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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_52313697_F | TCGTCGGCAGCGTCCGCATCACAAGGTGACCCTA | 59.7 | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313697_R | GTCTCGTGGGCTCGGCTCCTAATCCCAGGCTCCCT | 60.1 | 1.00 |
| mm4_intron_Rbp1_chr9_98429432_F | TCGTCGGCAGCGTCGAGGTGCACAGGACCATAGG | 59.8 | 0.49 |
| mm4_intron_Rbp1_chr9_98429432_R | GTCTCGTGGGCTCGGCCAGACTGGAAAAGTGGGCT | 59.8 | 0.49 |
| mm4_exon_Sqle_chr15_59315970_F | TCGTCGGCAGCGTCCTGGTGTTCCTGTCGCTGG | 60.6 | 0.36 |
| mm4_exon_Sqle_chr15_59315970_R | GTCTCGTGGGCTCGGTATCCGAGAAGGCAGCGAAC | 59.8 | 0.36 |
| mm3_exon_Ctsc_chr7_88278183_F | TCGTCGGCAGCGTCCCATCGAGTGGTGTTCCAGTT | 60.2 | 0.23 |
| mm3_exon_Ctsc_chr7_88278183_R | GTCTCGTGGGCTCGGGTGCAGACTCCCAAAAGCAC | 59.6 | 0.23 |
| mm4_intron_E2f7_chr10_110765978_F | TCGTCGGCAGCGTCCAAACCCAGACAGACCCCTT | 59.5 | 0.13 |
| mm4_intron_E2f7_chr10_110765978_R | GTCTCGTGGGCTCGGAAATGCACAGCGATCACACC | 59.4 | 0.13 |
| mm4_intergenic_Hnrnpa0|1700066J03Rik_chr13_58128755_F | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_Hnrnpa0|1700066J03Rik_chr13_58128755_R | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_Uvrag|Gm23479_chr7_98908413_F | TCGTCGGCAGCGTCGGCTCACAAAGGAACACAGC | 59.6 | 0.05 |
| mm4_intergenic_Uvrag|Gm23479_chr7_98908413_R | GTCTCGTGGGCTCGGCCCACAATACAGACATCTCTCCT | 59.2 | 0.05 |
| mm4_intergenic_Fndc3a|Cysltr2_chr14_72710556_F | TCGTCGGCAGCGTCGGACCAGCCGAATCTAGTGG | 59.8 | 0.04 |
| mm4_intergenic_Fndc3a|Cysltr2_chr14_72710556_R | GTCTCGTGGGCTCGGTAAGGAAATTGTCGCCGCCT | 60.0 | 0.04 |
| mm4_exon_Stard3nl_chr13_19395744_F | TCGTCGGCAGCGTCCGGAAAGACAGCCTACCTCC | 59.8 | 0.03 |
| mm4_exon_Stard3nl_chr13_19395744_R | GTCTCGTGGGCTCGGGTTGACGTCACGGACAGGG | 60.6 | 0.03 |
| mm4_exon_Strap_chr6_137735335_F | TCGTCGGCAGCGTCGCAGAGAAGGAGGACGACC | 59.4 | 0.02 |
| mm4_exon_Strap_chr6_137735335_R | GTCTCGTGGGCTCGGCTGAAGGCCAAATCCACCAC | 59.1 | 0.02 |
| mm4_intron_Lmna_chr3_88483080_F | TCGTCGGCAGCGTCGTGTGCATGCAATGGGAGTG | 60.1 | 0.02 |
| mm4_intron_Lmna_chr3_88483080_R | GTCTCGTGGGCTCGGGGAGTGGGGACTTGCTGATG | 60.3 | 0.02 |
| mm4_exon_Gm10676/Mypop_chr7_18992107_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Gm10676/Mypop_chr7_18992107_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Alms1_chr6_85587748_F | TCGTCGGCAGCGTCCCTGAGGACCTGCCGTGG | 62.0 | 0.01 |
| mm4_exon_Alms1_chr6_85587748_R | GTCTCGTGGGCTCGGTCTCCTCAGAGGTCAGGGC | 59.9 | 0.01 |
| mm4_exon_Cacna2d1_chr5_15934688_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Cacna2d1_chr5_15934688_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Calm2|Gm26005_chr17_87447157_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_Calm2|Gm26005_chr17_87447157_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Kcnq3_chr15_66286103_F | TCGTCGGCAGCGTCGACTTGCTCCACGTCTCCTG | 60.3 | 0.00 |
| mm4_exon_Kcnq3_chr15_66286103_R | GTCTCGTGGGCTCGGCGCTAACCCTGCTGGAGG | 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_52313697 |
CGCATCACAAGGTGACCCTAGCTCCCCACTGCCATCTCCGCGGTCGCCGTCGACACGGCGCTGGGGCTACCCGGCGCCTG CCTTGTCGCCTTAGCTCCTCTTCTCAGCCAAGATCCCAGGGAGCCTGGGATTAGGAG |
| mm4_intron_Rbp1_chr9_98429432 |
GAGGTGCACAGGACCATAGGTCACTGACTCTAGCCTACCTCCCCCGCCACCGTCGACATGGTCTAGGATTGCATGAGCTG TCTCTGTGTCCCTAGAGCTCTTAGCCCACTTTTCCAGTCTGG |
| mm4_exon_Sqle_chr15_59315970 |
CTGGTGTTCCTGTCGCTGGGCCTGGTGCTCTCCTACCGCTGTCGCCATCGACACGGGGGCCTCCTTGGCCGCCATCAGAG CGGCGCCCAGTTCGCTGCCTTCTCGGATA |
| mm3_exon_Ctsc_chr7_88278183 |
CCATCGAGTGGTGTTCCAGTTGAACTTGCTTTCTCTGCCATCTGCTCCGCGGGCGCCGTCAGCATGGGTCCCTGGACCCA CTCCTTGCGCGCCGTCCTGCTGCTGGTGCTTTTGGGAGTCTGCAC |
| mm4_intron_E2f7_chr10_110765978 |
CAAACCCAGACAGACCCCTTACTCCATACCTCCGAGGTTACCGTGGACAGGGAGGCAGAACGCACCCGGAGAGCGCAGGC CAGGGAGGGTGTGATCGCTGTGCATTT |
| mm4_intergenic_Uvrag|Gm23479_chr7_98908413 |
GGCTCACAAAGGAACACAGCATCTCCTTTGCTCAGACTGGAGCCTCTTGATTACAGCTCCGAGGTCGCCCACCACACGGG TCTGCAGTTTTCAGCAGAGAATACAGGAGAGATGTCTGTATTGTGGG |
| mm4_intergenic_Fndc3a|Cysltr2_chr14_72710556 |
GGACCAGCCGAATCTAGTGGAAACCCGTTACCTTGACGTCTGCCACCGCGGAGAGCGAAGCGGAGGCGGCGACAATTTCC TTA |
| mm4_exon_Stard3nl_chr13_19395744 |
CGGAAAGACAGCCTACCTCCTCCGCGGCGCGAGCCGCCCGCCGGCCTGCAGACACGGCTCCGCGGTCCCCGCCGGCGGGG CTGGGGCGGGGCTGCAGGGCGGGACCCTGTCCGTGACGTCAAC |
| mm4_exon_Strap_chr6_137735335 |
GCAGAGAAGGAGGACGACCGCGACCCTCGGTCCCCGCTCCGCGTCACCGCGCTCGCCGCCGCCATGGCCATGAGGCAGAC GCCGCTCACTTGCTCGGGCCACACGCGGCCCGTGGTGGATTTGGCCTTCAG |
| mm4_intron_Lmna_chr3_88483080 |
GTGTGCATGCAATGGGAGTGAGGAAGAGAGGCAGGGCTGGGGCCACCTCTGTCCTCCCAGCCCCTTCCCAGGACACAAAT CCAGAACCCTGTCCACTGCCACCGTGGAGTATCCATGGCATCAGCAAGTCCCCACTCC |
| mm4_exon_Alms1_chr6_85587748 |
CCTGAGGACCTGCCGTGGCCTGACGAGCTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNAACGCCTCGGCGGCGGCGACCGAGGAGGCCCTGACCTCTGAGGAGA |
| mm4_exon_Kcnq3_chr15_66286103 |
GACTTGCTCCACGTCTCCTGGCGCCAGCCCCACTTTCCGCTCCTCGTCGCCGGCCACCGCCGAGTCCCCTCCAGCAGGGT TAGCG |