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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_52313699_F | TCGTCGGCAGCGTCCGCATCACAAGGTGACCCTA | 59.7 | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313699_R | GTCTCGTGGGCTCGGCTCCTAATCCCAGGCTCCCT | 60.1 | 1.00 |
| mm4_intergenic_Srgap1|Tmem5_chr10_122047802_F | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_intergenic_Srgap1|Tmem5_chr10_122047802_R | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_exon_Yars_chr4_129189944_F | TCGTCGGCAGCGTCGGACTAGCGTGACAGTTCCC | 60.1 | 0.18 |
| mm4_exon_Yars_chr4_129189944_R | GTCTCGTGGGCTCGGGATGCAGCTTCTCCTCAGGG | 60.1 | 0.18 |
| mm3_exon_Sqle_chr15_59315972_F | TCGTCGGCAGCGTCCTGGTGTTCCTGTCGCTGG | 60.6 | 0.16 |
| mm3_exon_Sqle_chr15_59315972_R | GTCTCGTGGGCTCGGTATCCGAGAAGGCAGCGAAC | 59.8 | 0.16 |
| mm4_exon_Alms1_chr6_85587746_F | TCGTCGGCAGCGTCCCTGAGGACCTGCCGTGG | 62.0 | 0.13 |
| mm4_exon_Alms1_chr6_85587746_R | GTCTCGTGGGCTCGGTCTCCTCAGAGGTCAGGGC | 59.9 | 0.13 |
| mm4_exon_Gm10676/Mypop_chr7_18992105_F | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_exon_Gm10676/Mypop_chr7_18992105_R | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intron_Fgfr4_chr13_55153001_F | TCGTCGGCAGCGTCGCCACACACCGTACTCCC | 60.0 | 0.07 |
| mm4_intron_Fgfr4_chr13_55153001_R | GTCTCGTGGGCTCGGGCGGGTTACTGATTTACCGC | 59.3 | 0.07 |
| mm4_exon_Slc15a4_chr5_127617305_F | TCGTCGGCAGCGTCCAGCAGCTCGGCCAACAG | 61.4 | 0.06 |
| mm4_exon_Slc15a4_chr5_127617305_R | GTCTCGTGGGCTCGGGAGTCCTCAGTGAACCGGC | 60.0 | 0.06 |
| mm4_exon_Cacna2d1_chr5_15934686_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_exon_Cacna2d1_chr5_15934686_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_exon_Foxa2_chr2_148044629_F | TCGTCGGCAGCGTCAGACTCGGACTCAGGTGAGG | 60.3 | 0.04 |
| mm4_exon_Foxa2_chr2_148044629_R | GTCTCGTGGGCTCGGTATGTGGGCGCTGGAATGAG | 60.1 | 0.04 |
| mm4_intergenic_Gm20837|Gm21860_chrY_90744443_F | TCGTCGGCAGCGTCGGTACAACCTGGAGCGGC | 60.4 | 0.03 |
| mm4_intergenic_Gm20837|Gm21860_chrY_90744443_R | GTCTCGTGGGCTCGGGACGGATCTGGGACCCGAAG | 62.0 | 0.03 |
| mm4_intergenic_Erdr1|Gm21748_chrY_90828804_F | TCGTCGGCAGCGTCGCTCTGTCCCTGCACTTCTA | 59.1 | 0.03 |
| mm4_intergenic_Erdr1|Gm21748_chrY_90828804_R | GTCTCGTGGGCTCGGGACGGATCTGGGACCCGAAG | 62.0 | 0.03 |
| mm4_exon_Uncx_chr5_139547932_F | TCGTCGGCAGCGTCACGAATCAGGTGATCGGCTC | 59.8 | 0.02 |
| mm4_exon_Uncx_chr5_139547932_R | GTCTCGTGGGCTCGGTCACCCCAGGACCAGCAG | 60.9 | 0.02 |
| mm4_exon_Pou3f1_chr4_124658116_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_exon_Pou3f1_chr4_124658116_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Txlng_chrX_162828636_F | TCGTCGGCAGCGTCCCAAGCCCCGTTGTGGAT | 59.9 | 0.00 |
| mm4_intron_Txlng_chrX_162828636_R | GTCTCGTGGGCTCGGAGATAACAGCGCAGAAGCCG | 60.8 | 0.00 |
| mm4_intergenic_Tapt1|Ldb2_chr5_44226767_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Tapt1|Ldb2_chr5_44226767_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Calm2|Gm26005_chr17_87447159_F | TCGTCGGCAGCGTCCCTGGCAGGAGCTACTTTGC | 61.0 | 0.00 |
| mm4_intergenic_Calm2|Gm26005_chr17_87447159_R | GTCTCGTGGGCTCGGAGTGACGCATTGCAGGTACA | 59.9 | 0.00 |
| mm4_exon_Cxxc5_chr18_35858656_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Cxxc5_chr18_35858656_R | Primer3: not found at this Tm | N.d. | 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_52313699 |
CGCATCACAAGGTGACCCTAGCTCCCCACTGCCATCTCCGCGGTCGCCGTCGACACGGCGCTGGGGCTACCCGGCGCCTG CCTTGTCGCCTTAGCTCCTCTTCTCAGCCAAGATCCCAGGGAGCCTGGGATTAGGAG |
| mm4_exon_Yars_chr4_129189944 |
GGACTAGCGTGACAGTTCCCGGCACGCGGAGACGACGGCGAACGAGGAAAGGGCGCGGGTGCCGGCTGAGCGCGGGAAAC AGAGACAGCGGAGCCATGGGTGATGCTCCAAGCCCTGAGGAGAAGCTGCATC |
| mm3_exon_Sqle_chr15_59315972 |
CTGGTGTTCCTGTCGCTGGGCCTGGTGCTCTCCTACCGCTGTCGCCATCGACACGGGGGCCTCCTTGGCCGCCATCAGAG CGGCGCCCAGTTCGCTGCCTTCTCGGATA |
| mm4_exon_Alms1_chr6_85587746 |
CCTGAGGACCTGCCGTGGCCTGACGAGCTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNAACGCCTCGGCGGCGGCGACCGAGGAGGCCCTGACCTCTGAGGAGA |
| mm4_intron_Fgfr4_chr13_55153001 |
GCCACACACCGTACTCCCGGCCGCCGTGGGGACAGCGGCCGTGGAGCTCCCGAGCTGCCCCCTCTGTCCCGGGGACCTTG GCGCCCGCGCGGCGGGAGTGGTTGGGTGGAGGGGGCGGTAAATCAGTAACCCGC |
| mm4_exon_Slc15a4_chr5_127617305 |
CAGCAGCTCGGCCAACAGCACCGCCCCGCACGCCGCACGCCGCCCCGCGAACACCGCGGACGCCGCCGACACTGCCGGAC GCCGCGAACCCAACAGCGGCGCCCGCTCGCCCTCCATGGCCGGTTCACTGAGGACTC |
| mm4_exon_Foxa2_chr2_148044629 |
AGACTCGGACTCAGGTGAGGTCCCATGCCCGCCACGCCGGCCGCCCCGGCTGAGCCGCTCATGCCCGCCATGGCGCCGGC GCCCGGGGACATGCCAGCTAGCGACGGGCTCATTCCAGCGCCCACATA |
| mm4_intergenic_Gm20837|Gm21860_chrY_90744443 |
GGTACAACCTGGAGCGGCGGGCGCGGACCTGAACCCCCGTCTGCCCGGGGACGCCGCCGACACGGCCACACCGCTCGTCT GGAACCAGGGCTGGCCTTCGGGTCCCAGATCCGTC |
| mm4_intergenic_Erdr1|Gm21748_chrY_90828804 |
GCTCTGTCCCTGCACTTCTACAGGTACAACCTGGAGCGGCGGCCGCGGACCTGAACCCCCGTCTGCCCGGGGACGCCGCC GACACGGCCGCACCGCAGGTCTGGAACCGCGGCTGGCCTTCGGGTCCCAGATCCGTC |
| mm4_exon_Uncx_chr5_139547932 |
ACGAATCAGGTGATCGGCTCTTAGAGACATTGCTTTTCCCTCTTGCTTTTTGTTTTCTTCCTTTTATTATTATTTTTAAA GGCAAACGAAAACGCTGTGTCGATGGGGACCAAGGCGGCAGCTGCTGGTCCTGGGGTGA |
| mm4_intron_Txlng_chrX_162828636 |
CCAAGCCCCGTTGTGGATCCCGGGAGCCCCGCGGCGTCCCCCGCGTAGCCACCCGCGCAGTGTCGCGGGGGACCGCGGGG GGGCAAGGGGAAGGGGGGAAGGGGCGAAAGGCAGCCCCGGCTTCTGCGCTGTTATCT |
| mm4_intergenic_Calm2|Gm26005_chr17_87447159 |
CCTGGCAGGAGCTACTTTGCGGCGCGGAGGAGCACCGCGGTGGCGGTCGACCAGGCGAACCTCGTTGAGCTTGCTGCTGA TTGGCTGATTTGTACCTGCAATGCGTCACT |