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PCR primers for off-targets of CGCGGCGATATCATCATCCA TGG

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_5529636_F TCGTCGGCAGCGTCCACGATGGAGGGGAAGACG 59.8 1.00
ontarget_mm0__chr7 5.53 Mbp_chr7_5529636_R GTCTCGTGGGCTCGGCGGCTATTCTCGCAGCTCA 59.9 1.00
mm1__chr5 77.78 Mbp_chr5_77784884_F TCGTCGGCAGCGTCCACTGCCAGTCCACACCTG 60.5 0.92
mm1__chr5 77.78 Mbp_chr5_77784884_R GTCTCGTGGGCTCGGGGAGCCGTTGTCAATGACGA 60.6 0.92
mm2__chr22 41.08 Mbp_chr22_41075218_F TCGTCGGCAGCGTCACATGCCAGAGCTGTTGTCA 59.8 0.90
mm2__chr22 41.08 Mbp_chr22_41075218_R GTCTCGTGGGCTCGGCTTGGCTGCCTGTCCACA 59.8 0.90
mm3__chr22 15.72 Mbp_chr22_15721484_F TCGTCGGCAGCGTCGGGTCTACAACTGAATGAGCTCA 60.0 0.62
mm3__chr22 15.72 Mbp_chr22_15721484_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.62
mm3__chr14 19.40 Mbp_chr14_19402887_F TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC 60.1 0.62
mm3__chr14 19.40 Mbp_chr14_19402887_R GTCTCGTGGGCTCGGGGGTCTACAACTGAATGAGCTCA 60.0 0.62
mm3__chr14 19.00 Mbp_chr14_18998865_F TCGTCGGCAGCGTCGGGTCTACAACTGAATGAGCTCA 60.0 0.62
mm3__chr14 19.00 Mbp_chr14_18998865_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.62
mm2__chr2 131.26 Mbp_chr2_131263553_F TCGTCGGCAGCGTCAGGCTCTACAATTGAATGAGCTCA 60.0 0.62
mm2__chr2 131.26 Mbp_chr2_131263553_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.62
mm2__chr2 130.46 Mbp_chr2_130463922_F TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC 60.1 0.62
mm2__chr2 130.46 Mbp_chr2_130463922_R GTCTCGTGGGCTCGGAGGCTCTACAATTGAATGAGCTCA 60.0 0.62
mm4__chr8 84.95 Mbp_chr8_84948590_F TCGTCGGCAGCGTCCACTGCTTTGCCACTGGTTC 59.9 0.61
mm4__chr8 84.95 Mbp_chr8_84948590_R GTCTCGTGGGCTCGGCTGGCCTTGCACATACCAGA 60.0 0.61
mm4__chr18 62.44 Mbp_chr18_62442006_F TCGTCGGCAGCGTCACAGAGCCTTGTCCCTTGC 59.9 0.58
mm4__chr18 62.44 Mbp_chr18_62442006_R GTCTCGTGGGCTCGGATGCCAGAGCCATTGTCGAT 59.8 0.58
mm3__chr2 131.63 Mbp_chr2_131626136_F TCGTCGGCAGCGTCAGGCTCTACAATTGAATGAGCTCA 60.0 0.55
mm3__chr2 131.63 Mbp_chr2_131626136_R GTCTCGTGGGCTCGGAGAGCCGTTGTCAATGACGA 59.6 0.55
mm3__chr2 130.66 Mbp_chr2_130656747_F TCGTCGGCAGCGTCAGGCTCTACAATTGAATGAGCTCA 60.0 0.55
mm3__chr2 130.66 Mbp_chr2_130656747_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.55
mm3__chr2 130.08 Mbp_chr2_130075350_F TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC 60.1 0.55
mm3__chr2 130.08 Mbp_chr2_130075350_R GTCTCGTGGGCTCGGAGGCTCTACAATTGAATGAGCTCA 60.0 0.55
mm3__chr15 43.99 Mbp_chr15_43990163_F TCGTCGGCAGCGTCCACATGCCAGAGCCATTGTC 59.5 0.54
mm3__chr15 43.99 Mbp_chr15_43990163_R GTCTCGTGGGCTCGGCTGCTGAGACCGTGTCCAC 60.3 0.54
mm4__chrX 46.29 Mbp_chrX_46289107_F TCGTCGGCAGCGTCTTGCACATGCCAGAGCCTTT 60.8 0.53
mm4__chrX 46.29 Mbp_chrX_46289107_R GTCTCGTGGGCTCGGCTCCATCACCAGTCCACACC 60.0 0.53
mm1__chr1 223.86 Mbp_chr1_223864830_F TCGTCGGCAGCGTCATGCCGGAGCTGTTGTCAAT 60.3 0.52
mm1__chr1 223.86 Mbp_chr1_223864830_R GTCTCGTGGGCTCGGAAGACCATGTCTGCCAGGTG 59.9 0.52
mm4__chr15 66.28 Mbp_chr15_66280416_F TCGTCGGCAGCGTCGTACACACATGCCCCACTGT 60.2 0.52
mm4__chr15 66.28 Mbp_chr15_66280416_R GTCTCGTGGGCTCGGTGCACATCCGTGTATGCACT 60.0 0.52
mm3__chr5 98.34 Mbp_chr5_98338741_F TCGTCGGCAGCGTCCATCCATGCCCGCAGCTAG 60.9 0.50
mm3__chr5 98.34 Mbp_chr5_98338741_R GTCTCGTGGGCTCGGGCATTGTCACCTGTGAAGCC 59.7 0.50
mm2__chr9 100.73 Mbp_chr9_100731237_F TCGTCGGCAGCGTCAATACCACATCTGACCCGCG 60.1 0.50
mm2__chr9 100.73 Mbp_chr9_100731237_R GTCTCGTGGGCTCGGATGCCTGAGCCGTTGTCAAT 60.3 0.50
mm4__chr9 134.42 Mbp_chr9_134417588_F TCGTCGGCAGCGTCCCATGCACGAGTAGCCCAT 59.8 0.38
mm4__chr9 134.42 Mbp_chr9_134417588_R GTCTCGTGGGCTCGGGAGACAGGCCAAACTCCGAG 60.3 0.38
mm2__chr6 88.28 Mbp_chr6_88276968_F TCGTCGGCAGCGTCCTGGAGCAGTTGTCGACGAC 61.0 0.37
mm2__chr6 88.28 Mbp_chr6_88276968_R GTCTCGTGGGCTCGGCGAGCACAGAACCTTGTCCT 59.9 0.37
mm4__chr11 1.80 Mbp_chr11_1804074_F TCGTCGGCAGCGTCTTGCACATGCCAGAGGTGTT 60.4 0.36
mm4__chr11 1.80 Mbp_chr11_1804074_R GTCTCGTGGGCTCGGCCGTCTGTCCACACCCAC 59.9 0.36
mm4__chr14 78.96 Mbp_chr14_78957337_F TCGTCGGCAGCGTCGGTGGTGCGGAGAGGAAAA 59.9 0.36
mm4__chr14 78.96 Mbp_chr14_78957337_R GTCTCGTGGGCTCGGACTGTTTGCTCACTCAGTGACT 59.8 0.36
mm4__chr13 30.19 Mbp_chr13_30192754_F TCGTCGGCAGCGTCAGATGGGCTAAGACCAGATGTG 59.5 0.34
mm4__chr13 30.19 Mbp_chr13_30192754_R GTCTCGTGGGCTCGGTGCTGACTTGGCAAAAGCAT 58.9 0.34
mm4__chr2 241.97 Mbp_chr2_241972172_F TCGTCGGCAGCGTCATTTCTGTGGCCTGGGAAGG 59.9 0.33
mm4__chr2 241.97 Mbp_chr2_241972172_R GTCTCGTGGGCTCGGTGGAGCAGTTTGGTGGAACA 59.7 0.33
mm4__chr9 6.83 Mbp_chr9_6834453_F TCGTCGGCAGCGTCCACACCCGCTACCAGCTC 60.1 0.28
mm4__chr9 6.83 Mbp_chr9_6834453_R GTCTCGTGGGCTCGGACACGATGAAGGGGAAGACG 59.7 0.28
mm4__chr3 77.18 Mbp_chr3_77177393_F Primer3: not found at this Tm N.d. 0.18
mm4__chr3 77.18 Mbp_chr3_77177393_R Primer3: not found at this Tm N.d. 0.18
mm4__chr19 9.52 Mbp_chr19_9518147_F TCGTCGGCAGCGTCAAGAAATGCGACCGCTGAGA 60.0 0.13
mm4__chr19 9.52 Mbp_chr19_9518147_R GTCTCGTGGGCTCGGTGCCGGAGCCATTGTTAACT 59.9 0.13
mm4__chr18 78.19 Mbp_chr18_78189288_F Primer3: not found at this Tm N.d. 0.08
mm4__chr18 78.19 Mbp_chr18_78189288_R Primer3: not found at this Tm N.d. 0.08
mm4__chr21 6.10 Mbp_chr21_6103883_F Primer3: not found at this Tm N.d. 0.06
mm4__chr21 6.10 Mbp_chr21_6103883_R Primer3: not found at this Tm N.d. 0.06
mm4__chr21 43.43 Mbp_chr21_43434352_F Primer3: not found at this Tm N.d. 0.06
mm4__chr21 43.43 Mbp_chr21_43434352_R Primer3: not found at this Tm N.d. 0.06
mm4__chr5 131.66 Mbp_chr5_131660094_F TCGTCGGCAGCGTCTTCCACATGCCAGAGCCATT 59.9 0.05
mm4__chr5 131.66 Mbp_chr5_131660094_R GTCTCGTGGGCTCGGACCACCAAGACTGTGTCCAC 59.8 0.05
mm4__chr3 63.62 Mbp_chr3_63621604_F TCGTCGGCAGCGTCAGGGTTGGAGGAAGATGGGT 60.1 0.04
mm4__chr3 63.62 Mbp_chr3_63621604_R GTCTCGTGGGCTCGGGTTGCTGAGCATGAATGGGG 59.5 0.04
mm4__chr11 71.28 Mbp_chr11_71284374_F Primer3: not found at this Tm N.d. 0.04
mm4__chr11 71.28 Mbp_chr11_71284374_R Primer3: not found at this Tm N.d. 0.04
mm4__chr3 171.55 Mbp_chr3_171552127_F TCGTCGGCAGCGTCGCCCTTGGCTTCTAGAAACCA 60.2 0.02
mm4__chr3 171.55 Mbp_chr3_171552127_R GTCTCGTGGGCTCGGGCAGCACACAAGCAACAGTT 60.1 0.02
mm2__chr3 180.82 Mbp_chr3_180820541_F TCGTCGGCAGCGTCAAGCACAGAGTCTTGCCCTC 59.9 0.00
mm2__chr3 180.82 Mbp_chr3_180820541_R GTCTCGTGGGCTCGGTTGCACATGGGAGAGCCATT 59.9 0.00
mm4__chr2 12.77 Mbp_chr2_12769836_F TCGTCGGCAGCGTCATGTGGATGGGTTTCCAGGC 60.3 0.00
mm4__chr2 12.77 Mbp_chr2_12769836_R GTCTCGTGGGCTCGGGACAGCAGTGACTGTGAGCT 59.9 0.00
mm4__chr15 41.49 Mbp_chr15_41492677_F TCGTCGGCAGCGTCTGAGGGGAGAGAAGGGAAGG 59.9 0.00
mm4__chr15 41.49 Mbp_chr15_41492677_R GTCTCGTGGGCTCGGCACTCAGTGTCTGGGGATGG 59.7 0.00

Off-target amplicon sequences

Primers underlined, off-targets in bold.

ontarget_mm0__chr7 5.53 Mbp_chr7_5529636 CACGATGGAGGGGAAGACGGCCCGGGGGGCATCGTCGCCCGCGAAGCCGGCCTTGCACATGCCGGAGCCGTTGTCGACGA
CGAGCGCGGCGATATCATCATCCATGGTGAGCTGCGAGAATAGCCG
mm1__chr5 77.78 Mbp_chr5_77784884 CACTGCCAGTCCACACCTGCCACCAGCTCACCATGGATGATGATATCACCGCGCTCGTCATTGACAACGGCTCC
mm2__chr22 41.08 Mbp_chr22_41075218 ACATGCCAGAGCTGTTGTCAACAACGAGCGCAGCAATATCATCATCCATGGTGAGCTGGCTGGGGGTGTGGACAGGCAGC
CAAG
mm3__chr22 15.72 Mbp_chr22_15721484 GGGTCTACAACTGAATGAGCTCACCATGGATGATGATACTGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGG
CCGGCTTTGCAGGTGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm3__chr14 19.40 Mbp_chr14_19402887 GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCACCTGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA
GCACGGCAGTATCATCATCCATGGTGAGCTCATTCAGTTGTAGACCC
mm3__chr14 19.00 Mbp_chr14_18998865 GGGTCTACAACTGAATGAGCTCACCATGGATGATGATACTGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGG
CCGGCTTTGCAGGTGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm2__chr2 131.26 Mbp_chr2_131263553 AGGCTCTACAATTGAATGAGCTCACCATGGATGATGATACCGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAG
GCCGGCTTTGCGGGCGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm2__chr2 130.46 Mbp_chr2_130463922 GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCGCCCGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA
GCACGGCGGTATCATCATCCATGGTGAGCTCATTCAATTGTAGAGCCT
mm4__chr8 84.95 Mbp_chr8_84948590 CACTGCTTTGCCACTGGTTCACATCTGCCCTGAGCTCACCATGGATGATGATACCTCTGCACTTGTTGGCAACTGCTCTG
GTATGTGCAAGGCCAG
mm4__chr18 62.44 Mbp_chr18_62442006 ACAGAGCCTTGTCCCTTGCCACTCCGCCGCCCATCCACACCTGCTGCCAGCTCACCATGGATGATGATACTGTCGCCCTC
GTCATCGACAATGGCTCTGGCAT
mm3__chr2 131.63 Mbp_chr2_131626136 AGGCTCTACAATTGAATGAGCTCACCATGGATGATGATACCGCTGTGCTCGTCATTGACAACGGCTCT
mm3__chr2 130.66 Mbp_chr2_130656747 AGGCTCTACAATTGAATGAGCTCACCATGGATGATGATACCGCTGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAG
GCCGGCTTTGCGGGCGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm3__chr2 130.08 Mbp_chr2_130075350 GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCGCCCGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA
GCACAGCGGTATCATCATCCATGGTGAGCTCATTCAATTGTAGAGCCT
mm3__chr15 43.99 Mbp_chr15_43990163 CACATGCCAGAGCCATTGTCGACGACAAGCATGGTGATATCATCATCCATGGTGAGCTGGCGGCGGGTGTGGACGGGTGG
CAGAGAGGCGAGGGCAAGGCTCTGTGCTCGCAGGGTGGACACGGTCTCAGCAG
mm4__chrX 46.29 Mbp_chrX_46289107 TTGCACATGCCAGAGCCTTTGTTGAAGACGAGAGCAGTGATATCATCATTCATGGTGATCTGGCAGTGGGTGTGGACTGG
TGATGGAG
mm1__chr1 223.86 Mbp_chr1_223864830 ATGCCGGAGCTGTTGTCAATGACGAGCGCCGCGATATCATCATCCATGGTGAGCTGGCGGCGGGTGTGGACAGGCTGCAG
AGCAGCGAGGGCAAGGCTCTGTGCTCACCTGGCAGACATGGTCTT
mm4__chr15 66.28 Mbp_chr15_66280416 GTACACACATGCCCCACTGTGCACAGACACAGGCCAAGGCAATATAATCATCCATGGCACAATTTTGAAACTTCGTTCCC
AAATAGCCCATGGAACTGAATTTCTGATATCTGAGTGCATACACGGATGTGCA
mm3__chr5 98.34 Mbp_chr5_98338741 CATCCATGCCCGCAGCTAGCTCACCATGGATGATGATATCAGCACGCTCCTCATCAACAAGGGCTCTGGCATGTGCAAGG
CCGGCTTCACAGGTGACAATGC
mm2__chr9 100.73 Mbp_chr9_100731237 AATACCACATCTGACCCGCGAGCACAGAACCCCGCCCTTGCCGCTCTGCCGCCCAGTCCACACCCGCCGCCAGCTCACCA
TGGATGATGATATCGCCCCACTTGTCATTGACAACGGCTCAGGCAT
mm4__chr9 134.42 Mbp_chr9_134417588 CCATGCACGAGTAGCCCATNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTGCGGCCACATCATCATCCT
CGGCCACCTCTGCTGGGGCCTCAGCCGCCGTGTCCCCTCGGAGTTTGGCCTGTCTC
mm2__chr6 88.28 Mbp_chr6_88276968 CTGGAGCAGTTGTCGACGACGACGAGCGCGGTGATAGCATCATCCATGGTGAGCTGGCGGCGGGTGCGGACGCAAGGCGC
AGCGGCAAGGACAAGGTTCTGTGCTCG
mm4__chr11 1.80 Mbp_chr11_1804074 TTGCACATGCCAGAGGTGTTGTCTATGACAAGCAAGGTGATATTATCATCCATGGTGAGCTGGGTGGGTGTGGACAGACG
G
mm4__chr14 78.96 Mbp_chr14_78957337 GGTGGTGCGGAGAGGAAAAAGCCTTAAGTTAACCGTGGATGATGATGTGGCTGAGGGTGAGTATGACTATGGTGAAATTC
GTCTGTCTTTTCTCTCAGTCCTCTCAGTCACTGAGTGAGCAAACAGT
mm4__chr13 30.19 Mbp_chr13_30192754 AGATGGGCTAAGACCAGATGTGGGCCTGGAAAAATGGAGGGAAATGTAGCACTGAAAAGATATTGTGACCGGCAGCTGTA
TCATCATCCACGGCAGGTAAATATTAAAGCACTGAGAGGAATGCTTTTGCCAAGTCAGCA
mm4__chr2 241.97 Mbp_chr2_241972172 ATTTCTGTGGCCTGGGAAGGTGGGTGGCTACATCATCATCCATGGTCTCTGAAATATCCTGTGTTACCAAGGCCTGCTTG
TTCCACCAAACTGCTCCA
mm4__chr9 6.83 Mbp_chr9_6834453 CACACCCGCTACCAGCTCACCATAGATGATGATATTGCTACGCCCGTCGTCGACAACGGCTCCGGCATGTGGAAGGCTGG
CTTCGCAGGTGACGATGACCCCCGGCCGTCTTCCCCTTCATCGTGT
mm4__chr19 9.52 Mbp_chr19_9518147 AAGAAATGCGACCGCTGAGACTGCATCTATCCCGCGAGCACAAAGCCTTGCCCTGGCCACTCTGCCGCCTATACACGCCC
GCTGTCAGCTCACCATGGATGATGATATTGAGGTGCTTGTAGTTAACAATGGCTCCGGCA
mm4__chr5 131.66 Mbp_chr5_131660094 TTCCACATGCCAGAGCCATTGTCGACGACAAGCACAGCAATATCATTATCCATGGTGAACTGGTGGCAGGTGTGGATGGG
CGGCAGAGTGGTGAGGGTGAGGCTCTGTGCTTGCAGGGTGGACACAGTCTTGGTGGT
mm4__chr3 63.62 Mbp_chr3_63621604 AGGGTTGGAGGAAGATGGGTACAGGATGAGTTGCCAGAAGCCCTGGATGAGGATATGGCAGCCTGAGTTAGGATGGGGGC
CCCATTCATGCTCAGCAAC
mm4__chr3 171.55 Mbp_chr3_171552127 GCCCTTGGCTTCTAGAAACCACTTCGTTGGAAACTGTTTCCTAGTCCAGCCCTTGCATGTTGATATCGCCCGGGGGATCA
TTGTCTTTTCTTTTTTCCATTCATCTATGCTCATGACTTCAACTGTTGCTTGTGTGCTGC
mm2__chr3 180.82 Mbp_chr3_180820541 AAGCACAGAGTCTTGCCCTCGCCGCTCTGCCACGGGTCCACACCCACTGCCAGCTCATCATGGATAATGATATCGCCACG
CTCGTCATGGACAATGGCTCTCCCATGTGCAA
mm4__chr2 12.77 Mbp_chr2_12769836 ATGTGGATGGGTTTCCAGGCAGCTGCAGTCCTGCCCGCCCCCATACGCGGCGATGACTTCCTCCAAGGAAGAACTTGTGC
AACTGTGGAGCTGATCAGGCCGCCCGGGCTGCCTTTCAGCTCACAGTCACTGCTGTC
mm4__chr15 41.49 Mbp_chr15_41492677 TGAGGGGAGAGAAGGGAAGGAAAGCCCCTCCTGCGGCCATCTCAGCATCCAAGGCAATCCTTCTGGGGCTGCTCTGTCTC
CAGGGCCCCAGCCATCCCCAGACACTGAGTG

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.