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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_52313545_F | Primer3: not found at this Tm | N.d. | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313545_R | Primer3: not found at this Tm | N.d. | 1.00 |
| mm4_intergenic_Gm17644|Sulf1_chr1_12689122_F | TCGTCGGCAGCGTCTCACCTCCCTCTACACTCGG | 60.0 | 0.30 |
| mm4_intergenic_Gm17644|Sulf1_chr1_12689122_R | GTCTCGTGGGCTCGGACACTCAAAGCATCCTGTGTCT | 59.8 | 0.30 |
| mm4_exon_Catsper2/Pdia3_chr2_121414199_F | TCGTCGGCAGCGTCGGGCTCATGCTAGTCGAGTT | 59.5 | 0.20 |
| mm4_exon_Catsper2/Pdia3_chr2_121414199_R | GTCTCGTGGGCTCGGGCCAGGCCTGCTAGATAGTG | 59.9 | 0.20 |
| mm4_intron_Kdm3b_chr18_34787278_F | Primer3: not found at this Tm | N.d. | 0.20 |
| mm4_intron_Kdm3b_chr18_34787278_R | Primer3: not found at this Tm | N.d. | 0.20 |
| mm4_exon_B4galt3_chr1_171272542_F | TCGTCGGCAGCGTCTGTGCCTCCGATTTCTGACC | 60.0 | 0.13 |
| mm4_exon_B4galt3_chr1_171272542_R | GTCTCGTGGGCTCGGCTGATTCCACCCGGGGATTC | 60.1 | 0.13 |
| mm4_intergenic_Wnt7a|Gm4575_chr6_91422246_F | TCGTCGGCAGCGTCAGGGCACTCAAAGGTCGAAG | 59.9 | 0.11 |
| mm4_intergenic_Wnt7a|Gm4575_chr6_91422246_R | GTCTCGTGGGCTCGGTAGTGGCCTCCCATCTCCTC | 60.1 | 0.11 |
| mm4_exon_Npr3_chr15_11895766_F | TCGTCGGCAGCGTCAGCGTAGTCTCCACTGGTCA | 60.2 | 0.11 |
| mm4_exon_Npr3_chr15_11895766_R | GTCTCGTGGGCTCGGATTCCTCCTGCTGTGTGTGG | 59.9 | 0.11 |
| mm4_intergenic_Idi2|Gtpbp4_chr13_8968549_F | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intergenic_Idi2|Gtpbp4_chr13_8968549_R | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_exon_Syt8_chr7_142440209_F | TCGTCGGCAGCGTCCTTCTGGCCAACCCCTACAG | 60.0 | 0.09 |
| mm4_exon_Syt8_chr7_142440209_R | GTCTCGTGGGCTCGGGGCCAGAACACGATCCACTT | 60.3 | 0.09 |
| mm4_intergenic_AC113989.1|Gm24780_chr3_33313286_F | TCGTCGGCAGCGTCTCGGAAACCTTTAGCTGGTGT | 59.5 | 0.08 |
| mm4_intergenic_AC113989.1|Gm24780_chr3_33313286_R | GTCTCGTGGGCTCGGAGTGACTTGCGGAGAAGACA | 58.9 | 0.08 |
| mm4_exon_Zfp593/E130218I03Rik_chr4_134245579_F | TCGTCGGCAGCGTCGACACCCACACACAAGACCA | 60.1 | 0.07 |
| mm4_exon_Zfp593/E130218I03Rik_chr4_134245579_R | GTCTCGTGGGCTCGGGCAGTCTGTCCAATCCTGCT | 60.0 | 0.07 |
| mm4_intron_4930550C17Rik_chr13_56232450_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intron_4930550C17Rik_chr13_56232450_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm3_intergenic_Ptafr|Eya3_chr4_132587165_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm3_intergenic_Ptafr|Eya3_chr4_132587165_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intergenic_Fnbp1l|Pde5a_chr3_122628398_F | TCGTCGGCAGCGTCTCTCCCATCCGTGTCTCTGT | 59.9 | 0.01 |
| mm4_intergenic_Fnbp1l|Pde5a_chr3_122628398_R | GTCTCGTGGGCTCGGCCATGACTGCTGTGTTCCCA | 60.2 | 0.01 |
| mm4_intron_Pomt2_chr12_87122693_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Pomt2_chr12_87122693_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Cdkn2a_chr4_89282171_F | TCGTCGGCAGCGTCGATGGCTCTCCTCGAGTTCG | 59.9 | 0.00 |
| mm4_exon_Cdkn2a_chr4_89282171_R | GTCTCGTGGGCTCGGCAGGTCAGGAGCAGAGTGTG | 60.0 | 0.00 |
| mm4_intron_Foxo3_chr10_42192485_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Foxo3_chr10_42192485_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Rtn4r_chr16_18150640_F | TCGTCGGCAGCGTCCTGGCAGCCTTGGACTCTG | 60.3 | 0.00 |
| mm4_intron_Rtn4r_chr16_18150640_R | GTCTCGTGGGCTCGGGCTTCCTGTGGGCAGAGAAA | 60.2 | 0.00 |
| mm4_intron_Grlf1_chr7_16517419_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Grlf1_chr7_16517419_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_2610037D02Rik_chr15_96185620_F | TCGTCGGCAGCGTCACTCTGCTCTCTCTGGGCTT | 60.2 | 0.00 |
| mm4_intron_2610037D02Rik_chr15_96185620_R | GTCTCGTGGGCTCGGTGTGACTGGAGAGAGAGGTCA | 59.5 | 0.00 |
| mm4_intergenic_Rfwd3|Mlkl_chr8_111300252_F | TCGTCGGCAGCGTCCAGCTCTTCTGGACCCTGAG | 59.4 | 0.00 |
| mm4_intergenic_Rfwd3|Mlkl_chr8_111300252_R | GTCTCGTGGGCTCGGTGAGGGCGCCACTATTTCAG | 60.1 | 0.00 |
| mm4_intergenic_Gm24149|E030025P04Rik_chr11_109068884_F | TCGTCGGCAGCGTCTTGTGGGAGTTGGATGCTGG | 60.2 | 0.00 |
| mm4_intergenic_Gm24149|E030025P04Rik_chr11_109068884_R | GTCTCGTGGGCTCGGACGTGAGAGTCATGCCGATG | 60.1 | 0.00 |
These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.
| mm4_intergenic_Gm17644|Sulf1_chr1_12689122 |
TCACCTCCCTCTACACTCGGGCCATCTCTCTCAGCCTCCCTGATTACTGGGTTGGTATTCTTGGCCAAGTGGCATCATCA GGGGAAAAGACACAGGATGCTTTGAGTGT |
| mm4_exon_Catsper2/Pdia3_chr2_121414199 |
GGGCTCATGCTAGTCGAGTTCTTCGCCCCCTGGTGAGGCCACTTTGCGGGATCAGGGCCGGAGGACCGGCTGGGCGCGGG AGCGGTTGGGCCTACGCAGCGCGGGCGCCCTCCACTATCTAGCAGGCCTGGC |
| mm4_exon_B4galt3_chr1_171272542 |
TGTGCCTCCGATTTCTGACCTTGCCTTTCCCTACCAACAGTGGGTCCTGTGTCAGTATCCTTTAGCCCAGTGCCGTCACT AGCGGAGATTGTGGAGCGGAATCCCCGGGTGGAATCAG |
| mm4_intergenic_Wnt7a|Gm4575_chr6_91422246 |
AGGGCACTCAAAGGTCGAAGCTAGTAAGTCACCCGAGGTAGCAACTAGGACAGGCGATTGTGAAGACCTGAGGGAACAGT TGTGACCACTGGTGACGTCACACGGGCAACAGAGGAGATGGGAGGCCACTA |
| mm4_exon_Npr3_chr15_11895766 |
AGCGTAGTCTCCACTGGTCATGCCGTGTCTGTGCACCGCCAACATGATTCTCCGAATGGTGTCACCACTGGCACACATGA TCACCACTGGGGAGAGAAACAAAGCGTGAGTCACCCACACACAGCAGGAGGAAT |
| mm4_exon_Syt8_chr7_142440209 |
CTTCTGGCCAACCCCTACAGCACTGGGCAGACATGCTGGCCCATGCTCGGCGGCCCATCGCCCAGTGGCACCACCTGCGG TCCCCCAGGGAAGTGGATCGTGTTCTGGCC |
| mm4_intergenic_AC113989.1|Gm24780_chr3_33313286 | TCGGAAACCTTTAGCTGGTGTCCTCTGGTGACCCGACTCAGTGAGGGCCAGTTTTGATTGTCTTCTCCGCAAGTCACT |
| mm4_exon_Zfp593/E130218I03Rik_chr4_134245579 |
GACACCCACACACAAGACCAAGAAACAGGGAGTAGGGAAAAGACCCACACGTGTTGGCGCTTCCGGGCCGCCCAGTGACG TCACGAGAGGCCCGCCCGGGGGCGGAGCAGGATTGGACAGACTGC |
| mm4_intergenic_Fnbp1l|Pde5a_chr3_122628398 |
TCTCCCATCCGTGTCTCTGTCCCTGGCTCCCACTCCCAATCCATCGTCTGTATGCTCCGACCCCTGCTGACCCCACTAGG CCACGTGTAATAAGGACAGAATTACACATAAGTCTCTGGGAACACAGCAGTCATGG |
| mm4_exon_Cdkn2a_chr4_89282171 |
GATGGCTCTCCTCGAGTTCGCTTTCCTCGCGGAGATTCAGTGCCCGCCCAATCGCCCAGTCGTGTGACCAGTGGGTCCCT CCTTCCTTCCTCTGAGGATGGTGTGGGGGGGGGGGGGAGCCACACTCTGCTCCTGACCTG |
| mm4_intron_Rtn4r_chr16_18150640 |
CTGGCAGCCTTGGACTCTGCCCAGTCCCCTGGAGACCCCACTCGAGGAGAATTGGTTCCAGCATCAACTCTCAGCTCTTA GGACTCCTGTCTGATCATCTTTTTCTCTGCCCACAGGAAGC |
| mm4_intron_2610037D02Rik_chr15_96185620 |
ACTCTGCTCTCTCTGGGCTTCCTGGCTTGGCCGCTTCCAATCAGCCCCCATGGGTTTAATTAGCACTTTGATGCACACAG TTCCCAAAGTGGCGTCTCCAGAGGTGACCTCTCTCTCCAGTCACA |
| mm4_intergenic_Rfwd3|Mlkl_chr8_111300252 |
CAGCTCTTCTGGACCCTGAGGCGCCCCCGCTGGAGCCGCGGCTCGGCGAGCTCCTATTGGCCAGCCACTGCGGACGGCGA CCAATCTCTTCTCTTCTTCTTCCGTCGGTCGCCGCCAGCTGAAATAGTGGCGCCCTCA |
| mm4_intergenic_Gm24149|E030025P04Rik_chr11_109068884 |
TTGTGGGAGTTGGATGCTGGGTCTGGGAAGGGTCTGGGGCAGATGGACCACTGGGGACTCAACTCTGCGACCCCGGGGCT CCATCGGCATGACTCTCACGT |