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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_52314093_F | TCGTCGGCAGCGTCGTCGATGGACTAGCAGGCAG | 60.2 | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52314093_R | GTCTCGTGGGCTCGGAGCTACTAGGTTGCCCTTGG | 59.0 | 1.00 |
| mm4_intron_Nbea_chr3_56045181_F | Primer3: not found at this Tm | N.d. | 0.53 |
| mm4_intron_Nbea_chr3_56045181_R | Primer3: not found at this Tm | N.d. | 0.53 |
| mm4_intergenic_Gm3833|Fhit_chr14_10298016_F | Primer3: not found at this Tm | N.d. | 0.34 |
| mm4_intergenic_Gm3833|Fhit_chr14_10298016_R | Primer3: not found at this Tm | N.d. | 0.34 |
| mm4_intergenic_Gm22939|Cntnap2_chr6_43923645_F | Primer3: not found at this Tm | N.d. | 0.29 |
| mm4_intergenic_Gm22939|Cntnap2_chr6_43923645_R | Primer3: not found at this Tm | N.d. | 0.29 |
| mm4_intergenic_Gm22689|Gm9742_chr13_7629691_F | Primer3: not found at this Tm | N.d. | 0.29 |
| mm4_intergenic_Gm22689|Gm9742_chr13_7629691_R | Primer3: not found at this Tm | N.d. | 0.29 |
| mm4_intergenic_Id2|Gm22527_chr12_25385606_F | TCGTCGGCAGCGTCTGGAACCTGGCTCTGTGTTC | 59.8 | 0.24 |
| mm4_intergenic_Id2|Gm22527_chr12_25385606_R | GTCTCGTGGGCTCGGTAAGCTTGTGAAGACCGGGC | 60.3 | 0.24 |
| mm4_intergenic_Mib1|1010001N08Rik_chr18_10917819_F | Primer3: not found at this Tm | N.d. | 0.20 |
| mm4_intergenic_Mib1|1010001N08Rik_chr18_10917819_R | Primer3: not found at this Tm | N.d. | 0.20 |
| mm4_intergenic_Tnfaip3|Olig3_chr10_19295379_F | TCGTCGGCAGCGTCAGCGTCTAACAGATTCCAGTTCT | 59.4 | 0.17 |
| mm4_intergenic_Tnfaip3|Olig3_chr10_19295379_R | GTCTCGTGGGCTCGGTGAGTGGAACTTGTGAAAGGGA | 59.4 | 0.17 |
| mm4_intergenic_Btla|Cd200_chr16_45276215_F | Primer3: not found at this Tm | N.d. | 0.17 |
| mm4_intergenic_Btla|Cd200_chr16_45276215_R | Primer3: not found at this Tm | N.d. | 0.17 |
| mm4_intron_Grm8_chr6_27568347_F | Primer3: not found at this Tm | N.d. | 0.12 |
| mm4_intron_Grm8_chr6_27568347_R | Primer3: not found at this Tm | N.d. | 0.12 |
| mm4_intron_Grik4_chr9_42754623_F | Primer3: not found at this Tm | N.d. | 0.09 |
| mm4_intron_Grik4_chr9_42754623_R | Primer3: not found at this Tm | N.d. | 0.09 |
| mm4_intron_Gm13003_chr4_137160857_F | TCGTCGGCAGCGTCACAGTGCTCAAGGCTGTGTT | 60.1 | 0.09 |
| mm4_intron_Gm13003_chr4_137160857_R | GTCTCGTGGGCTCGGTTGGGCAAATCTCCATCCCC | 60.0 | 0.09 |
| mm4_intron_Afg3l1_chr8_123499298_F | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intron_Afg3l1_chr8_123499298_R | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intergenic_Otoa|Hs3st2_chr7_121252999_F | TCGTCGGCAGCGTCTAGGGGAGAAAACGATGGCC | 59.4 | 0.08 |
| mm4_intergenic_Otoa|Hs3st2_chr7_121252999_R | GTCTCGTGGGCTCGGTGCAGAGGGATGAAAGGCTG | 60.0 | 0.08 |
| mm4_exon_Brd4_chr17_32229439_F | TCGTCGGCAGCGTCCACAGGGAGGTTCAGCTTGA | 59.6 | 0.08 |
| mm4_exon_Brd4_chr17_32229439_R | GTCTCGTGGGCTCGGTGCTCAGAGTGGTGCTCAAG | 59.9 | 0.08 |
| mm2_exon_Nags/Gm11586_chr11_102145613_F | TCGTCGGCAGCGTCGGTTTGGTTGTCGTCATGGC | 60.0 | 0.08 |
| mm2_exon_Nags/Gm11586_chr11_102145613_R | GTCTCGTGGGCTCGGCTGCCACTCAGTCTCCGTG | 60.0 | 0.08 |
| mm4_intergenic_Gm23041|Nav3_chr10_109650419_F | TCGTCGGCAGCGTCCGCGGTGCAGATGTATCTCT | 59.9 | 0.08 |
| mm4_intergenic_Gm23041|Nav3_chr10_109650419_R | GTCTCGTGGGCTCGGTGCTCTCTCTCGTCACTCCA | 59.9 | 0.08 |
| mm4_intergenic_Rbbp6|Tnrc6a_chr7_123092064_F | TCGTCGGCAGCGTCGGCTAAGAACGTGGGCTCTT | 60.0 | 0.08 |
| mm4_intergenic_Rbbp6|Tnrc6a_chr7_123092064_R | GTCTCGTGGGCTCGGAGATGCCTCTGCTACCCTCA | 60.0 | 0.08 |
| mm4_intergenic_5430425J12Rik|Slc12a7_chr13_73759057_F | TCGTCGGCAGCGTCAAAGGCTGACTCGACTTCCA | 58.9 | 0.07 |
| mm4_intergenic_5430425J12Rik|Slc12a7_chr13_73759057_R | GTCTCGTGGGCTCGGATTTGACCTGATCTGGCGGG | 60.1 | 0.07 |
| mm4_intergenic_Gm25011|Gm23680_chr10_12123729_F | TCGTCGGCAGCGTCCCGTTTGAATGGGCGAATCC | 59.8 | 0.07 |
| mm4_intergenic_Gm25011|Gm23680_chr10_12123729_R | GTCTCGTGGGCTCGGTTAGCCCCTTTCCTTTGGCC | 60.2 | 0.07 |
| mm4_intergenic_Bloc1s2-ps|Cacnb4_chr2_52674843_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Bloc1s2-ps|Cacnb4_chr2_52674843_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intron_Sema5a_chr15_32645368_F | TCGTCGGCAGCGTCGCGTGGTGTGGTTAGCTCTT | 60.6 | 0.06 |
| mm4_intron_Sema5a_chr15_32645368_R | GTCTCGTGGGCTCGGACGCGTCTACAGACACTCAA | 59.0 | 0.06 |
| mm4_exon_Dusp27_chr1_166099804_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_exon_Dusp27_chr1_166099804_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intron_Bcor_chrX_12071471_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intron_Bcor_chrX_12071471_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intron_Gata2_chr6_88200919_F | TCGTCGGCAGCGTCGGTGTGGTGTGCCTCTGTAA | 59.8 | 0.05 |
| mm4_intron_Gata2_chr6_88200919_R | GTCTCGTGGGCTCGGGGTTTCTTCCTCCCCACAGG | 59.9 | 0.05 |
| mm4_intergenic_Gm4487|Gm24073_chr14_114949140_F | TCGTCGGCAGCGTCTGAAAGCTACAGAATGTTCGGA | 57.7 | 0.05 |
| mm4_intergenic_Gm4487|Gm24073_chr14_114949140_R | GTCTCGTGGGCTCGGTGGGGCTTTGTCAGAATGCA | 60.1 | 0.05 |
| mm4_intergenic_Gm23848|Trps1_chr15_50246622_F | TCGTCGGCAGCGTCGAACGACTCTGAGAAAGGCCA | 59.9 | 0.05 |
| mm4_intergenic_Gm23848|Trps1_chr15_50246622_R | GTCTCGTGGGCTCGGTGCAACTCTCCAAGGTCTCA | 58.5 | 0.05 |
| mm4_intergenic_Fam49b|Gm25628_chr15_64016515_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Fam49b|Gm25628_chr15_64016515_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_exon_Camkv_chr9_107947836_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Camkv_chr9_107947836_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Gnptab_chr10_88406676_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Gnptab_chr10_88406676_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Dpf2|Cdc42ep2_chr19_5914583_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Dpf2|Cdc42ep2_chr19_5914583_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Maf/Gm15655|Dynlrb2_chr8_116271201_F | TCGTCGGCAGCGTCGCCAGGCATATGCAGAAGGA | 60.1 | 0.03 |
| mm4_intergenic_Maf/Gm15655|Dynlrb2_chr8_116271201_R | GTCTCGTGGGCTCGGTCAGGAGCGCTCTGACTCTA | 59.7 | 0.03 |
| mm4_intergenic_Trim61/Trim60|Gm10664_chr8_65019128_F | TCGTCGGCAGCGTCAGAAGGTGATTGTGCCCAGG | 59.9 | 0.03 |
| mm4_intergenic_Trim61/Trim60|Gm10664_chr8_65019128_R | GTCTCGTGGGCTCGGGGGGTTGTTGCTTTGTTCTT | 57.0 | 0.03 |
| mm4_intergenic_Prkrir|Wnt11_chr7_98753045_F | TCGTCGGCAGCGTCTTAGACTTGCAGGCTCGGAG | 59.4 | 0.03 |
| mm4_intergenic_Prkrir|Wnt11_chr7_98753045_R | GTCTCGTGGGCTCGGATCCACAACTCCCAGTGTGG | 59.5 | 0.03 |
| mm4_exon_1700016D06Rik_chr8_11678667_F | TCGTCGGCAGCGTCCAGCACAGCAGGAAGACAGA | 59.9 | 0.02 |
| mm4_exon_1700016D06Rik_chr8_11678667_R | GTCTCGTGGGCTCGGGGGGAAGCTGAAGACCTTCC | 60.0 | 0.02 |
| mm4_intergenic_Gm23024|Gm24901_chr1_32877196_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_Gm23024|Gm24901_chr1_32877196_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_Rdh14|Pgk1-rs7_chr12_10768590_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_Rdh14|Pgk1-rs7_chr12_10768590_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_Efna2|Mum1_chr10_80226373_F | TCGTCGGCAGCGTCGAAAACTGGGGCGTGGACTA | 59.9 | 0.02 |
| mm4_intergenic_Efna2|Mum1_chr10_80226373_R | GTCTCGTGGGCTCGGCGCTGCTGGCCAATAAGAAG | 59.6 | 0.02 |
| mm4_intron_Cd200r3_chr16_44975002_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Cd200r3_chr16_44975002_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Kalrn_chr16_34161918_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Kalrn_chr16_34161918_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Itgam_chr7_128071398_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Itgam_chr7_128071398_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Arf3_chr15_98761723_F | TCGTCGGCAGCGTCATGTACTCAAGCGCAGGTCC | 60.1 | 0.00 |
| mm4_intron_Arf3_chr15_98761723_R | GTCTCGTGGGCTCGGAGGGAACTGTCTTTGCTGCC | 60.5 | 0.00 |
| mm4_intergenic_Gm24839|Tmem132c_chr5_126817907_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm24839|Tmem132c_chr5_126817907_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Mpzl1_chr1_165608464_F | TCGTCGGCAGCGTCGTGAGGGATGCGGTCCTTAC | 60.1 | 0.00 |
| mm4_exon_Mpzl1_chr1_165608464_R | GTCTCGTGGGCTCGGAAGGGAAGCTGACTTGCACA | 59.8 | 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_52314093 |
GTCGATGGACTAGCAGGCAGTGCTGCAGGGCTGGGCGCCGAGCCACGGTCTGCTGGAGCGGCCATGCTTGGCCCGGGACC CCCAGTCCCCTCCGCGGACAGCCTCTCTGGCCAAGGGCAACCTAGTAGCT |
| mm4_intergenic_Id2|Gm22527_chr12_25385606 |
TGGAACCTGGCTCTGTGTTCCCACCTGGCCTCCCCAGCAGAACGAGGAGCTGATCATAGCCAGAAATCTTCTTGATCATC ATCTCTATTTCTGAGGCTGAGATGGAACTGTGGCCCGGTCTTCACAAGCTTA |
| mm4_intergenic_Tnfaip3|Olig3_chr10_19295379 |
AGCGTCTAACAGATTCCAGTTCTTTCAGACCCTCGGTCTGCTGGAGGCTTCATTGCAAAGTTTACACCCTTTAGTGGAAA AATTAATTTCCCTTTCACAAGTTCCACTCA |
| mm4_intron_Gm13003_chr4_137160857 |
ACAGTGCTCAAGGCTGTGTTTAGCCAGGATAGGGCCTAGGCCCCCAGTCTGGTGGAGCTGGCATTGTGCCTCGCCCTCCC ATCCAAGGCCTGGGGGATGGAGATTTGCCCAA |
| mm4_intergenic_Otoa|Hs3st2_chr7_121252999 |
TAGGGGAGAAAACGATGGCCGTATTGAGCTTCAGTTCAGTGAAAAATGACCTCCTGATTTATTGGGGAATGATAGGGTGC TGGCCTCTCGGGCAGACCGGGGCAGATCCTGTCAACATGGCAGCCTTTCATCCCTCTGCA |
| mm4_exon_Brd4_chr17_32229439 |
CACAGGGAGGTTCAGCTTGACGGCATCCACGGGCTGCTGGAAAGGCCACGCAAACTGGTGTTTCCATAGTGTCTTGAGCA CCACTCTGAGCA |
| mm2_exon_Nags/Gm11586_chr11_102145613 |
GGTTTGGTTGTCGTCATGGCGACGGCGTGGGTGGCCACAGCCCTGCGGTCTGCTGCAGCGGCCAGAAGGTTGCGCAGCCC GGGAGGTCCTGGGGGCTCACGGAGACTGAGTGGCAG |
| mm4_intergenic_Gm23041|Nav3_chr10_109650419 |
CGCGGTGCAGATGTATCTCTGTGTAGAATGATATTCCATATTCATGAAATGGCAGCTTCAGCAGTCCTGGGACAAGTGAG AAAGTCACCAGAGCTGGAGTGACGAGAGAGAGCA |
| mm4_intergenic_Rbbp6|Tnrc6a_chr7_123092064 |
GGCTAAGAACGTGGGCTCTTGTAGCCAGATGGGGTTGGAGAAGCCAAGAGGAGCAGTGGCCTCTCTAGCAGACTGAGGTA CAGACTAAGATACAGAAGGCCATGAGGGTAGCAGAGGCATCT |
| mm4_intergenic_5430425J12Rik|Slc12a7_chr13_73759057 |
AAAGGCTGACTCGACTTCCATTTTCTTCCTGAGGGCAGCTCAAGCAGAGCGAGGCCCACTTCTACAGTCTGAATTCTGTA CATACAACCCCTGTTCCCGCCAGATCAGGTCAAAT |
| mm4_intergenic_Gm25011|Gm23680_chr10_12123729 |
CCGTTTGAATGGGCGAATCCATTGGTAGCCATCCTTGTGGACCTGATGGTAGCTCTAGCAGACCCAGGGCTTGGTTCCTA TGTCAACTCCATCTCCTTTAGGTGTAGGCCAAAGGAAAGGGGCTAA |
| mm4_intron_Sema5a_chr15_32645368 |
GCGTGGTGTGGTTAGCTCTTAGCTCCTGTCCCATGCCCACTCCAGCAGTCCAGGGGAAAGAAGCTAGATAGGCACATTCC TTTTTGGCCACGAGTTGTACTTTTTACGTATTTCTTGAGTGTCTGTAGACGCGT |
| mm4_intron_Gata2_chr6_88200919 |
GGTGTGGTGTGCCTCTGTAAAAGGGGTCCGTGGTCTGATAGAGGGGCCATGCTAGGGCGCCTGATTTAAGAATGGGTGGT GGTCATCTCTGATAGTCCACAGGCATTTGAAGTTCCCTGTGGGGAGGAAGAAACC |
| mm4_intergenic_Gm4487|Gm24073_chr14_114949140 |
TGAAAGCTACAGAATGTTCGGAATTTTAGGTTTCTCTGAGTATCCCGGGGTCTGACGGAGCGCCCATGAGAAATAGGAAA GAGCACCAATCCTATTTGATGCATTCTGACAAAGCCCCA |
| mm4_intergenic_Gm23848|Trps1_chr15_50246622 |
GAACGACTCTGAGAAAGGCCAACTATTCATATTAAGTATTTCTGTCAATGGGTCAAAAACATGACTCATGATGGCTTTTG GCAGCTCCAGCAGAGAGAGGGATGGTTACTTTTTCCAAATGAGACCTTGGAGAGTTGCA |
| mm4_intergenic_Maf/Gm15655|Dynlrb2_chr8_116271201 |
GCCAGGCATATGCAGAAGGAAAGCTTTCCCCAGGAAAGGCAAGAATCTAACAACAGCTAGAAGGAGAAACCCCCTCTCTG CTGGAAGGGCCATGACTCTTTAGAGTCAGAGCGCTCCTGA |
| mm4_intergenic_Trim61/Trim60|Gm10664_chr8_65019128 |
AGAAGGTGATTGTGCCCAGGTGCATCACCTCGACCCAGAGACACGCCTTAATGGGCACCAAGGGAAGACTGCGGTCTGCT GGAGCGTATCTATTGCCTTACGACATATCTAGATATAAAAGAACAAAGCAACAACCCC |
| mm4_intergenic_Prkrir|Wnt11_chr7_98753045 |
TTAGACTTGCAGGCTCGGAGCAACGTGATAATAGGACCAATTAAATTAATTTATATGGACTCTATGCTTTGGCCCCTCCC GCAGGCCGTGGTGGGCAGACTGACCCACCACACCTTGGCCCACACTGGGAGTTGTGGAT |
| mm4_exon_1700016D06Rik_chr8_11678667 |
CAGCACAGCAGGAAGACAGATAAGAAGGTCATTGCTCCTCTATTCGCTGTCATGGTCCCTCCAGCAGAAGGAGGAGGCCC TTCCCTGGAAGGTCTTCAGCTTCCCC |
| mm4_intergenic_Efna2|Mum1_chr10_80226373 |
GAAAACTGGGGCGTGGACTACGGGCTGAGGCGTGGTCTAGGGAGGCCGCCGCCGTCTGTGGGAGGGGCCATGATGTGAGC TCCATCTTCTTATTGGCCAGCAGCG |
| mm4_intron_Arf3_chr15_98761723 |
ATGTACTCAAGCGCAGGTCCAACATGCCAAAGGCCAACCCAATGGCCACTGCAGCACACAGTGGGGATACTATCCCCAAA CAGGTCTAAGACAAAATGGCAGCAAAGACAGTTCCCT |
| mm4_exon_Mpzl1_chr1_165608464 |
GTGAGGGATGCGGTCCTTACCGACACGGCGCTGTCGGTGCCGTCTGGCTGGAAGCTCCAGGAGACCGTGGTCAACCATCC AGTTGTGTTAGGAGAATCGAATGTGCAAGTCAGCTTCCCTT |