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PCR primers for off-targets of TCGGCGCGCCGGAAGTGGCC 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.

NamePrimer Sequence Tm CFD Score
ontarget_mm0__chr7 5.53 Mbp_chr7_5529707_F TCGTCGGCAGCGTCTGGTGAGCTGCGAGAATAGC 60.1 1.00
ontarget_mm0__chr7 5.53 Mbp_chr7_5529707_R GTCTCGTGGGCTCGGCGGCTTCCTTTGTCCCCAAT 60.6 1.00
mm4__chr7 39.95 Mbp_chr7_39950089_F Primer3: not found at this Tm N.d. 0.33
mm4__chr7 39.95 Mbp_chr7_39950089_R Primer3: not found at this Tm N.d. 0.33
mm4__chr10 73.79 Mbp_chr10_73788125_F Primer3: not found at this Tm N.d. 0.31
mm4__chr10 73.79 Mbp_chr10_73788125_R Primer3: not found at this Tm N.d. 0.31
mm4__chr9 129.84 Mbp_chr9_129835268_F TCGTCGGCAGCGTCGAGTTGTACAGCCGCCGC 61.4 0.22
mm4__chr9 129.84 Mbp_chr9_129835268_R GTCTCGTGGGCTCGGCTGACGCCAGGAGAGGGG 61.4 0.22
mm3__chr5 135.01 Mbp_chr5_135012998_F TCGTCGGCAGCGTCGGATGGCTCTTCAGGGTGAG 59.8 0.21
mm3__chr5 135.01 Mbp_chr5_135012998_R GTCTCGTGGGCTCGGTATCTACCCCAGCCCAGCTT 60.0 0.21
mm4__chr8 143.82 Mbp_chr8_143818330_F TCGTCGGCAGCGTCAAGCCCTAGACACAGGGACA 60.1 0.21
mm4__chr8 143.82 Mbp_chr8_143818330_R GTCTCGTGGGCTCGGGACTCCGTCACCATGAAGCA 60.0 0.21
mm3__chr16 88.47 Mbp_chr16_88473986_F TCGTCGGCAGCGTCGGTTCTCCGGAGTGAAGCAA 59.9 0.17
mm3__chr16 88.47 Mbp_chr16_88473986_R GTCTCGTGGGCTCGGTATCGACGCTGAAGGTTGGC 60.4 0.17
mm4__chr3 170.42 Mbp_chr3_170419651_F TCGTCGGCAGCGTCTAATTACTGCGGTCCCAGCC 59.8 0.17
mm4__chr3 170.42 Mbp_chr3_170419651_R GTCTCGTGGGCTCGGCACGCTCCTTCTCACCTCTG 60.1 0.17
mm4__chr9 21.97 Mbp_chr9_21968437_F TCGTCGGCAGCGTCGTCCAGCAGCTAGTCCACTG 60.1 0.15
mm4__chr9 21.97 Mbp_chr9_21968437_R GTCTCGTGGGCTCGGCTTTCTCTGCCCTCCGCC 60.1 0.15
mm4__chr1 898.94 Kbp_chr1_898937_F TCGTCGGCAGCGTCCGTCTTCCAGAACGCACTCT 60.0 0.15
mm4__chr1 898.94 Kbp_chr1_898937_R GTCTCGTGGGCTCGGAGCAAAGCTGAAATGAGCGC 60.1 0.15
mm4__chr17 76.74 Mbp_chr17_76736615_F Primer3: not found at this Tm N.d. 0.15
mm4__chr17 76.74 Mbp_chr17_76736615_R Primer3: not found at this Tm N.d. 0.15
mm4__chr19 18.94 Mbp_chr19_18941059_F Primer3: not found at this Tm N.d. 0.14
mm4__chr19 18.94 Mbp_chr19_18941059_R Primer3: not found at this Tm N.d. 0.14
mm4__chr8 22.97 Mbp_chr8_22973980_F TCGTCGGCAGCGTCCATCAACCTCACCACCCCTC 60.0 0.11
mm4__chr8 22.97 Mbp_chr8_22973980_R GTCTCGTGGGCTCGGAGTGATCACATGGCTCCCAG 59.4 0.11
mm4__chr7 44.89 Mbp_chr7_44885527_F Primer3: not found at this Tm N.d. 0.10
mm4__chr7 44.89 Mbp_chr7_44885527_R Primer3: not found at this Tm N.d. 0.10
mm4__chr1 95.12 Mbp_chr1_95117781_F Primer3: not found at this Tm N.d. 0.10
mm4__chr1 95.12 Mbp_chr1_95117781_R Primer3: not found at this Tm N.d. 0.10
mm4__chr5 128.08 Mbp_chr5_128083982_F Primer3: not found at this Tm N.d. 0.09
mm4__chr5 128.08 Mbp_chr5_128083982_R Primer3: not found at this Tm N.d. 0.09
mm4__chr8 10.05 Mbp_chr8_10054798_F Primer3: not found at this Tm N.d. 0.09
mm4__chr8 10.05 Mbp_chr8_10054798_R Primer3: not found at this Tm N.d. 0.09
mm2__chr7 101.32 Mbp_chr7_101321827_F TCGTCGGCAGCGTCTCTACTTGGCCGCTTTCGTT 59.9 0.08
mm2__chr7 101.32 Mbp_chr7_101321827_R GTCTCGTGGGCTCGGTTTGTGGCTTCTCTCCGACC 59.9 0.08
mm3__chr1 7.77 Mbp_chr1_7771212_F Primer3: not found at this Tm N.d. 0.08
mm3__chr1 7.77 Mbp_chr1_7771212_R Primer3: not found at this Tm N.d. 0.08
mm4__chr18 79.56 Mbp_chr18_79558528_F TCGTCGGCAGCGTCGAAACCTCATGGCATTGCGG 60.1 0.07
mm4__chr18 79.56 Mbp_chr18_79558528_R GTCTCGTGGGCTCGGCACCAACCAGACAGACCCTC 59.9 0.07
mm3__chr18 62.19 Mbp_chr18_62187624_F Primer3: not found at this Tm N.d. 0.07
mm3__chr18 62.19 Mbp_chr18_62187624_R Primer3: not found at this Tm N.d. 0.07
mm4__chr10 12.50 Mbp_chr10_12503284_F TCGTCGGCAGCGTCCTTGTGCCGCTGGGATTTTG 60.3 0.07
mm4__chr10 12.50 Mbp_chr10_12503284_R GTCTCGTGGGCTCGGCACTCATGTCGTTGAACGCA 59.1 0.07
mm4__chr10 45.00 Mbp_chr10_45001295_F TCGTCGGCAGCGTCGAACGGGAGGCTTGGACTG 60.3 0.06
mm4__chr10 45.00 Mbp_chr10_45001295_R GTCTCGTGGGCTCGGTGCCTAAGCATTTGTTCCACG 59.4 0.06
mm4__chr13 110.91 Mbp_chr13_110914277_F TCGTCGGCAGCGTCGGCTGTGCGGAACCAGAA 60.2 0.06
mm4__chr13 110.91 Mbp_chr13_110914277_R GTCTCGTGGGCTCGGATTTCGCAGAGCCGGGTATT 59.8 0.06
mm4__chr2 104.84 Mbp_chr2_104843149_F Primer3: not found at this Tm N.d. 0.05
mm4__chr2 104.84 Mbp_chr2_104843149_R Primer3: not found at this Tm N.d. 0.05
mm4__chr4 2.37 Mbp_chr4_2370711_F TCGTCGGCAGCGTCATCACTGTACCGGGAGAGGG 60.3 0.04
mm4__chr4 2.37 Mbp_chr4_2370711_R GTCTCGTGGGCTCGGGATGTGGTGTGCAATCTGGC 59.8 0.04
mm4__chr5 177.46 Mbp_chr5_177455523_F TCGTCGGCAGCGTCGTGAGTACCGACCTCCGC 59.5 0.04
mm4__chr5 177.46 Mbp_chr5_177455523_R GTCTCGTGGGCTCGGGAAGCCCTGAGGGTAAGCAG 60.1 0.04
mm4__chr17 81.24 Mbp_chr17_81238845_F TCGTCGGCAGCGTCCTTGGAAGGCCCGGCTTG 61.0 0.03
mm4__chr17 81.24 Mbp_chr17_81238845_R GTCTCGTGGGCTCGGAGCCATTGACCTTGCGTCTC 60.6 0.03
mm4__chr5 132.67 Mbp_chr5_132666613_F TCGTCGGCAGCGTCCCTCCCAGATGCCCATACTG 59.6 0.02
mm4__chr5 132.67 Mbp_chr5_132666613_R GTCTCGTGGGCTCGGGGTGAGGCTAGATAAGGGCAG 59.6 0.02
mm4__chr17 75.53 Mbp_chr17_75525691_F Primer3: not found at this Tm N.d. 0.02
mm4__chr17 75.53 Mbp_chr17_75525691_R Primer3: not found at this Tm N.d. 0.02
mm4__chr9 128.20 Mbp_chr9_128203691_F TCGTCGGCAGCGTCGGATCCCTGGAGTCCCCG 60.8 0.02
mm4__chr9 128.20 Mbp_chr9_128203691_R GTCTCGTGGGCTCGGGAGGGGCTCTCGTGGTGG 61.7 0.02
mm4__chr18 54.22 Mbp_chr18_54224375_F Primer3: not found at this Tm N.d. 0.01
mm4__chr18 54.22 Mbp_chr18_54224375_R Primer3: not found at this Tm N.d. 0.01
mm4__chr7 42.24 Mbp_chr7_42238109_F Primer3: not found at this Tm N.d. 0.01
mm4__chr7 42.24 Mbp_chr7_42238109_R Primer3: not found at this Tm N.d. 0.01
mm4__chr13 33.02 Mbp_chr13_33016350_F Primer3: not found at this Tm N.d. 0.00
mm4__chr13 33.02 Mbp_chr13_33016350_R Primer3: not found at this Tm N.d. 0.00

Off-target amplicon sequences

Primers underlined, off-targets in bold.

ontarget_mm0__chr7 5.53 Mbp_chr7_5529707 TGGTGAGCTGCGAGAATAGCCGGGCGCGCTGTGAGCCGAGGTCGCCCCCGCCCTGGCCACTTCCGGCGCGCCGAGTCCTT
AGGCCGCCAGGGGGCGCCGGCGCGCGCCCAGATTGGGGACAAAGGAAGCCG
mm4__chr9 129.84 Mbp_chr9_129835268 GAGTTGTACAGCCGCCGCGCCTCTGCGCAGCGGCCCAGGCTGCTTCCGGCGCGCGGCAGAGCGGTCAGAGCGCCTGCCCC
GTCTGGCCCCGCCCCCGGCACGCACAGTCCGCGTGGCCACGCCCCCTCTCCTGGCGTCAG
mm3__chr5 135.01 Mbp_chr5_135012998 GGATGGCTCTTCAGGGTGAGACCTGTTGAGGTGCTGTGGGGGAATCCTCTGAGGGCAGGAAATGCCTGGCCACTTCCGGC
ACGGAGAGAGCTTTGTGTCCATGCCCTGAAGCTGGGCTGGGGTAGATA
mm4__chr8 143.82 Mbp_chr8_143818330 AAGCCCTAGACACAGGGACACACCTGTCAGGCTGCGCGAGCCCAGGGGTGGGGGCGAGCCCGAAGTGGCCGGGGCGGACC
AAGCCTGCTGACCTTGTGCTTCATGGTGACGGAGTC
mm3__chr16 88.47 Mbp_chr16_88473986 GGTTCTCCGGAGTGAAGCAATCTCATGTAATCTATCAGTGGCTATCAGGCCCATCGGAGGGCCGGGAGTGGCCGGGCCCA
GAGATGGCGCCAACCTTCAGCGTCGATA
mm4__chr3 170.42 Mbp_chr3_170419651 TAATTACTGCGGTCCCAGCCCGCATCCTTCCACCGTCCGCTTCCCGAAGTGCTTCCCGCTCCCGCCCGGCCTCCCCCGGG
GCGCCGAGCCTTCGCGTTAGGTTCCGCCCGCAGAGGTGAGAAGGAGCGTG
mm4__chr9 21.97 Mbp_chr9_21968437 GTCCAGCAGCTAGTCCACTGCCCGCCTGGCTGCTCCAGGCGCGCCGACCGCTCAAGCGCTCCAGGTCCACCCGGCGGAGG
GCAGAGAAAG
mm4__chr1 898.94 Kbp_chr1_898937 CGTCTTCCAGAACGCACTCTGCAGGCACTCGGCAACGTGAAGTGTTCACGTCCTGTGATGCAGCCTCTGCTCCAGGCCAC
TTCCGGAACTGCGAGGGAACAGCTGTGGGCGCGCTCATTTCAGCTTTGCT
mm4__chr8 22.97 Mbp_chr8_22973980 CATCAACCTCACCACCCCTCCCACGCCCCACCATTTGAGTCAGATGAGACCCACCCCAGGCACCAGCCCTTCCAGGGAGT
CACCTGACCACTTCCGGGGCTCCCAGCCAGCTCCGCTGGGAGCCATGTGATCACT
mm2__chr7 101.32 Mbp_chr7_101321827 TCTACTTGGCCGCTTTCGTTTCCATGGCGACGGAGAGAGGCCCGCAGGGCCGACGGGACTCGGCCACTTCCGGCGCGCGG
GGCGGAAAGCGGGCGCGTCTGCGACTGGGAGGGGGTCGGAGAGAAGCCACAAA
mm4__chr18 79.56 Mbp_chr18_79558528 GAAACCTCATGGCATTGCGGCTGTGCTCAGAAGGCATGACCAGCCCCTCCGCCAGCCTGAAGTGGCCAGGCGTGTGCAGG
GGGCTTGAGGGGGATGGGGAAAGGCAGGGAGGGTCTGTCTGGTTGGTG
mm4__chr10 12.50 Mbp_chr10_12503284 CTTGTGCCGCTGGGATTTTGGGAGGAGCATTTGCTCCTCGCCCCGCCTGATGTGGCCAGGTGGCTGCGTGTAGTGTATTT
AAATGCGTTCAACGACATGAGTG
mm4__chr10 45.00 Mbp_chr10_45001295 GAACGGGAGGCTTGGACTGGGGGCCCGGGCGAGGCGCGGCCCGCGGCGGGTGGAACTGGGCGGGTGAGCAGCAGCCCCGG
CCCCTTCCGTCGCTCGGAGCGTTCGAGGACTAGATACGTGGAACAAATGCTTAGGCA
mm4__chr13 110.91 Mbp_chr13_110914277 GGCTGTGCGGAACCAGAAGGTCGACCGCGCCCGGGCATTCCCCGCGCGCCTCTGACCCCAGCTCGGCTCCCCGGAAGCCG
CCTGGGCGACTTCGCGGCCTAATACCCGGCTCTGCGAAAT
mm4__chr4 2.37 Mbp_chr4_2370711 ATCACTGTACCGGGAGAGGGGTGTCTCCCATGCCTGCTGCCACCCCCACCTCTCGGCTCCCCGGACGTGGCAAGGCCATG
GGACCACCTGCAGTGGGCCAGTCTGGGGCGCCAGATTGCACACCACATC
mm4__chr5 177.46 Mbp_chr5_177455523 GTGAGTACCGACCTCCGCCAGGGGGCGATCCGGGCCGCCGGAAGTGGGCGGGCGTTGGAGGGCTCGCTGCTTACCCTCAG
GGCTTC
mm4__chr17 81.24 Mbp_chr17_81238845 CTTGGAAGGCCCGGCTTGGAAGGCCCCGGGACGGCGCGGCGGGCGGGCAGCTCAGGGGCGTCGGCGCGGGCGAAGGGGCC
AGGAGCAGCTACCCGGCGCGGAGACGCAAGGTCAATGGCT
mm4__chr5 132.67 Mbp_chr5_132666613 CCTCCCAGATGCCCATACTGAGTTCCACCTCATCTGGCCACTTCCTGTGCCCCCAACTGCACTCAGATATCTCATGAGGT
CACTGATTATCAACTCCACTCCGTGCAGCAACGCATGTCTGCCCTTATCTAGCCTCACC
mm4__chr9 128.20 Mbp_chr9_128203691 GGATCCCTGGAGTCCCCGCCCGGGGCACTGACGGCGCGGCGACCTCGCAGCCCCCGACGCTGCACCCGCGGCCGGCGCGC
CCCCCACCCCCAGCCGGAGCGAGGAGGCCCTCCCCCCACCACGAGAGCCCCTC

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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