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PCR primers for off-targets of CTCGTCGTCGACAACGGCTC 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_5529613_F TCGTCGGCAGCGTCCACGATGGAGGGGAAGACG 59.8 1.00
ontarget_mm0__chr7 5.53 Mbp_chr7_5529613_R GTCTCGTGGGCTCGGGCTATTCTCGCAGCTCACCA 60.1 1.00
mm3__chr1 223.86 Mbp_chr1_223864807_F TCGTCGGCAGCGTCCATGGAAGGGAAGATGGCCC 60.4 0.87
mm3__chr1 223.86 Mbp_chr1_223864807_R GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT 60.1 0.87
mm2__chr5 77.78 Mbp_chr5_77784907_F TCGTCGGCAGCGTCCACCAGCTCACCATGGATGA 59.7 0.87
mm2__chr5 77.78 Mbp_chr5_77784907_R GTCTCGTGGGCTCGGAACGATGGAGGGGAAGACTG 59.0 0.87
mm2__chr22 15.72 Mbp_chr22_15721507_F TCGTCGGCAGCGTCACTGAATGAGCTCACCATGGA 59.0 0.87
mm2__chr22 15.72 Mbp_chr22_15721507_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.87
mm2__chr2 131.63 Mbp_chr2_131626159_F TCGTCGGCAGCGTCACCATGGATGATGATACCGCT 59.0 0.87
mm2__chr2 131.63 Mbp_chr2_131626159_R GTCTCGTGGGCTCGGCCTCCATCATGCCCTGGTG 60.1 0.87
mm2__chr2 131.26 Mbp_chr2_131263576_F TCGTCGGCAGCGTCTGGATGATGATACCGCCGTG 59.9 0.87
mm2__chr2 131.26 Mbp_chr2_131263576_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.87
mm2__chr2 130.66 Mbp_chr2_130656770_F TCGTCGGCAGCGTCACCATGGATGATGATACCGCT 59.0 0.87
mm2__chr2 130.66 Mbp_chr2_130656770_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.87
mm2__chr2 130.46 Mbp_chr2_130463899_F Primer3: not found at this Tm N.d. 0.87
mm2__chr2 130.46 Mbp_chr2_130463899_R Primer3: not found at this Tm N.d. 0.87
mm2__chr2 130.08 Mbp_chr2_130075327_F TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC 60.1 0.87
mm2__chr2 130.08 Mbp_chr2_130075327_R GTCTCGTGGGCTCGGACCATGGATGATGATACCGCT 59.0 0.87
mm2__chr14 19.40 Mbp_chr14_19402864_F TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC 60.1 0.87
mm2__chr14 19.40 Mbp_chr14_19402864_R GTCTCGTGGGCTCGGACTGAATGAGCTCACCATGGA 59.0 0.87
mm2__chr14 19.00 Mbp_chr14_18998888_F TCGTCGGCAGCGTCACTGAATGAGCTCACCATGGA 59.0 0.87
mm2__chr14 19.00 Mbp_chr14_18998888_R GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC 60.1 0.87
mm1__chr9 6.83 Mbp_chr9_6834476_F TCGTCGGCAGCGTCCCGCTACCAGCTCACCATAG 59.9 0.85
mm1__chr9 6.83 Mbp_chr9_6834476_R GTCTCGTGGGCTCGGACACGATGAAGGGGAAGACG 59.7 0.85
mm3__chr22 41.08 Mbp_chr22_41075195_F TCGTCGGCAGCGTCGATGGAAGGGAAGATGGCCC 60.1 0.81
mm3__chr22 41.08 Mbp_chr22_41075195_R GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT 60.1 0.81
mm3__chr9 100.73 Mbp_chr9_100731260_F TCGTCGGCAGCGTCGCCAGCTCACCATGGATGAT 60.1 0.60
mm3__chr9 100.73 Mbp_chr9_100731260_R GTCTCGTGGGCTCGGCTAGGGAGCTCCACGATGGA 60.4 0.60
mm4__chr3 169.77 Mbp_chr3_169769476_F Primer3: not found at this Tm N.d. 0.22
mm4__chr3 169.77 Mbp_chr3_169769476_R Primer3: not found at this Tm N.d. 0.22
mm3__chrX 46.29 Mbp_chrX_46289084_F TCGTCGGCAGCGTCTGGAGGGGAAGACAACTGGG 61.1 0.11
mm3__chrX 46.29 Mbp_chrX_46289084_R GTCTCGTGGGCTCGGCTCCATCACCAGTCCACACC 60.0 0.11
mm4__chr15 34.79 Mbp_chr15_34794758_F TCGTCGGCAGCGTCGGGAAGTTTACCTGGTGCCG 60.9 0.10
mm4__chr15 34.79 Mbp_chr15_34794758_R GTCTCGTGGGCTCGGATGTGTGACGACGAGGAGAC 59.4 0.10
mm4__chr2 16.08 Mbp_chr2_16083760_F TCGTCGGCAGCGTCAGTTAGAAGCCCGCAAGCAT 60.0 0.09
mm4__chr2 16.08 Mbp_chr2_16083760_R GTCTCGTGGGCTCGGAAGAGGACAAGGTGCTGGTG 59.8 0.09
mm4__chr4 140.15 Mbp_chr4_140154394_F TCGTCGGCAGCGTCCAACCACACTACCGCCCC 60.3 0.09
mm4__chr4 140.15 Mbp_chr4_140154394_R GTCTCGTGGGCTCGGAAGTTGATTTATTTCTGAGATCCCC 57.1 0.09
mm2__chr18 62.44 Mbp_chr18_62442029_F TCGTCGGCAGCGTCGCCAGCTCACCATGGATGAT 60.1 0.06
mm2__chr18 62.44 Mbp_chr18_62442029_R GTCTCGTGGGCTCGGATGATGGAGAGGAAGACGGC 59.2 0.06
mm4__chrX 53.14 Mbp_chrX_53143869_F TCGTCGGCAGCGTCGATGGAGGGGAACACAGCTC 60.1 0.05
mm4__chrX 53.14 Mbp_chrX_53143869_R GTCTCGTGGGCTCGGTGCTCTTCCACAGGTTGCAA 60.1 0.05
mm4__chr16 50.06 Mbp_chr16_50059187_F TCGTCGGCAGCGTCGAGGGGAACATGGTTCAGGG 60.0 0.05
mm4__chr16 50.06 Mbp_chr16_50059187_R GTCTCGTGGGCTCGGACTTCTCCACGATGGCGATG 60.1 0.05
mm2__chr5 131.66 Mbp_chr5_131660071_F TCGTCGGCAGCGTCATTATGGAGGGGAAGACGGC 59.2 0.04
mm2__chr5 131.66 Mbp_chr5_131660071_R GTCTCGTGGGCTCGGGCCACCAGTTCACCATGGAT 60.3 0.04
mm2__chr15 43.99 Mbp_chr15_43990140_F TCGTCGGCAGCGTCAATGAAGGGGAAGATGGCCC 59.7 0.04
mm2__chr15 43.99 Mbp_chr15_43990140_R GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT 60.1 0.04
mm4__chr7 289.59 Kbp_chr7_289586_F Primer3: not found at this Tm N.d. 0.04
mm4__chr7 289.59 Kbp_chr7_289586_R Primer3: not found at this Tm N.d. 0.04
mm4__chr5 80.30 Mbp_chr5_80302803_F TCGTCGGCAGCGTCATGATGAAGGGGAAGGCAGC 60.1 0.03
mm4__chr5 80.30 Mbp_chr5_80302803_R GTCTCGTGGGCTCGGAGCTCACCATGCATGACGAT 59.8 0.03
mm4__chr17 81.51 Mbp_chr17_81512310_F TCGTCGGCAGCGTCCCATCCACTCACCTGGTGTC 60.0 0.02
mm4__chr17 81.51 Mbp_chr17_81512310_R GTCTCGTGGGCTCGGATGGAAGAAGAGATCGCCGC 60.2 0.02
mm4__chr5 98.34 Mbp_chr5_98338764_F TCGTCGGCAGCGTCGCAGCTAGCTCACCATGGAT 59.8 0.02
mm4__chr5 98.34 Mbp_chr5_98338764_R GTCTCGTGGGCTCGGGCATTGTCACCTGTGAAGCC 59.7 0.02
mm4__chr6 46.20 Mbp_chr6_46204998_F TCGTCGGCAGCGTCATGGAAGAAGAGATCCCCGC 59.2 0.02
mm4__chr6 46.20 Mbp_chr6_46204998_R GTCTCGTGGGCTCGGATATGGCTCGGAGGGCATTG 59.9 0.02
mm4__chr1 229.43 Mbp_chr1_229433065_F TCGTCGGCAGCGTCAAGCCGGCTTTCACCAGG 60.2 0.01
mm4__chr1 229.43 Mbp_chr1_229433065_R GTCTCGTGGGCTCGGGCTGAGACTTCTGCGCTGAT 60.4 0.01
mm4__chr1 218.76 Mbp_chr1_218759728_F TCGTCGGCAGCGTCCAGCAGTCCCAGACTTGAGT 59.3 0.01
mm4__chr1 218.76 Mbp_chr1_218759728_R GTCTCGTGGGCTCGGCCCTGGCTAGACCCACCATA 60.4 0.01
mm2__chr6 88.28 Mbp_chr6_88276942_F TCGTCGGCAGCGTCCCCACAATGGAGGGGAAGAC 60.0 0.00
mm2__chr6 88.28 Mbp_chr6_88276942_R GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT 60.1 0.00

Off-target amplicon sequences

Primers underlined, off-targets in bold.

ontarget_mm0__chr7 5.53 Mbp_chr7_5529613 CACGATGGAGGGGAAGACGGCCCGGGGGGCATCGTCGCCCGCGAAGCCGGCCTTGCACATGCCGGAGCCGTTGTCGACGA
CGAG
CGCGGCGATATCATCATCCATGGTGAGCTGCGAGAATAGC
mm3__chr1 223.86 Mbp_chr1_223864807 CATGGAAGGGAAGATGGCCCAGGGGTCATCATCCCCTGTGAAGCCAGCCTTGCACATGCCGGAGCTGTTGTCAATGACGA
G
CGCCGCGATATCATCATCCATGGTGAGCTGGC
mm2__chr5 77.78 Mbp_chr5_77784907 CACCAGCTCACCATGGATGATGATATCACCGCGCTCGTCATTGACAACGGCTCCGGCATGTGCAAGGCCAGCTTCACGGG
CGACAATGCCGCCCGGGCAGTCTTCCCCTCCATCGTT
mm2__chr22 15.72 Mbp_chr22_15721507 ACTGAATGAGCTCACCATGGATGATGATACTGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTG
CAGGTGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm2__chr2 131.63 Mbp_chr2_131626159 ACCATGGATGATGATACCGCTGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTGCGGGCGACGATGC
CCCCCAGGCTGTCTTCCCTTCTATCGTGGGGCGCCCCAGGCACCAGGGCATGATGGAGG
mm2__chr2 131.26 Mbp_chr2_131263576 TGGATGATGATACCGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTGCGGGCGACGATGCCCCC
CGGGCTGTCTTCCCTTCCATC
mm2__chr2 130.66 Mbp_chr2_130656770 ACCATGGATGATGATACCGCTGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTGCGGGCGACGATGC
CCCCCGGGCTGTCTTCCCTTCCATC
mm2__chr2 130.08 Mbp_chr2_130075327 GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCGCCCGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA
G
CACAGCGGTATCATCATCCATGGT
mm2__chr14 19.40 Mbp_chr14_19402864 GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCACCTGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA
G
CACGGCAGTATCATCATCCATGGTGAGCTCATTCAGT
mm2__chr14 19.00 Mbp_chr14_18998888 ACTGAATGAGCTCACCATGGATGATGATACTGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTG
CAGGTGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC
mm1__chr9 6.83 Mbp_chr9_6834476 CCGCTACCAGCTCACCATAGATGATGATATTGCTACGCCCGTCGTCGACAACGGCTCCGGCATGTGGAAGGCTGGCTTCG
CAGGTGACGATGACCCCCGGCCGTCTTCCCCTTCATCGTGT
mm3__chr22 41.08 Mbp_chr22_41075195 GATGGAAGGGAAGATGGCCCAGGGGACATCGTCACCCGCGAAGCCGGCCTTGCACATGCCAGAGCTGTTGTCAACAACGA
G
CGCAGCAATATCATCATCCATGGTGAGCTGGC
mm3__chr9 100.73 Mbp_chr9_100731260 GCCAGCTCACCATGGATGATGATATCGCCCCACTTGTCATTGACAACGGCTCAGGCATGTGCAAGGCCGGCTTTGCAGGT
GATAATGTCCCCCTGGGCCATCTTCCCCCTCCATCGTGGAGCTCCCTAG
mm3__chrX 46.29 Mbp_chrX_46289084 TGGAGGGGAAGACAACTGGGGGGCATCATCGCCCATGAAGGCAGCCTTGCACATGCCAGAGCCTTTGTTGAAGACGAGAG
CAGTGATATCATCATTCATGGTGATCTGGCAGTGGGTGTGGACTGGTGATGGAG
mm4__chr15 34.79 Mbp_chr15_34794758 GGGAAGTTTACCTGGTGCCGCGGGCGGCCCACGATGGACGGGAAGACAGCGCGGGGCGCGTCATCGCCCGCAAAGCCGGC
CTTCACCAGCCCAGAGCCGTTGTCGCACACCAGGGCGGTGGTCTCCTCGTCGTCACACAT
mm4__chr2 16.08 Mbp_chr2_16083760 AGTTAGAAGCCCGCAAGCATTTGCCGAGGGTAGTGCGGGCATCTCAGGACTCCATCCTCCCCTCCTCGTGGACAGCGGTT
CCGG
GAGCCCCAACTCACTGGCTGGGGACTTGCCTCCCACACCAGCACCTTGTCCTCTT
mm4__chr4 140.15 Mbp_chr4_140154394 CAACCACACTACCGCCCCGCCGAGAAGCCCAGAGCCGTCGGCGACGGAGAGAAGAAAGATTCTGGGGGGATCTCAGAAAT
AAATCAACTT
mm2__chr18 62.44 Mbp_chr18_62442029 GCCAGCTCACCATGGATGATGATACTGTCGCCCTCGTCATCGACAATGGCTCTGGCATGTGCAGGGCTGGCTTCGCAGGC
GACAGTGCTCTCCAGGCCGTCTTCCTCTCCATCAT
mm4__chrX 53.14 Mbp_chrX_53143869 GATGGAGGGGAACACAGCTCAGGGGCCATCCATCCCCAGCAAAGCCAGCTTTGCACATGCCAGAGCCATTGTCAATGATG
AG
CGCGGCGATCTCTATTTCCATTGCAACCTGTGGAAGAGCA
mm4__chr16 50.06 Mbp_chr16_50059187 GAGGGGAACATGGTTCAGGGGGCATCGTCCCTAGCAAAGCCAGCTTTGCACCTGCCAGAGCCATTGTCAATGATGAGCAC
GATGATCTCTTCTTCCATCGCCATCGTGGAGAAGT
mm2__chr5 131.66 Mbp_chr5_131660071 ATTATGGAGGGGAAGACGGCCCGGGGGCATCGTTGCCCGTGAAGCAGACCTTCCACATGCCAGAGCCATTGTCGACGACA
AG
CACAGCAATATCATTATCCATGGTGAACTGGTGGC
mm2__chr15 43.99 Mbp_chr15_43990140 AATGAAGGGGAAGATGGCCCGGGGTGGGCATCGTCACCTGCAAAGCTGGCCTTGCACATGCCAGAGCCATTGTCGACGAC
AAG
CATGGTGATATCATCATCCATGGTGAGCTGGC
mm4__chr5 80.30 Mbp_chr5_80302803 ATGATGAAGGGGAAGGCAGCCCGGGGGACACCATCGCTCGCAAAGCTAGCCTTGCATATGCCAGAGCCATTGTTGAATAC
GAG
AGCAGTGATATCGTCATGCATGGTGAGCT
mm4__chr17 81.51 Mbp_chr17_81512310 CCATCCACTCACCTGGTGTCTGGGGCGCCCGACGATGGAAGGAAACACGGCTCGGGGAGCGTCGTCCCCAGCAAAACCAG
CTTTGCACATGCCGGAGCCATTGTCAATGACCAGCGCGGCGATCTCTTCTTCCAT
mm4__chr5 98.34 Mbp_chr5_98338764 GCAGCTAGCTCACCATGGATGATGATATCAGCACGCTCCTCATCAACAAGGGCTCTGGCATGTGCAAGGCCGGCTTCACA
GGTGACAATGC
mm4__chr6 46.20 Mbp_chr6_46204998 ATGGAAGAAGAGATCCCCGCGCTCTTCATTGACAATGGCTCCGGCATGTGAAAAGCAGCTTTGCTGGGACAATGCCCTCC
GAGCCATAT
mm4__chr1 229.43 Mbp_chr1_229433065 AAGCCGGCTTTCACCAGGCCGGAGCCATTGTCGCACACGAGGGCGGTGGTCTCGTCTTCGTCGCACATTGTGTCTAGTTT
CTGCAAGGACAGGGAGACCGCGAAGAGGCGCGGTGCATCAGCGCAGAAGTCTCAGC
mm4__chr1 218.76 Mbp_chr1_218759728 CAGCAGTCCCAGACTTGAGTAAGTGAAAATAGAGGTGGGACTCTGTTTCATTTTCAGGTAAAATCCCAAAAGACAACAAG
TATCCTCCTTGTCCACAATGGCTCAGGAGGAGTAAGTATCTATGGTGGGTCTAGCCAGGG
mm2__chr6 88.28 Mbp_chr6_88276942 CCCACAATGGAGGGGAAGACGGCCTGGGGAGCCTTGCGCATGCTGGAGCAGTTGTCGACGACGACGAGCGCGGTGATAGC
ATCATCCATGGTGAGCTGGC

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.