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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 |
---|---|---|---|
mm0_exon_Pop5_chr5_115238030_F | Primer3: not found at this Tm | N.d. | 1.00 |
mm0_exon_Pop5_chr5_115238030_R | Primer3: not found at this Tm | N.d. | 1.00 |
mm4_exon_Wiz/Gm22201_chr17_32388975_F | TCGTCGGCAGCGTCCAGTCCAGATGGTCGTTCCC | 60.1 | 0.27 |
mm4_exon_Wiz/Gm22201_chr17_32388975_R | GTCTCGTGGGCTCGGGAAGGGCGCATCCTGGGG | 62.8 | 0.27 |
mm4_intron_Dst_chr1_34285195_F | Primer3: not found at this Tm | N.d. | 0.23 |
mm4_intron_Dst_chr1_34285195_R | Primer3: not found at this Tm | N.d. | 0.23 |
mm4_intergenic_Pabpc1l2a-ps|Gm3928_chrX_103067555_F | Primer3: not found at this Tm | N.d. | 0.18 |
mm4_intergenic_Pabpc1l2a-ps|Gm3928_chrX_103067555_R | Primer3: not found at this Tm | N.d. | 0.18 |
mm4_intergenic_Gm21998|Pabpc1l2b-ps_chrX_103013356_F | Primer3: not found at this Tm | N.d. | 0.18 |
mm4_intergenic_Gm21998|Pabpc1l2b-ps_chrX_103013356_R | Primer3: not found at this Tm | N.d. | 0.18 |
mm4_exon_Wnt11_chr7_98835183_F | TCGTCGGCAGCGTCAGAGCCGAGCACAACTGAC | 60.0 | 0.12 |
mm4_exon_Wnt11_chr7_98835183_R | GTCTCGTGGGCTCGGTTCTTCCAGACGCAGCTCAG | 60.0 | 0.12 |
mm4_intergenic_Gm26622|Snx9_chr17_5841262_F | Primer3: not found at this Tm | N.d. | 0.11 |
mm4_intergenic_Gm26622|Snx9_chr17_5841262_R | Primer3: not found at this Tm | N.d. | 0.11 |
mm4_exon_Cdc34-ps_chr11_94742555_F | TCGTCGGCAGCGTCACATCATCCGGAAGCAGGTC | 59.8 | 0.10 |
mm4_exon_Cdc34-ps_chr11_94742555_R | GTCTCGTGGGCTCGGGTAGTCGAAGAGGTCCGAGC | 59.6 | 0.10 |
mm4_intergenic_Pigg|Gm10419_chr5_108366862_F | TCGTCGGCAGCGTCACTCCTCCGGGGATAGGATG | 59.8 | 0.10 |
mm4_intergenic_Pigg|Gm10419_chr5_108366862_R | GTCTCGTGGGCTCGGATCTTTGGGGCGACAACGG | 60.6 | 0.10 |
mm3_intron_Tcf20_chr15_82899597_F | Primer3: not found at this Tm | N.d. | 0.08 |
mm3_intron_Tcf20_chr15_82899597_R | Primer3: not found at this Tm | N.d. | 0.08 |
mm4_exon_Casz1_chr4_148938639_F | TCGTCGGCAGCGTCGCAAATACGAGGGCTGCATG | 59.9 | 0.06 |
mm4_exon_Casz1_chr4_148938639_R | GTCTCGTGGGCTCGGTGTGTGAGGTCATCTGGCTG | 59.6 | 0.06 |
mm4_exon_Thoc7_chr14_13961199_F | Primer3: not found at this Tm | N.d. | 0.05 |
mm4_exon_Thoc7_chr14_13961199_R | Primer3: not found at this Tm | N.d. | 0.05 |
mm4_exon_Tmem194_chr10_127666959_F | Primer3: not found at this Tm | N.d. | 0.04 |
mm4_exon_Tmem194_chr10_127666959_R | Primer3: not found at this Tm | N.d. | 0.04 |
mm4_exon_Taf10_chr7_105744284_F | TCGTCGGCAGCGTCTGCTAGTGGGCAGCGCAG | 62.7 | 0.03 |
mm4_exon_Taf10_chr7_105744284_R | GTCTCGTGGGCTCGGTCTGTTCGCCGCCTTTCC | 60.3 | 0.03 |
mm4_intron_Klf10_chr15_38299554_F | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intron_Klf10_chr15_38299554_R | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intron_Mmaa_chr8_79294468_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Mmaa_chr8_79294468_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Strn4|Prkd2_chr7_16842647_F | TCGTCGGCAGCGTCAAAGCCACCGGACTTGACAA | 60.1 | 0.00 |
mm4_intergenic_Strn4|Prkd2_chr7_16842647_R | GTCTCGTGGGCTCGGGCAGGCGCTTTGTCATTGAA | 60.0 | 0.00 |
These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.
mm4_exon_Wiz/Gm22201_chr17_32388975 |
CAGTCCAGATGGTCGTTCCCTACCCCACCTCCCGCAGGGGTCGCAGAGGTAGGGGCGGCGCTGCGCGGACGCACCCTGCG GGGAAGCCAGGGTCCCCAGGATGCGCCCTTC |
mm4_exon_Wnt11_chr7_98835183 |
AGAGCCGAGCACAACTGACCGCCTTGGCGTCCGCGCCCGAGTTGCCAGGATCCCCGGCTCCCTGCTGAGCTGCGTCTGGA AGAA |
mm4_exon_Cdc34-ps_chr11_94742555 |
ACATCATCCGGAAGCAGGTCCTGGGGACCAAGGTGGACGCAGAGCGCGATGGCGTGAAGGTGCCCACTACGCTGGCCGAG TACTGCGTGAAGACCAAGGCGCCGGCGCCGGATGAGGGCTCGGACCTCTTCGACTAC |
mm4_intergenic_Pigg|Gm10419_chr5_108366862 |
ACTCCTCCGGGGATAGGATGGAGGCCTCGCTGGGCCGTGGCTGCAGCCGCCGCCCCCTTTTGCCCCGGCGGCCGCGCCGG GTCCGGGAGGCTAGCGGCTGAGCGCGGAGCCCGGGTCCGCCCCGTTGTCGCCCCAAAGAT |
mm4_exon_Casz1_chr4_148938639 |
GCAAATACGAGGGCTGCATGTACAGCAAGGCCACCAACCATTTCCACTGCATCCGCGCCGGCTGCGGCTTCACCTTCACC TCCACCAGCCAGATGACCTCACACA |
mm4_exon_Taf10_chr7_105744284 |
TGCTAGTGGGCAGCGCAGCGGGAGCCGACACCAGGGGTGCCGGACCCGCGACCGAGGCGGCACAGGCGGTCGCCGCCTCG GGGTCCGCGCCGGAGCCGCTGCAGCTCATGGGGCCGGTGGGAAAGGCGGCGAACAGA |
mm4_intergenic_Strn4|Prkd2_chr7_16842647 |
AAAGCCACCGGACTTGACAACGATTTCCGACCCTTTCCCTGCGGTGGGCGCCGGATGCGTGCCAGTCTGACAGCTCACTC CTTCAATGACAAAGCGCCTGC |