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PCR primers for off-targets of TTCCGGCGCGCCGAGTCCTT 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_5529716_F TCGTCGGCAGCGTCTGGTGAGCTGCGAGAATAGC 60.1 1.00
ontarget_mm0__chr7 5.53 Mbp_chr7_5529716_R GTCTCGTGGGCTCGGCGGCTTCCTTTGTCCCCAAT 60.6 1.00
mm4__chr14 70.42 Mbp_chr14_70417095_F TCGTCGGCAGCGTCCACAGCGGTTTCGGTTTCAG 60.0 0.49
mm4__chr14 70.42 Mbp_chr14_70417095_R GTCTCGTGGGCTCGGAAACTGCAATCCATTGGCGG 59.7 0.49
mm4__chr2 119.49 Mbp_chr2_119488100_F Primer3: not found at this Tm N.d. 0.38
mm4__chr2 119.49 Mbp_chr2_119488100_R Primer3: not found at this Tm N.d. 0.38
mm4__chr1 207.67 Mbp_chr1_207670930_F TCGTCGGCAGCGTCACCTAGAAATTTTGTTGCTGGAGT 58.8 0.21
mm4__chr1 207.67 Mbp_chr1_207670930_R GTCTCGTGGGCTCGGTGCATACACCTTGGGAGCAT 59.3 0.21
mm4__chr22 49.20 Mbp_chr22_49195866_F TCGTCGGCAGCGTCGGCAACCTCCCTTGTCTGG 60.3 0.19
mm4__chr22 49.20 Mbp_chr22_49195866_R GTCTCGTGGGCTCGGTCAGTCTCGGGAGGAGTGAG 60.0 0.19
mm4__chr17 78.59 Mbp_chr17_78590923_F Primer3: not found at this Tm N.d. 0.10
mm4__chr17 78.59 Mbp_chr17_78590923_R Primer3: not found at this Tm N.d. 0.10
mm4__chr18 80.08 Mbp_chr18_80084090_F TCGTCGGCAGCGTCGGAACCGTGGAAGTGCTGAT 60.3 0.10
mm4__chr18 80.08 Mbp_chr18_80084090_R GTCTCGTGGGCTCGGGTTCCACTGACCTAGCAGGC 60.3 0.10
mm4__chr10 117.55 Mbp_chr10_117553513_F TCGTCGGCAGCGTCTTGAGAACCCTCTGACCCCA 60.1 0.06
mm4__chr10 117.55 Mbp_chr10_117553513_R GTCTCGTGGGCTCGGTGATGAATGGACCTTGCGCT 60.0 0.06
mm4__chr3 143.97 Mbp_chr3_143972920_F TCGTCGGCAGCGTCCTACGCGGAGACCAAGGAC 59.8 0.04
mm4__chr3 143.97 Mbp_chr3_143972920_R GTCTCGTGGGCTCGGCGCCTACCTGTAGCACCAG 59.8 0.04
mm4__chr2 71.28 Mbp_chr2_71276505_F TCGTCGGCAGCGTCCTGCAGGGGAGGAGCTGG 61.7 0.03
mm4__chr2 71.28 Mbp_chr2_71276505_R GTCTCGTGGGCTCGGCCTCAAGGCGCAGGACTC 60.1 0.03
mm4__chr11 909.45 Kbp_chr11_909450_F TCGTCGGCAGCGTCATAAGGTGGAGCGCTACTGC 59.8 0.02
mm4__chr11 909.45 Kbp_chr11_909450_R GTCTCGTGGGCTCGGTTCCATCAACACCACCGAGG 59.9 0.02
mm4__chrY 276.92 Kbp_chrY_276917_F TCGTCGGCAGCGTCCGTTGATTCACCCAGCAACC 59.7 0.02
mm4__chrY 276.92 Kbp_chrY_276917_R GTCTCGTGGGCTCGGCCAGGCGGGGTGAGACAC 62.3 0.02
mm4__chr19 19.54 Mbp_chr19_19544633_F TCGTCGGCAGCGTCGGCTTGTGGGAGGAGGAGA 60.6 0.01
mm4__chr19 19.54 Mbp_chr19_19544633_R GTCTCGTGGGCTCGGAGGCACGTCCAGATTGAACA 59.6 0.01
mm4__chr14 96.36 Mbp_chr14_96363777_F TCGTCGGCAGCGTCCGTGTTAAGAGGGGTCTGGG 59.7 0.01
mm4__chr14 96.36 Mbp_chr14_96363777_R GTCTCGTGGGCTCGGCTCGAGAAGGGCTCCAGTCC 61.6 0.01
mm4__chr18 75.20 Mbp_chr18_75204856_F TCGTCGGCAGCGTCGGGTGGACAAGTTCTCTCCG 60.0 0.00
mm4__chr18 75.20 Mbp_chr18_75204856_R GTCTCGTGGGCTCGGCCAAGGTCACGGTCAAGCC 60.9 0.00

Off-target amplicon sequences

Primers underlined, off-targets in bold.

ontarget_mm0__chr7 5.53 Mbp_chr7_5529716 TGGTGAGCTGCGAGAATAGCCGGGCGCGCTGTGAGCCGAGGTCGCCCCCGCCCTGGCCACTTCCGGCGCGCCGAGTCCTT
AGGCCGCCAGGGGGCGCCGGCGCGCGCCCAGATTGGGGACAAAGGAAGCCG
mm4__chr14 70.42 Mbp_chr14_70417095 CACAGCGGTTTCGGTTTCAGCAGCCTCGAGACCCGGAAAAGGAAGCCCGAAGGACTCGGAGCACTGGGAATGCTGGGAAT
CGTGGTTCCTACCGCCAATGGATTGCAGTTT
mm4__chr1 207.67 Mbp_chr1_207670930 ACCTAGAAATTTTGTTGCTGGAGTAATTTCAATTTTAAAATGCTTATGGATGCTTCCGGTGTGCACAGTCCTTTGGAATT
TAAAAGAAATGTAAGAGAAAGAGCTATGCTCCCAAGGTGTATGCA
mm4__chr22 49.20 Mbp_chr22_49195866 GGCAACCTCCCTTGTCTGGGGGTGGCTATGGGGCTTTGGCTGCGCCGAGTCCTTTGGTGAGTGCTGCCCCGCTCACTCCT
CCCGAGACTGA
mm4__chr18 80.08 Mbp_chr18_80084090 GGAACCGTGGAAGTGCTGATGGGGAAATGACTCAATGTCCTTTACCTGAAAATCCCTAGGCAGGGATTTCCTGCGTGCAG
AGTCCTAAGGATGCCTGCTAGGTCAGTGGAAC
mm4__chr10 117.55 Mbp_chr10_117553513 TTGAGAACCCTCTGACCCCAGCCGTGACCCCTCTCCGGGAGGTGGCTTCTCCCGCAGGACCCGGTGCGCCAGAAAACTCT
AGGCGGCGCCACGCTTGGAAGCGCAAGGTCCATTCATCA
mm4__chr3 143.97 Mbp_chr3_143972920 CTACGCGGAGACCAAGGACTCGGGCAGCTTCCTGCTTCGCAACCTCAAGGACTCGGAGCGCATGCAGCTGCTGCTGACCC
TGGCCTTCAACCCCGAGCCGCTGGTGCTACAGGTAGGCG
mm4__chr2 71.28 Mbp_chr2_71276505 CTGCAGGGGAGGAGCTGGGCCCCGCCCCCGGCTTCCCGCGCTCCGCGTCCATTGGCTGGCCTCGGGGGTGGGCGGGTCCC
GCCGCACGGAGTCCTGCGCCTTGAGG
mm4__chr11 909.45 Kbp_chr11_909450 ATAAGGTGGAGCGCTACTGCTCCCACTTTCCTGCGCTCCCAGTCCGTAGGTCCAGGACCTCCTCGGTGGTGTTGATGGAA
mm4__chrY 276.92 Kbp_chrY_276917 CGTTGATTCACCCAGCAACCATTCACCCCGTGCGTTGAATGTGCGCGTCACGGCCACTCACGCCTCTGGGACGTGAGTCT
CCCGAGGACTCAGCGGGCGGGAAGGTGTAGGTGTCTCACCCCGCCTGG
mm4__chr19 19.54 Mbp_chr19_19544633 GGCTTGTGGGAGGAGGAGAGCGGCTTCCGGCGCGAGGGGTCCTCGGGCCCCCGGGTGCGCCGGGAGGAGCGCGTCTTCCT
GGTGGGCAACGTGGAGATCCGGGAGCGGCGCCTGTTCAATCTGGACGTGCCT
mm4__chr14 96.36 Mbp_chr14_96363777 CGTGTTAAGAGGGGTCTGGGGGCCGGTCTGGCCGGCCTGGCTCTCCCGGCTCCCCTCAGACCTCGGCGCCCCGGAAACGC
ACACTTGGGGCTCCGCCGTCTCCGCTGGGAGGACTGGAGCCCTTCTCGAG
mm4__chr18 75.20 Mbp_chr18_75204856 GGGTGGACAAGTTCTCTCCGAGTCCCGTGCACTCGGCGCGCCGGAGCGCAGTGGCCGACAGACAGGGCCCGCCGGCGGCT
TGACCGTGACCTTGG

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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