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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 |
|---|---|---|---|
| mm0_exon_Pop5_chr5_115238025_F | TCGTCGGCAGCGTCGAGCGTGGAGATTGACAGGG | 60.4 | 1.00 |
| mm0_exon_Pop5_chr5_115238025_R | GTCTCGTGGGCTCGGTTACCTGTGCTTGAACCGCA | 60.1 | 1.00 |
| mm4_exon_Ergic3_chr2_156008073_F | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_exon_Ergic3_chr2_156008073_R | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_exon_Zfp691_chr4_119170640_F | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_exon_Zfp691_chr4_119170640_R | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_intergenic_Cdkal1|E2f3_chr13_29868984_F | TCGTCGGCAGCGTCGTTTGTGCTGCTTCACCTGG | 59.9 | 0.23 |
| mm4_intergenic_Cdkal1|E2f3_chr13_29868984_R | GTCTCGTGGGCTCGGCTTTCTCAGGAGAAGTGCGGT | 59.9 | 0.23 |
| mm4_intergenic_Gm22825|Zfhx3_chr8_107860184_F | TCGTCGGCAGCGTCTGCCTTGGAAATGCGGTAGT | 59.9 | 0.20 |
| mm4_intergenic_Gm22825|Zfhx3_chr8_107860184_R | GTCTCGTGGGCTCGGACAAACTGTGCACCAGACCA | 60.0 | 0.20 |
| mm3_exon_Mtmr12_chr15_12205031_F | Primer3: not found at this Tm | N.d. | 0.20 |
| mm3_exon_Mtmr12_chr15_12205031_R | Primer3: not found at this Tm | N.d. | 0.20 |
| mm4_intergenic_Gxylt1|Yaf2_chr15_93275331_F | Primer3: not found at this Tm | N.d. | 0.19 |
| mm4_intergenic_Gxylt1|Yaf2_chr15_93275331_R | Primer3: not found at this Tm | N.d. | 0.19 |
| mm4_exon_Cnot8_chr11_58104177_F | TCGTCGGCAGCGTCGTCGAGCTGGGTTTCTGGAG | 60.3 | 0.18 |
| mm4_exon_Cnot8_chr11_58104177_R | GTCTCGTGGGCTCGGCCTAACCTGCTTACCCTGGC | 60.1 | 0.18 |
| mm4_exon_Keap1_chr9_21239044_F | TCGTCGGCAGCGTCCTTGGAGAAGGGCACGCG | 61.1 | 0.13 |
| mm4_exon_Keap1_chr9_21239044_R | GTCTCGTGGGCTCGGGGGTGGGACGGAGGTGAG | 61.3 | 0.13 |
| mm4_intergenic_Aldh1l2|A230046K03Rik_chr10_83543847_F | Primer3: not found at this Tm | N.d. | 0.11 |
| mm4_intergenic_Aldh1l2|A230046K03Rik_chr10_83543847_R | Primer3: not found at this Tm | N.d. | 0.11 |
| mm4_intron_Dus3l_chr17_56764985_F | Primer3: not found at this Tm | N.d. | 0.11 |
| mm4_intron_Dus3l_chr17_56764985_R | Primer3: not found at this Tm | N.d. | 0.11 |
| mm4_exon_Glra1_chr11_55607952_F | TCGTCGGCAGCGTCCAGCCAGGCAGCTAAGGTAC | 60.4 | 0.07 |
| mm4_exon_Glra1_chr11_55607952_R | GTCTCGTGGGCTCGGCCTCCAGCGAGTAGAGCTTC | 59.6 | 0.07 |
| mm4_intron_H2afv_chr11_6444181_F | TCGTCGGCAGCGTCCTCAGAACTCGCAGGGCTAC | 60.1 | 0.07 |
| mm4_intron_H2afv_chr11_6444181_R | GTCTCGTGGGCTCGGCTGTTCCGGACTCCGCCG | 62.7 | 0.07 |
| mm4_intergenic_Plekho2|Pif1_chr9_65580936_F | TCGTCGGCAGCGTCTGAAGAAACAGGCTCGGAGA | 58.6 | 0.06 |
| mm4_intergenic_Plekho2|Pif1_chr9_65580936_R | GTCTCGTGGGCTCGGGCATGCAGCACACCTGACTA | 60.6 | 0.06 |
| mm4_intergenic_Gm26839|Efcab11_chr12_99627940_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Gm26839|Efcab11_chr12_99627940_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Mir181c|Gm26887_chr8_84183687_F | TCGTCGGCAGCGTCGAGAACAAAGCGCCCCGG | 61.4 | 0.06 |
| mm4_intergenic_Mir181c|Gm26887_chr8_84183687_R | GTCTCGTGGGCTCGGGAGGCCTCCATTGTTCCCG | 60.4 | 0.06 |
| mm4_intron_Cd63_chr10_128910497_F | TCGTCGGCAGCGTCCTCTACGTTCTCCTGCTGGC | 60.1 | 0.04 |
| mm4_intron_Cd63_chr10_128910497_R | GTCTCGTGGGCTCGGCCTCCAGCTGCCTTCATCTC | 60.1 | 0.04 |
| mm4_exon_Basp1_chr15_25364553_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Basp1_chr15_25364553_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Pdss2|Bend3_chr10_43478917_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Pdss2|Bend3_chr10_43478917_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Eif3j1_chr2_122028941_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Eif3j1_chr2_122028941_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Mex3a_chr3_88532618_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Mex3a_chr3_88532618_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Tada1_chr1_166379300_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Tada1_chr1_166379300_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Klf10_chr15_38299559_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Klf10_chr15_38299559_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Adss|Gm16432_chr1_177983519_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Adss|Gm16432_chr1_177983519_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Desi2_chr1_178187771_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Desi2_chr1_178187771_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Olig2_chr16_91227065_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_exon_Olig2_chr16_91227065_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Kbtbd12_chr6_88627607_F | TCGTCGGCAGCGTCCCAGAGGTAACAGGTGCTCG | 60.1 | 0.02 |
| mm4_intron_Kbtbd12_chr6_88627607_R | GTCTCGTGGGCTCGGCTCTGGGGTCTGTGTGGAAC | 59.9 | 0.02 |
| mm4_exon_Sp9_chr2_73273466_F | TCGTCGGCAGCGTCACTATGGCGGCCTTTTCTCC | 60.1 | 0.02 |
| mm4_exon_Sp9_chr2_73273466_R | GTCTCGTGGGCTCGGTTGGAGATGAAGGCCGACTG | 59.7 | 0.02 |
| mm4_intergenic_Dgkh|Vwa8_chr14_78725735_F | TCGTCGGCAGCGTCGCTAGGACCTTACCTCCCCA | 60.0 | 0.02 |
| mm4_intergenic_Dgkh|Vwa8_chr14_78725735_R | GTCTCGTGGGCTCGGACCTCCCGGAAAATGCTCTG | 60.0 | 0.02 |
| mm4_intron_Mthfd1l_chr10_3973741_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Mthfd1l_chr10_3973741_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_exon_Etv6_chr6_134035805_F | TCGTCGGCAGCGTCTCCAGGAAGGAAAACATTCGAGA | 59.6 | 0.02 |
| mm4_exon_Etv6_chr6_134035805_R | GTCTCGTGGGCTCGGGGCCTGGCGTGTCTCCTC | 62.4 | 0.02 |
| mm4_intron_Car10_chr11_93098740_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Car10_chr11_93098740_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_exon_Slc24a3_chr2_145168002_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Slc24a3_chr2_145168002_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Hsdl1_chr8_119575124_F | TCGTCGGCAGCGTCAATACCTGCAGCGAGAGGC | 59.8 | 0.01 |
| mm4_exon_Hsdl1_chr8_119575124_R | GTCTCGTGGGCTCGGGATCACGTGGGTAGCGGAC | 60.2 | 0.01 |
| mm4_intergenic_Tbx1|4930588K23Rik_chr16_18588887_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Tbx1|4930588K23Rik_chr16_18588887_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Usp12|Rpl21_chr5_146795012_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Usp12|Rpl21_chr5_146795012_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm3_intergenic_Gm26887|Mir23a_chr8_84198423_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm3_intergenic_Gm26887|Mir23a_chr8_84198423_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Otogl|Ppp1r12a_chr10_108162129_F | TCGTCGGCAGCGTCGAAGTTACGCTCAGCCGGG | 60.8 | 0.01 |
| mm4_intergenic_Otogl|Ppp1r12a_chr10_108162129_R | GTCTCGTGGGCTCGGGGGCCTCTCTTCCTACCACA | 60.6 | 0.01 |
| mm4_exon_Zbtb22_chr17_33918564_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Zbtb22_chr17_33918564_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Gm17669|Cep76_chr18_67610394_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm17669|Cep76_chr18_67610394_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Syt3|Lrrc4b_chr7_44428938_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Syt3|Lrrc4b_chr7_44428938_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Fam83d|Dhx35_chr2_158794780_F | TCGTCGGCAGCGTCGAAGGCGTGGCTTCTTTTCG | 60.1 | 0.00 |
| mm4_intergenic_Fam83d|Dhx35_chr2_158794780_R | GTCTCGTGGGCTCGGAGAAGACTCCGCCCCTCTAG | 60.1 | 0.00 |
| mm3_intergenic_Mrps6/Slc5a3|Slc5a3_chr16_92058651_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm3_intergenic_Mrps6/Slc5a3|Slc5a3_chr16_92058651_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_4930404H24Rik|8430427H17Rik_chr2_153528277_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_4930404H24Rik|8430427H17Rik_chr2_153528277_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Brsk2_chr7_141950181_F | TCGTCGGCAGCGTCGCAGTATGTGGGGCCCTAC | 59.8 | 0.00 |
| mm4_intron_Brsk2_chr7_141950181_R | GTCTCGTGGGCTCGGGTCAGCACGGTCTCCCTG | 59.7 | 0.00 |
| mm4_intergenic_Gadd45b|Gng7_chr10_80936648_F | TCGTCGGCAGCGTCCAGCCTTTCTCTGGTGCCC | 60.6 | 0.00 |
| mm4_intergenic_Gadd45b|Gng7_chr10_80936648_R | GTCTCGTGGGCTCGGCGTTCACAAAGGGAGGGTCT | 59.6 | 0.00 |
| mm4_intergenic_Sav1|Gm23579_chr12_69987136_F | TCGTCGGCAGCGTCACGTCCGGTAAACTTTCCCC | 59.9 | 0.00 |
| mm4_intergenic_Sav1|Gm23579_chr12_69987136_R | GTCTCGTGGGCTCGGCCACACCCGGCCCGTTAG | 62.7 | 0.00 |
| mm4_intron_Zfp407_chr18_84589365_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Zfp407_chr18_84589365_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Rnf114/Spata2_chr2_167492664_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Rnf114/Spata2_chr2_167492664_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Rab3ip_chr10_116950258_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Rab3ip_chr10_116950258_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Ppp1r37_chr7_19561983_F | TCGTCGGCAGCGTCGTAGGAGACTCAGCTTCGGC | 59.8 | 0.00 |
| mm4_exon_Ppp1r37_chr7_19561983_R | GTCTCGTGGGCTCGGGTGAGGAGGAGGAGGCGG | 61.1 | 0.00 |
| mm3_intergenic_Pigg|Gm10419_chr5_108366867_F | TCGTCGGCAGCGTCACTCCTCCGGGGATAGGATG | 59.8 | 0.00 |
| mm3_intergenic_Pigg|Gm10419_chr5_108366867_R | GTCTCGTGGGCTCGGATCTTTGGGGCGACAACGG | 60.6 | 0.00 |
These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.
| mm0_exon_Pop5_chr5_115238025 |
GAGCGTGGAGATTGACAGGGCACGCTGCCTGTCCGAGCCTAGTGCCGCCTTCCTCCCGCATCCGGCGCGGACGCCATGGT GCGGTTCAAGCACAGGTAA |
| mm4_intergenic_Cdkal1|E2f3_chr13_29868984 |
GTTTGTGCTGCTTCACCTGGCCCCACGCTTCCGGTGCAGACGCAGTGAATGACATTTACTGCGTTCCCTTATTTTACAAT CTACTCTCCTTATTCTGACCCAGTGTTATACCGCACTTCTCCTGAGAAAG |
| mm4_intergenic_Gm22825|Zfhx3_chr8_107860184 |
TGCCTTGGAAATGCGGTAGTTAGTGAATAAGTGTTCATGTCCATAGGACGCTGCTTCTGCGCCGGATGCTTGAGGGTCCA TATTAAAGACAGACTTGGTCTGGTGCACAGTTTGT |
| mm4_exon_Cnot8_chr11_58104177 |
GTCGAGCTGGGTTTCTGGAGCGGATCCGGGGCAAGAGGGAGGAGAGCTCGGGGTCAAGGGCCGCTGCCGCCCGCACTCGG CGGGGCCGCCAGCCAGGGTAAGCAGGTTAGG |
| mm4_exon_Keap1_chr9_21239044 |
CTTGGAGAAGGGCACGCGCTTCCCCCCACCGCGTGCACGGCCCCACGCGCGACCGCGGGCAGCTCCCGGAGCTGGCTCCC CACCGCATCCTCCGCGGGCGCTCGCTCACCTCCGTCCCACCC |
| mm4_exon_Glra1_chr11_55607952 |
CAGCCAGGCAGCTAAGGTACCGCGGACAGCGGCAGTCGTGCAGGTTCAGCACCACGGAGAGCGTCCAGCCCGGCTGCGGG CGGGAAGAGCGCGGCTGAAGCTCTACTCGCTGGAGG |
| mm4_intron_H2afv_chr11_6444181 |
CTCAGAACTCGCAGGGCTACAGCCGGACCTCCTCTGTGCGGCGCAGCACCCGCCCCCCTGCTTCTGCCTCCCGCGTCCGG CGCGCCCCCCCAACCCCCGGCGGAGTCCGGAACAG |
| mm4_intergenic_Plekho2|Pif1_chr9_65580936 |
TGAAGAAACAGGCTCGGAGAGTGACTGTCACAGCCAAACCTCCAGCATCCCGCGCGCGCGCGNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNTATCTTTCCTAGTCAGGTGTGCTGCATGC |
| mm4_intergenic_Mir181c|Gm26887_chr8_84183687 |
GAGAACAAAGCGCCCCGGTCGGCCCGGCCCAGGCGCTCGCGACCGCGGCGGGTGCAGGCGGCGTCCCGGGCCACACGGCC GGGAACAATGGAGGCCTC |
| mm4_intron_Cd63_chr10_128910497 |
CTCTACGTTCTCCTGCTGGCCTTCTGCGTGAGTAGTGCGGGGAGAGCGANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNA GTCTTGGAGCGGGAGCCGTGAAGGGTGAGGGACCCAGAGATGAAGGCAGCTGGAGG |
| mm4_intron_Kbtbd12_chr6_88627607 |
CCAGAGGTAACAGGTGCTCGGCCTCCGCGCGGGAAGCGGCCGGGACACGCGCTGCAGTGTGCGCCTGCGCGGGGTCTTCC CGCGCCTGTTCCACACAGACCCCAGAG |
| mm4_exon_Sp9_chr2_73273466 |
ACTATGGCGGCCTTTTCTCCAACTNNNNNNNNNNNNNNNNNNNNNNNNNCGGCGTGTCCCCGCAGGAGGCGGGCGGCCAG TCGGCCTTCATCTCCAA |
| mm4_intergenic_Dgkh|Vwa8_chr14_78725735 |
GCTAGGACCTTACCTCCCCACCTCTGCCCGGGGGTTCGGGTCCTTTCGGCCTCCTCTACAGTGCTGTTTAGTTTCGGGCC CACCCCCATCCGGCGCACACGGAAGAGCAGAGCATTTTCCGGGAGGT |
| mm4_exon_Etv6_chr6_134035805 |
TCCAGGAAGGAAAACATTCGAGAAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNTGGGAGGAGACACGCCAGGCC |
| mm4_exon_Hsdl1_chr8_119575124 |
AATACCTGCAGCGAGAGGCGCGGTCTCCGAAGCCCGTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACACCACCGC AGGTCCGCTACCCACGTGATC |
| mm4_intergenic_Otogl|Ppp1r12a_chr10_108162129 |
GAAGTTACGCTCAGCCGGGCCGCGGCTGACGGACTGACGGGCGGGCGGCCGTGCCGGGGGCGGGAGGGCGTCTGTGGTAG GAAGAGAGGCCC |
| mm4_intergenic_Fam83d|Dhx35_chr2_158794780 |
GAAGGCGTGGCTTCTTTTCGCTCTACCCGCCCCCCGCAGCCCCCGCGGAGGCTGCTGGCGGGCCACGCCCCTTGAGCCCG GAGGGACTCGCGCGGGTCTAGAGGGGCGGAGTCTTCT |
| mm4_intron_Brsk2_chr7_141950181 |
GCAGTATGTGGGGCCCTACCGGCTGGAGAAGACGCTGGGCAAGGGGCAGACAGGTGCGTNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGGGGCCAGGGAGACCGTGCTGAC |
| mm4_intergenic_Gadd45b|Gng7_chr10_80936648 |
CAGCCTTTCTCTGGTGCCCGCCAGGCCGGGTGTCTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNTTCGCACAGACCCTCCCTTTGTGAACG |
| mm4_intergenic_Sav1|Gm23579_chr12_69987136 |
ACGTCCGGTAAACTTTCCCCGCGCCGCTGGGAATGCTGGGAAAGCCGACGCGCANNNNNNNNNNNNNNNNNNNNNNNNNN NNACGCGGCTGCGTGCGGCGACTCGCGACGGGCGGGCGGCTAACGGGCCGGGTGTGG |
| mm4_exon_Ppp1r37_chr7_19561983 |
GTAGGAGACTCAGCTTCGGCGGAGGCCTCCTCGGTCTCGGCCTCCGCGTCCGCGTCGGCGTCCGCGCCCGGCCCGGGCGG AGCCTCCTGCGGCGGGATCTCCATCGCCGCCGCCTCCTCCTCCTCAC |
| mm3_intergenic_Pigg|Gm10419_chr5_108366867 |
ACTCCTCCGGGGATAGGATGGAGGCCTCGCTGGGCCGTGGCTGCAGCCGCCGCCCCCTTTTGCCCCGGCGGCCGCGCCGG GTCCGGGAGGCTAGCGGCTGAGCGCGGAGCCCGGGTCCGCCCCGTTGTCGCCCCAAAGAT |