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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_115238029_F | TCGTCGGCAGCGTCGAGCGTGGAGATTGACAGGG | 60.4 | 1.00 |
| mm0_exon_Pop5_chr5_115238029_R | GTCTCGTGGGCTCGGTTACCTGTGCTTGAACCGCA | 60.1 | 1.00 |
| mm3_exon_Cdc34-ps_chr11_94742556_F | TCGTCGGCAGCGTCACATCATCCGGAAGCAGGTC | 59.8 | 0.27 |
| mm3_exon_Cdc34-ps_chr11_94742556_R | GTCTCGTGGGCTCGGGTAGTCGAAGAGGTCCGAGC | 59.6 | 0.27 |
| mm4_intron_St3gal1_chr15_67107187_F | TCGTCGGCAGCGTCGCTTGGAAGGCAGGCAAATC | 60.1 | 0.26 |
| mm4_intron_St3gal1_chr15_67107187_R | GTCTCGTGGGCTCGGAAGCTGCCACCTACCTTCTG | 59.6 | 0.26 |
| mm4_exon_Tcp10b_chr17_13061333_F | TCGTCGGCAGCGTCATGGATGCCAGTCTACCCCT | 60.0 | 0.22 |
| mm4_exon_Tcp10b_chr17_13061333_R | GTCTCGTGGGCTCGGCCCTGAGAGTCAAAGGCACC | 60.3 | 0.22 |
| mm4_intergenic_Gm22382|Gm22169_chr9_68363613_F | TCGTCGGCAGCGTCACGTTACTCACACCCTTGCC | 60.2 | 0.17 |
| mm4_intergenic_Gm22382|Gm22169_chr9_68363613_R | GTCTCGTGGGCTCGGTGGAGCATGTTGCCCTTAGG | 60.0 | 0.17 |
| mm4_intergenic_Gm10561|Mars2_chr1_55237140_F | TCGTCGGCAGCGTCAGGAGTCAACGCTTGCCG | 60.3 | 0.11 |
| mm4_intergenic_Gm10561|Mars2_chr1_55237140_R | GTCTCGTGGGCTCGGCACTGGAGGCGGTGTTCC | 60.3 | 0.11 |
| mm4_intergenic_Gm23026|Exoc6_chr19_37497079_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Gm23026|Exoc6_chr19_37497079_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Casz1_chr4_148938640_F | TCGTCGGCAGCGTCGCAAATACGAGGGCTGCATG | 59.9 | 0.03 |
| mm4_exon_Casz1_chr4_148938640_R | GTCTCGTGGGCTCGGTGTGTGAGGTCATCTGGCTG | 59.6 | 0.03 |
| mm4_intergenic_Gm15425|Pde10a_chr17_8677657_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intergenic_Gm15425|Pde10a_chr17_8677657_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_intron_Dst_chr1_34285194_F | TCGTCGGCAGCGTCTTGGCAGGCCTTCAGAAGAG | 59.9 | 0.00 |
| mm4_intron_Dst_chr1_34285194_R | GTCTCGTGGGCTCGGACCACAAGCTGCTCACAGAT | 59.6 | 0.00 |
| mm4_intron_Tcf20_chr15_82899596_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Tcf20_chr15_82899596_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Trim11|Obscn_chr11_58992177_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Trim11|Obscn_chr11_58992177_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Pabpc1l2a-ps|Gm3928_chrX_103067554_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Pabpc1l2a-ps|Gm3928_chrX_103067554_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm21998|Pabpc1l2b-ps_chrX_103013357_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gm21998|Pabpc1l2b-ps_chrX_103013357_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Thoc7_chr14_13961200_F | TCGTCGGCAGCGTCGGCCTCACCGTCAGTCAC | 60.0 | 0.00 |
| mm4_exon_Thoc7_chr14_13961200_R | GTCTCGTGGGCTCGGCCATGACTGTTTACCCGGCT | 60.0 | 0.00 |
| mm4_exon_Sirt1_chr10_63338907_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Sirt1_chr10_63338907_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Decr1_chr4_15924258_F | TCGTCGGCAGCGTCCAGGCTTCCTCTTGACCAGA | 59.0 | 0.00 |
| mm4_exon_Decr1_chr4_15924258_R | GTCTCGTGGGCTCGGATGCTGAGAGTGGATCAGGC | 59.5 | 0.00 |
| mm4_exon_Atp11a_chr8_12832630_F | TCGTCGGCAGCGTCGGATGATGTCGATGGTCCCC | 59.9 | 0.00 |
| mm4_exon_Atp11a_chr8_12832630_R | GTCTCGTGGGCTCGGATCCTGACTCACCTCTGCAC | 59.0 | 0.00 |
These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.
| mm0_exon_Pop5_chr5_115238029 |
GAGCGTGGAGATTGACAGGGCACGCTGCCTGTCCGAGCCTAGTGCCGCCTTCCTCCCGCATCCGGCGCGGACGCCATGGT GCGGTTCAAGCACAGGTAA |
| mm3_exon_Cdc34-ps_chr11_94742556 |
ACATCATCCGGAAGCAGGTCCTGGGGACCAAGGTGGACGCAGAGCGCGATGGCGTGAAGGTGCCCACTACGCTGGCCGAG TACTGCGTGAAGACCAAGGCGCCGGCGCCGGATGAGGGCTCGGACCTCTTCGACTAC |
| mm4_intron_St3gal1_chr15_67107187 |
GCTTGGAAGGCAGGCAAATCATTCGTCATCCAAGCTGACTCTTGACACATCAGATCATTTCAGAACCTCATTTCCTCATC TGGCACGGAGGCCAAGACACATGGCCACAGAAGGTAGGTGGCAGCTT |
| mm4_exon_Tcp10b_chr17_13061333 |
ATGGATGCCAGTCTACCCCTGCAGAAAACCATGGGGCCCGGGCCAGATGAGGGTGCAGTGGGTTATGAATAAGCTCTGCT ATAGGTGCCTTTGACTCTCAGGG |
| mm4_intergenic_Gm22382|Gm22169_chr9_68363613 |
ACGTTACTCACACCCTTGCCCCTCACACAGGAACAGACCTTAGCTCTTCTGTTCTCATCCCCATCCTGTGCAGACTCCAT GGCTCTACCACCATTTGTGGAGTAAGTCAGCCTCCCTAAGGGCAACATGCTCCA |
| mm4_intergenic_Gm10561|Mars2_chr1_55237140 |
AGGAGTCAACGCTTGCCGTTCATTGGCTTCCGCGCCGGAAGCGGCGGTACCGTCCAGCTGCGCACGCGCCCTTTCCCGTG CTCGGGAACACCGCCTCCAGTG |
| mm4_exon_Casz1_chr4_148938640 |
GCAAATACGAGGGCTGCATGTACAGCAAGGCCACCAACCATTTCCACTGCATCCGCGCCGGCTGCGGCTTCACCTTCACC TCCACCAGCCAGATGACCTCACACA |
| mm4_intron_Dst_chr1_34285194 | TTGGCAGGCCTTCAGAAGAGCGTGAAGCCACAGCCAGCGCGCACGCCAGGGCTGACTTTATCTGTGAGCAGCTTGTGGT |
| mm4_exon_Thoc7_chr14_13961200 |
GGCCTCACCGTCAGTCACGGCTCCCATGGCGTNNNNNNNNNNNNNNNNNNNNNAGCTGAGGCGGCGGTTGGCGGCGAAGG TCAAACTCCCACAATGCAGCCGGGTAAACAGTCATGG |
| mm4_exon_Decr1_chr4_15924258 |
CAGGCTTCCTCTTGACCAGAGAACTTTATCTATTTTTGGCAACACAATTAACTCAGTAGTGCTCACTTATTCATGGCTTC CACGCCTGATTTGGCTGAAGAACTTGGCATTACAAAGCCTGATCCACTCTCAGCAT |
| mm4_exon_Atp11a_chr8_12832630 |
GGATGATGTCGATGGTCCCCAGAAATCTCCAGATGCAAAATCCTGTGTGTACATATCGTCCTCGCCTGATGAGGTTGCAC TGGTCGAAGGCGTGCAGAGGTGAGTCAGGAT |