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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_5529618_F | TCGTCGGCAGCGTCCACGATGGAGGGGAAGACG | 59.8 | 1.00 |
ontarget_mm0__chr7 5.53 Mbp_chr7_5529618_R | GTCTCGTGGGCTCGGGCTATTCTCGCAGCTCACCA | 60.1 | 1.00 |
mm3__chr15 43.99 Mbp_chr15_43990145_F | TCGTCGGCAGCGTCAATGAAGGGGAAGATGGCCC | 59.7 | 0.84 |
mm3__chr15 43.99 Mbp_chr15_43990145_R | GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT | 60.1 | 0.84 |
mm2__chr5 77.78 Mbp_chr5_77784902_F | TCGTCGGCAGCGTCCACCAGCTCACCATGGATGA | 59.7 | 0.50 |
mm2__chr5 77.78 Mbp_chr5_77784902_R | GTCTCGTGGGCTCGGAACGATGGAGGGGAAGACTG | 59.0 | 0.50 |
mm3__chr22 15.72 Mbp_chr22_15721502_F | TCGTCGGCAGCGTCACTGAATGAGCTCACCATGGA | 59.0 | 0.47 |
mm3__chr22 15.72 Mbp_chr22_15721502_R | GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC | 60.1 | 0.47 |
mm3__chr2 131.26 Mbp_chr2_131263571_F | TCGTCGGCAGCGTCTGAGCTCACCATGGATGATGA | 58.8 | 0.47 |
mm3__chr2 131.26 Mbp_chr2_131263571_R | GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC | 60.1 | 0.47 |
mm3__chr2 130.46 Mbp_chr2_130463904_F | TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC | 60.1 | 0.47 |
mm3__chr2 130.46 Mbp_chr2_130463904_R | GTCTCGTGGGCTCGGTGAGCTCACCATGGATGATGA | 58.8 | 0.47 |
mm3__chr14 19.40 Mbp_chr14_19402869_F | TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC | 60.1 | 0.47 |
mm3__chr14 19.40 Mbp_chr14_19402869_R | GTCTCGTGGGCTCGGACTGAATGAGCTCACCATGGA | 59.0 | 0.47 |
mm3__chr14 19.00 Mbp_chr14_18998883_F | TCGTCGGCAGCGTCACTGAATGAGCTCACCATGGA | 59.0 | 0.47 |
mm3__chr14 19.00 Mbp_chr14_18998883_R | GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC | 60.1 | 0.47 |
mm4__chr6 88.28 Mbp_chr6_88276950_F | TCGTCGGCAGCGTCCCCACAATGGAGGGGAAGAC | 60.0 | 0.45 |
mm4__chr6 88.28 Mbp_chr6_88276950_R | GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT | 60.1 | 0.45 |
mm4__chr1 156.15 Mbp_chr1_156154649_F | TCGTCGGCAGCGTCCCTGGGCCTTTTCCTCTTCC | 60.3 | 0.32 |
mm4__chr1 156.15 Mbp_chr1_156154649_R | GTCTCGTGGGCTCGGCTTCCTGCTGTCGGACACTT | 59.9 | 0.32 |
mm4__chr9 100.73 Mbp_chr9_100731255_F | TCGTCGGCAGCGTCGCCAGCTCACCATGGATGAT | 60.1 | 0.23 |
mm4__chr9 100.73 Mbp_chr9_100731255_R | GTCTCGTGGGCTCGGCTAGGGAGCTCCACGATGGA | 60.4 | 0.23 |
mm3__chr3 180.82 Mbp_chr3_180820559_F | TCGTCGGCAGCGTCAAGCACAGAGTCTTGCCCTC | 59.9 | 0.19 |
mm3__chr3 180.82 Mbp_chr3_180820559_R | GTCTCGTGGGCTCGGCTAAGCTGGCCTTGCACATG | 59.5 | 0.19 |
mm3__chrY 18.15 Mbp_chrY_18147899_F | Primer3: not found at this Tm | N.d. | 0.11 |
mm3__chrY 18.15 Mbp_chrY_18147899_R | Primer3: not found at this Tm | N.d. | 0.11 |
mm3__chrY 17.76 Mbp_chrY_17758086_F | TCGTCGGCAGCGTCAACGGTGGAGGGAAACATGG | 60.2 | 0.11 |
mm3__chrY 17.76 Mbp_chrY_17758086_R | GTCTCGTGGGCTCGGTCCTCTGTCCCACTCCTCTG | 59.9 | 0.11 |
mm4__chr6 46.20 Mbp_chr6_46204993_F | TCGTCGGCAGCGTCAGATGCTCAGTCGTCACCAC | 59.7 | 0.10 |
mm4__chr6 46.20 Mbp_chr6_46204993_R | GTCTCGTGGGCTCGGAAGCTGCTTTTCACATGCCG | 60.0 | 0.10 |
mm1__chr9 6.83 Mbp_chr9_6834471_F | TCGTCGGCAGCGTCCCGCTACCAGCTCACCATAG | 59.9 | 0.04 |
mm1__chr9 6.83 Mbp_chr9_6834471_R | GTCTCGTGGGCTCGGACACGATGAAGGGGAAGACG | 59.7 | 0.04 |
mm4__chrX 53.14 Mbp_chrX_53143874_F | TCGTCGGCAGCGTCGATGGAGGGGAACACAGCTC | 60.1 | 0.03 |
mm4__chrX 53.14 Mbp_chrX_53143874_R | GTCTCGTGGGCTCGGTGCTCTTCCACAGGTTGCAA | 60.1 | 0.03 |
mm4__chr10 133.21 Mbp_chr10_133207245_F | TCGTCGGCAGCGTCCCTCCCAGTGATCGTGAACA | 59.3 | 0.03 |
mm4__chr10 133.21 Mbp_chr10_133207245_R | GTCTCGTGGGCTCGGCAGGATGCGGGCCTTCTG | 60.5 | 0.03 |
mm4__chr4 91.92 Mbp_chr4_91920489_F | Primer3: not found at this Tm | N.d. | 0.01 |
mm4__chr4 91.92 Mbp_chr4_91920489_R | Primer3: not found at this Tm | N.d. | 0.01 |
mm4__chr10 84.20 Mbp_chr10_84195508_F | TCGTCGGCAGCGTCGGTGTCCGTCTGTTCTGTGT | 59.8 | 0.01 |
mm4__chr10 84.20 Mbp_chr10_84195508_R | GTCTCGTGGGCTCGGCCATGTTTCCGTTGGTGCTG | 60.0 | 0.01 |
mm4__chr8 4.26 Mbp_chr8_4258832_F | TCGTCGGCAGCGTCTAAGGGCACAGCAGCAGAAA | 59.8 | 0.00 |
mm4__chr8 4.26 Mbp_chr8_4258832_R | GTCTCGTGGGCTCGGACGGCCAGGTAGAGATGAGA | 59.7 | 0.00 |
mm4__chr17 81.51 Mbp_chr17_81512315_F | TCGTCGGCAGCGTCAAACCAGCTTTGCACATGCC | 60.2 | 0.00 |
mm4__chr17 81.51 Mbp_chr17_81512315_R | GTCTCGTGGGCTCGGCCGCCTGAGTCCATCCTTTT | 60.0 | 0.00 |
Primers underlined, off-targets in bold.
ontarget_mm0__chr7 5.53 Mbp_chr7_5529618 |
CACGATGGAGGGGAAGACGGCCCGGGGGGCATCGTCGCCCGCGAAGCCGGCCTTGCACATGCCGGAGCCGTTGTCGACGA CGAGCGCGGCGATATCATCATCCATGGTGAGCTGCGAGAATAGC |
mm3__chr15 43.99 Mbp_chr15_43990145 |
AATGAAGGGGAAGATGGCCCGGGGTGGGCATCGTCACCTGCAAAGCTGGCCTTGCACATGCCAGAGCCATTGTCGACGAC AAGCATGGTGATATCATCATCCATGGTGAGCTGGC |
mm2__chr5 77.78 Mbp_chr5_77784902 |
CACCAGCTCACCATGGATGATGATATCACCGCGCTCGTCATTGACAACGGCTCCGGCATGTGCAAGGCCAGCTTCACGGG CGACAATGCCGCCCGGGCAGTCTTCCCCTCCATCGTT |
mm3__chr22 15.72 Mbp_chr22_15721502 |
ACTGAATGAGCTCACCATGGATGATGATACTGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTG CAGGTGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC |
mm3__chr2 131.26 Mbp_chr2_131263571 |
TGAGCTCACCATGGATGATGATACCGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTGCGGGCG ACGATGCCCCCCGGGCTGTCTTCCCTTCCATC |
mm3__chr2 130.46 Mbp_chr2_130463904 |
GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCGCCCGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA GCACGGCGGTATCATCATCCATGGTGAGCTCA |
mm3__chr14 19.40 Mbp_chr14_19402869 |
GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCACCTGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA GCACGGCAGTATCATCATCCATGGTGAGCTCATTCAGT |
mm3__chr14 19.00 Mbp_chr14_18998883 |
ACTGAATGAGCTCACCATGGATGATGATACTGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTG CAGGTGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC |
mm4__chr6 88.28 Mbp_chr6_88276950 |
CCCACAATGGAGGGGAAGACGGCCTGGGGAGCCTTGCGCATGCTGGAGCAGTTGTCGACGACGACGAGCGCGGTGATAGC ATCATCCATGGTGAGCTGGC |
mm4__chr1 156.15 Mbp_chr1_156154649 |
CCTGGGCCTTTTCCTCTTCCAACTGCTTCAGCTGCTGCTGCCGACGACGACCGCGGGGGGAGGCGGGCAGGGGCCCATGC CCAGGGTCAGATACTATGCAGGTAAGTGTCCGACAGCAGGAAG |
mm4__chr9 100.73 Mbp_chr9_100731255 |
GCCAGCTCACCATGGATGATGATATCGCCCCACTTGTCATTGACAACGGCTCAGGCATGTGCAAGGCCGGCTTTGCAGGT GATAATGTCCCCCTGGGCCATCTTCCCCCTCCATCGTGGAGCTCCCTAG |
mm3__chr3 180.82 Mbp_chr3_180820559 |
AAGCACAGAGTCTTGCCCTCGCCGCTCTGCCACGGGTCCACACCCACTGCCAGCTCATCATGGATAATGATATCGCCACG CTCGTCATGGACAATGGCTCTCCCATGTGCAAGGCCAGCTTAG |
mm3__chrY 17.76 Mbp_chrY_17758086 |
AACGGTGGAGGGAAACATGGCTTGGGGAGCATCGTCCCCTGCAAAGCCAGCTTTGCACATGCAAGAGCCATTGTCAATGA CGAGCGCAGCGATCTCTTCTTCTACTGCGATGGACAGAGGAGTGGGACAGAGGA |
mm4__chr6 46.20 Mbp_chr6_46204993 |
AGATGCTCAGTCGTCACCACCAGCACGTGCACCATGTCCTCCCGCTGCTGCGTGCTCCTCTGCCAGTGGCAATGGAAGAA GAGATCCCCGCGCTCTTCATTGACAATGGCTCCGGCATGTGAAAAGCAGCTT |
mm1__chr9 6.83 Mbp_chr9_6834471 |
CCGCTACCAGCTCACCATAGATGATGATATTGCTACGCCCGTCGTCGACAACGGCTCCGGCATGTGGAAGGCTGGCTTCG CAGGTGACGATGACCCCCGGCCGTCTTCCCCTTCATCGTGT |
mm4__chrX 53.14 Mbp_chrX_53143874 |
GATGGAGGGGAACACAGCTCAGGGGCCATCCATCCCCAGCAAAGCCAGCTTTGCACATGCCAGAGCCATTGTCAATGATG AGCGCGGCGATCTCTATTTCCATTGCAACCTGTGGAAGAGCA |
mm4__chr10 133.21 Mbp_chr10_133207245 |
CCTCCCAGTGATCGTGAACATCGCGGCCGCACCCTGCGACACGCTGGACTTCAGCCCCCTGGACGAGTCCTCCTCGCTCA TCTTCTACAACGTCAACAAGCACCCGGGCGGCCGGCAGAAGGCCCGCATCCTG |
mm4__chr10 84.20 Mbp_chr10_84195508 |
GGTGTCCGTCTGTTCTGTGTTCTTTTTCCAGCTCAGGCCAACTTCGCCCCGCACTTCTTCGACAACGGGGTCGGCAGCAC CAACGGAAACATGG |
mm4__chr8 4.26 Mbp_chr8_4258832 |
TAAGGGCACAGCAGCAGAAAGATCAGGAGCCCCACTCCTCATGGACAACGGCTGCAAAAGTCTCATCTCTACCTGGCCGT |
mm4__chr17 81.51 Mbp_chr17_81512315 |
AAACCAGCTTTGCACATGCCGGAGCCATTGTCAATGACCAGCGCGGCGATCTCTTCTTCCATTGCGACCTGCCCGGAAAA GGATGGACTCAGGCGG |