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PCR primers for off-targets of GCCGCGCTCGTCGTCGACAA 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_5529619_F TCGTCGGCAGCGTCCACGATGGAGGGGAAGACG 59.8 1.00
ontarget_mm0__chr7 5.53 Mbp_chr7_5529619_R GTCTCGTGGGCTCGGGCTATTCTCGCAGCTCACCA 60.1 1.00
mm3__chr9 6.83 Mbp_chr9_6834470_F TCGTCGGCAGCGTCCCGCTACCAGCTCACCATAG 59.9 0.61
mm3__chr9 6.83 Mbp_chr9_6834470_R GTCTCGTGGGCTCGGACACGATGAAGGGGAAGACG 59.7 0.61
mm3__chr18 62.44 Mbp_chr18_62442023_F TCGTCGGCAGCGTCGCCAGCTCACCATGGATGAT 60.1 0.57
mm3__chr18 62.44 Mbp_chr18_62442023_R GTCTCGTGGGCTCGGATGATGGAGAGGAAGACGGC 59.2 0.57
mm4__chr15 43.99 Mbp_chr15_43990146_F TCGTCGGCAGCGTCAATGAAGGGGAAGATGGCCC 59.7 0.45
mm4__chr15 43.99 Mbp_chr15_43990146_R GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT 60.1 0.45
mm3__chr5 131.66 Mbp_chr5_131660077_F TCGTCGGCAGCGTCCAGACCTTCCACATGCCAGA 59.6 0.38
mm3__chr5 131.66 Mbp_chr5_131660077_R GTCTCGTGGGCTCGGCCAAGACTGTGTCCACCCTG 60.2 0.38
mm4__chr4 91.92 Mbp_chr4_91920490_F Primer3: not found at this Tm N.d. 0.25
mm4__chr4 91.92 Mbp_chr4_91920490_R Primer3: not found at this Tm N.d. 0.25
mm4__chr13 25.17 Mbp_chr13_25170605_F TCGTCGGCAGCGTCTCTGAGGGTGGATCGACAGA 59.6 0.15
mm4__chr13 25.17 Mbp_chr13_25170605_R GTCTCGTGGGCTCGGCAGAGGACGCTTCCAGGAC 59.7 0.15
mm4__chr10 84.20 Mbp_chr10_84195507_F TCGTCGGCAGCGTCGGTGTCCGTCTGTTCTGTGT 59.8 0.10
mm4__chr10 84.20 Mbp_chr10_84195507_R GTCTCGTGGGCTCGGCCATGTTTCCGTTGGTGCTG 60.0 0.10
mm3__chrX 53.14 Mbp_chrX_53143875_F TCGTCGGCAGCGTCGATGGAGGGGAACACAGCTC 60.1 0.05
mm3__chrX 53.14 Mbp_chrX_53143875_R GTCTCGTGGGCTCGGTGCTCTTCCACAGGTTGCAA 60.1 0.05
mm3__chr5 77.78 Mbp_chr5_77784901_F TCGTCGGCAGCGTCCACCAGCTCACCATGGATGA 59.7 0.05
mm3__chr5 77.78 Mbp_chr5_77784901_R GTCTCGTGGGCTCGGAACGATGGAGGGGAAGACTG 59.0 0.05
mm3__chrY 18.15 Mbp_chrY_18147898_F Primer3: not found at this Tm N.d. 0.04
mm3__chrY 18.15 Mbp_chrY_18147898_R Primer3: not found at this Tm N.d. 0.04
mm3__chrY 17.76 Mbp_chrY_17758087_F TCGTCGGCAGCGTCAACGGTGGAGGGAAACATGG 60.2 0.04
mm3__chrY 17.76 Mbp_chrY_17758087_R GTCTCGTGGGCTCGGTCCTCTGTCCCACTCCTCTG 59.9 0.04
mm3__chr3 180.82 Mbp_chr3_180820558_F TCGTCGGCAGCGTCAAGCACAGAGTCTTGCCCTC 59.9 0.04
mm3__chr3 180.82 Mbp_chr3_180820558_R GTCTCGTGGGCTCGGCTAAGCTGGCCTTGCACATG 59.5 0.04
mm4__chr4 6.56 Mbp_chr4_6563612_F Primer3: not found at this Tm N.d. 0.03
mm4__chr4 6.56 Mbp_chr4_6563612_R Primer3: not found at this Tm N.d. 0.03
mm3__chr22 15.72 Mbp_chr22_15721501_F TCGTCGGCAGCGTCACTGAATGAGCTCACCATGGA 59.0 0.03
mm3__chr22 15.72 Mbp_chr22_15721501_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.03
mm3__chr2 131.26 Mbp_chr2_131263570_F TCGTCGGCAGCGTCTGAGCTCACCATGGATGATGA 58.8 0.03
mm3__chr2 131.26 Mbp_chr2_131263570_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.03
mm3__chr2 130.46 Mbp_chr2_130463905_F TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC 60.1 0.03
mm3__chr2 130.46 Mbp_chr2_130463905_R GTCTCGTGGGCTCGGTGAGCTCACCATGGATGATGA 58.8 0.03
mm3__chr14 19.40 Mbp_chr14_19402870_F TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC 60.1 0.03
mm3__chr14 19.40 Mbp_chr14_19402870_R GTCTCGTGGGCTCGGACTGAATGAGCTCACCATGGA 59.0 0.03
mm3__chr14 19.00 Mbp_chr14_18998882_F TCGTCGGCAGCGTCACTGAATGAGCTCACCATGGA 59.0 0.03
mm3__chr14 19.00 Mbp_chr14_18998882_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.03
mm3__chr17 81.51 Mbp_chr17_81512316_F TCGTCGGCAGCGTCAAACCAGCTTTGCACATGCC 60.2 0.03
mm3__chr17 81.51 Mbp_chr17_81512316_R GTCTCGTGGGCTCGGCCGCCTGAGTCCATCCTTTT 60.0 0.03
mm4__chr6 46.20 Mbp_chr6_46204992_F TCGTCGGCAGCGTCAGATGCTCAGTCGTCACCAC 59.7 0.03
mm4__chr6 46.20 Mbp_chr6_46204992_R GTCTCGTGGGCTCGGAAGCTGCTTTTCACATGCCG 60.0 0.03
mm4__chr17 81.65 Mbp_chr17_81647980_F TCGTCGGCAGCGTCCTCCTGCTGCATCGACCC 60.2 0.03
mm4__chr17 81.65 Mbp_chr17_81647980_R GTCTCGTGGGCTCGGCCCTCCAGGTACACCACTCT 60.2 0.03
mm4__chr7 63.63 Mbp_chr7_63627890_F Primer3: not found at this Tm N.d. 0.03
mm4__chr7 63.63 Mbp_chr7_63627890_R Primer3: not found at this Tm N.d. 0.03
mm4__chr7 56.95 Mbp_chr7_56949044_F Primer3: not found at this Tm N.d. 0.03
mm4__chr7 56.95 Mbp_chr7_56949044_R Primer3: not found at this Tm N.d. 0.03
mm4__chr7 5.28 Mbp_chr7_5283133_F Primer3: not found at this Tm N.d. 0.03
mm4__chr7 5.28 Mbp_chr7_5283133_R Primer3: not found at this Tm N.d. 0.03
mm4__chr2 131.63 Mbp_chr2_131626153_F TCGTCGGCAGCGTCTGAGCTCACCATGGATGATGA 58.8 0.02
mm4__chr2 131.63 Mbp_chr2_131626153_R GTCTCGTGGGCTCGGAGAAGGGAAGACAGCCTGGG 61.2 0.02
mm4__chr2 130.66 Mbp_chr2_130656764_F TCGTCGGCAGCGTCTGAGCTCACCATGGATGATGA 58.8 0.02
mm4__chr2 130.66 Mbp_chr2_130656764_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.02
mm4__chr2 130.08 Mbp_chr2_130075333_F TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC 60.1 0.02
mm4__chr2 130.08 Mbp_chr2_130075333_R GTCTCGTGGGCTCGGTGAGCTCACCATGGATGATGA 58.8 0.02
mm4__chrX 154.37 Mbp_chrX_154374445_F TCGTCGGCAGCGTCGCGTTTTCCGGCCTCTCT 60.0 0.01
mm4__chrX 154.37 Mbp_chrX_154374445_R GTCTCGTGGGCTCGGGGAACAGCAGGTGAGGTCTC 60.0 0.01

Off-target amplicon sequences

Primers underlined, off-targets in bold.

ontarget_mm0__chr7 5.53 Mbp_chr7_5529619 CACGATGGAGGGGAAGACGGCCCGGGGGGCATCGTCGCCCGCGAAGCCGGCCTTGCACATGCCGGAGCCGTTGTCGACGA
CGAGCGCGGCGATATCATCATCCATGGTGAGCTGCGAGAATAGC
mm3__chr9 6.83 Mbp_chr9_6834470 CCGCTACCAGCTCACCATAGATGATGATATTGCTACGCCCGTCGTCGACAACGGCTCCGGCATGTGGAAGGCTGGCTTCG
CAGGTGACGATGACCCCCGGCCGTCTTCCCCTTCATCGTGT
mm3__chr18 62.44 Mbp_chr18_62442023 GCCAGCTCACCATGGATGATGATACTGTCGCCCTCGTCATCGACAATGGCTCTGGCATGTGCAGGGCTGGCTTCGCAGGC
GACAGTGCTCTCCAGGCCGTCTTCCTCTCCATCAT
mm4__chr15 43.99 Mbp_chr15_43990146 AATGAAGGGGAAGATGGCCCGGGGTGGGCATCGTCACCTGCAAAGCTGGCCTTGCACATGCCAGAGCCATTGTCGACGAC
AAGCATGGTGATATCATCATCCATGGTGAGCTGGC
mm3__chr5 131.66 Mbp_chr5_131660077 CAGACCTTCCACATGCCAGAGCCATTGTCGACGACAAGCACAGCAATATCATTATCCATGGTGAACTGGTGGCAGGTGTG
GATGGGCGGCAGAGTGGTGAGGGTGAGGCTCTGTGCTTGCAGGGTGGACACAGTCTTGG
mm4__chr13 25.17 Mbp_chr13_25170605 TCTGAGGGTGGATCGACAGAGGCAGGGTCTGGGGCGGCGGGCGCCCGGCCGCTGCCGCCTTCGGAGTCGACAAGGGGGAC
CGTCTTTACGGGAACGGCCCCCGTCCTGGAAGCGTCCTCTG
mm4__chr10 84.20 Mbp_chr10_84195507 GGTGTCCGTCTGTTCTGTGTTCTTTTTCCAGCTCAGGCCAACTTCGCCCCGCACTTCTTCGACAACGGGGTCGGCAGCAC
CAACGGAAACATGG
mm3__chrX 53.14 Mbp_chrX_53143875 GATGGAGGGGAACACAGCTCAGGGGCCATCCATCCCCAGCAAAGCCAGCTTTGCACATGCCAGAGCCATTGTCAATGATG
AGCGCGGCGATCTCTATTTCCATTGCAACCTGTGGAAGAGCA
mm3__chr5 77.78 Mbp_chr5_77784901 CACCAGCTCACCATGGATGATGATATCACCGCGCTCGTCATTGACAACGGCTCCGGCATGTGCAAGGCCAGCTTCACGGG
CGACAATGCCGCCCGGGCAGTCTTCCCCTCCATCGTT
mm3__chrY 17.76 Mbp_chrY_17758087 AACGGTGGAGGGAAACATGGCTTGGGGAGCATCGTCCCCTGCAAAGCCAGCTTTGCACATGCAAGAGCCATTGTCAATGA
CGAGCGCAGCGATCTCTTCTTCTACTGCGATGGACAGAGGAGTGGGACAGAGGA
mm3__chr3 180.82 Mbp_chr3_180820558 AAGCACAGAGTCTTGCCCTCGCCGCTCTGCCACGGGTCCACACCCACTGCCAGCTCATCATGGATAATGATATCGCCACG
CTCGTCATGGACAATGGCTCTCCCATGTGCAAGGCCAGCTTAG
mm3__chr22 15.72 Mbp_chr22_15721501 ACTGAATGAGCTCACCATGGATGATGATACTGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTG
CAGGTGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm3__chr2 131.26 Mbp_chr2_131263570 TGAGCTCACCATGGATGATGATACCGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTGCGGGCG
ACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm3__chr2 130.46 Mbp_chr2_130463905 GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCGCCCGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA
GCACGGCGGTATCATCATCCATGGTGAGCTCA
mm3__chr14 19.40 Mbp_chr14_19402870 GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCACCTGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA
GCACGGCAGTATCATCATCCATGGTGAGCTCATTCAGT
mm3__chr14 19.00 Mbp_chr14_18998882 ACTGAATGAGCTCACCATGGATGATGATACTGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTG
CAGGTGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm3__chr17 81.51 Mbp_chr17_81512316 AAACCAGCTTTGCACATGCCGGAGCCATTGTCAATGACCAGCGCGGCGATCTCTTCTTCCATTGCGACCTGCCCGGAAAA
GGATGGACTCAGGCGG
mm4__chr6 46.20 Mbp_chr6_46204992 AGATGCTCAGTCGTCACCACCAGCACGTGCACCATGTCCTCCCGCTGCTGCGTGCTCCTCTGCCAGTGGCAATGGAAGAA
GAGATCCCCGCGCTCTTCATTGACAATGGCTCCGGCATGTGAAAAGCAGCTT
mm4__chr17 81.65 Mbp_chr17_81647980 CTCCTGCTGCATCGACCCCCGCGAAGATGGAGCCTCTGTCAACGACCAGTGCGGCTTCGGGGTCCTGCGCCTGGATGCGG
ACGCAGCTCAGAGAGTGGTGTACCTGGAGGG
mm4__chr2 131.63 Mbp_chr2_131626153 TGAGCTCACCATGGATGATGATACCGCTGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTGCGGGCG
ACGATGCCCCCCAGGCTGTCTTCCCTTCT
mm4__chr2 130.66 Mbp_chr2_130656764 TGAGCTCACCATGGATGATGATACCGCTGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTGCGGGCG
ACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm4__chr2 130.08 Mbp_chr2_130075333 GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCGCCCGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA
GCACAGCGGTATCATCATCCATGGTGAGCTCA
mm4__chrX 154.37 Mbp_chrX_154374445 GCGTTTTCCGGCCTCTCTGCCCGCCTTCTCCAACCCCGCCCCCGGCCGACGAGGAGCGCGGCGACCCTACGGCGCGAAGC
CGGGCCGCGGAGACCTCACCTGCTGTTCC

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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CRISPR/Cas9 Guide Designer for chordate vertebrate ecdysozoans lophotrochozoans protostomes spongi corals plants butterflies metazoans genomes fruitflies insects nematodes mammals.