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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_115238047_F | Primer3: not found at this Tm | N.d. | 1.00 |
| mm0_exon_Pop5_chr5_115238047_R | Primer3: not found at this Tm | N.d. | 1.00 |
| mm1_intron_Cars2_chr8_11540099_F | Primer3: not found at this Tm | N.d. | 0.87 |
| mm1_intron_Cars2_chr8_11540099_R | Primer3: not found at this Tm | N.d. | 0.87 |
| mm4_intergenic_Gm14580|Gm14628_chrX_43819277_F | Primer3: not found at this Tm | N.d. | 0.81 |
| mm4_intergenic_Gm14580|Gm14628_chrX_43819277_R | Primer3: not found at this Tm | N.d. | 0.81 |
| mm4_intergenic_Gm24232|Gm23987_chr15_69551276_F | Primer3: not found at this Tm | N.d. | 0.61 |
| mm4_intergenic_Gm24232|Gm23987_chr15_69551276_R | Primer3: not found at this Tm | N.d. | 0.61 |
| mm4_intergenic_Gm15585|Ppil2_chr16_17095609_F | Primer3: not found at this Tm | N.d. | 0.40 |
| mm4_intergenic_Gm15585|Ppil2_chr16_17095609_R | Primer3: not found at this Tm | N.d. | 0.40 |
| mm4_intergenic_Tmem74|Trhr_chr15_43887799_F | Primer3: not found at this Tm | N.d. | 0.36 |
| mm4_intergenic_Tmem74|Trhr_chr15_43887799_R | Primer3: not found at this Tm | N.d. | 0.36 |
| mm4_intergenic_Gm13519|Gm13527_chr2_56861253_F | TCGTCGGCAGCGTCCTGACTTTCCTAGAGACTGGCA | 59.1 | 0.33 |
| mm4_intergenic_Gm13519|Gm13527_chr2_56861253_R | GTCTCGTGGGCTCGGACAACAGTGAGGGCATCTGT | 59.2 | 0.33 |
| mm4_intergenic_Gm11515|Tac4_chr11_95258446_F | Primer3: not found at this Tm | N.d. | 0.31 |
| mm4_intergenic_Gm11515|Tac4_chr11_95258446_R | Primer3: not found at this Tm | N.d. | 0.31 |
| mm4_intergenic_Rgs17|Gm10945_chr10_6294684_F | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_intergenic_Rgs17|Gm10945_chr10_6294684_R | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_intron_Gnrhr_chr5_86185653_F | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_intron_Gnrhr_chr5_86185653_R | Primer3: not found at this Tm | N.d. | 0.25 |
| mm4_intron_Frg1_chr8_41405043_F | Primer3: not found at this Tm | N.d. | 0.24 |
| mm4_intron_Frg1_chr8_41405043_R | Primer3: not found at this Tm | N.d. | 0.24 |
| mm4_intergenic_Gm23531|Gm26181_chr8_42681547_F | Primer3: not found at this Tm | N.d. | 0.22 |
| mm4_intergenic_Gm23531|Gm26181_chr8_42681547_R | Primer3: not found at this Tm | N.d. | 0.22 |
| mm4_intron_Trpm3_chr19_22916543_F | TCGTCGGCAGCGTCTTCTCTGCACTGGGTTCTGC | 60.2 | 0.21 |
| mm4_intron_Trpm3_chr19_22916543_R | GTCTCGTGGGCTCGGAGCTACACAAAGTCCCAGGC | 59.9 | 0.21 |
| mm4_intergenic_Gm22104|Gm25550_chr15_15523608_F | Primer3: not found at this Tm | N.d. | 0.19 |
| mm4_intergenic_Gm22104|Gm25550_chr15_15523608_R | Primer3: not found at this Tm | N.d. | 0.19 |
| mm4_intergenic_Gm20705|Foxp1/Gm20696_chr6_99391776_F | Primer3: not found at this Tm | N.d. | 0.17 |
| mm4_intergenic_Gm20705|Foxp1/Gm20696_chr6_99391776_R | Primer3: not found at this Tm | N.d. | 0.17 |
| mm4_intron_Pramef6_chr4_143898034_F | TCGTCGGCAGCGTCTTTAGCAAGCCTCTTACCCC | 57.1 | 0.17 |
| mm4_intron_Pramef6_chr4_143898034_R | GTCTCGTGGGCTCGGGGACCTTGAAGGATCTTTGGC | 58.9 | 0.17 |
| mm4_intergenic_Gm13080|Gm13083_chr4_143614926_F | Primer3: not found at this Tm | N.d. | 0.17 |
| mm4_intergenic_Gm13080|Gm13083_chr4_143614926_R | Primer3: not found at this Tm | N.d. | 0.17 |
| mm4_intergenic_Gm9915|2610017I09Rik_chr1_42485580_F | TCGTCGGCAGCGTCAAGTTAGCGTTCCAAGACCA | 57.0 | 0.15 |
| mm4_intergenic_Gm9915|2610017I09Rik_chr1_42485580_R | GTCTCGTGGGCTCGGGGCCACAAGTTTCAGGCATG | 60.0 | 0.15 |
| mm4_intron_Tfeb_chr17_47743750_F | TCGTCGGCAGCGTCTCCCTTCTGATGCTGGGGTA | 59.9 | 0.14 |
| mm4_intron_Tfeb_chr17_47743750_R | GTCTCGTGGGCTCGGGCCAGACTCCGTTCTCCTCT | 60.9 | 0.14 |
| mm4_intron_Tstd2_chr4_46119695_F | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intron_Tstd2_chr4_46119695_R | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intron_Ubp1_chr9_113964673_F | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intron_Ubp1_chr9_113964673_R | Primer3: not found at this Tm | N.d. | 0.10 |
| mm4_intron_Grb10_chr11_11934865_F | Primer3: not found at this Tm | N.d. | 0.09 |
| mm4_intron_Grb10_chr11_11934865_R | Primer3: not found at this Tm | N.d. | 0.09 |
| mm4_intergenic_Actrt2|Gm13113_chr4_154737039_F | TCGTCGGCAGCGTCAACCCAGGGAAACAGAACCC | 59.8 | 0.09 |
| mm4_intergenic_Actrt2|Gm13113_chr4_154737039_R | GTCTCGTGGGCTCGGGCCCTTTCTCCCCAACTCTC | 60.0 | 0.09 |
| mm4_intron_Hpse2_chr19_42844222_F | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intron_Hpse2_chr19_42844222_R | Primer3: not found at this Tm | N.d. | 0.08 |
| mm4_intergenic_Gm12439|Gm12438_chr4_48736766_F | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_Gm12439|Gm12438_chr4_48736766_R | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_Gpatch2|Esrrg_chr1_187379592_F | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intergenic_Gpatch2|Esrrg_chr1_187379592_R | Primer3: not found at this Tm | N.d. | 0.07 |
| mm4_intron_Numb_chr12_83911533_F | TCGTCGGCAGCGTCACATCCTCATCTGCACTTGCT | 59.7 | 0.06 |
| mm4_intron_Numb_chr12_83911533_R | GTCTCGTGGGCTCGGCTGGCACTCAGTGGTAGTCC | 59.7 | 0.06 |
| mm4_intergenic_Atp1a1|Mab21l3_chr3_101690191_F | TCGTCGGCAGCGTCGCCAACAGTTCCTGAGAGCT | 59.9 | 0.06 |
| mm4_intergenic_Atp1a1|Mab21l3_chr3_101690191_R | GTCTCGTGGGCTCGGGTAGGTCTGTGCTGTGCTGT | 59.9 | 0.06 |
| mm4_intergenic_Gm23117|Gm5083_chr13_43914150_F | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Gm23117|Gm5083_chr13_43914150_R | Primer3: not found at this Tm | N.d. | 0.06 |
| mm4_intergenic_Rpl8|Zfp647_chr15_76909627_F | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Rpl8|Zfp647_chr15_76909627_R | Primer3: not found at this Tm | N.d. | 0.05 |
| mm4_intergenic_Has2os|Gm26178_chr15_56896462_F | Primer3: not found at this Tm | N.d. | 0.03 |
| mm4_intergenic_Has2os|Gm26178_chr15_56896462_R | Primer3: not found at this Tm | N.d. | 0.03 |
| mm3_exon_Suclg1_chr6_73263940_F | TCGTCGGCAGCGTCCTTTTGCTGCTGCTGCCATT | 60.3 | 0.03 |
| mm3_exon_Suclg1_chr6_73263940_R | GTCTCGTGGGCTCGGATTAGCCTCGTCGTTCCCTG | 59.5 | 0.03 |
| mm3_intergenic_Id2|Gm22527_chr12_25389524_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm3_intergenic_Id2|Gm22527_chr12_25389524_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_exon_Slitrk1_chr14_108911648_F | Primer3: not found at this Tm | N.d. | 0.02 |
| mm4_exon_Slitrk1_chr14_108911648_R | Primer3: not found at this Tm | N.d. | 0.02 |
| mm3_intergenic_Slc44a5|Lhx8_chr3_154283376_F | TCGTCGGCAGCGTCGGCTAGATCAGACCTCCAGG | 58.6 | 0.02 |
| mm3_intergenic_Slc44a5|Lhx8_chr3_154283376_R | GTCTCGTGGGCTCGGTTCTCCAATGCCCAGCACAT | 59.9 | 0.02 |
| mm4_intergenic_Kdm4c|SNORA43_chr4_74465085_F | TCGTCGGCAGCGTCAGGATGTTTGATAAGTGAGTGGA | 57.2 | 0.01 |
| mm4_intergenic_Kdm4c|SNORA43_chr4_74465085_R | GTCTCGTGGGCTCGGAGCTGTTCCTAAAGCCACCT | 58.9 | 0.01 |
| mm3_intron_1810046K07Rik_chr9_51319639_F | TCGTCGGCAGCGTCGGAGAATCTTCTGCTTGGACCA | 60.0 | 0.01 |
| mm3_intron_1810046K07Rik_chr9_51319639_R | GTCTCGTGGGCTCGGTCCCAGCCATCTAACCTACCA | 59.9 | 0.01 |
| mm3_intergenic_Wdfy4|Gm25498_chr14_33203118_F | TCGTCGGCAGCGTCTGGCCTATCTGCTCAGGTCT | 60.0 | 0.01 |
| mm3_intergenic_Wdfy4|Gm25498_chr14_33203118_R | GTCTCGTGGGCTCGGCGCCAAGGTTAAACACAGCC | 60.0 | 0.01 |
| mm4_intergenic_St8sia2|AC114591.1_chr7_74022245_F | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intergenic_St8sia2|AC114591.1_chr7_74022245_R | Primer3: not found at this Tm | N.d. | 0.01 |
| mm4_intron_Pfkfb1_chrX_150603710_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Pfkfb1_chrX_150603710_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Disc1|Sipa1l2_chr8_125385358_F | TCGTCGGCAGCGTCCTGGGTGCCCTAGTTTCCTG | 60.0 | 0.00 |
| mm4_intergenic_Disc1|Sipa1l2_chr8_125385358_R | GTCTCGTGGGCTCGGAGACCCCTCATGAGCTTCCT | 59.9 | 0.00 |
| mm4_intron_Cnnm2_chr19_46796956_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Cnnm2_chr19_46796956_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Grin2a_chr16_9856932_F | TCGTCGGCAGCGTCCCCATTCTGTCCCCATTCCC | 60.1 | 0.00 |
| mm4_intron_Grin2a_chr16_9856932_R | GTCTCGTGGGCTCGGGGTTGCTGAGCCAGAAAAGC | 60.0 | 0.00 |
| mm4_intron_Ect2l_chr10_18173551_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Ect2l_chr10_18173551_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Cuedc1_chr11_88143364_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intron_Cuedc1_chr11_88143364_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Lingo1|Odf3l1_chr9_56694013_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Lingo1|Odf3l1_chr9_56694013_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Hspa12a|Mir3086_chr19_58895425_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Hspa12a|Mir3086_chr19_58895425_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gad1-ps|Csl_chr10_99661805_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_Gad1-ps|Csl_chr10_99661805_R | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_intergenic_B3galt5|Igsf5_chr16_96328860_F | TCGTCGGCAGCGTCGCCTTTGCACCGAAAGTCAG | 60.0 | 0.00 |
| mm4_intergenic_B3galt5|Igsf5_chr16_96328860_R | GTCTCGTGGGCTCGGTCACACTGAAACCACACCCA | 59.3 | 0.00 |
| mm4_exon_Tmem206_chr1_191340856_F | Primer3: not found at this Tm | N.d. | 0.00 |
| mm4_exon_Tmem206_chr1_191340856_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.
| mm4_intergenic_Gm13519|Gm13527_chr2_56861253 |
CTGACTTTCCTAGAGACTGGCATGTACAGTCCTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTAAAACAGATGCCCTCACTGTTGT |
| mm4_intron_Trpm3_chr19_22916543 |
TTCTCTGCACTGGGTTCTGCCATTCAGAGGGCCTTCCGTTTCTTGAACAGTACCATGGACTTGTAAGCTGAAAAAACAGA AATAAATTTGCCTGGGACTTTGTGTAGCT |
| mm4_intron_Pramef6_chr4_143898034 |
TTTAGCAAGCCTCTTACCCCCCCCCCCTTCCCAATTACATAAACCATGATGTGGTCCAATCACTGGTCTGGTGACATTGA AAACCTTGTTATTCAGATATCTTTATCTTTCTCATTGCCAAAGATCCTTCAAGGTCC |
| mm4_intergenic_Gm9915|2610017I09Rik_chr1_42485580 | AAGTTAGCGTTCCAAGACCATCCAGAGCTTGACCAGCACCATTGTGCCTATCGACATGCCTGAAACTTGTGGCC |
| mm4_intron_Tfeb_chr17_47743750 |
TCCCTTCTGATGCTGGGGTATCAAACCTAGAGCCTCACACAGTTCCCAGAGCTTGACCCTCACCGTGGTCCTGGTCTTAA CTGGGAGAATAAGGAAGAGGAGAACGGAGTCTGGC |
| mm4_intergenic_Actrt2|Gm13113_chr4_154737039 | AACCCAGGGAAACAGAACCCAGCTGTCCCATGGTGAGGTTTCAGCTCAGGCCTGAGAGTTGGGGAGAAAGGGC |
| mm4_intron_Numb_chr12_83911533 |
ACATCCTCATCTGCACTTGCTATTATTTCTGTCCCTCCCCACCATGTTGGGGATCAAGCCCAGGGCTACATCCATACAGG GGACTACCACTGAGTGCCAG |
| mm4_intergenic_Atp1a1|Mab21l3_chr3_101690191 |
GCCAACAGTTCCTGAGAGCTTTTGAGCAATAAGTCCTGTGCTGAAAACTCACCATGGTGATACAGCACAGCACAGACCTA C |
| mm3_exon_Suclg1_chr6_73263940 |
CTTTTGCTGCTGCTGCCATTAATGAAGCAATCGACGCGGAGATTCCCTTGGTTGTGTGCATTACGGAAGGTATTCCGCAG CAGGATATGGTGCGGGTCAAGCACAGACTGACACGCCAGGGAACGACGAGGCTAAT |
| mm3_intergenic_Slc44a5|Lhx8_chr3_154283376 |
GGCTAGATCAGACCTCCAGGTACAGATCTGTGCTTGAACAGGACCCTGGGTAACTCCAGCACACACCCAAGCTTCAGATG TGCTGGGCATTGGAGAA |
| mm4_intergenic_Kdm4c|SNORA43_chr4_74465085 |
AGGATGTTTGATAAGTGAGTGGATATTGTCAGAATTTATCTCTTGTGATTTTTGGAAGATTTCCCTGGTGGGGTTAAGGC ACAGGGAACTCATTGTTTATATAGATGAAAATGCAGAAGTAGGTGGCTTTAGGAACAGCT |
| mm3_intron_1810046K07Rik_chr9_51319639 |
GGAGAATCTTCTGCTTGGACCACCCTGGTGGTGTTCAAGCACAGAATTAGTGGTTGAACTAGACATAGTTTAAAATATTT TTAAGCTTGAAATTGCTGTGGTAGGTTAGATGGCTGGGA |
| mm3_intergenic_Wdfy4|Gm25498_chr14_33203118 |
TGGCCTATCTGCTCAGGTCTCTAGCTGCTCATGGTGCTGTTCAACCACAGACTGGACGACTCGATGCTGAATTACAGTCT CTCTGGGCTGTGTTTAACCTTGGCG |
| mm4_intergenic_Disc1|Sipa1l2_chr8_125385358 |
CTGGGTGCCCTAGTTTCCTGCCAGTCCCTGAAACCCACCATGGCTTCCTTGGCATCCTCGGTGCCCATTGGGGGACATGG TTTGGACTTCCACTTTCCTTTCTGTTGCTCTCTATAAGGAAGCTCATGAGGGGTCT |
| mm4_intron_Grin2a_chr16_9856932 |
CCCATTCTGTCCCCATTCCCTCTAGACATAGCCTCCTGGGCATGAACCACAGCATGGCATTAGTGCTTTTCTGGCTCAGC AACC |
| mm4_intergenic_B3galt5|Igsf5_chr16_96328860 |
GCCTTTGCACCGAAAGTCAGCCATGGTGCGTTTCCTCCACAGGGTCCCTGACACTCATGGAGGCGTGGGTGTGGTTTCAG TGTGA |