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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.
Name | Primer Sequence | Tm | CFD Score |
---|---|---|---|
ontarget_mm0__chr7 5.53 Mbp_chr7_5529707_F | TCGTCGGCAGCGTCTGGTGAGCTGCGAGAATAGC | 60.1 | 1.00 |
ontarget_mm0__chr7 5.53 Mbp_chr7_5529707_R | GTCTCGTGGGCTCGGCGGCTTCCTTTGTCCCCAAT | 60.6 | 1.00 |
mm4__chr7 39.95 Mbp_chr7_39950089_F | Primer3: not found at this Tm | N.d. | 0.33 |
mm4__chr7 39.95 Mbp_chr7_39950089_R | Primer3: not found at this Tm | N.d. | 0.33 |
mm4__chr10 73.79 Mbp_chr10_73788125_F | Primer3: not found at this Tm | N.d. | 0.31 |
mm4__chr10 73.79 Mbp_chr10_73788125_R | Primer3: not found at this Tm | N.d. | 0.31 |
mm4__chr9 129.84 Mbp_chr9_129835268_F | TCGTCGGCAGCGTCGAGTTGTACAGCCGCCGC | 61.4 | 0.22 |
mm4__chr9 129.84 Mbp_chr9_129835268_R | GTCTCGTGGGCTCGGCTGACGCCAGGAGAGGGG | 61.4 | 0.22 |
mm3__chr5 135.01 Mbp_chr5_135012998_F | TCGTCGGCAGCGTCGGATGGCTCTTCAGGGTGAG | 59.8 | 0.21 |
mm3__chr5 135.01 Mbp_chr5_135012998_R | GTCTCGTGGGCTCGGTATCTACCCCAGCCCAGCTT | 60.0 | 0.21 |
mm4__chr8 143.82 Mbp_chr8_143818330_F | TCGTCGGCAGCGTCAAGCCCTAGACACAGGGACA | 60.1 | 0.21 |
mm4__chr8 143.82 Mbp_chr8_143818330_R | GTCTCGTGGGCTCGGGACTCCGTCACCATGAAGCA | 60.0 | 0.21 |
mm3__chr16 88.47 Mbp_chr16_88473986_F | TCGTCGGCAGCGTCGGTTCTCCGGAGTGAAGCAA | 59.9 | 0.17 |
mm3__chr16 88.47 Mbp_chr16_88473986_R | GTCTCGTGGGCTCGGTATCGACGCTGAAGGTTGGC | 60.4 | 0.17 |
mm4__chr3 170.42 Mbp_chr3_170419651_F | TCGTCGGCAGCGTCTAATTACTGCGGTCCCAGCC | 59.8 | 0.17 |
mm4__chr3 170.42 Mbp_chr3_170419651_R | GTCTCGTGGGCTCGGCACGCTCCTTCTCACCTCTG | 60.1 | 0.17 |
mm4__chr9 21.97 Mbp_chr9_21968437_F | TCGTCGGCAGCGTCGTCCAGCAGCTAGTCCACTG | 60.1 | 0.15 |
mm4__chr9 21.97 Mbp_chr9_21968437_R | GTCTCGTGGGCTCGGCTTTCTCTGCCCTCCGCC | 60.1 | 0.15 |
mm4__chr1 898.94 Kbp_chr1_898937_F | TCGTCGGCAGCGTCCGTCTTCCAGAACGCACTCT | 60.0 | 0.15 |
mm4__chr1 898.94 Kbp_chr1_898937_R | GTCTCGTGGGCTCGGAGCAAAGCTGAAATGAGCGC | 60.1 | 0.15 |
mm4__chr17 76.74 Mbp_chr17_76736615_F | Primer3: not found at this Tm | N.d. | 0.15 |
mm4__chr17 76.74 Mbp_chr17_76736615_R | Primer3: not found at this Tm | N.d. | 0.15 |
mm4__chr19 18.94 Mbp_chr19_18941059_F | Primer3: not found at this Tm | N.d. | 0.14 |
mm4__chr19 18.94 Mbp_chr19_18941059_R | Primer3: not found at this Tm | N.d. | 0.14 |
mm4__chr8 22.97 Mbp_chr8_22973980_F | TCGTCGGCAGCGTCCATCAACCTCACCACCCCTC | 60.0 | 0.11 |
mm4__chr8 22.97 Mbp_chr8_22973980_R | GTCTCGTGGGCTCGGAGTGATCACATGGCTCCCAG | 59.4 | 0.11 |
mm4__chr7 44.89 Mbp_chr7_44885527_F | Primer3: not found at this Tm | N.d. | 0.10 |
mm4__chr7 44.89 Mbp_chr7_44885527_R | Primer3: not found at this Tm | N.d. | 0.10 |
mm4__chr1 95.12 Mbp_chr1_95117781_F | Primer3: not found at this Tm | N.d. | 0.10 |
mm4__chr1 95.12 Mbp_chr1_95117781_R | Primer3: not found at this Tm | N.d. | 0.10 |
mm4__chr5 128.08 Mbp_chr5_128083982_F | Primer3: not found at this Tm | N.d. | 0.09 |
mm4__chr5 128.08 Mbp_chr5_128083982_R | Primer3: not found at this Tm | N.d. | 0.09 |
mm4__chr8 10.05 Mbp_chr8_10054798_F | Primer3: not found at this Tm | N.d. | 0.09 |
mm4__chr8 10.05 Mbp_chr8_10054798_R | Primer3: not found at this Tm | N.d. | 0.09 |
mm2__chr7 101.32 Mbp_chr7_101321827_F | TCGTCGGCAGCGTCTCTACTTGGCCGCTTTCGTT | 59.9 | 0.08 |
mm2__chr7 101.32 Mbp_chr7_101321827_R | GTCTCGTGGGCTCGGTTTGTGGCTTCTCTCCGACC | 59.9 | 0.08 |
mm3__chr1 7.77 Mbp_chr1_7771212_F | Primer3: not found at this Tm | N.d. | 0.08 |
mm3__chr1 7.77 Mbp_chr1_7771212_R | Primer3: not found at this Tm | N.d. | 0.08 |
mm4__chr18 79.56 Mbp_chr18_79558528_F | TCGTCGGCAGCGTCGAAACCTCATGGCATTGCGG | 60.1 | 0.07 |
mm4__chr18 79.56 Mbp_chr18_79558528_R | GTCTCGTGGGCTCGGCACCAACCAGACAGACCCTC | 59.9 | 0.07 |
mm3__chr18 62.19 Mbp_chr18_62187624_F | Primer3: not found at this Tm | N.d. | 0.07 |
mm3__chr18 62.19 Mbp_chr18_62187624_R | Primer3: not found at this Tm | N.d. | 0.07 |
mm4__chr10 12.50 Mbp_chr10_12503284_F | TCGTCGGCAGCGTCCTTGTGCCGCTGGGATTTTG | 60.3 | 0.07 |
mm4__chr10 12.50 Mbp_chr10_12503284_R | GTCTCGTGGGCTCGGCACTCATGTCGTTGAACGCA | 59.1 | 0.07 |
mm4__chr10 45.00 Mbp_chr10_45001295_F | TCGTCGGCAGCGTCGAACGGGAGGCTTGGACTG | 60.3 | 0.06 |
mm4__chr10 45.00 Mbp_chr10_45001295_R | GTCTCGTGGGCTCGGTGCCTAAGCATTTGTTCCACG | 59.4 | 0.06 |
mm4__chr13 110.91 Mbp_chr13_110914277_F | TCGTCGGCAGCGTCGGCTGTGCGGAACCAGAA | 60.2 | 0.06 |
mm4__chr13 110.91 Mbp_chr13_110914277_R | GTCTCGTGGGCTCGGATTTCGCAGAGCCGGGTATT | 59.8 | 0.06 |
mm4__chr2 104.84 Mbp_chr2_104843149_F | Primer3: not found at this Tm | N.d. | 0.05 |
mm4__chr2 104.84 Mbp_chr2_104843149_R | Primer3: not found at this Tm | N.d. | 0.05 |
mm4__chr4 2.37 Mbp_chr4_2370711_F | TCGTCGGCAGCGTCATCACTGTACCGGGAGAGGG | 60.3 | 0.04 |
mm4__chr4 2.37 Mbp_chr4_2370711_R | GTCTCGTGGGCTCGGGATGTGGTGTGCAATCTGGC | 59.8 | 0.04 |
mm4__chr5 177.46 Mbp_chr5_177455523_F | TCGTCGGCAGCGTCGTGAGTACCGACCTCCGC | 59.5 | 0.04 |
mm4__chr5 177.46 Mbp_chr5_177455523_R | GTCTCGTGGGCTCGGGAAGCCCTGAGGGTAAGCAG | 60.1 | 0.04 |
mm4__chr17 81.24 Mbp_chr17_81238845_F | TCGTCGGCAGCGTCCTTGGAAGGCCCGGCTTG | 61.0 | 0.03 |
mm4__chr17 81.24 Mbp_chr17_81238845_R | GTCTCGTGGGCTCGGAGCCATTGACCTTGCGTCTC | 60.6 | 0.03 |
mm4__chr5 132.67 Mbp_chr5_132666613_F | TCGTCGGCAGCGTCCCTCCCAGATGCCCATACTG | 59.6 | 0.02 |
mm4__chr5 132.67 Mbp_chr5_132666613_R | GTCTCGTGGGCTCGGGGTGAGGCTAGATAAGGGCAG | 59.6 | 0.02 |
mm4__chr17 75.53 Mbp_chr17_75525691_F | Primer3: not found at this Tm | N.d. | 0.02 |
mm4__chr17 75.53 Mbp_chr17_75525691_R | Primer3: not found at this Tm | N.d. | 0.02 |
mm4__chr9 128.20 Mbp_chr9_128203691_F | TCGTCGGCAGCGTCGGATCCCTGGAGTCCCCG | 60.8 | 0.02 |
mm4__chr9 128.20 Mbp_chr9_128203691_R | GTCTCGTGGGCTCGGGAGGGGCTCTCGTGGTGG | 61.7 | 0.02 |
mm4__chr18 54.22 Mbp_chr18_54224375_F | Primer3: not found at this Tm | N.d. | 0.01 |
mm4__chr18 54.22 Mbp_chr18_54224375_R | Primer3: not found at this Tm | N.d. | 0.01 |
mm4__chr7 42.24 Mbp_chr7_42238109_F | Primer3: not found at this Tm | N.d. | 0.01 |
mm4__chr7 42.24 Mbp_chr7_42238109_R | Primer3: not found at this Tm | N.d. | 0.01 |
mm4__chr13 33.02 Mbp_chr13_33016350_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4__chr13 33.02 Mbp_chr13_33016350_R | Primer3: not found at this Tm | N.d. | 0.00 |
Primers underlined, off-targets in bold.
ontarget_mm0__chr7 5.53 Mbp_chr7_5529707 |
TGGTGAGCTGCGAGAATAGCCGGGCGCGCTGTGAGCCGAGGTCGCCCCCGCCCTGGCCACTTCCGGCGCGCCGAGTCCTT AGGCCGCCAGGGGGCGCCGGCGCGCGCCCAGATTGGGGACAAAGGAAGCCG |
mm4__chr9 129.84 Mbp_chr9_129835268 |
GAGTTGTACAGCCGCCGCGCCTCTGCGCAGCGGCCCAGGCTGCTTCCGGCGCGCGGCAGAGCGGTCAGAGCGCCTGCCCC GTCTGGCCCCGCCCCCGGCACGCACAGTCCGCGTGGCCACGCCCCCTCTCCTGGCGTCAG |
mm3__chr5 135.01 Mbp_chr5_135012998 |
GGATGGCTCTTCAGGGTGAGACCTGTTGAGGTGCTGTGGGGGAATCCTCTGAGGGCAGGAAATGCCTGGCCACTTCCGGC ACGGAGAGAGCTTTGTGTCCATGCCCTGAAGCTGGGCTGGGGTAGATA |
mm4__chr8 143.82 Mbp_chr8_143818330 |
AAGCCCTAGACACAGGGACACACCTGTCAGGCTGCGCGAGCCCAGGGGTGGGGGCGAGCCCGAAGTGGCCGGGGCGGACC AAGCCTGCTGACCTTGTGCTTCATGGTGACGGAGTC |
mm3__chr16 88.47 Mbp_chr16_88473986 |
GGTTCTCCGGAGTGAAGCAATCTCATGTAATCTATCAGTGGCTATCAGGCCCATCGGAGGGCCGGGAGTGGCCGGGCCCA GAGATGGCGCCAACCTTCAGCGTCGATA |
mm4__chr3 170.42 Mbp_chr3_170419651 |
TAATTACTGCGGTCCCAGCCCGCATCCTTCCACCGTCCGCTTCCCGAAGTGCTTCCCGCTCCCGCCCGGCCTCCCCCGGG GCGCCGAGCCTTCGCGTTAGGTTCCGCCCGCAGAGGTGAGAAGGAGCGTG |
mm4__chr9 21.97 Mbp_chr9_21968437 |
GTCCAGCAGCTAGTCCACTGCCCGCCTGGCTGCTCCAGGCGCGCCGACCGCTCAAGCGCTCCAGGTCCACCCGGCGGAGG GCAGAGAAAG |
mm4__chr1 898.94 Kbp_chr1_898937 |
CGTCTTCCAGAACGCACTCTGCAGGCACTCGGCAACGTGAAGTGTTCACGTCCTGTGATGCAGCCTCTGCTCCAGGCCAC TTCCGGAACTGCGAGGGAACAGCTGTGGGCGCGCTCATTTCAGCTTTGCT |
mm4__chr8 22.97 Mbp_chr8_22973980 |
CATCAACCTCACCACCCCTCCCACGCCCCACCATTTGAGTCAGATGAGACCCACCCCAGGCACCAGCCCTTCCAGGGAGT CACCTGACCACTTCCGGGGCTCCCAGCCAGCTCCGCTGGGAGCCATGTGATCACT |
mm2__chr7 101.32 Mbp_chr7_101321827 |
TCTACTTGGCCGCTTTCGTTTCCATGGCGACGGAGAGAGGCCCGCAGGGCCGACGGGACTCGGCCACTTCCGGCGCGCGG GGCGGAAAGCGGGCGCGTCTGCGACTGGGAGGGGGTCGGAGAGAAGCCACAAA |
mm4__chr18 79.56 Mbp_chr18_79558528 |
GAAACCTCATGGCATTGCGGCTGTGCTCAGAAGGCATGACCAGCCCCTCCGCCAGCCTGAAGTGGCCAGGCGTGTGCAGG GGGCTTGAGGGGGATGGGGAAAGGCAGGGAGGGTCTGTCTGGTTGGTG |
mm4__chr10 12.50 Mbp_chr10_12503284 |
CTTGTGCCGCTGGGATTTTGGGAGGAGCATTTGCTCCTCGCCCCGCCTGATGTGGCCAGGTGGCTGCGTGTAGTGTATTT AAATGCGTTCAACGACATGAGTG |
mm4__chr10 45.00 Mbp_chr10_45001295 |
GAACGGGAGGCTTGGACTGGGGGCCCGGGCGAGGCGCGGCCCGCGGCGGGTGGAACTGGGCGGGTGAGCAGCAGCCCCGG CCCCTTCCGTCGCTCGGAGCGTTCGAGGACTAGATACGTGGAACAAATGCTTAGGCA |
mm4__chr13 110.91 Mbp_chr13_110914277 |
GGCTGTGCGGAACCAGAAGGTCGACCGCGCCCGGGCATTCCCCGCGCGCCTCTGACCCCAGCTCGGCTCCCCGGAAGCCG CCTGGGCGACTTCGCGGCCTAATACCCGGCTCTGCGAAAT |
mm4__chr4 2.37 Mbp_chr4_2370711 |
ATCACTGTACCGGGAGAGGGGTGTCTCCCATGCCTGCTGCCACCCCCACCTCTCGGCTCCCCGGACGTGGCAAGGCCATG GGACCACCTGCAGTGGGCCAGTCTGGGGCGCCAGATTGCACACCACATC |
mm4__chr5 177.46 Mbp_chr5_177455523 |
GTGAGTACCGACCTCCGCCAGGGGGCGATCCGGGCCGCCGGAAGTGGGCGGGCGTTGGAGGGCTCGCTGCTTACCCTCAG GGCTTC |
mm4__chr17 81.24 Mbp_chr17_81238845 |
CTTGGAAGGCCCGGCTTGGAAGGCCCCGGGACGGCGCGGCGGGCGGGCAGCTCAGGGGCGTCGGCGCGGGCGAAGGGGCC AGGAGCAGCTACCCGGCGCGGAGACGCAAGGTCAATGGCT |
mm4__chr5 132.67 Mbp_chr5_132666613 |
CCTCCCAGATGCCCATACTGAGTTCCACCTCATCTGGCCACTTCCTGTGCCCCCAACTGCACTCAGATATCTCATGAGGT CACTGATTATCAACTCCACTCCGTGCAGCAACGCATGTCTGCCCTTATCTAGCCTCACC |
mm4__chr9 128.20 Mbp_chr9_128203691 |
GGATCCCTGGAGTCCCCGCCCGGGGCACTGACGGCGCGGCGACCTCGCAGCCCCCGACGCTGCACCCGCGGCCGGCGCGC CCCCCACCCCCAGCCGGAGCGAGGAGGCCCTCCCCCCACCACGAGAGCCCCTC |