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PCR primers for off-targets of CGCCGGCGCGCGCCCAGATT 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.

NamePrimer Sequence Tm CFD Score
ontarget_mm0__chr7 5.53 Mbp_chr7_5529750_F Primer3: not found at this Tm N.d. 1.00
ontarget_mm0__chr7 5.53 Mbp_chr7_5529750_R Primer3: not found at this Tm N.d. 1.00
mm4__chr16 871.16 Kbp_chr16_871159_F TCGTCGGCAGCGTCCACCACCCGACTTTCTCCTG 60.3 0.29
mm4__chr16 871.16 Kbp_chr16_871159_R GTCTCGTGGGCTCGGGATGCAGAGGGACATCCGAG 59.9 0.29
mm4__chr9 93.83 Mbp_chr9_93826536_F TCGTCGGCAGCGTCACGCAGGTAAGGTAAAGCCA 59.3 0.19
mm4__chr9 93.83 Mbp_chr9_93826536_R GTCTCGTGGGCTCGGACGAGCTGCTGAAAGCCC 60.3 0.19
mm4__chr19 1.41 Mbp_chr19_1408419_F TCGTCGGCAGCGTCCCAAACTGTGGCGTGGGG 61.2 0.15
mm4__chr19 1.41 Mbp_chr19_1408419_R GTCTCGTGGGCTCGGGAGTGGGATAGGTTGGGCG 59.8 0.15
mm4__chr2 171.20 Mbp_chr2_171204333_F Primer3: not found at this Tm N.d. 0.15
mm4__chr2 171.20 Mbp_chr2_171204333_R Primer3: not found at this Tm N.d. 0.15
mm3__chr1 44.81 Mbp_chr1_44813244_F TCGTCGGCAGCGTCGACGAGGCTGAACTGGTCC 60.0 0.15
mm3__chr1 44.81 Mbp_chr1_44813244_R GTCTCGTGGGCTCGGCCTCCTTCCGTGCCCTTG 60.0 0.15
mm4__chr1 121.52 Mbp_chr1_121519418_F TCGTCGGCAGCGTCTTCTCCTCCTCCAGTGCCTT 60.1 0.07
mm4__chr1 121.52 Mbp_chr1_121519418_R GTCTCGTGGGCTCGGCCCTGTACCCATCACACCTG 59.7 0.07
mm4__chr22 30.29 Mbp_chr22_30289366_F TCGTCGGCAGCGTCGCGATCCTAATCCTCCGACC 59.7 0.06
mm4__chr22 30.29 Mbp_chr22_30289366_R GTCTCGTGGGCTCGGATCAAGCTGCTCTTCCTGCC 60.3 0.06
mm4__chr17 81.40 Mbp_chr17_81396043_F TCGTCGGCAGCGTCACTCGAAGACAGGACAGGGA 59.8 0.03
mm4__chr17 81.40 Mbp_chr17_81396043_R GTCTCGTGGGCTCGGGCGAATGTTGGGGTTCGTTC 60.1 0.03
mm4__chr22 33.92 Mbp_chr22_33920413_F Primer3: not found at this Tm N.d. 0.03
mm4__chr22 33.92 Mbp_chr22_33920413_R Primer3: not found at this Tm N.d. 0.03
mm4__chr19 18.23 Mbp_chr19_18225717_F TCGTCGGCAGCGTCTGGACCGAGTGCTTAGAGGA 59.9 0.03
mm4__chr19 18.23 Mbp_chr19_18225717_R GTCTCGTGGGCTCGGATGAACTCTGGGTGTCCACG 59.6 0.03
mm4__chr10 133.22 Mbp_chr10_133220543_F TCGTCGGCAGCGTCGTGAGGAGGGGCTCAGGC 61.7 0.03
mm4__chr10 133.22 Mbp_chr10_133220543_R GTCTCGTGGGCTCGGGAGCCCCTCCTCTCCTGG 60.4 0.03
mm4__chr10 133.22 Mbp_chr10_133220351_F Primer3: not found at this Tm N.d. 0.03
mm4__chr10 133.22 Mbp_chr10_133220351_R Primer3: not found at this Tm N.d. 0.03
mm4__chrX 154.37 Mbp_chrX_154374380_F TCGTCGGCAGCGTCGGACACTCACTCCCAGATGC 60.1 0.02
mm4__chrX 154.37 Mbp_chrX_154374380_R GTCTCGTGGGCTCGGGGCAGAGAGGCCGGAAAAC 61.0 0.02
mm4__chr1 3.06 Mbp_chr1_3063768_F TCGTCGGCAGCGTCGAGGCTCGCCATTTTCGTTG 60.1 0.01
mm4__chr1 3.06 Mbp_chr1_3063768_R GTCTCGTGGGCTCGGGGAAGGTGGCAGAGCGAC 60.4 0.01
mm4__chr10 133.22 Mbp_chr10_133220799_F TCGTCGGCAGCGTCCAGGAGACGAGGGGCTCA 60.3 0.01
mm4__chr10 133.22 Mbp_chr10_133220799_R GTCTCGTGGGCTCGGAGCAAGGACAGGAGGAGAGG 60.6 0.01
mm4__chr4 8.16 Mbp_chr4_8159400_F Primer3: not found at this Tm N.d. 0.01
mm4__chr4 8.16 Mbp_chr4_8159400_R Primer3: not found at this Tm N.d. 0.01
mm3__chr21 44.01 Mbp_chr21_44012825_F Primer3: not found at this Tm N.d. 0.01
mm3__chr21 44.01 Mbp_chr21_44012825_R Primer3: not found at this Tm N.d. 0.01
mm4__chr1 226.74 Mbp_chr1_226738211_F Primer3: not found at this Tm N.d. 0.00
mm4__chr1 226.74 Mbp_chr1_226738211_R Primer3: not found at this Tm N.d. 0.00
mm4__chr16 400.82 Kbp_chr16_400823_F TCGTCGGCAGCGTCGATGTATGAGCCAGGCAGGG 60.2 0.00
mm4__chr16 400.82 Kbp_chr16_400823_R GTCTCGTGGGCTCGGTTCTCCTTTGGCGGGATCAC 60.0 0.00
mm4__chr19 1.59 Mbp_chr19_1592775_F Primer3: not found at this Tm N.d. 0.00
mm4__chr19 1.59 Mbp_chr19_1592775_R Primer3: not found at this Tm N.d. 0.00
mm4__chr4 1.01 Mbp_chr4_1011270_F TCGTCGGCAGCGTCCGGAGGTGTCTCGAGAGTTG 59.8 0.00
mm4__chr4 1.01 Mbp_chr4_1011270_R GTCTCGTGGGCTCGGCCTAGGACCGGCCAGGAG 60.8 0.00
mm4__chr12 111.93 Mbp_chr12_111932950_F Primer3: not found at this Tm N.d. 0.00
mm4__chr12 111.93 Mbp_chr12_111932950_R Primer3: not found at this Tm N.d. 0.00
mm4__chr16 641.95 Kbp_chr16_641947_F TCGTCGGCAGCGTCAGTCCGTTAAGTGCGAGCC 60.0 0.00
mm4__chr16 641.95 Kbp_chr16_641947_R GTCTCGTGGGCTCGGGTGATGGTGTACATGACGCG 59.3 0.00
mm4__chr2 85.54 Mbp_chr2_85539106_F Primer3: not found at this Tm N.d. 0.00
mm4__chr2 85.54 Mbp_chr2_85539106_R Primer3: not found at this Tm N.d. 0.00
mm4__chr17 31.49 Mbp_chr17_31487801_F Primer3: not found at this Tm N.d. 0.00
mm4__chr17 31.49 Mbp_chr17_31487801_R Primer3: not found at this Tm N.d. 0.00
mm4__chr9 127.54 Mbp_chr9_127540771_F TCGTCGGCAGCGTCCATCCAGCTCATGTCCACCA 59.7 0.00
mm4__chr9 127.54 Mbp_chr9_127540771_R GTCTCGTGGGCTCGGCTCGTGGGTGAGGTGTGTC 60.0 0.00
mm4__chr13 24.68 Mbp_chr13_24680445_F TCGTCGGCAGCGTCTCAATCCTGGACTCTCCCGA 59.6 0.00
mm4__chr13 24.68 Mbp_chr13_24680445_R GTCTCGTGGGCTCGGCACCAGCCTTAAATGCCGC 59.8 0.00
mm4__chr11 113.45 Mbp_chr11_113452472_F TCGTCGGCAGCGTCTCCATGTTGAGGAGCAAGGG 59.6 0.00
mm4__chr11 113.45 Mbp_chr11_113452472_R GTCTCGTGGGCTCGGGGGTGGATCTGAATCAGCAGT 59.7 0.00
mm4__chr10 97.33 Mbp_chr10_97334058_F Primer3: not found at this Tm N.d. 0.00
mm4__chr10 97.33 Mbp_chr10_97334058_R Primer3: not found at this Tm N.d. 0.00
mm4__chr10 22.32 Mbp_chr10_22320591_F Primer3: not found at this Tm N.d. 0.00
mm4__chr10 22.32 Mbp_chr10_22320591_R Primer3: not found at this Tm N.d. 0.00
mm3__chr7 143.36 Mbp_chr7_143362633_F Primer3: not found at this Tm N.d. 0.00
mm3__chr7 143.36 Mbp_chr7_143362633_R Primer3: not found at this Tm N.d. 0.00

Off-target amplicon sequences

Primers underlined, off-targets in bold.

mm4__chr16 871.16 Kbp_chr16_871159 CACCACCCGACTTTCTCCTGCCCTTGGGCAGGGGCCCCCAGCTGAGCCACCTACCGAATCTGGGCTCGGGCCGGGCCCCT
CGGATGTCCCTCTGCATC
mm4__chr9 93.83 Mbp_chr9_93826536 ACGCAGGTAAGGTAAAGCCAGGCAGACCCGAACCTGGACGCGCACCTGCGGGGGAGGGGGTATGCGCGGGGCGGGTTGCC
GCCCCTCCCGGGAGTACGGGCGGAGGCAGGGGCGGGCGGGGGCTTTCAGCAGCTCGT
mm4__chr19 1.41 Mbp_chr19_1408419 CCAAACTGTGGCGTGGGGGGTGGTCCGGGGCGCCCCCAGCGCGCGCCCAAACTTGGCGGGCGGCGCACGTGGCGAGCCGA
GGCGAGGGTTCCGGTTCTGCCACCGCCCAACCTATCCCACTC
mm3__chr1 44.81 Mbp_chr1_44813244 GACGAGGCTGAACTGGTCCGCGCGGCCCGAAGCTGGGCGCGCGTGGGCGCGGTGAGTGGGGCTGGGGGAGCGCAAGGGCA
CGGAAGGAGG
mm4__chr1 121.52 Mbp_chr1_121519418 TTCTCCTCCTCCAGTGCCTTCTCGCCGCCGCGCGCCCAAGCTCGGCGGACGGCAGTGCCCCAGGTGTGATGGGTACAGGG
mm4__chr22 30.29 Mbp_chr22_30289366 GCGATCCTAATCCTCCGACCCTGGCCCGGAGCGAGAGCAGAGCCCCCAGCCCCTATCTGCGCTCCCGAACCCGGGCGCGC
ACCGGCTGCGGCGGGGCAGGAAGAGCAGCTTGAT
mm4__chr17 81.40 Mbp_chr17_81396043 ACTCGAAGACAGGACAGGGACCGCCAGGCTGCGTCTGGCGCGAGCCGCGCGGGTGGCACCGGGCGGACAGTAGGCGAAGT
CCGGGCGCCGCCAGCGTGCGCCCAGTTCTGGGTGACCCACGAACGAACCCCAACATTCGC
mm4__chr19 18.23 Mbp_chr19_18225717 TGGACCGAGTGCTTAGAGGATGCAGCGCAAGCAGCTGCCGGGCGCAGGCGCGCGCTCAGCCTGGGACTCCCGAACGCCCT
GCCTCGAAAACCCCCTCAATTCAGGTCTTCCTCCGTGGACACCCAGAGTTCAT
mm4__chr10 133.22 Mbp_chr10_133220543 GTGAGGAGGGGCTCAGGCGGCCGCGCGCCCAGGTGAGGAGGGGCTCAGGCGGGCGCGCGCCCAGGTGAGGAGGGGCTCAG
GCGGGCGCGCGCCCAGGAGAGGAGGGGCTC
mm4__chrX 154.37 Mbp_chrX_154374380 GGACACTCACTCCCAGATGCCAGGGGAGCCTCCATCGGCTCACGCATCCCAGCTGAACCTGGCCCGGACCTGGGCGCGAG
CTGGCGCCTGCGTTTTCCGGCCTCTCTGCC
mm4__chr1 3.06 Mbp_chr1_3063768 GAGGCTCGCCATTTTCGTTGCTGCCAGGGCCCCGCCCGCGCGCGGCCGAATTGGGATCTGAAGGCAGCGGCAGCGTCTAC
GCAAGCAGCGTTCTGGAGCGTCGCTCTGCCACCTTCC
mm4__chr10 133.22 Mbp_chr10_133220799 CAGGAGACGAGGGGCTCAGGCGGGCGCGCGCCCAGGTCAGGAGGGGCTCAGGTGGCCGCGCATCCAGCAGAGGAGGGGCT
CAGATGGGCGCATGCCCAGGTGGGGCACGCTCAGGTGGCACCTCTCCTCCTGTCCTTGCT
mm4__chr16 400.82 Kbp_chr16_400823 GATGTATGAGCCAGGCAGGGGTCCAGAGACCACCAGCCTGCAATCCGGAGAACCCAAGCTAGGCGCGAGCCGGTGCTGTG
ATCCCGCCAAAGGAGAA
mm4__chr4 1.01 Mbp_chr4_1011270 CGGAGGTGTCTCGAGAGTTGAGGACCCGACCCGGGCGCGCGCGGGCGCAGGTATGGGGAGGGCGCGGCGGCTCCTGGCCG
GTCCTAGG
mm4__chr16 641.95 Kbp_chr16_641947 AGTCCGTTAAGTGCGAGCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGTGTGGGAGCGGCGCGTCATGTA
CACCATCAC
mm4__chr9 127.54 Mbp_chr9_127540771 CATCCAGCTCATGTCCACCATGGGGGCTGCACGCCGGCGCCAGCCCTGGTTTGGCCCCAGTGGAGGGCGCTGTGCCAGAC
CCGTTCCTGGCTCATCCGGTCTCTCCAGTGGGCTCCAGGGACACACCTCACCCACGAG
mm4__chr13 24.68 Mbp_chr13_24680445 TCAATCCTGGACTCTCCCGACCCCTAGCTGTCGGTCCCCCTCCCTGCGCGCGCGCCGGCGGGTTTCCTACCCTCCGAGGC
GTCCGCTGGCCTGCGCCCTGGCGGGGACGTGGGCGGGGCGGGCGGCATTTAAGGCTGGTG
mm4__chr11 113.45 Mbp_chr11_113452472 TCCATGTTGAGGAGCAAGGGCTAGGGCCACTGGTGTGCATGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNACATTGGGGGACAGGAAATGAGACACTGCTGATTCAGATCCACCC

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_gYvicTzp9e5VPFC9YwLR.txt --name MY_EXPERIMENT

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