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PCR primers for off-targets of GCCGTTGTCGACGACGAGCG CGG

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_5529618_F TCGTCGGCAGCGTCCACGATGGAGGGGAAGACG 59.8 1.00
ontarget_mm0__chr7 5.53 Mbp_chr7_5529618_R GTCTCGTGGGCTCGGGCTATTCTCGCAGCTCACCA 60.1 1.00
mm3__chr15 43.99 Mbp_chr15_43990145_F TCGTCGGCAGCGTCAATGAAGGGGAAGATGGCCC 59.7 0.84
mm3__chr15 43.99 Mbp_chr15_43990145_R GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT 60.1 0.84
mm2__chr5 77.78 Mbp_chr5_77784902_F TCGTCGGCAGCGTCCACCAGCTCACCATGGATGA 59.7 0.50
mm2__chr5 77.78 Mbp_chr5_77784902_R GTCTCGTGGGCTCGGAACGATGGAGGGGAAGACTG 59.0 0.50
mm3__chr22 15.72 Mbp_chr22_15721502_F TCGTCGGCAGCGTCACTGAATGAGCTCACCATGGA 59.0 0.47
mm3__chr22 15.72 Mbp_chr22_15721502_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.47
mm3__chr2 131.26 Mbp_chr2_131263571_F TCGTCGGCAGCGTCTGAGCTCACCATGGATGATGA 58.8 0.47
mm3__chr2 131.26 Mbp_chr2_131263571_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.47
mm3__chr2 130.46 Mbp_chr2_130463904_F TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC 60.1 0.47
mm3__chr2 130.46 Mbp_chr2_130463904_R GTCTCGTGGGCTCGGTGAGCTCACCATGGATGATGA 58.8 0.47
mm3__chr14 19.40 Mbp_chr14_19402869_F TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC 60.1 0.47
mm3__chr14 19.40 Mbp_chr14_19402869_R GTCTCGTGGGCTCGGACTGAATGAGCTCACCATGGA 59.0 0.47
mm3__chr14 19.00 Mbp_chr14_18998883_F TCGTCGGCAGCGTCACTGAATGAGCTCACCATGGA 59.0 0.47
mm3__chr14 19.00 Mbp_chr14_18998883_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.47
mm4__chr6 88.28 Mbp_chr6_88276950_F TCGTCGGCAGCGTCCCCACAATGGAGGGGAAGAC 60.0 0.45
mm4__chr6 88.28 Mbp_chr6_88276950_R GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT 60.1 0.45
mm4__chr1 156.15 Mbp_chr1_156154649_F TCGTCGGCAGCGTCCCTGGGCCTTTTCCTCTTCC 60.3 0.32
mm4__chr1 156.15 Mbp_chr1_156154649_R GTCTCGTGGGCTCGGCTTCCTGCTGTCGGACACTT 59.9 0.32
mm4__chr9 100.73 Mbp_chr9_100731255_F TCGTCGGCAGCGTCGCCAGCTCACCATGGATGAT 60.1 0.23
mm4__chr9 100.73 Mbp_chr9_100731255_R GTCTCGTGGGCTCGGCTAGGGAGCTCCACGATGGA 60.4 0.23
mm3__chr3 180.82 Mbp_chr3_180820559_F TCGTCGGCAGCGTCAAGCACAGAGTCTTGCCCTC 59.9 0.19
mm3__chr3 180.82 Mbp_chr3_180820559_R GTCTCGTGGGCTCGGCTAAGCTGGCCTTGCACATG 59.5 0.19
mm3__chrY 18.15 Mbp_chrY_18147899_F Primer3: not found at this Tm N.d. 0.11
mm3__chrY 18.15 Mbp_chrY_18147899_R Primer3: not found at this Tm N.d. 0.11
mm3__chrY 17.76 Mbp_chrY_17758086_F TCGTCGGCAGCGTCAACGGTGGAGGGAAACATGG 60.2 0.11
mm3__chrY 17.76 Mbp_chrY_17758086_R GTCTCGTGGGCTCGGTCCTCTGTCCCACTCCTCTG 59.9 0.11
mm4__chr6 46.20 Mbp_chr6_46204993_F TCGTCGGCAGCGTCAGATGCTCAGTCGTCACCAC 59.7 0.10
mm4__chr6 46.20 Mbp_chr6_46204993_R GTCTCGTGGGCTCGGAAGCTGCTTTTCACATGCCG 60.0 0.10
mm1__chr9 6.83 Mbp_chr9_6834471_F TCGTCGGCAGCGTCCCGCTACCAGCTCACCATAG 59.9 0.04
mm1__chr9 6.83 Mbp_chr9_6834471_R GTCTCGTGGGCTCGGACACGATGAAGGGGAAGACG 59.7 0.04
mm4__chrX 53.14 Mbp_chrX_53143874_F TCGTCGGCAGCGTCGATGGAGGGGAACACAGCTC 60.1 0.03
mm4__chrX 53.14 Mbp_chrX_53143874_R GTCTCGTGGGCTCGGTGCTCTTCCACAGGTTGCAA 60.1 0.03
mm4__chr10 133.21 Mbp_chr10_133207245_F TCGTCGGCAGCGTCCCTCCCAGTGATCGTGAACA 59.3 0.03
mm4__chr10 133.21 Mbp_chr10_133207245_R GTCTCGTGGGCTCGGCAGGATGCGGGCCTTCTG 60.5 0.03
mm4__chr4 91.92 Mbp_chr4_91920489_F Primer3: not found at this Tm N.d. 0.01
mm4__chr4 91.92 Mbp_chr4_91920489_R Primer3: not found at this Tm N.d. 0.01
mm4__chr10 84.20 Mbp_chr10_84195508_F TCGTCGGCAGCGTCGGTGTCCGTCTGTTCTGTGT 59.8 0.01
mm4__chr10 84.20 Mbp_chr10_84195508_R GTCTCGTGGGCTCGGCCATGTTTCCGTTGGTGCTG 60.0 0.01
mm4__chr8 4.26 Mbp_chr8_4258832_F TCGTCGGCAGCGTCTAAGGGCACAGCAGCAGAAA 59.8 0.00
mm4__chr8 4.26 Mbp_chr8_4258832_R GTCTCGTGGGCTCGGACGGCCAGGTAGAGATGAGA 59.7 0.00
mm4__chr17 81.51 Mbp_chr17_81512315_F TCGTCGGCAGCGTCAAACCAGCTTTGCACATGCC 60.2 0.00
mm4__chr17 81.51 Mbp_chr17_81512315_R GTCTCGTGGGCTCGGCCGCCTGAGTCCATCCTTTT 60.0 0.00

Off-target amplicon sequences

Primers underlined, off-targets in bold.

ontarget_mm0__chr7 5.53 Mbp_chr7_5529618 CACGATGGAGGGGAAGACGGCCCGGGGGGCATCGTCGCCCGCGAAGCCGGCCTTGCACATGCCGGAGCCGTTGTCGACGA
CGAGCGCGG
CGATATCATCATCCATGGTGAGCTGCGAGAATAGC
mm3__chr15 43.99 Mbp_chr15_43990145 AATGAAGGGGAAGATGGCCCGGGGTGGGCATCGTCACCTGCAAAGCTGGCCTTGCACATGCCAGAGCCATTGTCGACGAC
AAGCATGG
TGATATCATCATCCATGGTGAGCTGGC
mm2__chr5 77.78 Mbp_chr5_77784902 CACCAGCTCACCATGGATGATGATATCACCGCGCTCGTCATTGACAACGGCTCCGGCATGTGCAAGGCCAGCTTCACGGG
CGACAATGCCGCCCGGGCAGTCTTCCCCTCCATCGTT
mm3__chr22 15.72 Mbp_chr22_15721502 ACTGAATGAGCTCACCATGGATGATGATACTGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTG
CAGGTGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm3__chr2 131.26 Mbp_chr2_131263571 TGAGCTCACCATGGATGATGATACCGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTGCGGGCG
ACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm3__chr2 130.46 Mbp_chr2_130463904 GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCGCCCGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA
GCACGG
CGGTATCATCATCCATGGTGAGCTCA
mm3__chr14 19.40 Mbp_chr14_19402869 GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCACCTGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA
GCACGG
CAGTATCATCATCCATGGTGAGCTCATTCAGT
mm3__chr14 19.00 Mbp_chr14_18998883 ACTGAATGAGCTCACCATGGATGATGATACTGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTG
CAGGTGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm4__chr6 88.28 Mbp_chr6_88276950 CCCACAATGGAGGGGAAGACGGCCTGGGGAGCCTTGCGCATGCTGGAGCAGTTGTCGACGACGACGAGCGCGGTGATAGC
ATCATCCATGGTGAGCTGGC
mm4__chr1 156.15 Mbp_chr1_156154649 CCTGGGCCTTTTCCTCTTCCAACTGCTTCAGCTGCTGCTGCCGACGACGACCGCGGGGGGAGGCGGGCAGGGGCCCATGC
CCAGGGTCAGATACTATGCAGGTAAGTGTCCGACAGCAGGAAG
mm4__chr9 100.73 Mbp_chr9_100731255 GCCAGCTCACCATGGATGATGATATCGCCCCACTTGTCATTGACAACGGCTCAGGCATGTGCAAGGCCGGCTTTGCAGGT
GATAATGTCCCCCTGGGCCATCTTCCCCCTCCATCGTGGAGCTCCCTAG
mm3__chr3 180.82 Mbp_chr3_180820559 AAGCACAGAGTCTTGCCCTCGCCGCTCTGCCACGGGTCCACACCCACTGCCAGCTCATCATGGATAATGATATCGCCACG
CTCGTCATGGACAATGGC
TCTCCCATGTGCAAGGCCAGCTTAG
mm3__chrY 17.76 Mbp_chrY_17758086 AACGGTGGAGGGAAACATGGCTTGGGGAGCATCGTCCCCTGCAAAGCCAGCTTTGCACATGCAAGAGCCATTGTCAATGA
CGAGCGCAG
CGATCTCTTCTTCTACTGCGATGGACAGAGGAGTGGGACAGAGGA
mm4__chr6 46.20 Mbp_chr6_46204993 AGATGCTCAGTCGTCACCACCAGCACGTGCACCATGTCCTCCCGCTGCTGCGTGCTCCTCTGCCAGTGGCAATGGAAGAA
GAGATCCCCGCGCTCTTCATTGACAATGGCTCCGGCATGTGAAAAGCAGCTT
mm1__chr9 6.83 Mbp_chr9_6834471 CCGCTACCAGCTCACCATAGATGATGATATTGCTACGCCCGTCGTCGACAACGGCTCCGGCATGTGGAAGGCTGGCTTCG
CAGGTGACGATGACCCCCGGCCGTCTTCCCCTTCATCGTGT
mm4__chrX 53.14 Mbp_chrX_53143874 GATGGAGGGGAACACAGCTCAGGGGCCATCCATCCCCAGCAAAGCCAGCTTTGCACATGCCAGAGCCATTGTCAATGATG
AGCGCGG
CGATCTCTATTTCCATTGCAACCTGTGGAAGAGCA
mm4__chr10 133.21 Mbp_chr10_133207245 CCTCCCAGTGATCGTGAACATCGCGGCCGCACCCTGCGACACGCTGGACTTCAGCCCCCTGGACGAGTCCTCCTCGCTCA
TCTTCTACAACGTC
AACAAGCACCCGGGCGGCCGGCAGAAGGCCCGCATCCTG
mm4__chr10 84.20 Mbp_chr10_84195508 GGTGTCCGTCTGTTCTGTGTTCTTTTTCCAGCTCAGGCCAACTTCGCCCCGCACTTCTTCGACAACGGGGTCGGCAGCAC
CAACGGAAACATGG
mm4__chr8 4.26 Mbp_chr8_4258832 TAAGGGCACAGCAGCAGAAAGATCAGGAGCCCCACTCCTCATGGACAACGGCTGCAAAAGTCTCATCTCTACCTGGCCGT
mm4__chr17 81.51 Mbp_chr17_81512315 AAACCAGCTTTGCACATGCCGGAGCCATTGTCAATGACCAGCGCGGCGATCTCTTCTTCCATTGCGACCTGCCCGGAAAA
GGATGGACTCAGGCGG

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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