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PCR primers for off-targets of AGGTACCTGCGGCGACAGCG GGG

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.

Maximum amplicon length:     Primer Tm:

NamePrimer Sequence Tm CFD Score
mm0_exon_Pop5_chr5_115238117_F TCGTCGGCAGCGTCTGCGGTTCAAGCACAGGTAA 60.1 1.00
mm0_exon_Pop5_chr5_115238117_R GTCTCGTGGGCTCGGGTCGTCCAGGCTCAAACGG 61.0 1.00
mm4_intergenic_Zfp619|Gm26526/Gm2058_chr7_39552544_F Primer3: not found at this Tm N.d. 0.42
mm4_intergenic_Zfp619|Gm26526/Gm2058_chr7_39552544_R Primer3: not found at this Tm N.d. 0.42
mm4_intergenic_B230323A14Rik|Bnip2_chr9_69951466_F TCGTCGGCAGCGTCAAGTGGCTTTGACCCCTTCA 59.4 0.40
mm4_intergenic_B230323A14Rik|Bnip2_chr9_69951466_R GTCTCGTGGGCTCGGGGAACACAACAGAGGGCCAG 60.8 0.40
mm4_intron_Nup214_chr2_32022646_F TCGTCGGCAGCGTCCTGTATCGCTCCCTGCAGAG 59.9 0.30
mm4_intron_Nup214_chr2_32022646_R GTCTCGTGGGCTCGGTGAATTTGGTTGCCTGGTGG 58.9 0.30
mm4_intron_Sv2c_chr13_95995520_F TCGTCGGCAGCGTCAAAGAGGGACGGATCTTGCA 59.0 0.29
mm4_intron_Sv2c_chr13_95995520_R GTCTCGTGGGCTCGGGCATTCCTTGAGCAACAGGC 60.1 0.29
mm4_intron_Fam160a1_chr3_85677754_F Primer3: not found at this Tm N.d. 0.25
mm4_intron_Fam160a1_chr3_85677754_R Primer3: not found at this Tm N.d. 0.25
mm4_exon_Adcy4_chr14_55769266_F TCGTCGGCAGCGTCTGACCTTGATGCTGCCTCTG 60.0 0.22
mm4_exon_Adcy4_chr14_55769266_R GTCTCGTGGGCTCGGGGCCCCTCTACACTCCCTTA 60.0 0.22
mm4_exon_Arhgef11_chr3_87729635_F Primer3: not found at this Tm N.d. 0.17
mm4_exon_Arhgef11_chr3_87729635_R Primer3: not found at this Tm N.d. 0.17
mm4_intron_Wnt3_chr11_103780428_F TCGTCGGCAGCGTCGGGGATGACTCAGAGGGTGA 60.3 0.15
mm4_intron_Wnt3_chr11_103780428_R GTCTCGTGGGCTCGGGGAGGGGACGAGGTAGAAGT 60.0 0.15
mm3_intergenic_Synpo2|Sec24d_chr3_123238503_F Primer3: not found at this Tm N.d. 0.15
mm3_intergenic_Synpo2|Sec24d_chr3_123238503_R Primer3: not found at this Tm N.d. 0.15
mm4_exon_Usp12_chr5_146794593_F Primer3: not found at this Tm N.d. 0.15
mm4_exon_Usp12_chr5_146794593_R Primer3: not found at this Tm N.d. 0.15
mm4_exon_Scel_chr14_103562466_F Primer3: not found at this Tm N.d. 0.15
mm4_exon_Scel_chr14_103562466_R Primer3: not found at this Tm N.d. 0.15
mm4_intergenic_9230109A22Rik|Basp1_chr15_25223863_F Primer3: not found at this Tm N.d. 0.14
mm4_intergenic_9230109A22Rik|Basp1_chr15_25223863_R Primer3: not found at this Tm N.d. 0.14
mm4_intergenic_Gm26766|Gm16136_chr15_37207522_F Primer3: not found at this Tm N.d. 0.13
mm4_intergenic_Gm26766|Gm16136_chr15_37207522_R Primer3: not found at this Tm N.d. 0.13
mm4_intron_Arhgef16_chr4_154290910_F TCGTCGGCAGCGTCTGTGAACCCCAGAGCTCCTA 59.8 0.13
mm4_intron_Arhgef16_chr4_154290910_R GTCTCGTGGGCTCGGCTCCCGGTATCCAGCCAAAA 59.7 0.13
mm4_exon_Dcst1_chr3_89364262_F TCGTCGGCAGCGTCCACCAGTGCCTGCTCCTAG 59.7 0.12
mm4_exon_Dcst1_chr3_89364262_R GTCTCGTGGGCTCGGCAGTCATACAGGGGCTGCTC 60.1 0.12
mm3_intergenic_Pax7|Gm1667_chr4_139905437_F Primer3: not found at this Tm N.d. 0.10
mm3_intergenic_Pax7|Gm1667_chr4_139905437_R Primer3: not found at this Tm N.d. 0.10
mm4_intergenic_n-R5s198|Gm13652_chr2_75015472_F TCGTCGGCAGCGTCTGACTCTCGCACCACATAGC 59.8 0.10
mm4_intergenic_n-R5s198|Gm13652_chr2_75015472_R GTCTCGTGGGCTCGGACAGACCTGTACGCCAGAAC 59.6 0.10
mm4_intergenic_Mir5112|4930445N18Rik_chr18_82753477_F Primer3: not found at this Tm N.d. 0.09
mm4_intergenic_Mir5112|4930445N18Rik_chr18_82753477_R Primer3: not found at this Tm N.d. 0.09
mm4_intergenic_Rinl|Hnrnpl_chr7_28800636_F Primer3: not found at this Tm N.d. 0.07
mm4_intergenic_Rinl|Hnrnpl_chr7_28800636_R Primer3: not found at this Tm N.d. 0.07
mm4_exon_A430078I02Rik/C87436_chr6_86472253_F TCGTCGGCAGCGTCACACCGGTAGAGGGGAGAAG 60.3 0.07
mm4_exon_A430078I02Rik/C87436_chr6_86472253_R GTCTCGTGGGCTCGGGGAAGAACAGTGGCGGTGT 60.2 0.07
mm4_exon_Uspl1_chr5_149184474_F Primer3: not found at this Tm N.d. 0.07
mm4_exon_Uspl1_chr5_149184474_R Primer3: not found at this Tm N.d. 0.07
mm3_exon_Mycs_chrX_5468579_F TCGTCGGCAGCGTCCACACCTGCTGTCCCACTG 60.5 0.06
mm3_exon_Mycs_chrX_5468579_R GTCTCGTGGGCTCGGGGGAAGCCAGAAAGTGTGGT 60.1 0.06
mm4_intergenic_Gm25985|Gm6404_chr13_115474607_F Primer3: not found at this Tm N.d. 0.06
mm4_intergenic_Gm25985|Gm6404_chr13_115474607_R Primer3: not found at this Tm N.d. 0.06
mm4_intergenic_Gm24526|Gm25064_chr9_63068003_F TCGTCGGCAGCGTCCTGGAAGTCGGACCACATGT 59.6 0.06
mm4_intergenic_Gm24526|Gm25064_chr9_63068003_R GTCTCGTGGGCTCGGTCCCTTCTCAGTAGCACCCC 60.6 0.06
mm4_intergenic_Slc28a1|Pde8a_chr7_81199913_F Primer3: not found at this Tm N.d. 0.05
mm4_intergenic_Slc28a1|Pde8a_chr7_81199913_R Primer3: not found at this Tm N.d. 0.05
mm3_intergenic_Adcy6|Cacnb3_chr15_98609166_F TCGTCGGCAGCGTCCATGTCCTCCAGCACTTCCC 60.3 0.05
mm3_intergenic_Adcy6|Cacnb3_chr15_98609166_R GTCTCGTGGGCTCGGCAGCAGGTAAGCGTGGGG 60.4 0.05
mm4_intergenic_Gpm6a|Adam29_chr8_55605442_F TCGTCGGCAGCGTCCTTGCAGCAATGGGAAGCAG 60.1 0.04
mm4_intergenic_Gpm6a|Adam29_chr8_55605442_R GTCTCGTGGGCTCGGTCAACTGCATGTTTAAATGGGAA 57.2 0.04
mm4_intergenic_Egln1|Tsnax_chr8_124958622_F TCGTCGGCAGCGTCGCTCTCTGCCACCTTGATGT 60.0 0.04
mm4_intergenic_Egln1|Tsnax_chr8_124958622_R GTCTCGTGGGCTCGGAGACTGGGGTGGGGTCATTA 59.8 0.04
mm4_intron_Stk24_chr14_121294471_F TCGTCGGCAGCGTCGTGGCCACACCTCTCCTAAG 59.7 0.04
mm4_intron_Stk24_chr14_121294471_R GTCTCGTGGGCTCGGGGTGGGAGGGCAAAAGAGAG 60.3 0.04
mm4_intron_Adam19_chr11_46139254_F Primer3: not found at this Tm N.d. 0.04
mm4_intron_Adam19_chr11_46139254_R Primer3: not found at this Tm N.d. 0.04
mm4_exon_Tnp2_chr16_10788243_F TCGTCGGCAGCGTCAGTGCGTGTTGCTCAGGTAA 59.8 0.03
mm4_exon_Tnp2_chr16_10788243_R GTCTCGTGGGCTCGGGGAAACGGACTCACAGAGCT 59.6 0.03
mm4_intergenic_Gm14005|Gm14006_chr2_128513926_F Primer3: not found at this Tm N.d. 0.03
mm4_intergenic_Gm14005|Gm14006_chr2_128513926_R Primer3: not found at this Tm N.d. 0.03
mm4_exon_Celsr1_chr15_85959181_F TCGTCGGCAGCGTCGCGTCCTCCTACCTTGTTGT 59.6 0.03
mm4_exon_Celsr1_chr15_85959181_R GTCTCGTGGGCTCGGCTCTTGCTGCACAGGTGAGA 59.9 0.03
mm4_intron_Nomo1_chr7_46058213_F TCGTCGGCAGCGTCAGGTCCTCCGGTCTCTCTAC 59.4 0.02
mm4_intron_Nomo1_chr7_46058213_R GTCTCGTGGGCTCGGGCTGCAGAGGAGTACACATGT 60.0 0.02
mm4_intergenic_Gm15082|Atp2b2_chr6_113940496_F TCGTCGGCAGCGTCAAGGCTAGGAGGAGGGATGG 60.1 0.02
mm4_intergenic_Gm15082|Atp2b2_chr6_113940496_R GTCTCGTGGGCTCGGCCATGCTACTGTGTCCAGCA 60.0 0.02
mm4_intergenic_Gm13322|Nebl_chr2_17219388_F Primer3: not found at this Tm N.d. 0.02
mm4_intergenic_Gm13322|Nebl_chr2_17219388_R Primer3: not found at this Tm N.d. 0.02
mm4_intergenic_Uncx|Micall2_chr5_139571511_F Primer3: not found at this Tm N.d. 0.02
mm4_intergenic_Uncx|Micall2_chr5_139571511_R Primer3: not found at this Tm N.d. 0.02
mm4_intergenic_Cep41|Gm14528_chr6_30706811_F Primer3: not found at this Tm N.d. 0.01
mm4_intergenic_Cep41|Gm14528_chr6_30706811_R Primer3: not found at this Tm N.d. 0.01
mm4_intron_Myo3b_chr2_70119689_F TCGTCGGCAGCGTCGGTCCTTCTGTGTCCATCCG 60.1 0.00
mm4_intron_Myo3b_chr2_70119689_R GTCTCGTGGGCTCGGAACCAGCCTGTCAGTCAGTG 59.8 0.00

Off-target amplicon sequences with primers

These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.

mm0_exon_Pop5_chr5_115238117 TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGAC
mm4_intergenic_B230323A14Rik|Bnip2_chr9_69951466 AAGTGGCTTTGACCCCTTCATCTGTCATCATTATGGGGAAGAGCCCGAGCCCCCAATGTATCATTTGTTTATGACAGTAA
ACACAACAAAGAAAGTGCCTGCGGTGACAGCATGGCTGCTGGCCCTCTGTTGTGTTCC
mm4_intron_Nup214_chr2_32022646 CTGTATCGCTCCCTGCAGAGCATCTGCTGTGAGTGTGGTGCTTCCCTGGGGTGAAGGAGAAGTAGTTGTGAAGAAGGCCT
TCATCCCTGTTGTCGCCACAGGGACCTCAGCCAGGAGGCCACCAGGCAACCAAATTCA
mm4_intron_Sv2c_chr13_95995520 AAAGAGGGACGGATCTTGCATAATAACCAAGGCCCTTGCCAGCCTCCCCCTGCTGTCGCTGCAGGGCCCTATGGCTGCCT
CCTCGGCTGCCTTCCAGCTGCCTGTTGCTCAAGGAATGC
mm4_exon_Adcy4_chr14_55769266 TGACCTTGATGCTGCCTCTGCTATAGCACGTGTAACCCAGGGACTGCAGGGCCCGCGCTGTCTCCTCAGTCACCTGCAAT
TGGAAGAGGGGTGGGTAAGGGAGTGTAGAGGGGCC
mm4_intron_Wnt3_chr11_103780428 GGGGATGACTCAGAGGGTGATTTATAGACGGAAAGCAGAAAGCTTGGCTCCTGGCAGAAGCCTGCTGCTTAATTACATTC
TTTGGGGGGTGGAGGTATGTGGGGCGACAGCAAGGGAGAGACTTCTACCTCGTCCCCTCC
mm4_intron_Arhgef16_chr4_154290910 TGTGAACCCCAGAGCTCCTAGACACACTGCCCACCCTGCTGTCGGGGCAGGTCCCTACCTTATTTTTGGCTGGATACCGG
GAG
mm4_exon_Dcst1_chr3_89364262 CACCAGTGCCTGCTCCTAGCAGGAAAGCAGTTACTGGAAACTCCCCTGGCTGTCGCCACAGGAACCGGATCCAGGAAACT
GGAAGACTCCAGAGGAGCAGCCCCTGTATGACTG
mm4_intergenic_n-R5s198|Gm13652_chr2_75015472 TGACTCTCGCACCACATAGCTCTGCTTTTTTATTGCGCCAATAACCTATCTGTCTCCTCAGGTACCTCTCTGCTGTGCAG
AGCTGGTGAGAGCTCACACACGGGTTCTGGCGTACAGGTCTGT
mm4_exon_A430078I02Rik/C87436_chr6_86472253 ACACCGGTAGAGGGGAGAAGCCGGAACAGCACGCGGTCACGTGGCCCCGCGAGGTGACGTCACCACGCTGCCGCCGCACG
TCGCTTCCAGCCACACCGCCACTGTTCTTCC
mm3_exon_Mycs_chrX_5468579 CACACCTGCTGTCCCACTGCGCTCATGGCCAGCAGAGCTGGCGGCAGCTGCCGCTCCGGGGGCCAAGGTGCCTGCGCCGA
CGGCGAGGGACCGGTAGCCCCCTGGCAGCTTCTCGCTCACCACACTTTCTGGCTTCCC
mm4_intergenic_Gm24526|Gm25064_chr9_63068003 CTGGAAGTCGGACCACATGTTTCTGTTACACACCCATCACTCTCACTGGCAATGGAATACCATGGTTGGAATCTATGGTG
TCATCCAGAGGTCCCTGCGGTGAAGGCGTGGTCCCAGCTGGGGGTGCTACTGAGAAGGGA
mm3_intergenic_Adcy6|Cacnb3_chr15_98609166 CATGTCCTCCAGCACTTCCCAGGCGCCCTGCAACTCTTGGGACTCCGCTGTCGCCGCAGGAGCCGATCCCCCACGCTTAC
CTGCTG
mm4_intergenic_Gpm6a|Adam29_chr8_55605442 CTTGCAGCAATGGGAAGCAGCAGGTACCAGCAGGCACAGCGGGGTTTACTTGCTAGTGTTAGAAGAACTTTCTCTATTAA
GTTTGTGTTTTCATTCAGCTTACTATTTCCCATTTAAACATGCAGTTGA
mm4_intergenic_Egln1|Tsnax_chr8_124958622 GCTCTCTGCCACCTTGATGTCTTGCAGAGACACCCTTAAAGTCTTAGCAGCTCGTTCCCCTCGCTGTCTCCCCAGCTCCC
TGCTAATGACCCCACCCCAGTCT
mm4_intron_Stk24_chr14_121294471 GTGGCCACACCTCTCCTAAGGATCCGCCTGCCACTTCACTATTAGTCCCTTTGGGGCCTCAGTGTCTCCGCAGGTTCCTC
CTTCTGGCCATGACTCTAAGTTTGTACTCTCTTTTGCCCTCCCACC
mm4_exon_Tnp2_chr16_10788243 AGTGCGTGTTGCTCAGGTAAGCCATGACCGCCTGGGCAGCCTTGCAGTGTTCCCTTGTCCCCGCTTGCCCTGCAGGTACC
TGAGCTACGCCTCTTAGCTCTGTGAGTCCGTTTCC
mm4_exon_Celsr1_chr15_85959181 GCGTCCTCCTACCTTGTTGTAGTACTGCACCAGCACCGAATGCCACCGCCCATCGCTCACACCTCCAGGAACCTGCGGTG
TCACCGTGGTTGTGGTCTCACCTGTGCAGCAAGAG
mm4_intron_Nomo1_chr7_46058213 AGGTCCTCCGGTCTCTCTACAGATACCTGCGGTGANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNTGCACAATTCTGTGTTTAGTACATGTGTACTCCTCTGCAGC
mm4_intergenic_Gm15082|Atp2b2_chr6_113940496 AAGGCTAGGAGGAGGGATGGCTGATATGGGGGCCACACACAGAGGTCCCTGAGGCTTCAGCGGGGCCTTAAGAGCTTTAG
CTTCCTGTGTTTGGGTATCTGGCAGGGTAGTGGTGCTGGACACAGTAGCATGG
mm4_intron_Myo3b_chr2_70119689 GGTCCTTCTGTGTCCATCCGAGTGGAGGTAGCTGCTGCGTCAGGGAGGGTAGGGAATGAATGCGCTAGGCCTCCAGGCTG
ACCAGGCTGCTGGTGTCCTAAGGAGGCAGGCACTGACTGACAGGCTGGTT

Input file for Crispresso

Crispresso, written by Luca Pinello, is a software package to quantify the Cas9-induced mutations on off- or on-targets.

Click here to download an amplicon input file for Crispresso. For each off-target, it includes the off-target name, its PCR amplicon and the guide sequence. Keep a copy of this file.

After sequencing, run CRISPRessoPooled. The tool will map the reads to the amplicons and analyse the mutations:
CRISPRessoPooled -r1 Reads1.fastq.gz -r2 Reads2.fastq.gz -f crisporAmplicons_ozJbN0W08qlgEBn3RvZK.txt --name MY_EXPERIMENT

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