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PCR primers for off-targets of GCCGAGTCCTTAGGCCGCCA GGG

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_5529725_F TCGTCGGCAGCGTCTGGTGAGCTGCGAGAATAGC 60.1 1.00
ontarget_mm0__chr7 5.53 Mbp_chr7_5529725_R GTCTCGTGGGCTCGGCGGCTTCCTTTGTCCCCAAT 60.6 1.00
mm3__chr15 40.95 Mbp_chr15_40952430_F TCGTCGGCAGCGTCTTCAGCTGCCTCTGTTTCCC 60.2 0.43
mm3__chr15 40.95 Mbp_chr15_40952430_R GTCTCGTGGGCTCGGGTCTTCTCCCTCGGCTTCTG 59.8 0.43
mm4__chr16 18.74 Mbp_chr16_18743875_F Primer3: not found at this Tm N.d. 0.39
mm4__chr16 18.74 Mbp_chr16_18743875_R Primer3: not found at this Tm N.d. 0.39
mm3__chr13 85.01 Mbp_chr13_85013119_F Primer3: not found at this Tm N.d. 0.34
mm3__chr13 85.01 Mbp_chr13_85013119_R Primer3: not found at this Tm N.d. 0.34
mm4__chr3 37.77 Mbp_chr3_37771188_F TCGTCGGCAGCGTCAGGACTGACTGGGCAGAGAA 60.1 0.33
mm4__chr3 37.77 Mbp_chr3_37771188_R GTCTCGTGGGCTCGGCCGGCCAATGACCAACAATG 60.1 0.33
mm3__chr17 80.10 Mbp_chr17_80095019_F Primer3: not found at this Tm N.d. 0.32
mm3__chr17 80.10 Mbp_chr17_80095019_R Primer3: not found at this Tm N.d. 0.32
mm4__chr12 110.74 Mbp_chr12_110742903_F TCGTCGGCAGCGTCCTCCTTCTCTCCCCACTGGA 59.9 0.30
mm4__chr12 110.74 Mbp_chr12_110742903_R GTCTCGTGGGCTCGGTGGCCCATAAAGAAGTCCCG 59.7 0.30
mm4__chr19 18.05 Mbp_chr19_18049040_F Primer3: not found at this Tm N.d. 0.22
mm4__chr19 18.05 Mbp_chr19_18049040_R Primer3: not found at this Tm N.d. 0.22
mm4__chr1 240.00 Mbp_chr1_239997995_F Primer3: not found at this Tm N.d. 0.17
mm4__chr1 240.00 Mbp_chr1_239997995_R Primer3: not found at this Tm N.d. 0.17
mm4__chr2 72.91 Mbp_chr2_72905984_F Primer3: not found at this Tm N.d. 0.17
mm4__chr2 72.91 Mbp_chr2_72905984_R Primer3: not found at this Tm N.d. 0.17
mm4__chr15 22.16 Mbp_chr15_22157925_F Primer3: not found at this Tm N.d. 0.12
mm4__chr15 22.16 Mbp_chr15_22157925_R Primer3: not found at this Tm N.d. 0.12
mm4__chr15 21.72 Mbp_chr15_21715302_F Primer3: not found at this Tm N.d. 0.12
mm4__chr15 21.72 Mbp_chr15_21715302_R Primer3: not found at this Tm N.d. 0.12
mm4__chr12 3.75 Mbp_chr12_3753216_F Primer3: not found at this Tm N.d. 0.12
mm4__chr12 3.75 Mbp_chr12_3753216_R Primer3: not found at this Tm N.d. 0.12
mm4__chr8 21.58 Mbp_chr8_21578725_F Primer3: not found at this Tm N.d. 0.11
mm4__chr8 21.58 Mbp_chr8_21578725_R Primer3: not found at this Tm N.d. 0.11
mm4__chrX 71.16 Mbp_chrX_71163384_F Primer3: not found at this Tm N.d. 0.11
mm4__chrX 71.16 Mbp_chrX_71163384_R Primer3: not found at this Tm N.d. 0.11
mm3__chr1 2.48 Mbp_chr1_2483173_F TCGTCGGCAGCGTCAGCACCTCCTCGACTTTTGG 59.9 0.11
mm3__chr1 2.48 Mbp_chr1_2483173_R GTCTCGTGGGCTCGGCAGCAGGTGTCCGCCTAC 60.1 0.11
mm4__chr3 49.87 Mbp_chr3_49867897_F TCGTCGGCAGCGTCAAGGGACACAGGCACAAAGG 60.4 0.11
mm4__chr3 49.87 Mbp_chr3_49867897_R GTCTCGTGGGCTCGGGCCCAGGGATTTAGAGCACA 59.7 0.11
mm4__chr20 50.25 Mbp_chr20_50246451_F TCGTCGGCAGCGTCCCCACTGACCGGTTTTGGAT 60.2 0.10
mm4__chr20 50.25 Mbp_chr20_50246451_R GTCTCGTGGGCTCGGCTCACAACCAGGAAGCCAGA 59.6 0.10
mm4__chr18 51.20 Mbp_chr18_51196565_F TCGTCGGCAGCGTCTTCTCTCGTCTCCCCACCC 60.3 0.10
mm4__chr18 51.20 Mbp_chr18_51196565_R GTCTCGTGGGCTCGGGAAAGAGCGTCAACACCACC 59.4 0.10
mm4__chr7 148.04 Mbp_chr7_148035406_F Primer3: not found at this Tm N.d. 0.09
mm4__chr7 148.04 Mbp_chr7_148035406_R Primer3: not found at this Tm N.d. 0.09
mm4__chr2 66.45 Mbp_chr2_66446410_F TCGTCGGCAGCGTCTTCCACTCCAAGAACGCGTT 60.1 0.09
mm4__chr2 66.45 Mbp_chr2_66446410_R GTCTCGTGGGCTCGGCCTAGCTAATCTCGGCTCGC 60.1 0.09
mm3__chr2 129.67 Mbp_chr2_129669479_F TCGTCGGCAGCGTCCAAGGAACCCGCATGAGCTA 60.1 0.09
mm3__chr2 129.67 Mbp_chr2_129669479_R GTCTCGTGGGCTCGGGATGATGGGGCAGCCACTG 60.8 0.09
mm4__chr6 161.83 Mbp_chr6_161832682_F TCGTCGGCAGCGTCGTGTATACCCGCGGAACTGT 59.8 0.08
mm4__chr6 161.83 Mbp_chr6_161832682_R GTCTCGTGGGCTCGGCAGGATGGCGCATGTCATTG 59.9 0.08
mm4__chr21 40.80 Mbp_chr21_40802726_F Primer3: not found at this Tm N.d. 0.07
mm4__chr21 40.80 Mbp_chr21_40802726_R Primer3: not found at this Tm N.d. 0.07
mm4__chr12 105.43 Mbp_chr12_105434714_F Primer3: not found at this Tm N.d. 0.07
mm4__chr12 105.43 Mbp_chr12_105434714_R Primer3: not found at this Tm N.d. 0.07
mm3__chr17 63.70 Mbp_chr17_63700683_F Primer3: not found at this Tm N.d. 0.07
mm3__chr17 63.70 Mbp_chr17_63700683_R Primer3: not found at this Tm N.d. 0.07
mm4__chr4 8.47 Mbp_chr4_8473257_F TCGTCGGCAGCGTCCCTGAGCATGACCCATGAGG 60.1 0.07
mm4__chr4 8.47 Mbp_chr4_8473257_R GTCTCGTGGGCTCGGCCATACCAGCCAGCATGGAT 59.8 0.07
mm4__chr6 154.90 Mbp_chr6_154900975_F TCGTCGGCAGCGTCCATCTCTGCTGTCCCCTTGG 60.1 0.06
mm4__chr6 154.90 Mbp_chr6_154900975_R GTCTCGTGGGCTCGGACCCATTCTTCCTTCTGCCA 58.9 0.06
mm4__chr6 168.40 Mbp_chr6_168397187_F TCGTCGGCAGCGTCGCAGACAGAGCTCCAGAAGG 60.1 0.04
mm4__chr6 168.40 Mbp_chr6_168397187_R GTCTCGTGGGCTCGGATTTGCTGGAAGGGTCCCTG 59.9 0.04
mm4__chr9 136.44 Mbp_chr9_136439894_F Primer3: not found at this Tm N.d. 0.04
mm4__chr9 136.44 Mbp_chr9_136439894_R Primer3: not found at this Tm N.d. 0.04
mm4__chr15 66.95 Mbp_chr15_66948303_F Primer3: not found at this Tm N.d. 0.04
mm4__chr15 66.95 Mbp_chr15_66948303_R Primer3: not found at this Tm N.d. 0.04
mm4__chr2 128.75 Mbp_chr2_128750938_F TCGTCGGCAGCGTCAAGGGCATTTGCTTGGAGGT 60.1 0.04
mm4__chr2 128.75 Mbp_chr2_128750938_R GTCTCGTGGGCTCGGAACTCCTCTAGGCCCACCC 60.3 0.04
mm4__chr1 2.88 Mbp_chr1_2878772_F TCGTCGGCAGCGTCGAAGGGCAGCAGCGTTTCT 60.9 0.04
mm4__chr1 2.88 Mbp_chr1_2878772_R GTCTCGTGGGCTCGGCTGCCAGAGGTGGGAAGTG 60.0 0.04
mm4__chr6 125.96 Mbp_chr6_125956945_F TCGTCGGCAGCGTCGGCGACGTCTCGAGGTAAAA 60.1 0.03
mm4__chr6 125.96 Mbp_chr6_125956945_R GTCTCGTGGGCTCGGCGCTGCGGTTCACCTGTT 60.9 0.03
mm4__chr1 9.27 Mbp_chr1_9272461_F TCGTCGGCAGCGTCTCTCTGGACTCAGGCCTTGG 60.9 0.02
mm4__chr1 9.27 Mbp_chr1_9272461_R GTCTCGTGGGCTCGGCTCTCCTCTCCTCCATGCCA 60.3 0.02
mm4__chr16 67.45 Mbp_chr16_67447397_F TCGTCGGCAGCGTCCCTGGCAGAGATAAGGCTGG 59.8 0.02
mm4__chr16 67.45 Mbp_chr16_67447397_R GTCTCGTGGGCTCGGATGGTGGAGTGGCTTTCTGG 59.9 0.02
mm4__chr1 29.12 Mbp_chr1_29123673_F TCGTCGGCAGCGTCACAGCCCGACAGAAGATGC 60.0 0.02
mm4__chr1 29.12 Mbp_chr1_29123673_R GTCTCGTGGGCTCGGCCAGGCGGGAGTGTGGAC 62.3 0.02
mm4__chr7 53.37 Mbp_chr7_53367503_F Primer3: not found at this Tm N.d. 0.01
mm4__chr7 53.37 Mbp_chr7_53367503_R Primer3: not found at this Tm N.d. 0.01
mm4__chr2 20.53 Mbp_chr2_20525358_F TCGTCGGCAGCGTCCTGCAGGGTCAGATGTGGAG 60.1 0.01
mm4__chr2 20.53 Mbp_chr2_20525358_R GTCTCGTGGGCTCGGACAAACCAACATCCCAGGGG 60.1 0.01
mm3__chr7 2.61 Mbp_chr7_2607986_F TCGTCGGCAGCGTCGGCAGCCTTAAGACCCTGTT 59.9 0.01
mm3__chr7 2.61 Mbp_chr7_2607986_R GTCTCGTGGGCTCGGCTCCTCCTTCCCTGACTCCA 59.9 0.01
mm4__chr1 156.60 Mbp_chr1_156595897_F TCGTCGGCAGCGTCAGGGTCCAAGTCAAAGCGAG 59.9 0.01
mm4__chr1 156.60 Mbp_chr1_156595897_R GTCTCGTGGGCTCGGGAAGATAGGCAGCTCCCCAC 59.8 0.01
mm4__chr10 132.95 Mbp_chr10_132948534_F TCGTCGGCAGCGTCGCTTCCAGAAGGACTCAGCC 60.3 0.00
mm4__chr10 132.95 Mbp_chr10_132948534_R GTCTCGTGGGCTCGGGCTGAGTCCTTGGGCAGTAA 59.6 0.00
mm3__chr8 131.04 Mbp_chr8_131043392_F TCGTCGGCAGCGTCTGGCAATGGAAGGCATCACT 59.9 0.00
mm3__chr8 131.04 Mbp_chr8_131043392_R GTCTCGTGGGCTCGGCTGCATCCAGCCCCTCATTT 60.3 0.00
mm4__chr10 96.50 Mbp_chr10_96502623_F TCGTCGGCAGCGTCCAGTGTGGTATCTGCACCCA 59.6 0.00
mm4__chr10 96.50 Mbp_chr10_96502623_R GTCTCGTGGGCTCGGGCTGGTTTCCTGTCCTGTCC 60.6 0.00
mm4__chr20 62.96 Mbp_chr20_62963018_F TCGTCGGCAGCGTCCTAGGGCTAAGGCAGACACC 59.5 0.00
mm4__chr20 62.96 Mbp_chr20_62963018_R GTCTCGTGGGCTCGGCACCTTTGAGAACCGCGTTC 59.7 0.00
mm3__chr16 58.99 Mbp_chr16_58987258_F Primer3: not found at this Tm N.d. 0.00
mm3__chr16 58.99 Mbp_chr16_58987258_R Primer3: not found at this Tm N.d. 0.00
mm4__chr11 126.70 Mbp_chr11_126702551_F TCGTCGGCAGCGTCGCTTCCACCAGCTTCCTCTC 60.3 0.00
mm4__chr11 126.70 Mbp_chr11_126702551_R GTCTCGTGGGCTCGGGCCAGCCTTTCTAGAGTGCA 60.0 0.00
mm4__chr7 78.90 Mbp_chr7_78895225_F Primer3: not found at this Tm N.d. 0.00
mm4__chr7 78.90 Mbp_chr7_78895225_R Primer3: not found at this Tm N.d. 0.00

Off-target amplicon sequences

Primers underlined, off-targets in bold.

ontarget_mm0__chr7 5.53 Mbp_chr7_5529725 TGGTGAGCTGCGAGAATAGCCGGGCGCGCTGTGAGCCGAGGTCGCCCCCGCCCTGGCCACTTCCGGCGCGCCGAGTCCTT
AGGCCGCCAGGGGGCGCCGGCGCGCGCCCAGATTGGGGACAAAGGAAGCCG
mm3__chr15 40.95 Mbp_chr15_40952430 TTCAGCTGCCTCTGTTTCCCTTTTACAGTCAGTGACATCCCCTGAGGGCAGTCCCTTGCTGCGTATTGGGTCAACCCCCG
GCGGCCTGTGGACTCGGCTGGGAGGCCAGAAGCCGAGGGAGAAGAC
mm4__chr3 37.77 Mbp_chr3_37771188 AGGACTGACTGGGCAGAGAAGAGCAAGGGGGCGATGGCCACAGAGTCCTTAGCCAGCCAAGGAAATGACATTGTTGGTCA
TTGGCCGG
mm4__chr12 110.74 Mbp_chr12_110742903 CTCCTTCTCTCCCCACTGGAACCACGAGAAGAACAAAATGGCCGCCGACTCCTTAGACAGCCTCGGGCGGCGCACGGATG
CGCGCGGGGAGTGCAGCGGCCCGGCCGGGACTTCTTTATGGGCCA
mm3__chr1 2.48 Mbp_chr1_2483173 AGCACCTCCTCGACTTTTGGAGGTATTTCCTGCTTCTCAGGGCACCCAGTCTGGGGCTTCCGTGGAGGCCTGAGGACTCA
GCCAGTGTAGGCGGACACCTGCTG
mm4__chr3 49.87 Mbp_chr3_49867897 AAGGGACACAGGCACAAAGGGCAGGAAAGGGGAGGTGGACGGGAGTTCTGAAGGGGGCGGATGGGTGCCCAGCAGAGCCC
TAAGGCCTCCATGGCGATGTGCTCTAAATCCCTGGGC
mm4__chr20 50.25 Mbp_chr20_50246451 CCCACTGACCGGTTTTGGATGTAGTTGGAGATGGAACTGGCCCTCTAGGCTTTGTCCCCTGGAGGCCTAGGGACTCTGTC
TTCAGGGCAGTGCTGGCTGTCACTTTGGCCCAGCCCCTCTGGCTTCCTGGTTGTGAG
mm4__chr18 51.20 Mbp_chr18_51196565 TTCTCTCGTCTCCCCACCCACGCCCGGGGCCATGCCCAGCTGGACCTCCCCACCCCTGCACTCACCCTGGCGGCCGACGA
TCTCGGCGACGTGCTCGGAGCTGGGCACCGGGACGCACTCGGTGGTGTTGACGCTCTTTC
mm4__chr2 66.45 Mbp_chr2_66446410 TTCCACTCCAAGAACGCGTTGGGAGAGAGGGGAGAGCGGAGCAGAGTCTCTAGGCCCCCAGGGCCGCCTCCCGGGGTGGC
CACGCCTGCCGGTAGCGAGCCGAGATTAGCTAGG
mm3__chr2 129.67 Mbp_chr2_129669479 CAAGGAACCCGCATGAGCTAGCCGAGTCCTGAGACTGCCAGGGTCCACACACCCTTCCCGGGTGAGGTGTTGGCTCGGTG
GGCCTGTGCACCAGGTGGGCAGGACCCCAGTGGCTGCCCCATCATC
mm4__chr6 161.83 Mbp_chr6_161832682 GTGTATACCCGCGGAACTGTAATACAGGCCGATCCCTAAGGCCTCCATGGTGTCTTACAGCCACACAGTGTGACACAATG
ACATGCGCCATCCTG
mm4__chr4 8.47 Mbp_chr4_8473257 CCTGAGCATGACCCATGAGGGCCACAAGGGCCAGCCCTGCCTCTGTCCCTGCCCCTTCCTGAGGCCGCCAAGGGGCGGCG
CGCTGCATCCATGCTGGCTGGTATGG
mm4__chr6 154.90 Mbp_chr6_154900975 CATCTCTGCTGTCCCCTTGGTCCCCAGGGCGGCCTGAGAACGCGGCTGGCAGAAGGAAGAATGGGT
mm4__chr6 168.40 Mbp_chr6_168397187 GCAGACAGAGCTCCAGAAGGAGTGCGGTCCAGCTCACCCTGGAGGCCTCAGGGCTCAGCCGCCCACGGCCAGAGGTGAGC
ACATCACAACGTCCTGGACGGTGCCCTCCTCCAGGGACCCTTCCAGCAAAT
mm4__chr2 128.75 Mbp_chr2_128750938 AAGGGCATTTGCTTGGAGGTCAAGAGTTGAACTTGAGCTCCAGCTTTTTTTTGGCTACTGACCTCTGGTCATGCCATCGC
GGCCTCAGGACTCTGAGTTCCGTCCTCTGGGAGGCATCCCAGGGTGGGCCTAGAGGAGTT
mm4__chr1 2.88 Mbp_chr1_2878772 GAAGGGCAGCAGCGTTTCTGTGCCACGGCCGGGTCCTTCAGCCCCCAAGGGGACAGGGAGGAAGCCGCCTCACTTCCCAC
CTCTGGCAG
mm4__chr6 125.96 Mbp_chr6_125956945 GGCGACGTCTCGAGGTAAAACGGAGGAGGTGCGGGACGCGGAGACTGCGCGGGCCCGGTAGCCCTGGANNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAACAGGTGAACCGCAGCG
mm4__chr1 9.27 Mbp_chr1_9272461 TCTCTGGACTCAGGCCTTGGTGAGCTGCAGCAGCCATCTCAGCGCCACGGAGGCCTAAGGACAAGGCTGGCATGGAGGAG
AGGAGAG
mm4__chr16 67.45 Mbp_chr16_67447397 CCTGGCAGAGATAAGGCTGGCCCAGCAGTCTGCCTTTCTCCCAAGGCCGAGGCCTTCGGCAGGCAGGGAAATCCTCCTCT
TCCTCCTTGCCTGGCTTCAGCAGCTGCCTCCCTGCCCCCAGAAAGCCACTCCACCAT
mm4__chr1 29.12 Mbp_chr1_29123673 ACAGCCCGACAGAAGATGCTGGGGCCGAGTCCCCAGGACTCCAGGGTCCCGCCACCGCCCGCGTCCACACTCCCGCCTGG
mm4__chr2 20.53 Mbp_chr2_20525358 CTGCAGGGTCAGATGTGGAGTGTCCCCTGCAGGCCTGAGGACTCAGCACTAGTGAAGGGCTCTCAGCAGCTTGCTCAACA
GGCACTGTCCCCTGGGATGTTGGTTTGT
mm3__chr7 2.61 Mbp_chr7_2607986 GGCAGCCTTAAGACCCTGTTGAGGAGGGGAGTGTGGCGTGCGGGGCTGTTTCACGTGGGAAGTGTCAGGCGAAGGGCTTT
GCTGTGCTGAGTCCTTAGGTCTCCAGGGTGGAGTCAGGGAAGGAGGAG
mm4__chr1 156.60 Mbp_chr1_156595897 AGGGTCCAAGTCAAAGCGAGGGTATCGGGGGAAATGGGCCTAGTCCTTAAGCCTGCAGGGAAAGTTGGTGGAGGGTAGGA
GTGGGGAGCTGCCTATCTTC
mm4__chr10 132.95 Mbp_chr10_132948534 GCTTCCAGAAGGACTCAGCCATCACCGCCCAAGGACTCAGCCGTCATGGCCCAAGGACTTGGCCATCACCGCCCAAGGAC
TCGGCCATTACTGCCCAAGGACTCAGC
mm3__chr8 131.04 Mbp_chr8_131043392 TGGCAATGGAAGGCATCACTTTATTTTCTCACTTCTTTCTCCTTGGGGCCCTAAGGACTCAGCCTGAATAAATGAGGGGC
TGGATGCAG
mm4__chr10 96.50 Mbp_chr10_96502623 CAGTGTGGTATCTGCACCCATGAGGCCCAGTCCTTAGTGAGCCAAGGAGGACAGGACAGGAAACCAGC
mm4__chr20 62.96 Mbp_chr20_62963018 CTAGGGCTAAGGCAGACACCCAGGAAGACCTGCTGGGCACAGGTCAAGGCAGGGTGCAGGAGCAGCCGAGTCTTTGGGTG
GCCAGGGGGCTCTGGAGGAGGCCGTGTGGAGGGTCGTTCAGAACGCGGTTCTCAAAGGTG
mm4__chr11 126.70 Mbp_chr11_126702551 GCTTCCACCAGCTTCCTCTCACCGTGGAGCCCTAAGGCCTCAGCAGACACGCTGACTTCTTTTNNNNNNNNNNNNNNNNN
NNNNNNNNNNCACAGCGGCTGCTGAATGGAAAACACTTGCACTCTAGAAAGGCTGGC

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.