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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_115238028_F | TCGTCGGCAGCGTCGAGCGTGGAGATTGACAGGG | 60.4 | 1.00 |
| mm0_exon_Pop5_chr5_115238028_R | GTCTCGTGGGCTCGGTTACCTGTGCTTGAACCGCA | 60.1 | 1.00 |
| mm4_exon_Trappc10_chr10_78200699_F | TCGTCGGCAGCGTCCTTCCTGTGGAGCTCTGGTG | 60.0 | 0.20 |
| mm4_exon_Trappc10_chr10_78200699_R | GTCTCGTGGGCTCGGAAGCATCGGTCTTCCCACTG | 60.0 | 0.20 |
| mm4_exon_Cdc34-ps_chr11_94742557_F | TCGTCGGCAGCGTCCCGAGTACTGCGTGAAGACC | 60.4 | 0.18 |
| mm4_exon_Cdc34-ps_chr11_94742557_R | GTCTCGTGGGCTCGGGACTCTTCGGGCCAAAGTCA | 59.9 | 0.18 |
| mm4_exon_Tada1_chr1_166379303_F | Primer3: not found at this Tm | N.d. | 0.15 |
| mm4_exon_Tada1_chr1_166379303_R | Primer3: not found at this Tm | N.d. | 0.15 |
| mm4_exon_BC042782_chr4_95075771_F | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_exon_BC042782_chr4_95075771_R | Primer3: not found at this Tm | N.d. | 0.07 |
| mm3_intergenic_Aldh1l2|A230046K03Rik_chr10_83543844_F | TCGTCGGCAGCGTCGCCCTGAGAACAGTCGTGAA | 59.9 | 0.06 |
| mm3_intergenic_Aldh1l2|A230046K03Rik_chr10_83543844_R | GTCTCGTGGGCTCGGCTGGGAAAGCGAGTCGACC | 60.4 | 0.06 |
| mm3_intergenic_Tmem132c|Slc15a4_chr5_127578328_F | TCGTCGGCAGCGTCCACCTCCAAAAGCCCTTTGC | 59.9 | 0.05 |
| mm3_intergenic_Tmem132c|Slc15a4_chr5_127578328_R | GTCTCGTGGGCTCGGGTGGGCCAGGAGCTGATAAT | 59.5 | 0.05 |
| mm4_intron_Zfp429_chr13_67396367_F | TCGTCGGCAGCGTCCCTGATTCTGAGTTTCTTTAGAGGG | 58.8 | 0.02 |
| mm4_intron_Zfp429_chr13_67396367_R | GTCTCGTGGGCTCGGAGCATGCCTCCTTTGCTCTT | 59.9 | 0.02 |
| mm4_exon_Pole4_chr6_82652816_F | TCGTCGGCAGCGTCCTCTTCCTCTCTGGGCGTCC | 61.6 | 0.01 |
| mm4_exon_Pole4_chr6_82652816_R | GTCTCGTGGGCTCGGAGAGGCGTGGTCTCTACCC | 60.3 | 0.01 |
| mm4_exon_Ccnl1_chr3_65957618_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Ccnl1_chr3_65957618_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.
| mm0_exon_Pop5_chr5_115238028 |
GAGCGTGGAGATTGACAGGGCACGCTGCCTGTCCGAGCCTAGTGCCGCCTTCCTCCCGCATCCGGCGCGGACGCCATGGT GCGGTTCAAGCACAGGTAA |
| mm4_exon_Trappc10_chr10_78200699 |
CTTCCTGTGGAGCTCTGGTGTCCCCGGAACACTGGTGTCCCCGCCAGATGCTGGAGCTGAAGGTAAAGACAGAACCTCCA GCTCAAACTCGATCACGTGGTACGCAGGTGCAGTGGGAAGACCGATGCTT |
| mm4_exon_Cdc34-ps_chr11_94742557 |
CCGAGTACTGCGTGAAGACCAAGGCGCCGGCGCCGGATGAGGGCTCGGACCTCTTCGACTACTACGTGGACGGCGAAGTG GAGGAGGCCGACAGCTGCTTTGGGGATGAAGAGGATGACTTTGGCCCGAAGAGTC |
| mm3_intergenic_Aldh1l2|A230046K03Rik_chr10_83543844 |
GCCCTGAGAACAGTCGTGAACGGCAGCGAGACCAGGAAAGGATGCAGGATGCCAACGGCAGCCATCACTATGGCGGCCGC GCCGGGAGCAGGCGGTCGACTCGCTTTCCCAG |
| mm3_intergenic_Tmem132c|Slc15a4_chr5_127578328 |
CACCTCCAAAAGCCCTTTGCTTCTCCTTCAAAAGATCTCCTGCAGCCGGGGCCGACGCCATTAGGGTGAATTATCAGCTC CTGGCCCAC |
| mm4_intron_Zfp429_chr13_67396367 |
CCTGATTCTGAGTTTCTTTAGAGGGAAAACATGAAACTGAATAGAGAAACAATTTCATTAACAAATGTGGTCCACGCCTG ATGCCGGTGCATTTCTAACAGTCCCACAAGAGCAAAGGAGGCATGCT |
| mm4_exon_Pole4_chr6_82652816 |
CTCTTCCTCTCTGGGCGTCCCGCTCCCAGCAGCCGCCGCTGCCGCCATCCCCTCCCGAAACGTGTGGCGCGCGGTCGCTG ACTGAGCGCGCGCACGCGATTTCGGCCCCTTTCCCGCGCCGGGTAGAGACCACGCCTCT |