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PCR primers for off-targets of CCCGTGCTTGAACCGCACCA TGG

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
mm1_exon_Pop5_chr5_115238047_F Primer3: not found at this Tm N.d. 0.68
mm1_exon_Pop5_chr5_115238047_R Primer3: not found at this Tm N.d. 0.68
mm2_intron_Cars2_chr8_11540099_F Primer3: not found at this Tm N.d. 0.64
mm2_intron_Cars2_chr8_11540099_R Primer3: not found at this Tm N.d. 0.64
mm4_intron_Dnah5_chr15_28306179_F TCGTCGGCAGCGTCTGCTTTGGGGAAGTTCCACT 59.4 0.56
mm4_intron_Dnah5_chr15_28306179_R GTCTCGTGGGCTCGGAACACCCACATGCACACCAA 60.6 0.56
mm4_intron_Tstd2_chr4_46119695_F TCGTCGGCAGCGTCCTTCGAGCCTGCTAAGTGCT 60.1 0.28
mm4_intron_Tstd2_chr4_46119695_R GTCTCGTGGGCTCGGCCGGCCGAGGGATTTATGTT 60.1 0.28
mm4_intergenic_Kdm4c|SNORA43_chr4_74465085_F TCGTCGGCAGCGTCAGGATGTTTGATAAGTGAGTGGA 57.2 0.15
mm4_intergenic_Kdm4c|SNORA43_chr4_74465085_R GTCTCGTGGGCTCGGAGCTGTTCCTAAAGCCACCT 58.9 0.15
mm4_exon_Tmem206_chr1_191340856_F TCGTCGGCAGCGTCGGAACTGCACCACTCAGAGG 60.3 0.13
mm4_exon_Tmem206_chr1_191340856_R GTCTCGTGGGCTCGGGGGAAGAAAGGCTGGAGTCC 60.0 0.13
mm4_intergenic_Gm9801|Ndn_chr7_62317308_F Primer3: not found at this Tm N.d. 0.12
mm4_intergenic_Gm9801|Ndn_chr7_62317308_R Primer3: not found at this Tm N.d. 0.12
mm4_intergenic_Gm25758|Adamts3_chr5_89843550_F TCGTCGGCAGCGTCGAAATGTTCAGCCACGCCC 59.7 0.10
mm4_intergenic_Gm25758|Adamts3_chr5_89843550_R GTCTCGTGGGCTCGGGCGGTTTGTTTGACTTGCCT 59.8 0.10
mm4_intron_Copb1_chr7_114241651_F TCGTCGGCAGCGTCCAATAAAGTGCTGCCCGTCC 59.5 0.07
mm4_intron_Copb1_chr7_114241651_R GTCTCGTGGGCTCGGTGGGGCCTGAGTTCACTAGT 60.1 0.07
mm4_intergenic_Olfr921|Olfr922_chr9_38783507_F Primer3: not found at this Tm N.d. 0.07
mm4_intergenic_Olfr921|Olfr922_chr9_38783507_R Primer3: not found at this Tm N.d. 0.07
mm4_intergenic_Gm25937|Cxcl13_chr5_95935336_F Primer3: not found at this Tm N.d. 0.07
mm4_intergenic_Gm25937|Cxcl13_chr5_95935336_R Primer3: not found at this Tm N.d. 0.07
mm4_intron_1810046K07Rik_chr9_51319639_F TCGTCGGCAGCGTCGGAGAATCTTCTGCTTGGACCA 60.0 0.06
mm4_intron_1810046K07Rik_chr9_51319639_R GTCTCGTGGGCTCGGTCCCAGCCATCTAACCTACCA 59.9 0.06
mm4_intergenic_Gm26288|Atp10a_chr7_58312027_F TCGTCGGCAGCGTCCAAAGGGCAGGTTTGGAAGC 59.9 0.06
mm4_intergenic_Gm26288|Atp10a_chr7_58312027_R GTCTCGTGGGCTCGGTGCACCACATTTCCTGCAGA 60.1 0.06
mm4_intron_Chst8_chr7_34698902_F TCGTCGGCAGCGTCCGATCCTGGCTCATGGAGTC 59.9 0.05
mm4_intron_Chst8_chr7_34698902_R GTCTCGTGGGCTCGGCCACTCACCTGTCGGACTTC 60.0 0.05
mm4_exon_Gm11620_chr11_105165300_F TCGTCGGCAGCGTCGCACCGGAAGAAGATGTCCT 59.7 0.04
mm4_exon_Gm11620_chr11_105165300_R GTCTCGTGGGCTCGGCTCATAGCACGGGAGCTGC 60.5 0.04
mm4_intergenic_Gm23278|Tdrd3_chr14_87385274_F Primer3: not found at this Tm N.d. 0.04
mm4_intergenic_Gm23278|Tdrd3_chr14_87385274_R Primer3: not found at this Tm N.d. 0.04
mm4_intron_Phactr2_chr10_13225223_F TCGTCGGCAGCGTCCGTGTGTGCAGTGTGTATGA 58.5 0.03
mm4_intron_Phactr2_chr10_13225223_R GTCTCGTGGGCTCGGTGGTCAACACACAAAGCTCCT 60.0 0.03
mm4_intron_Col26a1_chr5_136846613_F Primer3: not found at this Tm N.d. 0.03
mm4_intron_Col26a1_chr5_136846613_R Primer3: not found at this Tm N.d. 0.03
mm4_intergenic_Gm5839|Gm25440_chr18_49530424_F TCGTCGGCAGCGTCTTTCCTGAAGACCCAGTCGC 59.9 0.03
mm4_intergenic_Gm5839|Gm25440_chr18_49530424_R GTCTCGTGGGCTCGGTCTCACCCGACTGATCATGT 58.1 0.03
mm4_intron_Galnt9_chr5_110579458_F TCGTCGGCAGCGTCCACGGTGGTAAGATGGCCAA 60.3 0.01
mm4_intron_Galnt9_chr5_110579458_R GTCTCGTGGGCTCGGCGTTCCATTGCTGGGAGGAT 60.1 0.01
mm3_intergenic_Bai1|Mroh4_chr15_74604987_F TCGTCGGCAGCGTCCCTGGCTGCTCATTCTGAGT 59.7 0.01
mm3_intergenic_Bai1|Mroh4_chr15_74604987_R GTCTCGTGGGCTCGGCCGGAGCTCTATTCGCAACA 60.1 0.01
mm4_intron_Pfkfb1_chrX_150603710_F Primer3: not found at this Tm N.d. 0.01
mm4_intron_Pfkfb1_chrX_150603710_R Primer3: not found at this Tm N.d. 0.01
mm4_intron_Slc9a2_chr1_40730505_F TCGTCGGCAGCGTCTGGGGAAGGGAGAAGAAACA 58.1 0.00
mm4_intron_Slc9a2_chr1_40730505_R GTCTCGTGGGCTCGGAGAGCATGCTCACTGAAGCA 59.6 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.

mm4_intron_Dnah5_chr15_28306179 TGCTTTGGGGAAGTTCCACTGTGTATCCTCTAGCCCACCATGGTGTAGTTCAAGCATGAGTTGGTGTGCATGTGGGTGTT
mm4_intron_Tstd2_chr4_46119695 CTTCGAGCCTGCTAAGTGCTGGAGACTTGAGAAGCTCCGGCTTTATACTTACAACCCCAGTGCTGGAGCTGCACCAGGGA
ACATAAATCCCTCGGCCGG
mm4_intergenic_Kdm4c|SNORA43_chr4_74465085 AGGATGTTTGATAAGTGAGTGGATATTGTCAGAATTTATCTCTTGTGATTTTTGGAAGATTTCCCTGGTGGGGTTAAGGC
ACAGGGAACTCATTGTTTATATAGATGAAAATGCAGAAGTAGGTGGCTTTAGGAACAGCT
mm4_exon_Tmem206_chr1_191340856 GGAACTGCACCACTCAGAGGATCAATTACACCCACCCCTTCTTCAACCACACCATGGTAATGCCTGCTTCCCTGGGTTCT
GGTAGCCACACACCCTGAGGACTCCAGCCTTTCTTCCC
mm4_intergenic_Gm25758|Adamts3_chr5_89843550 GAAATGTTCAGCCACGCCCAGAGAACCTGGGGAAAAAGCCATCCTCAGAGTCCCTGCTTGAACTCCACCAAGGAGGAGTT
CATAAAGAGGAGTCCTCCATAAGCCAGTCATGTGGAACAAGGCAAGTCAAACAAACCGC
mm4_intron_Copb1_chr7_114241651 CAATAAAGTGCTGCCCGTCCCTCCCCCAGCTTTAACAGCACCAAGGGCAAACCCAGGGCCTGTGTGTACCAAATAGTGTC
CTACTAGTGAACTCAGGCCCCA
mm4_intron_1810046K07Rik_chr9_51319639 GGAGAATCTTCTGCTTGGACCACCCTGGTGGTGTTCAAGCACAGAATTAGTGGTTGAACTAGACATAGTTTAAAATATTT
TTAAGCTTGAAATTGCTGTGGTAGGTTAGATGGCTGGGA
mm4_intergenic_Gm26288|Atp10a_chr7_58312027 CAAAGGGCAGGTTTGGAAGCATGGGCCCTTGGTGAGGTTCTAGCAGGAGAAACAGTGCCTTGAAATCTGACTCCTCCACA
TCTGCAGGAAATGTGGTGCA
mm4_intron_Chst8_chr7_34698902 CGATCCTGGCTCATGGAGTCCACCTATGCCTCCTGTTTGAACCTCACCAGGGTCACACTGCCTGGGTCGCTGAACGTGGT
GCTGCCAAGCTGAAAGAAGTCCGACAGGTGAGTGG
mm4_exon_Gm11620_chr11_105165300 GCACCGGAAGAAGATGTCCTCAGCGCTCTCGCTCGCATCACCCGGAGCCTGAAGCTGCTGGCGAGACAACAGCAGGCGCT
GCAGGCCCTCGTGCGGCTCCAGCACGTGCGCGTCCACGGGCAGCTCCCGTGCTATGAG
mm4_intron_Phactr2_chr10_13225223 CGTGTGTGCAGTGTGTATGAATGCAAATGAGTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNATAGAGGAGCTTTGTGTGTTGACCA
mm4_intergenic_Gm5839|Gm25440_chr18_49530424 TTTCCTGAAGACCCAGTCGCAGTGGACAACTTTTACAGATGCTCTCCCTTGGTGAGATGCAAGCATGGGTTACATGATCA
GTCGGGTGAGA
mm4_intron_Galnt9_chr5_110579458 CACGGTGGTAAGATGGCCAAAGCCCAGAGTCCCAGCTGGCTGTGATGCCCCAAATCTGGAACAGTTCTCTGGGGCTGTGC
TTGAACCGCTCCCAGGATGGTGCCTGGGCTGGGGTGATCCTCCCAGCAATGGAACG
mm3_intergenic_Bai1|Mroh4_chr15_74604987 CCTGGCTGCTCATTCTGAGTGTTGTTTGCAAAGACCTCGTGCTTCAGCCGCACCAAGATTTGTAGTGTCTATGTGCACTG
TTGCGAATAGAGCTCCGG
mm4_intron_Slc9a2_chr1_40730505 TGGGGAAGGGAGAAGAAACATGAAAAAGAAAGAAAACAGAAGTTCTGGAATGCAGATGTTTCTATAATGTTTGCCTCAGA
ACTGAAGCCTTGGTGCCGTTCCAGACCGGGGTGGTCATTGCTTCAGTGAGCATGCTCT

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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