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PCR primers for off-targets of GAGCCGGTCGATGGACTAGC AGG

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
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52314045_F Primer3: not found at this Tm N.d. 1.00
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52314045_R Primer3: not found at this Tm N.d. 1.00
mm4_intergenic_Tln2|Mir190_chr9_67231573_F TCGTCGGCAGCGTCTCTGCAGACTCACGGTTCAC 59.9 0.45
mm4_intergenic_Tln2|Mir190_chr9_67231573_R GTCTCGTGGGCTCGGTCCTGAGCCTCACCTAGACC 60.0 0.45
mm4_intergenic_Tln2|Mir190_chr9_67231511_F TCGTCGGCAGCGTCCCATCTGCAGACTCACGGTT 60.0 0.45
mm4_intergenic_Tln2|Mir190_chr9_67231511_R GTCTCGTGGGCTCGGGGTCTATGCGTGAGTCTGCA 59.8 0.45
mm4_intergenic_Gm25564|Dcdc2c_chr12_28442710_F Primer3: not found at this Tm N.d. 0.35
mm4_intergenic_Gm25564|Dcdc2c_chr12_28442710_R Primer3: not found at this Tm N.d. 0.35
mm4_intron_Lrrc17_chr5_21574773_F Primer3: not found at this Tm N.d. 0.31
mm4_intron_Lrrc17_chr5_21574773_R Primer3: not found at this Tm N.d. 0.31
mm3_intron_1700112E06Rik_chr14_22306800_F Primer3: not found at this Tm N.d. 0.31
mm3_intron_1700112E06Rik_chr14_22306800_R Primer3: not found at this Tm N.d. 0.31
mm4_intergenic_Gm23831|1700017G19Rik_chr3_40427935_F Primer3: not found at this Tm N.d. 0.28
mm4_intergenic_Gm23831|1700017G19Rik_chr3_40427935_R Primer3: not found at this Tm N.d. 0.28
mm4_intergenic_Ptprm|Gm22041_chr17_67016368_F TCGTCGGCAGCGTCTTCTGACCCAGTCCAGGACA 60.1 0.23
mm4_intergenic_Ptprm|Gm22041_chr17_67016368_R GTCTCGTGGGCTCGGAGATGATGGCAGGAGGAGGT 60.0 0.23
mm4_intron_Slc22a8_chr19_8596390_F TCGTCGGCAGCGTCTTTGTGGGACAGAGACTGGC 59.8 0.22
mm4_intron_Slc22a8_chr19_8596390_R GTCTCGTGGGCTCGGCAGGTCCCACAGAGTCCAAC 59.9 0.22
mm3_intron_Gpam_chr19_55086184_F TCGTCGGCAGCGTCGACTGAATGCCACTGTGGGA 59.9 0.17
mm3_intron_Gpam_chr19_55086184_R GTCTCGTGGGCTCGGTCGACCTGCCAGGAGTTCTA 59.9 0.17
mm4_exon_Kctd4_chr14_75963459_F TCGTCGGCAGCGTCAAGGCATCCCAAACAACGTG 59.3 0.17
mm4_exon_Kctd4_chr14_75963459_R GTCTCGTGGGCTCGGTCGGTGGAAATGTGATGGCA 59.9 0.17
mm4_intron_Snapc3_chr4_83434647_F Primer3: not found at this Tm N.d. 0.16
mm4_intron_Snapc3_chr4_83434647_R Primer3: not found at this Tm N.d. 0.16
mm4_intergenic_Gm24062|Gm10259_chr3_24855607_F TCGTCGGCAGCGTCTGAGTCCTCTCAAACTTGTAGCT 59.1 0.16
mm4_intergenic_Gm24062|Gm10259_chr3_24855607_R GTCTCGTGGGCTCGGTGCACAGTATCTGGGTAGGT 57.7 0.16
mm4_intron_Ppfia2_chr10_106519153_F Primer3: not found at this Tm N.d. 0.15
mm4_intron_Ppfia2_chr10_106519153_R Primer3: not found at this Tm N.d. 0.15
mm4_exon_Cacna1i_chr15_80398090_F TCGTCGGCAGCGTCAATCAACGTCCCTGGCAGAG 60.0 0.14
mm4_exon_Cacna1i_chr15_80398090_R GTCTCGTGGGCTCGGCCGGAAGTTAGAGTCACAGCA 59.7 0.14
mm4_exon_Iqsec1_chr6_90716448_F TCGTCGGCAGCGTCCAGGTTCACGCAGCCCTAG 60.4 0.14
mm4_exon_Iqsec1_chr6_90716448_R GTCTCGTGGGCTCGGCATTCCTCAGGCCGGACTTG 60.7 0.14
mm3_intergenic_Cers3|Adamts17_chr7_66830831_F Primer3: not found at this Tm N.d. 0.14
mm3_intergenic_Cers3|Adamts17_chr7_66830831_R Primer3: not found at this Tm N.d. 0.14
mm4_intron_Pgbd5_chr8_124415220_F TCGTCGGCAGCGTCGTCCATCAACCAGCACTCCA 59.9 0.13
mm4_intron_Pgbd5_chr8_124415220_R GTCTCGTGGGCTCGGAAGAGATTTGGCCGGTGAGG 60.0 0.13
mm4_intergenic_Adra2a|Gpam_chr19_54436957_F TCGTCGGCAGCGTCAATCCAGCCAGGGAGAGGAT 60.0 0.13
mm4_intergenic_Adra2a|Gpam_chr19_54436957_R GTCTCGTGGGCTCGGGGCAGTGTTAAAAGAATTACTGGGA 59.5 0.13
mm4_intergenic_Gm23892|Gm25382_chr10_16928424_F Primer3: not found at this Tm N.d. 0.07
mm4_intergenic_Gm23892|Gm25382_chr10_16928424_R Primer3: not found at this Tm N.d. 0.07
mm4_intron_Lphn3_chr5_81181477_F TCGTCGGCAGCGTCACTCTGTTCTTTCCCCAGGC 59.5 0.07
mm4_intron_Lphn3_chr5_81181477_R GTCTCGTGGGCTCGGGCCCAAACAGAAAACACGCT 59.8 0.07
mm4_intron_Sgcz_chr8_37616434_F TCGTCGGCAGCGTCTGAAGCAGAACAGACAGGCG 60.6 0.04
mm4_intron_Sgcz_chr8_37616434_R GTCTCGTGGGCTCGGTGCAGTAATGGCCTGTGGTT 59.8 0.04
mm4_intron_Ankrd2_chr19_42039992_F Primer3: not found at this Tm N.d. 0.04
mm4_intron_Ankrd2_chr19_42039992_R Primer3: not found at this Tm N.d. 0.04
mm4_intergenic_Spock3|Gm5350_chr8_63570610_F Primer3: not found at this Tm N.d. 0.03
mm4_intergenic_Spock3|Gm5350_chr8_63570610_R Primer3: not found at this Tm N.d. 0.03
mm4_intergenic_Gm25148|Tifab_chr13_56171306_F Primer3: not found at this Tm N.d. 0.00
mm4_intergenic_Gm25148|Tifab_chr13_56171306_R Primer3: not found at this Tm N.d. 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_intergenic_Tln2|Mir190_chr9_67231573 TCTGCAGACTCACGGTTCACCAGCGAGTCCATCTGCAGACTCACGGTTCACCAGCTAGTCCATCTGCAGACTCACGCATA
GACCTGGATAAAGATGGATTTTGTGCCTTGGTTGGTCTAGGTGAGGCTCAGGA
mm4_intergenic_Tln2|Mir190_chr9_67231511 CCATCTGCAGACTCACGGTTCACCAGCTAGTCCATCTGCAGACTCACGGTTCACCAGCGAGTCCATCTGCAGACTCACGG
TTCACCAGCTAGTCCATCTGCAGACTCACGCATAGACC
mm4_intergenic_Ptprm|Gm22041_chr17_67016368 TTCTGACCCAGTCCAGGACATGGAGACACCAGTAGTGCCAGCCAGTTAGTCCATTCACCTGCTCAGAGCTGTTCTGTTTC
CAGGCTTCAGCTGTTCACCCTCCTACCTCCTCCTGCCATCATCT
mm4_intron_Slc22a8_chr19_8596390 TTTGTGGGACAGAGACTGGCCTGTAGAGAGTTTTAGTCTAGCAGTAGAGTAGCTCCAGGATGGAGGTAGCAGGTCGATGG
AATGGCGGGTTTTGACAACAGAGAATAGGGAGTTGGACTCTGTGGGACCTG
mm3_intron_Gpam_chr19_55086184 GACTGAATGCCACTGTGGGAGGCCCTGCTTGTCCAGCGACCGGCCCCTCCCTGCAGTTTCTCTCTACATTCACTGCATTA
GAACTCCTGGCAGGTCGA
mm4_exon_Kctd4_chr14_75963459 AAGGCATCCCAAACAACGTGCCCAGCCAGCTAGTCCAGAGCCCGGCTGCTAAGCAAGTACTGTGAGCTAACGGTATGTAA
ATCTATCACTAAAGATGTCCTTCTCTGGGGTGTTCTGCCATCACATTTCCACCGA
mm4_intergenic_Gm24062|Gm10259_chr3_24855607 TGAGTCCTCTCAAACTTGTAGCTATCCAGTCAGTCCATCTACAGGCTCTTCTGGATTATCATTTCTTCCAAAAATAAGTA
TACTCTATTCAAACCTACCCAGATACTGTGCA
mm4_exon_Cacna1i_chr15_80398090 AATCAACGTCCCTGGCAGAGCCAGGGCTGAACTGGGGCCTGGGAGGAGGCAGAGGCCGCCTTGTCCAGCAAGTCCCTAGC
CCGGCTCTGCTGCTGTGACTCTAACTTCCGG
mm4_exon_Iqsec1_chr6_90716448 CAGGTTCACGCAGCCCTAGGCCTGCTAGTCCAATCACCGGCCCGGTGCGACCCTTCCGCCGCCGCCTGCCAGGCGTAGCT
CAAGTCCGGCCTGAGGAATG
mm4_intron_Pgbd5_chr8_124415220 GTCCATCAACCAGCACTCCAACCTCATTGGCAAGCCCGCCTGTCCATCAACCGGCACTCTAACCTCACCGGCCAAATCTC
TT
mm4_intergenic_Adra2a|Gpam_chr19_54436957 AATCCAGCCAGGGAGAGGATGGCTTTTATAGGTACAGATACACAACTCTATACAGCTCCAGGTAGTGCATAGACCGTCTC
CCAACATCCCAGTAATTCTTTTAACACTGCC
mm4_intron_Lphn3_chr5_81181477 ACTCTGTTCTTTCCCCAGGCTTTGTTGAGCTGTTTATCAATGAAAAACTCAGCTTACACTACTTTGGCTCAGAGCAGGTC
TATGGATAAGCAGGGTCCCACGGATGCATCCTGAGCGTGTTTTCTGTTTGGGC
mm4_intron_Sgcz_chr8_37616434 TGAAGCAGAACAGACAGGCGCTGAGCTGGTAGATGGAGAAGCTGGCTATGTGAAACTCAACCACAGGCCATTACTGCA

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_mN1FkRligAThyqKv3eg3.txt --name MY_EXPERIMENT

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