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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_52314007_F | TCGTCGGCAGCGTCAACCGCCAGAGAAGATGGTG | 60.0 | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52314007_R | GTCTCGTGGGCTCGGCTGCCTGCTAGTCCATCGAC | 60.2 | 1.00 |
| mm4_intron_Myo16_chr8_10439861_F | TCGTCGGCAGCGTCCTGAGTGCCAGGAGAAAGCA | 59.9 | 0.80 |
| mm4_intron_Myo16_chr8_10439861_R | GTCTCGTGGGCTCGGTGACAGGTTGATTAGGAAGGGG | 59.4 | 0.80 |
| mm4_intron_Ttc28_chr5_111158440_F | TCGTCGGCAGCGTCCTCTCCTCGGCTTCACAGAC | 59.8 | 0.48 |
| mm4_intron_Ttc28_chr5_111158440_R | GTCTCGTGGGCTCGGGCAGACAGCAGAACTAGGCA | 60.0 | 0.48 |
| mm4_intergenic_Chchd6|Txnrd3_chr6_89643923_F | TCGTCGGCAGCGTCCTGTGGAGGCCTGGAGGT | 60.6 | 0.36 |
| mm4_intergenic_Chchd6|Txnrd3_chr6_89643923_R | GTCTCGTGGGCTCGGTTGAGAACGCTACGCACTGG | 60.6 | 0.36 |
| mm4_exon_Dab1_chr4_104367158_F | TCGTCGGCAGCGTCTCAGCAGAGTCCTAGAGGGG | 59.7 | 0.28 |
| mm4_exon_Dab1_chr4_104367158_R | GTCTCGTGGGCTCGGGGGAGTAGAGACCGATCGGA | 59.8 | 0.28 |
| mm4_intergenic_Wnt2|Asz1_chr6_18031519_F | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_intergenic_Wnt2|Asz1_chr6_18031519_R | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_intergenic_5730405O15Rik|Usp9x_chrX_13058675_F | Primer3: not found at this Tm | N.d. | 0.22 |
| mm4_intergenic_5730405O15Rik|Usp9x_chrX_13058675_R | Primer3: not found at this Tm | N.d. | 0.22 |
| mm4_intergenic_Gm26474|RP24-351J24.2_chr5_120313446_F | TCGTCGGCAGCGTCTCCCCAGAGGAAGAGTGGAG | 59.9 | 0.20 |
| mm4_intergenic_Gm26474|RP24-351J24.2_chr5_120313446_R | GTCTCGTGGGCTCGGCCATTAGCCTGCTCAGTCCC | 60.1 | 0.20 |
| mm4_exon_Podnl1_chr8_84127804_F | TCGTCGGCAGCGTCTGCTGTTACCCTTGCAGCTT | 60.1 | 0.18 |
| mm4_exon_Podnl1_chr8_84127804_R | GTCTCGTGGGCTCGGCTAAGGCTACCGGAGTGCAG | 59.8 | 0.18 |
| mm4_intergenic_Mpp5|Gm25225_chr12_78749152_F | Primer3: not found at this Tm | N.d. | 0.18 |
| mm4_intergenic_Mpp5|Gm25225_chr12_78749152_R | Primer3: not found at this Tm | N.d. | 0.18 |
| mm4_exon_Slc38a7_chr8_95837185_F | TCGTCGGCAGCGTCCCCCGAGGTACTGAGCACA | 60.9 | 0.17 |
| mm4_exon_Slc38a7_chr8_95837185_R | GTCTCGTGGGCTCGGAGGGTCAGTGGGTCTCAGAA | 59.8 | 0.17 |
| mm3_intron_Elmo1_chr13_20091379_F | TCGTCGGCAGCGTCTAGAGAGAGGCTAGCGTGGG | 60.1 | 0.11 |
| mm3_intron_Elmo1_chr13_20091379_R | GTCTCGTGGGCTCGGTTCCTCTTCCTCTAAGCCGC | 59.1 | 0.11 |
| mm4_intergenic_1700011I03Rik|Slc12a2_chr18_57878612_F | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intergenic_1700011I03Rik|Slc12a2_chr18_57878612_R | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intron_Dnajc13_chr9_104236428_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intron_Dnajc13_chr9_104236428_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm3_exon_Chic2_chr5_75044632_F | TCGTCGGCAGCGTCCAACCACAGCAACAGCCC | 59.2 | 0.04 |
| mm3_exon_Chic2_chr5_75044632_R | GTCTCGTGGGCTCGGGAGACCGTCCGCGTGAAC | 60.8 | 0.04 |
| mm4_intergenic_A530013C23Rik|Gm14321_chr2_167730472_F | TCGTCGGCAGCGTCCTGAGCTGAGACACACCCAG | 60.0 | 0.03 |
| mm4_intergenic_A530013C23Rik|Gm14321_chr2_167730472_R | GTCTCGTGGGCTCGGTCTGGGTCCTATGTGGGTGT | 59.8 | 0.03 |
| mm4_intron_Gm26839_chr12_99614960_F | TCGTCGGCAGCGTCTATGCCTGAAGCCTCCCTCT | 60.0 | 0.03 |
| mm4_intron_Gm26839_chr12_99614960_R | GTCTCGTGGGCTCGGACAGAGCAGACAGGTTTGGA | 58.8 | 0.03 |
| mm4_intergenic_Nkx1-1|Fam53a_chr5_33545899_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_Nkx1-1|Fam53a_chr5_33545899_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Chst15_chr7_132286972_F | TCGTCGGCAGCGTCGTCACAGAGACCAGGGAGGA | 60.2 | 0.02 |
| mm4_intron_Chst15_chr7_132286972_R | GTCTCGTGGGCTCGGGGAGGAAGTCTGATCTTCTGGG | 59.5 | 0.02 |
| mm4_exon_Rem1_chr2_152633625_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_exon_Rem1_chr2_152633625_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Scarb1_chr5_125288649_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Scarb1_chr5_125288649_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_exon_Gm14597_chrX_53579875_F | TCGTCGGCAGCGTCGAGCATGAAGGCCTGGATGT | 60.1 | 0.02 |
| mm4_exon_Gm14597_chrX_53579875_R | GTCTCGTGGGCTCGGCTGAGGGCTGCCAAGTAGTC | 60.1 | 0.02 |
| mm4_exon_4933416I08Rik_chrX_53686595_F | TCGTCGGCAGCGTCCTGAGGGCTGCCAAGTAGTC | 60.1 | 0.02 |
| mm4_exon_4933416I08Rik_chrX_53686595_R | GTCTCGTGGGCTCGGCTGCCACCACAATTCGGAGA | 60.3 | 0.02 |
| mm4_intron_Pitpnc1_chr11_107299079_F | TCGTCGGCAGCGTCGGTAACCACTCTGCCCTCAC | 60.0 | 0.02 |
| mm4_intron_Pitpnc1_chr11_107299079_R | GTCTCGTGGGCTCGGAGTCAGTCAAGGTTCTGCCG | 59.9 | 0.02 |
| mm4_intergenic_Gm23708|G6pd2_chr5_60726239_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Gm23708|G6pd2_chr5_60726239_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm3_intron_Pan3_chr5_147431202_F | TCGTCGGCAGCGTCAGCCCTCCTTCCTTCTTCCT | 59.8 | 0.01 |
| mm3_intron_Pan3_chr5_147431202_R | GTCTCGTGGGCTCGGATTACACCACGACCGTCCG | 59.7 | 0.01 |
| mm4_intergenic_Gm24696|Myc_chr15_61951975_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Gm24696|Myc_chr15_61951975_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Camta1_chr4_151859281_F | TCGTCGGCAGCGTCGATGACACTGCTGAGCGAGA | 59.8 | 0.01 |
| mm4_intron_Camta1_chr4_151859281_R | GTCTCGTGGGCTCGGCCTTCGGAAAAGTGCTTGCC | 60.0 | 0.01 |
| mm4_intergenic_Gm17689|A630095E13Rik_chr9_36626842_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Gm17689|A630095E13Rik_chr9_36626842_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Eya2|Zmynd8_chr2_165783915_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Eya2|Zmynd8_chr2_165783915_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Slc8a1|Rpl31-ps25_chr17_81672240_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Slc8a1|Rpl31-ps25_chr17_81672240_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Cacnb3_chr15_98634877_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Cacnb3_chr15_98634877_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Fbn1_chr2_125336173_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Fbn1_chr2_125336173_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_BC034090_chr1_155226594_F | TCGTCGGCAGCGTCACATTCTCCCATGGTGCCTG | 60.0 | 0.00 |
| mm4_intron_BC034090_chr1_155226594_R | GTCTCGTGGGCTCGGGATGTGCTCATCCTGCAGGG | 60.8 | 0.00 |
| mm4_intergenic_Vwa5b1|Ubxn10_chr4_138640686_F | TCGTCGGCAGCGTCTTGGGTCATGGCCAATGGAA | 59.8 | 0.00 |
| mm4_intergenic_Vwa5b1|Ubxn10_chr4_138640686_R | GTCTCGTGGGCTCGGACCAAGAATTCAGCTGCCCT | 59.5 | 0.00 |
| mm4_intergenic_Mageb4|Gm8839_chrX_86266354_F | TCGTCGGCAGCGTCTGTGGATATTGGGGAAGCGG | 59.8 | 0.00 |
| mm4_intergenic_Mageb4|Gm8839_chrX_86266354_R | GTCTCGTGGGCTCGGCTGCCAGTCTGCACCTCTAC | 60.1 | 0.00 |
| mm4_intergenic_Gm25787|Gm23345_chr7_111835189_F | TCGTCGGCAGCGTCCTTCTGAGGAGCGGGTGTAC | 59.8 | 0.00 |
| mm4_intergenic_Gm25787|Gm23345_chr7_111835189_R | GTCTCGTGGGCTCGGGGAGGGAATGGCACTTTGGA | 59.9 | 0.00 |
| mm4_intergenic_Gm24601|Gm26501_chr6_95006542_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm24601|Gm26501_chr6_95006542_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm23505|Gm13494_chr2_51530885_F | TCGTCGGCAGCGTCTGCTTTGAACAATGCTCAGCT | 59.0 | 0.00 |
| mm4_intergenic_Gm23505|Gm13494_chr2_51530885_R | GTCTCGTGGGCTCGGAGGACTCAGGCAACAATATTCCA | 59.7 | 0.00 |
| mm4_intergenic_Api5|Gm13788_chr2_94474049_F | TCGTCGGCAGCGTCCCCTTTCTACCTGCCACCTG | 60.0 | 0.00 |
| mm4_intergenic_Api5|Gm13788_chr2_94474049_R | GTCTCGTGGGCTCGGAGCTGAGACCTGTCTAGCAG | 58.5 | 0.00 |
| mm4_exon_Znrf3_chr11_5278012_F | TCGTCGGCAGCGTCATGTGCAATCTGGACCCTGG | 60.0 | 0.00 |
| mm4_exon_Znrf3_chr11_5278012_R | GTCTCGTGGGCTCGGAGGAAGAGTTTGGCCGAGTG | 59.9 | 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_52314007 |
AACCGCCAGAGAAGATGGTGCCCCACGGTTGCCTGAGCCCGCGAGCCGGCCCTCCGACTTCCCGGGAGCGTGGCGGGGGA GGCCAGGAGGAGGAGCCGGTCGATGGACTAGCAGGCAG |
| mm4_intron_Myo16_chr8_10439861 |
CTGAGTGCCAGGAGAAAGCACTGAAAGGCTGCTGTATGGGAACCCTGCCATGCTCCCGGGAGATCATTCTGAGTAACTAG CCCCCTTCCTAATCAACCTGTCA |
| mm4_intron_Ttc28_chr5_111158440 |
CTCTCCTCGGCTTCACAGACTTACGTCTTACTGTGCTGCCTGCCCGAGCATAGCTATGCCAGCCACGCTCCTGAGAAGAT GGCAGAGTTCCCTTGCCTAGTTCTGCTGTCTGC |
| mm4_intergenic_Chchd6|Txnrd3_chr6_89643923 |
CTGTGGAGGCCTGGAGGTGGGGCAAAGTTTTCCCGGGGGCGTGGCCGGTCGGTAGGCGGGCCCTCTCCGTGGGTCGCGCG GTCGGGGGCGTGGCCCCGTAGCTGTCCGGTGCGCCCGCGGCCAGTGCGTAGCGTTCTCAA |
| mm4_exon_Dab1_chr4_104367158 |
TCAGCAGAGTCCTAGAGGGGGCTGCAGCGGCTGCCTAGGAGCGTGGCGGGCTCCCAGCGCAACCCCCAACCCCACCCCCT GCGACGAACCGCAGGGCGGGGACCCCAAGCTCTTCGCTCCGATCGGTCTCTACTCCC |
| mm4_intergenic_Gm26474|RP24-351J24.2_chr5_120313446 |
TCCCCAGAGGAAGAGTGGAGGGGTATTCTAGAGTAGTTCCCTCCAAGTCAGCCCCCTGCCATGCTCATGGGCAGTCGGGG GAGCAGGCCACCGCCTGAGCGCCCAGAGGGAGGGACTGAGCAGGCTAATGG |
| mm4_exon_Podnl1_chr8_84127804 |
TGCTGTTACCCTTGCAGCTTTCTGTAGCGCCCCAGTTCCTGCCACGCTCCCTGCGTGTCGCCGATCTGGCTGCCAATGAA GTGGTGGAGATCTTTCCTCTCACCTTTGGGGAGAAGCCTGCACTCCGGTAGCCTTAG |
| mm4_exon_Slc38a7_chr8_95837185 |
CCCCGAGGTACTGAGCACAGTCCTGCCACGCTCCGCGGAGGACGCCAGCCCTCTGCAGATGCATTTTTTTGTTGTTGTTC TTTTTCCAGAGGGAAGTCAGAATAGACTCTCTCTTCTGAGACCCACTGACCCT |
| mm3_intron_Elmo1_chr13_20091379 |
TAGAGAGAGGCTAGCGTGGGAGGGGGCGTACCGCGCGTCTTCCCGGGAGAGCGGCGGGCATGGGGCGCCAGGGAGGGAGC GAGCGAGCTGCGCGCCGCGGTAGCCCCGCGGCTTAGAGGAAGAGGAA |
| mm3_exon_Chic2_chr5_75044632 |
CAACCACAGCAACAGCCCGAGCCACCCGCAGCAGGAGCAGCCGGCTACCGCATCGCGAGACTTCCCGGGAGCNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGTTCACGCGGACGGTCTC |
| mm4_intergenic_A530013C23Rik|Gm14321_chr2_167730472 |
CTGAGCTGAGACACACCCAGCCTCCCACCTGCCCTGCTCCCGGGAAGGAGGGACCCGCCAATGACAGGAGGAAGCAATAG AGCCTCTGTCCTCACGCTCTGTCACACCCACATAGGACCCAGA |
| mm4_intron_Gm26839_chr12_99614960 |
TATGCCTGAAGCCTCCCTCTTCCCATACCATCAGTCCCTCACTCACTACGGCTCTGGCCTCCTGCCACCTTCCCAGGAAG TCCATTTTCCAAACCTGTCTGCTCTGT |
| mm4_intron_Chst15_chr7_132286972 | GTCACAGAGACCAGGGAGGACCTGCCACACCCCCGTGAAGTCAAGGCTTATTCCCAGAAGATCAGACTTCCTCC |
| mm4_exon_Gm14597_chrX_53579875 |
GAGCATGAAGGCCTGGATGTTGTGACCCCCATGGTGCCACCTTTTGAAGACCTGCCTCACTCCTGGGAAGTGGTACCAGA TGACTACTTGGCAGCCCTCAG |
| mm4_exon_4933416I08Rik_chrX_53686595 |
CTGAGGGCTGCCAAGTAGTCATCTGGTACCACTTCCCAGGAGTGAGGCAGGTCTTCAAAAGGTGGCACCATGGGGGTCAC GACATCCCGGCCTTCATGCTCCTGCTGCAGCTGTTTCTCCGAATTGTGGTGGCAG |
| mm4_intron_Pitpnc1_chr11_107299079 |
GGTAACCACTCTGCCCTCACACCCATTTCCTGGGACTTCCAGGGAGGGAGGCTGGTCATCACGAGTCAAGACGCTGGCAC CCTTGAAGCCCCACCCTCCCCCCGGCAGAACCTTGACTGACT |
| mm3_intron_Pan3_chr5_147431202 |
AGCCCTCCTTCCTTCTTCCTGTTTCCTCTCTGGGCTCCGCCGCTCGGGGCAAACCCCGGAGCTAGGGGAGGCGGGTTCCC GGGAGCGTGGGTGGACGGACGGTCGTGGTGTAAT |
| mm4_intron_Camta1_chr4_151859281 |
GATGACACTGCTGAGCGAGAGCACGAGACAGGGAGGTGAGCACCTGAGCAGAGGCAGGGCTGTGGGCCCCAGAGAAAGAA CCTGGAAACCCTGCCACCTTCCCGGGAAGCAGCGGCAAGCACTTTTCCGAAGG |
| mm4_intron_BC034090_chr1_155226594 |
ACATTCTCCCATGGTGCCTGACGGTACCAACAATCCACAAGGTTACCCCCAGTCAGGCTCCCGGGAAGACACGTGACAAG CCCCAAGCCCTGCAGGATGAGCACATC |
| mm4_intergenic_Vwa5b1|Ubxn10_chr4_138640686 |
TTGGGTCATGGCCAATGGAAGTACCACGTCTTCAAGGGAGCTTGGCTGGAGCCACAGTATCTCACTGCCAGCCTAGGGCA GCTGAATTCTTGGT |
| mm4_intergenic_Mageb4|Gm8839_chrX_86266354 |
TGTGGATATTGGGGAAGCGGTCTTACCTGTGTCTCTGGTTAGAGCAGACTTCCTGGGAGACTGGCAGGGCTGTGGGTACA GGTAGCAAGCATGCTGCTCTGCCACAGGTACAGGTAGAGGTGCAGACTGGCAG |
| mm4_intergenic_Gm25787|Gm23345_chr7_111835189 |
CTTCTGAGGAGCGGGTGTACTCCCCAGGCAAGCTCCTGGGAAGTAGATTTTGGACCAAGGTAAGGAGGGAAACCTCCGAG ATGAGTAAAAGAAAATGGATTTGGGGGGAAGTACCTGTTCCAAAGTGCCATTCCCTCC |
| mm4_intergenic_Gm23505|Gm13494_chr2_51530885 |
TGCTTTGAACAATGCTCAGCTCCTGCCAGGGTCCCGGGAAGAGGTAGGTGCTAGGTGCCCACATTGGCTATCGTATTTTG GAATATTGTTGCCTGAGTCCT |
| mm4_intergenic_Api5|Gm13788_chr2_94474049 |
CCCTTTCTACCTGCCACCTGAAGCAGCCGTTAGTTACTTATGCTTCTGTTCCTCTGTTCCCATCGACAACTCTTCAAGCC CCAGGCAAGTTCCCGGGAAGACGCCATCATTGCTTCTGCTAGACAGGTCTCAGCT |
| mm4_exon_Znrf3_chr11_5278012 |
ATGTGCAATCTGGACCCTGGGATGACTTCCTGGGAGCCTGACTGGGAAGAAGGGTCATTTCTGTCTCTGGCAACCCAGGT GGTCCCCATTCTGTAACCGAGTTTTCAGACCCACTCGGCCAAACTCTTCCT |