACCCAGACTGGGCCTCCTGGAAAGGAGAAAGCCGGGTGC TCTGCTGTACATTTGGTGCCTGTTCTGCCCCACGCTATAGCCAGGATG |
| mm4_intergenic_Gm2373|5330416C01Rik_chr13_97613658 |
ATGATGCCTGGAGCCCAAAAACGTCGTGTGGGGCAGATGGCTGGCTATATTCCCCAAGACTGGGCCTCAGGGAAGGTTTG AGATATGGATAAAATATAATGTAAAAGTCATCAGAAGCCATGGACTTGGATGC |
| mm4_intron_Col6a2_chr10_76603946 |
CCACACGTGTGCCTATGAGAGAAGATCAGCATGAACAGTGTCCACCATGACTGGGCCACCTGGAATGAATCACGGGGTGT GTAGCAGAGCCATGA |
| mm3_intron_Stard8_chrX_99061058 |
CGGCATCCTGCTTATCCTCCTGTTGTGTCCTTCCTGGTGGCTCAGTCTTGGACCCCACTTCGCCCATGGCAATGGCCTCA CATCTGTT |
| mm4_intergenic_Rfwd2|Gm10530_chr1_159501911 |
GCAGGTCTAGAGCTAAGGCCTTAATGAGTTCTCCATTGGACCATTTCCTTCCCTAGGATCTGCTGTCATTCAAACAGAAT ATGCTAAGCATGGTCGTGGCCGGTAGAGCAGTCTTGGCTTCGTGCTTTGGGGAGTCC |
| mm3_exon_Mdm1_chr10_118159651 |
AGTGTGACCGACATGTGCTTTCCATGGGGTCTTCCAGGTGGCGCAGTCTTAGTGTCTCCATCTAAAGTGAAGCCACCAGG CCTCGAGCAGAGGAGGAGAGCGTCCTCCCAAGATG |
| mm2_intergenic_Gm26435|Snx19_chr9_30427191 |
GAGCTTGTTGTGGCTGCTGGCCGCGGCGGTCGGAAGGATCCCCACCCCCCCAAGAGTTCGTTCCCAGGGGCGCAGTCTGA GCACGGGTCCCCCAGCGACGTGTGACCCGATCTGCCGGCTGCTGCTTCGGAGTTCGTA |
| mm4_intron_Gm14114_chr2_146839264 |
CCCCGGTTCCTTCTTTCCAAGGGCGTTTTTAGCCTCAGAAACATCCTAGACTGCGGCAGAGGGAAGGAGATTTAAGATCT GACAGTTCCCTGCCTGCGACTGTTTTCCTGTGACC |
| mm3_intron_Nudt9_chr5_104062131 |
GGCTGTTTGGTGATGGGGTAAGAGACGGCTTTTCTGTTTACCTTGCCGCGTGACGGTCCCGGTGCCGCAGCCTTGGGCTG ACTTGCCCTGTGTGTCGTGAGCGTGTGTTGTT |
| mm4_intergenic_Kl|Stard13_chr5_151022580 |
GATCAGGCAGAGCTGGTCTCTCTCTCCTCTTTATTTTTTACTACATATGTTTTTGAAAATTCCTTGTATTATTGTTTTCA ATGTCGGCCCGTTCTCTGTGGCACATTCTGGGCTGAAATTCTTCCATGACCATGTGTGG |
| mm4_intergenic_Rapgef5|Gm24741_chr12_117528937 |
ACACACATGGTTCTCTCTGGCATTCTCGGTGAGGCAGTCTGGGAAATGAGTTTTGTGTGACATAATCTGTCTTCTGATCA GCTAGCTACTCAGCTCTGATCCTCAGAGAGGCCAGGCTCAGGTCTGTCTA |
| mm4_intron_Chrna4_chr2_181042035 |
GCTCAGCTCTGTTGCCTGTTTATCTTCACAGCTTCTAGAGACAGGCTTTGTGCCCAGCCTTCCAGGTTGAGCAGTCTGGG ACAGTGGCTCGCACCAGCGGGCAGTCTCTCTCGGGCAGTCTCTC |
| mm4_intron_Lmx1b_chr2_33567118 |
GATAGCAGTGGGTCATGGGGGAGGAACAGACCCACAATAAGCAAAAGGGGGGCGTTGGCCCCAGACTGCCCCACCCAGAA CCCCTCACCTTCCGACAGGGCTTGGAGGAGACCTCAAAGGATGCCTTGAAAGCTCTTCGC |
| mm3_exon_Tsnaxip1_chr8_105844366 |
TAACATGCTGGGCCCTTCAGATCAGGGTACAGAGAGAAGAGCTCAAACCTGCATTCCTATCTTCCTTTCCATGTGGCGCA GTCTTGAAGACGTGACCCTACTGAAGATGCGTGGG |
| mm4_intron_Mylk_chr16_34868160 |
GCAACCTCAGCCATCCTCAAATACCCCCCATCCCTACATCAGGTGTCACCAGGCCCTGTTATGTCATCTCCTACACAGTT CTTTCCTTTCCATTCCCAGTGGCACTGTCTGGGTCCTAGCTGCTATCATGATCACCCGCC |
| mm4_exon_Mpc1/4930506C21Rik_chr17_8297941 |
TCTCCCTGCCAAACTGCATCTGCAGCCACAGCCCAGACAGCCCCACTGGGAATGGTGCTAGTCCACGGCTCTCATGGAGT CCTAGCACAGACACCCA |