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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_52314228_F | TCGTCGGCAGCGTCAGAAATCGAGGTGAGCTGCA | 59.3 | 1.00 |
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52314228_R | GTCTCGTGGGCTCGGGAAGGGAACTGTGGGTGGAG | 59.9 | 1.00 |
mm4_intergenic_RP23-264N13.2|Lrrc1_chr9_77426541_F | TCGTCGGCAGCGTCACGCACACAGGACAGATGAG | 60.0 | 0.12 |
mm4_intergenic_RP23-264N13.2|Lrrc1_chr9_77426541_R | GTCTCGTGGGCTCGGGGCCCCTCCAAGTAACTCAC | 60.0 | 0.12 |
mm4_intron_Ctnna3_chr10_64527497_F | TCGTCGGCAGCGTCTCATTCTTCCCCAGGCACAT | 58.9 | 0.09 |
mm4_intron_Ctnna3_chr10_64527497_R | GTCTCGTGGGCTCGGGCAGGTAAAGATTGCTGCTGT | 59.1 | 0.09 |
mm4_intergenic_Klrb1c|Klrb1b_chr6_128799517_F | Primer3: not found at this Tm | N.d. | 0.09 |
mm4_intergenic_Klrb1c|Klrb1b_chr6_128799517_R | Primer3: not found at this Tm | N.d. | 0.09 |
mm4_intron_Socs5_chr17_87113924_F | TCGTCGGCAGCGTCGCCAGTATAGAGCCAGTGCC | 60.2 | 0.07 |
mm4_intron_Socs5_chr17_87113924_R | GTCTCGTGGGCTCGGCAAGGGGTGTTCCTAAGCCC | 60.3 | 0.07 |
mm4_intergenic_Rps18-ps2|Gm23423_chr4_9031046_F | Primer3: not found at this Tm | N.d. | 0.06 |
mm4_intergenic_Rps18-ps2|Gm23423_chr4_9031046_R | Primer3: not found at this Tm | N.d. | 0.06 |
mm4_intron_Chrna4_chr2_181042034_F | TCGTCGGCAGCGTCGCTCAGCTCTGTTGCCTGTT | 60.8 | 0.03 |
mm4_intron_Chrna4_chr2_181042034_R | GTCTCGTGGGCTCGGGAGAGACTGCCCGAGAGAGA | 60.1 | 0.03 |
mm3_exon_Tfr2_chr5_137574692_F | Primer3: not found at this Tm | N.d. | 0.03 |
mm3_exon_Tfr2_chr5_137574692_R | Primer3: not found at this Tm | N.d. | 0.03 |
mm4_intergenic_Mllt1|Acer1_chr17_56941434_F | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intergenic_Mllt1|Acer1_chr17_56941434_R | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intergenic_Rnf14|Gnpda1_chr18_38321903_F | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_intergenic_Rnf14|Gnpda1_chr18_38321903_R | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_intron_Dag1_chr9_108263345_F | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_intron_Dag1_chr9_108263345_R | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_intergenic_Clptm1|Apoc2_chr7_19665050_F | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_intergenic_Clptm1|Apoc2_chr7_19665050_R | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_intron_Kazald1_chr19_45075887_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Kazald1_chr19_45075887_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Itsn2_chr12_4697985_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Itsn2_chr12_4697985_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_exon_2510039O18Rik_chr4_147944712_F | TCGTCGGCAGCGTCCCGTGAACCTGACCCTGTAC | 60.0 | 0.00 |
mm4_exon_2510039O18Rik_chr4_147944712_R | GTCTCGTGGGCTCGGTAGTTGAGCGTTGCCTCCAT | 59.3 | 0.00 |
mm4_intron_Lmx1b_chr2_33567119_F | TCGTCGGCAGCGTCGATAGCAGTGGGTCATGGGG | 59.8 | 0.00 |
mm4_intron_Lmx1b_chr2_33567119_R | GTCTCGTGGGCTCGGGCGAAGAGCTTTCAAGGCAT | 59.1 | 0.00 |
mm4_intron_Hvcn1_chr5_122236442_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Hvcn1_chr5_122236442_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Slc9a4|Slc9a2_chr1_40667659_F | TCGTCGGCAGCGTCGCAGGTGCTGGTGTCAGTAA | 60.2 | 0.00 |
mm4_intergenic_Slc9a4|Slc9a2_chr1_40667659_R | GTCTCGTGGGCTCGGCTGCTCTCAGGGAAGGCCT | 60.9 | 0.00 |
mm4_intergenic_Rapgef5|Gm24741_chr12_117528936_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Rapgef5|Gm24741_chr12_117528936_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_exon_Cacna1b_chr2_24610117_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_exon_Cacna1b_chr2_24610117_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_exon_4930404I05Rik/Paxbp1_chr16_91015439_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_exon_4930404I05Rik/Paxbp1_chr16_91015439_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_52314228 |
AGAAATCGAGGTGAGCTGCACCCCAGACTGCGCCACCGGGAACGCCGAGTACCAGCACAGCAAAGGTAGCCACCTTGCCC CTCCGCTCCCCGGTCCGCCCACTCCACCCACAGTTCCCTTC |
mm4_intergenic_RP23-264N13.2|Lrrc1_chr9_77426541 |
ACGCACACAGGACAGATGAGCCCACTTCCGGAGAATATTTTCTCAGGTGTTCAGGGTGGCCCAGTCAGGCAGCCTTTTTC AAACTGCTTATTAGAATGGGCATCTTTGTGAGTTACTTGGAGGGGCC |
mm4_intron_Ctnna3_chr10_64527497 | TCATTCTTCCCCAGGCACATGAGTTCCCGGTGTCACACTCTGGCACACACAGCAGCAATCTTTACCTGC |
mm4_intron_Socs5_chr17_87113924 |
GCCAGTATAGAGCCAGTGCCATCCATGTGCCGTTCCCGGTGGTTAAGTCCGGCAGCAAGGAGGGGCTTAGGAACACCCCT TG |
mm4_intron_Chrna4_chr2_181042034 |
GCTCAGCTCTGTTGCCTGTTTATCTTCACAGCTTCTAGAGACAGGCTTTGTGCCCAGCCTTCCAGGTTGAGCAGTCTGGG ACAGTGGCTCGCACCAGCGGGCAGTCTCTCTCGGGCAGTCTCTC |
mm4_exon_2510039O18Rik_chr4_147944712 |
CCGTGAACCTGACCCTGTACTACATGCTCTCCTGCTCTCCAGCCCCACTGCTCAGCCCCTCTCTGAGCCACAGGGAACGC GAGCAGATGGAGGCAACGCTCAACTA |
mm4_intron_Lmx1b_chr2_33567119 |
GATAGCAGTGGGTCATGGGGGAGGAACAGACCCACAATAAGCAAAAGGGGGGCGTTGGCCCCAGACTGCCCCACCCAGAA CCCCTCACCTTCCGACAGGGCTTGGAGGAGACCTCAAAGGATGCCTTGAAAGCTCTTCGC |
mm4_intergenic_Slc9a4|Slc9a2_chr1_40667659 | GCAGGTGCTGGTGTCAGTAAAAGCACTCTGCCTGGCTGTCCCACCGGGAATGCAGGCCTTCCCTGAGAGCAG |