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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_52313705_F | TCGTCGGCAGCGTCCGCATCACAAGGTGACCCTA | 59.7 | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313705_R | GTCTCGTGGGCTCGGCTCCTAATCCCAGGCTCCCT | 60.1 | 1.00 |
| mm4_intergenic_Lfng|Ttyh3_chr5_140617038_F | TCGTCGGCAGCGTCCCTCTGCTCTGGTCGTCAAA | 59.6 | 0.26 |
| mm4_intergenic_Lfng|Ttyh3_chr5_140617038_R | GTCTCGTGGGCTCGGGAATGGGAAGCTCACCCTGG | 60.3 | 0.26 |
| mm4_exon_Epas1_chr17_86826574_F | TCGTCGGCAGCGTCAGCAGCCCTGAGGACTACTA | 59.3 | 0.17 |
| mm4_exon_Epas1_chr17_86826574_R | GTCTCGTGGGCTCGGGGAGCCCTCTGTCACCCA | 60.6 | 0.17 |
| mm4_intron_Tia1_chr6_86404608_F | TCGTCGGCAGCGTCGCCTCTCTGCAGCCTATTGT | 59.8 | 0.09 |
| mm4_intron_Tia1_chr6_86404608_R | GTCTCGTGGGCTCGGGCTCTAAGCATCCTGGCGT | 59.8 | 0.09 |
| mm4_intergenic_Scml4|Sobp_chr10_42962671_F | TCGTCGGCAGCGTCCACTCAGGCTTGCTCTGTCA | 59.9 | 0.09 |
| mm4_intergenic_Scml4|Sobp_chr10_42962671_R | GTCTCGTGGGCTCGGGTTGTTGGTTCGTTCCCGTG | 59.9 | 0.09 |
| mm4_exon_Slc27a4_chr2_29812305_F | TCGTCGGCAGCGTCCACACCTGACTGGCTTCCTC | 60.3 | 0.06 |
| mm4_exon_Slc27a4_chr2_29812305_R | GTCTCGTGGGCTCGGTTGTTGTTGGCACCCTGGTT | 60.6 | 0.06 |
| mm4_intron_Zbtb21_chr16_97962131_F | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_intron_Zbtb21_chr16_97962131_R | Primer3: not found at this Tm | N.d. | 0.04 |
| mm4_exon_Nr2f6_chr8_71381899_F | TCGTCGGCAGCGTCTTCCCTGGATCGTCTCCCC | 60.3 | 0.04 |
| mm4_exon_Nr2f6_chr8_71381899_R | GTCTCGTGGGCTCGGGATGGAACGCGGGTGTCA | 60.0 | 0.04 |
| mm4_intergenic_AI854517|Mir9-3_chr7_79505201_F | TCGTCGGCAGCGTCTCTGTCGGTCCCCTCTGG | 59.9 | 0.03 |
| mm4_intergenic_AI854517|Mir9-3_chr7_79505201_R | GTCTCGTGGGCTCGGGAGAAACGGGCCTCCCATTC | 60.7 | 0.03 |
| mm4_intron_Usp33_chr3_152360910_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intron_Usp33_chr3_152360910_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intron_Snx9_chr17_5841586_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intron_Snx9_chr17_5841586_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm3_intron_Ldhd_chr8_111628284_F | TCGTCGGCAGCGTCAGGCTGATGGTTAGACCCCT | 59.9 | 0.02 |
| mm3_intron_Ldhd_chr8_111628284_R | GTCTCGTGGGCTCGGTTCCTGGAGTTCCACGGTTC | 59.6 | 0.02 |
| mm4_intron_Eif4e_chr3_138527733_F | TCGTCGGCAGCGTCGAGAGAGCCGGAGAGGGAG | 60.5 | 0.01 |
| mm4_intron_Eif4e_chr3_138527733_R | GTCTCGTGGGCTCGGTCCTCCGCAGCTCCAGAG | 60.7 | 0.01 |
| mm4_intergenic_Nckap1|Gm13688_chr2_80581442_F | TCGTCGGCAGCGTCGAAGTTGTCCCCGCTCCC | 60.0 | 0.00 |
| mm4_intergenic_Nckap1|Gm13688_chr2_80581442_R | GTCTCGTGGGCTCGGGGGAGGAAAACAAGCTCGGA | 59.9 | 0.00 |
| mm4_intron_Slc2a9_chr5_38403849_F | TCGTCGGCAGCGTCAATGAGGTCAGCAGCCTCAC | 60.0 | 0.00 |
| mm4_intron_Slc2a9_chr5_38403849_R | GTCTCGTGGGCTCGGTGTCCTTGGCAGTGAGATGA | 58.6 | 0.00 |
| mm4_exon_Ptprs_chr17_56422212_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Ptprs_chr17_56422212_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_52313705 |
CGCATCACAAGGTGACCCTAGCTCCCCACTGCCATCTCCGCGGTCGCCGTCGACACGGCGCTGGGGCTACCCGGCGCCTG CCTTGTCGCCTTAGCTCCTCTTCTCAGCCAAGATCCCAGGGAGCCTGGGATTAGGAG |
| mm4_intergenic_Lfng|Ttyh3_chr5_140617038 |
CCTCTGCTCTGGTCGTCAAAAGCCCCTTGCAGCTGGCACAGAGCTGGCAGCCACAGAGGTGTGGTACAGCGGCACCAACC TCGCCGCCGGCATGGAGCTGGGGGCCCCAGCCAGGGTGAGCTTCCCATTC |
| mm4_exon_Epas1_chr17_86826574 |
AGCAGCCCTGAGGACTACTATTCATCCTTGGAGAATCCCTTGAAGATCGAAGTGATTGAGAAGCTTTTCGCCATGGACAC GGAGCCGAGGGACCCGGGCAGTACCCAGGTGGGCCCCGCGCGTGGGTGACAGAGGGCTCC |
| mm4_intron_Tia1_chr6_86404608 |
GCCTCTCTGCAGCCTATTGTTCTCCGTGGACGCCGCGCTGTGGTGGTTCCTCCCTCCCGTGCGCACGCCAGGATGCTTAG AGC |
| mm4_intergenic_Scml4|Sobp_chr10_42962671 |
CACTCAGGCTTGCTCTGTCACCTACTTGACCACATGGCCCGCGCCACGGCGCTGAGGCAGAAAAGTAGTCTACCAGTGGG TCCGGCACGGGAACGAACCAACAAC |
| mm4_exon_Slc27a4_chr2_29812305 |
CACACCTGACTGGCTTCCTCGTCCCTAGGTCAGCCAGGCCAGCTGGTGGGTCGCATCATCCAGCAGGACCCTCTGCGCCG TTTCGACGGGTACCTCAACCAGGGTGCCAACAACAA |
| mm4_exon_Nr2f6_chr8_71381899 |
TTCCCTGGATCGTCTCCCCGCCGAGCGCTCGCGCGGCCGGCCTCGGCCTGCGCCTGGGCCCGAGCCTCGGCCTCGCCCTG GCGCTNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGACACCCGCGTTCCATC |
| mm4_intergenic_AI854517|Mir9-3_chr7_79505201 |
TCTGTCGGTCCCCTCTGGCTCTCCGCGTGCGCCCGGGGCTGCGGGAGCGGAGTCCGCTCAGGCCCCCGCGCCGGGTCGGC GGCGGCACGTGGGGCCCACAGCGCGGCAGCGGTGCCGGCGAATGGGAGGCCCGTTTCTC |
| mm3_intron_Ldhd_chr8_111628284 |
AGGCTGATGGTTAGACCCCTACACTGGGACAGCCTCAGACCTCACAGCGCCGTGTCCCCGGCGCACCTGTGCGCTGCAGC TGTTCCGCCAGTGTCTGCTGGGAACCGTGGAACTCCAGGAA |
| mm4_intron_Eif4e_chr3_138527733 |
GAGAGAGCCGGAGAGGGAGCCCGAGGCAGCGGCGCCTCGCCGTCGCCGGCGACCCGCCGCCGGGGGTCCTCGTGCGGGCC GCGCTCTGGAGCTGCGGAGGA |
| mm4_intergenic_Nckap1|Gm13688_chr2_80581442 |
GAAGTTGTCCCCGCTCCCCACGCCCCAGGGCCCGCACCGCCGTGGGGACGGGGACGGTGTGTTTCGGGAGACTTTGGCCC CGAGAGGAGCCTGGCGGCTCCGAGCTTGTTTTCCTCCC |
| mm4_intron_Slc2a9_chr5_38403849 | AATGAGGTCAGCAGCCTCACTGTCTCCGTCTACAGGGCTCTGTGGGTTCATCTCACTGCCAAGGACA |