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PCR primers for off-targets of CTGGTACTCGGCGTTCCCGG 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
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52314238_F Primer3: not found at this Tm N.d. 1.00
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52314238_R Primer3: not found at this Tm N.d. 1.00
mm4_intergenic_Zswim6|Smim15_chr13_107938155_F TCGTCGGCAGCGTCGCTCCTCGCTGACTTGCTAA 60.1 0.57
mm4_intergenic_Zswim6|Smim15_chr13_107938155_R GTCTCGTGGGCTCGGTGTGTGCAATATCATTTGGCCA 59.1 0.57
mm4_intron_1110057K04Rik_chr12_8243745_F TCGTCGGCAGCGTCCGGGATTTACTTGGTGCTTGG 59.5 0.53
mm4_intron_1110057K04Rik_chr12_8243745_R GTCTCGTGGGCTCGGTCTGAAACAGCGCGGTTACT 59.9 0.53
mm4_exon_Cdh6_chr15_13053929_F TCGTCGGCAGCGTCTCGGCATCACTGGCTTTGAT 60.0 0.34
mm4_exon_Cdh6_chr15_13053929_R GTCTCGTGGGCTCGGAGTGTCTGCCTCACCATGTG 59.9 0.34
mm3_exon_Tmem65_chr15_58822997_F Primer3: not found at this Tm N.d. 0.31
mm3_exon_Tmem65_chr15_58822997_R Primer3: not found at this Tm N.d. 0.31
mm4_exon_Itpkb/Gm5069_chr1_180330579_F TCGTCGGCAGCGTCGCGGTAGTGTCTCCAACCAA 59.9 0.28
mm4_exon_Itpkb/Gm5069_chr1_180330579_R GTCTCGTGGGCTCGGGGGTGCAACGAAGCTGAATG 60.1 0.28
mm4_exon_Vps37b_chr5_124004774_F TCGTCGGCAGCGTCCCCCTTTACTGCATCCACCC 60.3 0.18
mm4_exon_Vps37b_chr5_124004774_R GTCTCGTGGGCTCGGACCCTACTGGAACCACTCTGT 60.1 0.18
mm4_intergenic_Gm25660|Slc27a6_chr18_58387266_F TCGTCGGCAGCGTCCAGTTCAGATTGCGCACAGG 59.8 0.16
mm4_intergenic_Gm25660|Slc27a6_chr18_58387266_R GTCTCGTGGGCTCGGGGACTTGTGGGTAGGACAGG 59.3 0.16
mm4_intergenic_Cat|Gm13996_chr2_103538154_F TCGTCGGCAGCGTCCAACCAGAGCCTGCTACACA 59.9 0.15
mm4_intergenic_Cat|Gm13996_chr2_103538154_R GTCTCGTGGGCTCGGAAGTTGGTAACTGGGATATTGGA 57.2 0.15
mm4_intergenic_Fkrp|Strn4_chr7_16816908_F TCGTCGGCAGCGTCCAAGCCCAGAGAGCCTGTC 60.0 0.15
mm4_intergenic_Fkrp|Strn4_chr7_16816908_R GTCTCGTGGGCTCGGTTTATTCGCGCAGACCCCAT 60.1 0.15
mm4_exon_Dmgdh_chr13_93674520_F TCGTCGGCAGCGTCCACTGCCACCTCGCCATG 61.1 0.14
mm4_exon_Dmgdh_chr13_93674520_R GTCTCGTGGGCTCGGCCTGCTCCTCTTCCCACCC 61.6 0.14
mm3_exon_Hoxb1_chr11_96366824_F TCGTCGGCAGCGTCCTGACGTGGCTTTTCTCCCT 59.9 0.11
mm3_exon_Hoxb1_chr11_96366824_R GTCTCGTGGGCTCGGTGGTGAAGTTTGTGCGGAGA 59.8 0.11
mm4_exon_BC025920/Gm26896_chr10_81608927_F TCGTCGGCAGCGTCATGTGTCGTGAGTGGAGCAG 60.0 0.11
mm4_exon_BC025920/Gm26896_chr10_81608927_R GTCTCGTGGGCTCGGTAGGCTTGGGAGTGGAAGGT 60.1 0.11
mm4_intergenic_Fbxl2|Susd5_chr9_114047563_F Primer3: not found at this Tm N.d. 0.05
mm4_intergenic_Fbxl2|Susd5_chr9_114047563_R Primer3: not found at this Tm N.d. 0.05
mm4_intron_Ubr4_chr4_139420618_F TCGTCGGCAGCGTCTTAGCGGTGGAGGAGGAAGA 59.9 0.04
mm4_intron_Ubr4_chr4_139420618_R GTCTCGTGGGCTCGGAGAAGGCAGCAGGACTCAAC 59.9 0.04
mm4_intergenic_Nek2|1700034H15Rik_chr1_191841216_F TCGTCGGCAGCGTCACAAGCTATGCCTCAGCCTG 60.1 0.03
mm4_intergenic_Nek2|1700034H15Rik_chr1_191841216_R GTCTCGTGGGCTCGGTGCCTTGTGTGAGTTCTGCT 59.8 0.03
mm4_intergenic_Ccl25|Cers4_chr8_4405092_F TCGTCGGCAGCGTCCTGGGCCAGTATATGCAGGG 59.9 0.03
mm4_intergenic_Ccl25|Cers4_chr8_4405092_R GTCTCGTGGGCTCGGTCTCAGGCGTGGTACTGAGA 59.9 0.03
mm4_intergenic_Vash2|Mfsd7b_chr1_190988237_F TCGTCGGCAGCGTCTTTTGAGTGTCTCTCCCAGAAA 57.2 0.02
mm4_intergenic_Vash2|Mfsd7b_chr1_190988237_R GTCTCGTGGGCTCGGTGGGGAATTGGTGCTCAGT 58.8 0.02
mm4_intron_Gabbr2_chr4_46881504_F Primer3: not found at this Tm N.d. 0.02
mm4_intron_Gabbr2_chr4_46881504_R Primer3: not found at this Tm N.d. 0.02
mm4_intergenic_Itpkb/Gm5069|Itpkb_chr1_180331025_F Primer3: not found at this Tm N.d. 0.00
mm4_intergenic_Itpkb/Gm5069|Itpkb_chr1_180331025_R Primer3: not found at this Tm N.d. 0.00
mm4_exon_Grin2c_chr11_115250438_F Primer3: not found at this Tm N.d. 0.00
mm4_exon_Grin2c_chr11_115250438_R Primer3: not found at this Tm N.d. 0.00
mm4_exon_Dync1li2_chr8_104442831_F TCGTCGGCAGCGTCCAACTGCAGCCACAAAGAGG 59.6 0.00
mm4_exon_Dync1li2_chr8_104442831_R GTCTCGTGGGCTCGGCGCTGGATAACGGAGCTTCT 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.

mm4_intergenic_Zswim6|Smim15_chr13_107938155 GCTCCTCGCTGACTTGCTAAATAGCAAAAATTGGTACTTGGCATTCCCAGTGGATGTGGGAAAAGATAATAAGTACCCCA
AATGTAAAGAAAATCGAAATGAAACTAATTATGGCCAAATGATATTGCACACA
mm4_intron_1110057K04Rik_chr12_8243745 CGGGATTTACTTGGTGCTTGGGGGTCGTATCTCTCAGAGATAGCAGATCCTAATCCTGTGGTACCTGGCGTTCCCTGGGG
AAAGTGCACAGTAACCGCGCTGTTTCAGA
mm4_exon_Cdh6_chr15_13053929 TCGGCATCACTGGCTTTGATCCTGCCAATTGGCGTTCCCGGTGGAGAGGACTCGGGGGTCTTAAACTGGTAGGTACCTGT
CAATCACCAAACACATGGTGAGGCAGACACT
mm4_exon_Itpkb/Gm5069_chr1_180330579 GCGGTAGTGTCTCCAACCAAGCACAGAGCAACGCAGGGTCGGCCTGGCGGTGTCCCCTCCGGGATCGCCTAGTCCCTGCG
CCGGTACATTCAGCTTCGTTGCACCC
mm4_exon_Vps37b_chr5_124004774 CCCCTTTACTGCATCCACCCGGAGCTCCCATGTGGCTGCGGCCCGAGACTGGCAGTGGGCGTTCCTGGTGGCAGAGGTAC
TGAAGTGGCACATACAGCTTTGTCTCTTTACAGAGTGGTTCCAGTAGGGT
mm4_intergenic_Gm25660|Slc27a6_chr18_58387266 CAGTTCAGATTGCGCACAGGGGCTGTTATGACACCTGGTATTCGGGGATCCCAGCGGGGCAGTATCGAGTAGACACCTTC
TGAGAGGCTGATTGGTATTTCTGAAACTTTCCTGTCCTACCCACAAGTCC
mm4_intergenic_Cat|Gm13996_chr2_103538154 CAACCAGAGCCTGCTACACATACTGGGGTAGCGATTCCAACACAATCTGTCTACTCCTGGTTATCGGGGTTCCTGGGGGC
ATTGCTGGATGTAATCCAATATCCCAGTTACCAACTT
mm4_intergenic_Fkrp|Strn4_chr7_16816908 CAAGCCCAGAGAGCCTGTCTGGGCGATGCGACTCGGGGTTCCCGGGGGACAGAGTGCTGAGAAAGTGGATGGGGTCTGCG
CGAATAAA
mm4_exon_Dmgdh_chr13_93674520 CACTGCCACCTCGCCATGCTCCGCCCGGGCGCGCTGCGGCTGCGGGGCCTCGCGCTGCGGGGCTCCCCCAGGCGCCCAAG
CTCCGCTGGCCTCCGGGAAGGGTGAGTACCCGATGGCTGCCGGGTGGGAAGAGGAGCAGG
mm3_exon_Hoxb1_chr11_96366824 CTGACGTGGCTTTTCTCCCTGCCCCGCCCCTAGCGAAGGTGTCCGAGCTGGGACTGGGCGCTCCCGGCGGTCTCCGCACA
AACTTCACCA
mm4_exon_BC025920/Gm26896_chr10_81608927 ATGTGTCGTGAGTGGAGCAGAGCCTTCTGGTACTCTGTGTCCCCGAGGGCACACTGGATGACACATTTGGGAACCAGTCC
TTACTATGTGAGCAGTGTGGGAAAACCTTCCACTCCCAAGCCTA
mm4_intron_Ubr4_chr4_139420618 TTAGCGGTGGAGGAGGAAGACTCCCAGGCTGAGGACTCTGTAAGTGCTCTAGGGAAGGGAATGGCTCTTCAGCCTGTCCA
GTGTGCTCTGGTACTCTGCCTCCTCGGTGGGCTTGAGGACGTTGAGTCCTGCTGCCTTCT
mm4_intergenic_Nek2|1700034H15Rik_chr1_191841216 ACAAGCTATGCCTCAGCCTGGGGCAGGTCCCCAGCACCTTGGCCTCCGAGGACGCCCAGTACAAGCTTGGTACATCCTCT
GACATTTAGACAGCAGAACTCACACAAGGCA
mm4_intergenic_Ccl25|Cers4_chr8_4405092 CTGGGCCAGTATATGCAGGGCTGCAGACAAAGGAATCCCTGGCCGGGTCCCAGTCGGTCAGGCGCCCTCTGGCAACTTCT
GGACCTCTCCTCCTCCGCGTTCCCGGTGGTCACCCCTTCTCAGTACCACGCCTGAGA
mm4_intergenic_Vash2|Mfsd7b_chr1_190988237 TTTTGAGTGTCTCTCCCAGAAACTGGTACTCGTCGTTTCAGCTGGACTGAGCACCAATTCCCCA
mm4_exon_Dync1li2_chr8_104442831 CAACTGCAGCCACAAAGAGGGCCCTCCCCCGGGCCCGCCGAGGACCCGCAAGGANNNNNNNNNNNNNNNNNNNNNNNNNN
NNAACGCCGCAGAAGCTCCGTTATCCAGCG

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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