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PCR primers for off-targets of CGCCGAGTCCTTAGGCCGCC AGG

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_5529724_F TCGTCGGCAGCGTCTGGTGAGCTGCGAGAATAGC 60.1 1.00
ontarget_mm0__chr7 5.53 Mbp_chr7_5529724_R GTCTCGTGGGCTCGGCGGCTTCCTTTGTCCCCAAT 60.6 1.00
mm3__chr15 40.95 Mbp_chr15_40952431_F TCGTCGGCAGCGTCTTCAGCTGCCTCTGTTTCCC 60.2 0.71
mm3__chr15 40.95 Mbp_chr15_40952431_R GTCTCGTGGGCTCGGGTCTTCTCCCTCGGCTTCTG 59.8 0.71
mm4__chr7 151.14 Mbp_chr7_151137342_F TCGTCGGCAGCGTCCCACTAACCTCCAGCACCTG 60.0 0.40
mm4__chr7 151.14 Mbp_chr7_151137342_R GTCTCGTGGGCTCGGCACCCAACACTGAGACCACA 59.8 0.40
mm4__chr14 99.59 Mbp_chr14_99586879_F TCGTCGGCAGCGTCGCCCTACCTATAGCACACCC 59.3 0.39
mm4__chr14 99.59 Mbp_chr14_99586879_R GTCTCGTGGGCTCGGCAGGTCCCCGGTAGGAGG 60.4 0.39
mm4__chr5 374.67 Kbp_chr5_374670_F TCGTCGGCAGCGTCCTCAGAGACCAGGGAGGGAG 60.3 0.33
mm4__chr5 374.67 Kbp_chr5_374670_R GTCTCGTGGGCTCGGCTGACGAGCTAAGGTGTCCG 60.1 0.33
mm4__chr14 90.61 Mbp_chr14_90613707_F TCGTCGGCAGCGTCATAGCCCAGTCACCTCACCT 59.9 0.33
mm4__chr14 90.61 Mbp_chr14_90613707_R GTCTCGTGGGCTCGGCTGGGGTCCTTCCAGTTTCC 59.9 0.33
mm4__chr17 17.85 Mbp_chr17_17845006_F TCGTCGGCAGCGTCGGCCTTGAGAGTCCAGGTTT 59.5 0.30
mm4__chr17 17.85 Mbp_chr17_17845006_R GTCTCGTGGGCTCGGGCCTGTCCTGAGATCTCTGC 59.8 0.30
mm4__chr10 103.60 Mbp_chr10_103602120_F TCGTCGGCAGCGTCGTGAGTCTGAACCCTGGCTG 60.3 0.28
mm4__chr10 103.60 Mbp_chr10_103602120_R GTCTCGTGGGCTCGGCTCCTTTAGCACTGCTCGCT 60.1 0.28
mm4__chr14 103.55 Mbp_chr14_103554283_F TCGTCGGCAGCGTCCCTGCTGGTGACTGGCATAA 60.0 0.26
mm4__chr14 103.55 Mbp_chr14_103554283_R GTCTCGTGGGCTCGGTGCACAGATTTTGGGGTGGG 60.8 0.26
mm4__chr17 18.63 Mbp_chr17_18634916_F TCGTCGGCAGCGTCCCTCTGCTCGCTCACCCG 62.5 0.25
mm4__chr17 18.63 Mbp_chr17_18634916_R GTCTCGTGGGCTCGGCAGGAGTCAGTGGCAGGC 60.0 0.25
mm4__chr17 15.75 Mbp_chr17_15749781_F TCGTCGGCAGCGTCTAGGAGGCTGCCCTGGAG 60.0 0.25
mm4__chr17 15.75 Mbp_chr17_15749781_R GTCTCGTGGGCTCGGCACGCCTCTGCTCGCTCA 62.4 0.25
mm4__chr1 35.57 Mbp_chr1_35574176_F Primer3: not found at this Tm N.d. 0.24
mm4__chr1 35.57 Mbp_chr1_35574176_R Primer3: not found at this Tm N.d. 0.24
mm4__chr10 78.33 Mbp_chr10_78327745_F Primer3: not found at this Tm N.d. 0.24
mm4__chr10 78.33 Mbp_chr10_78327745_R Primer3: not found at this Tm N.d. 0.24
mm4__chr11 1.51 Mbp_chr11_1508063_F TCGTCGGCAGCGTCCTGGAGCTGGCGAGAAGTC 60.1 0.24
mm4__chr11 1.51 Mbp_chr11_1508063_R GTCTCGTGGGCTCGGTTTCTGTTTGAGCCCCTGCA 60.1 0.24
mm4__chr16 58.99 Mbp_chr16_58987259_F Primer3: not found at this Tm N.d. 0.23
mm4__chr16 58.99 Mbp_chr16_58987259_R Primer3: not found at this Tm N.d. 0.23
mm4__chr2 129.67 Mbp_chr2_129669478_F TCGTCGGCAGCGTCATGTTTTGCCAAGGAACCCG 59.3 0.22
mm4__chr2 129.67 Mbp_chr2_129669478_R GTCTCGTGGGCTCGGGATGATGGGGCAGCCACTG 60.8 0.22
mm4__chr22 37.02 Mbp_chr22_37018467_F TCGTCGGCAGCGTCCATAGAAGCTGCCCAGGTGT 59.7 0.21
mm4__chr22 37.02 Mbp_chr22_37018467_R GTCTCGTGGGCTCGGCCTACGAGATGATGCTGCCC 60.3 0.21
mm4__chr19 344.33 Kbp_chr19_344335_F Primer3: not found at this Tm N.d. 0.20
mm4__chr19 344.33 Kbp_chr19_344335_R Primer3: not found at this Tm N.d. 0.20
mm4__chr11 104.76 Mbp_chr11_104758701_F Primer3: not found at this Tm N.d. 0.18
mm4__chr11 104.76 Mbp_chr11_104758701_R Primer3: not found at this Tm N.d. 0.18
mm4__chr1 155.21 Mbp_chr1_155207410_F TCGTCGGCAGCGTCTCCAGCTACCTCTCCCACAT 59.6 0.17
mm4__chr1 155.21 Mbp_chr1_155207410_R GTCTCGTGGGCTCGGGTACTGTAGGTCCCTGGGCT 60.3 0.17
mm4__chr7 2.61 Mbp_chr7_2607985_F TCGTCGGCAGCGTCGGCAGCCTTAAGACCCTGTT 59.9 0.16
mm4__chr7 2.61 Mbp_chr7_2607985_R GTCTCGTGGGCTCGGCTCCTCCTTCCCTGACTCCA 59.9 0.16
mm4__chr22 37.02 Mbp_chr22_37024460_F TCGTCGGCAGCGTCTCGACATCGACCAGTGCAG 59.7 0.16
mm4__chr22 37.02 Mbp_chr22_37024460_R GTCTCGTGGGCTCGGGTAGATCACGACGTGGGTGG 60.1 0.16
mm4__chr3 44.58 Mbp_chr3_44581073_F Primer3: not found at this Tm N.d. 0.14
mm4__chr3 44.58 Mbp_chr3_44581073_R Primer3: not found at this Tm N.d. 0.14
mm4__chr17 17.04 Mbp_chr17_17042766_F Primer3: not found at this Tm N.d. 0.14
mm4__chr17 17.04 Mbp_chr17_17042766_R Primer3: not found at this Tm N.d. 0.14
mm4__chr12 113.46 Mbp_chr12_113463179_F Primer3: not found at this Tm N.d. 0.13
mm4__chr12 113.46 Mbp_chr12_113463179_R Primer3: not found at this Tm N.d. 0.13
mm4__chr22 41.91 Mbp_chr22_41909974_F Primer3: not found at this Tm N.d. 0.12
mm4__chr22 41.91 Mbp_chr22_41909974_R Primer3: not found at this Tm N.d. 0.12
mm4__chr5 112.45 Mbp_chr5_112445010_F Primer3: not found at this Tm N.d. 0.12
mm4__chr5 112.45 Mbp_chr5_112445010_R Primer3: not found at this Tm N.d. 0.12
mm4__chr19 5.04 Mbp_chr19_5041583_F TCGTCGGCAGCGTCGTACTTTCCCAGTCCCTGCC 60.0 0.12
mm4__chr19 5.04 Mbp_chr19_5041583_R GTCTCGTGGGCTCGGCTGAGCCCAAGTCAGGACC 59.7 0.12
mm3__chr15 66.98 Mbp_chr15_66975543_F Primer3: not found at this Tm N.d. 0.11
mm3__chr15 66.98 Mbp_chr15_66975543_R Primer3: not found at this Tm N.d. 0.11
mm4__chr1 240.00 Mbp_chr1_239997996_F Primer3: not found at this Tm N.d. 0.10
mm4__chr1 240.00 Mbp_chr1_239997996_R Primer3: not found at this Tm N.d. 0.10
mm4__chr17 43.88 Mbp_chr17_43879955_F TCGTCGGCAGCGTCGCTGTAACCCTGACCTTCCC 60.0 0.10
mm4__chr17 43.88 Mbp_chr17_43879955_R GTCTCGTGGGCTCGGTCTGAGGCACTGTAACCTGC 59.6 0.10
mm4__chr1 24.10 Mbp_chr1_24101899_F Primer3: not found at this Tm N.d. 0.09
mm4__chr1 24.10 Mbp_chr1_24101899_R Primer3: not found at this Tm N.d. 0.09
mm4__chr11 69.92 Mbp_chr11_69916090_F Primer3: not found at this Tm N.d. 0.09
mm4__chr11 69.92 Mbp_chr11_69916090_R Primer3: not found at this Tm N.d. 0.09
mm4__chr5 138.93 Mbp_chr5_138927927_F TCGTCGGCAGCGTCACTGTCAGTGCTGGCATTGT 60.1 0.08
mm4__chr5 138.93 Mbp_chr5_138927927_R GTCTCGTGGGCTCGGGTGAGAACAGTGGACGCTCA 59.9 0.08
mm4__chr8 128.07 Mbp_chr8_128069591_F Primer3: not found at this Tm N.d. 0.08
mm4__chr8 128.07 Mbp_chr8_128069591_R Primer3: not found at this Tm N.d. 0.08
mm4__chr12 131.88 Mbp_chr12_131879624_F Primer3: not found at this Tm N.d. 0.08
mm4__chr12 131.88 Mbp_chr12_131879624_R Primer3: not found at this Tm N.d. 0.08
mm4__chr7 10.53 Mbp_chr7_10527549_F Primer3: not found at this Tm N.d. 0.08
mm4__chr7 10.53 Mbp_chr7_10527549_R Primer3: not found at this Tm N.d. 0.08
mm4__chr17 63.70 Mbp_chr17_63700682_F Primer3: not found at this Tm N.d. 0.07
mm4__chr17 63.70 Mbp_chr17_63700682_R Primer3: not found at this Tm N.d. 0.07
mm4__chr7 56.05 Mbp_chr7_56052230_F TCGTCGGCAGCGTCCGGAGAACAAAGCGGCTCTA 60.1 0.06
mm4__chr7 56.05 Mbp_chr7_56052230_R GTCTCGTGGGCTCGGGGGGCTAGTTATGTCCAGCA 59.1 0.06
mm4__chr2 219.25 Mbp_chr2_219250524_F Primer3: not found at this Tm N.d. 0.06
mm4__chr2 219.25 Mbp_chr2_219250524_R Primer3: not found at this Tm N.d. 0.06
mm4__chr19 49.89 Mbp_chr19_49888611_F TCGTCGGCAGCGTCGACAGCAGCCTGGAGATCC 59.8 0.06
mm4__chr19 49.89 Mbp_chr19_49888611_R GTCTCGTGGGCTCGGCCTGGCTCGGGCTAAAGTG 60.4 0.06
mm4__chr19 57.32 Mbp_chr19_57317580_F TCGTCGGCAGCGTCACACACGTTGAGCACCACTA 59.5 0.06
mm4__chr19 57.32 Mbp_chr19_57317580_R GTCTCGTGGGCTCGGGCTCAGGCCCCTTGAAGC 60.7 0.06
mm4__chr2 178.19 Mbp_chr2_178194287_F Primer3: not found at this Tm N.d. 0.06
mm4__chr2 178.19 Mbp_chr2_178194287_R Primer3: not found at this Tm N.d. 0.06
mm4__chr19 6.21 Mbp_chr19_6212209_F TCGTCGGCAGCGTCCTGTTGGCGCTAGTCTGAGT 59.7 0.06
mm4__chr19 6.21 Mbp_chr19_6212209_R GTCTCGTGGGCTCGGCTTTGCCGTTCCAAGGATGC 60.1 0.06
mm4__chr18 51.20 Mbp_chr18_51196566_F TCGTCGGCAGCGTCTTCTCTCGTCTCCCCACCC 60.3 0.05
mm4__chr18 51.20 Mbp_chr18_51196566_R GTCTCGTGGGCTCGGGAAAGAGCGTCAACACCACC 59.4 0.05
mm4__chr4 76.42 Mbp_chr4_76421362_F TCGTCGGCAGCGTCCATCCGAGCGTCGAAGTCC 60.5 0.05
mm4__chr4 76.42 Mbp_chr4_76421362_R GTCTCGTGGGCTCGGCAGGGTAGTTTCTGGGGCAG 60.0 0.05
mm4__chr19 30.32 Mbp_chr19_30322473_F TCGTCGGCAGCGTCTCCACTGTGCTTGTCCATCC 59.9 0.05
mm4__chr19 30.32 Mbp_chr19_30322473_R GTCTCGTGGGCTCGGGCAAGCATGTAACACACGCA 60.0 0.05
mm4__chr17 81.46 Mbp_chr17_81461124_F TCGTCGGCAGCGTCCACCCATATTTGGCAACGGC 60.1 0.05
mm4__chr17 81.46 Mbp_chr17_81461124_R GTCTCGTGGGCTCGGCACCCTCTCGGCCTTCTTAG 59.5 0.05
mm4__chr10 6.09 Mbp_chr10_6089059_F Primer3: not found at this Tm N.d. 0.03
mm4__chr10 6.09 Mbp_chr10_6089059_R Primer3: not found at this Tm N.d. 0.03
mm4__chr7 1.36 Mbp_chr7_1360765_F TCGTCGGCAGCGTCTTGGGGAGAATGCAGGTTCC 59.9 0.03
mm4__chr7 1.36 Mbp_chr7_1360765_R GTCTCGTGGGCTCGGGCCCATGCCCACTTCACA 60.2 0.03
mm4__chr4 56.05 Mbp_chr4_56049219_F Primer3: not found at this Tm N.d. 0.02
mm4__chr4 56.05 Mbp_chr4_56049219_R Primer3: not found at this Tm N.d. 0.02
mm4__chr13 98.75 Mbp_chr13_98752550_F Primer3: not found at this Tm N.d. 0.02
mm4__chr13 98.75 Mbp_chr13_98752550_R Primer3: not found at this Tm N.d. 0.02
mm4__chr10 133.23 Mbp_chr10_133229581_F Primer3: not found at this Tm N.d. 0.01
mm4__chr10 133.23 Mbp_chr10_133229581_R Primer3: not found at this Tm N.d. 0.01
mm4__chr9 127.94 Mbp_chr9_127944879_F TCGTCGGCAGCGTCACGATCTTCTCCACGATGGC 59.8 0.00
mm4__chr9 127.94 Mbp_chr9_127944879_R GTCTCGTGGGCTCGGCTGTTACTGCCCTGACCGTT 59.9 0.00
mm4__chr11 3.84 Mbp_chr11_3840824_F Primer3: not found at this Tm N.d. 0.00
mm4__chr11 3.84 Mbp_chr11_3840824_R Primer3: not found at this Tm N.d. 0.00
mm4__chr19 49.65 Mbp_chr19_49652772_F Primer3: not found at this Tm N.d. 0.00
mm4__chr19 49.65 Mbp_chr19_49652772_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_5529724 TGGTGAGCTGCGAGAATAGCCGGGCGCGCTGTGAGCCGAGGTCGCCCCCGCCCTGGCCACTTCCGGCGCGCCGAGTCCTT
AGGCCGCCAGGGGGCGCCGGCGCGCGCCCAGATTGGGGACAAAGGAAGCCG
mm3__chr15 40.95 Mbp_chr15_40952431 TTCAGCTGCCTCTGTTTCCCTTTTACAGTCAGTGACATCCCCTGAGGGCAGTCCCTTGCTGCGTATTGGGTCAACCCCCG
GCGGCCTGTGGACTCGGCTGGGAGGCCAGAAGCCGAGGGAGAAGAC
mm4__chr7 151.14 Mbp_chr7_151137342 CCACTAACCTCCAGCACCTGGCGGGCTCCTCCGCTTCAAGGTTGAAGCACCTCAGGTTCGGCCCCCGTGTTCTTAGACCG
CCTGGGGCCATTTCTTTGTGGTCTCAGTGTTGGGTG
mm4__chr14 99.59 Mbp_chr14_99586879 GCCCTACCTATAGCACACCCCAAATCCCACCAGGCGGCCTCAGGGCTGGGCACCACAGCCAAGCCCGCAGCCACGGGCGG
CACTCCCGCGCTCACAGAGGGCCTCTGCCCTCCTACCGGGGACCTG
mm4__chr5 374.67 Kbp_chr5_374670 CTCAGAGACCAGGGAGGGAGGGGAACAGAGAGGCCGAGTCCCAAGGCCACCCGGTGCCACCGTCTCCGGGCTTCTGGCTG
CCTCACCTCCTCACTCGGGCCCTCGCCCCCCACGGACACCTTAGCTCGTCAG
mm4__chr14 90.61 Mbp_chr14_90613707 ATAGCCCAGTCACCTCACCTCTTCTCTGCCGGGCCCTTAGGCTGCCAGGAGCACTTCCCAAGGAGGGAGGCAGGAAGGGA
GAGAAAACCATTCCTGGTGAGTGTTGTAGTAGAGCGAAAGGAAACTGGAAGGACCCCAG
mm4__chr17 17.85 Mbp_chr17_17845006 GGCCTTGAGAGTCCAGGTTTCCTGACAGAGCCGCACCCCCTTCCTGGACCAGGCAGCCTAAGGACTGTGCTCCCCAGATG
GTGGGGTCTGGGAGCAGAGATCTCAGGACAGGC
mm4__chr10 103.60 Mbp_chr10_103602120 GTGAGTCTGAACCCTGGCTGGCAGCCCGTTCGGCCAGGCGGCCCTTGGCCTCGGCGGCAGCGGAGCGAGCAGTGCTAAAG
GAG
mm4__chr14 103.55 Mbp_chr14_103554283 CCTGCTGGTGACTGGCATAAAAAAAGGGCACAAGCTGAAGAGACCTAGAACTAGGGACACAGAGTCCTAAGGCTGCCTGG
TGACCCCCACCCCCACCCCAAAATCTGTGCA
mm4__chr17 18.63 Mbp_chr17_18634916 CCTCTGCTCGCTCACCCGCCCGGCTGCCTTAGGGGTCGGCGCTGCCCAGCGGGCTCCAGCGCAGCCTCCTACCCGGCATC
CCACCGGCCGCGCCTGCCACTGACTCCTG
mm4__chr17 15.75 Mbp_chr17_15749781 TAGGAGGCTGCCCTGGAGCCCGCTGGGCAGCGCCGACCCCTAAGGCAGCCGGGCGGGTGAGCGAGCAGAGGCGTG
mm4__chr11 1.51 Mbp_chr11_1508063 CTGGAGCTGGCGAGAAGTCAGCCCGAAGCGCCCACTCGCCGGCTCCTGAGGACGCCTGGCTGTGGGGACCCTCTTGGTCA
CGGGCCTGACCCCGGGTGCTGCCATGGCCACCTGGCCTGCAGGGGCTCAAACAGAAA
mm4__chr2 129.67 Mbp_chr2_129669478 ATGTTTTGCCAAGGAACCCGCATGAGCTAGCCGAGTCCTGAGACTGCCAGGGTCCACACACCCTTCCCGGGTGAGGTGTT
GGCTCGGTGGGCCTGTGCACCAGGTGGGCAGGACCCCAGTGGCTGCCCCATCATC
mm4__chr22 37.02 Mbp_chr22_37018467 CATAGAAGCTGCCCAGGTGTTCACCATCATACACCACCACGTGCGTGTGGTTGCTGATGCCCAGGCGGCCCACATACTCG
GCGAAGCCAGCCTCGCTGGGCAGCATCATCTCGTAGG
mm4__chr1 155.21 Mbp_chr1_155207410 TCCAGCTACCTCTCCCACATCCATCGGCCCATCCCACCACCAGACCTCCGGGAGAGCCCAGGCTTCTTGGTCCCCACCCC
CGCCCTGTCCTTGGGCAGCCAGGAGCCCAGGGGGACGAAGCCCAGGGACCTACAGTAC
mm4__chr7 2.61 Mbp_chr7_2607985 GGCAGCCTTAAGACCCTGTTGAGGAGGGGAGTGTGGCGTGCGGGGCTGTTTCACGTGGGAAGTGTCAGGCGAAGGGCTTT
GCTGTGCTGAGTCCTTAGGTCTCCAGGGTGGAGTCAGGGAAGGAGGAG
mm4__chr22 37.02 Mbp_chr22_37024460 TCGACATCGACCAGTGCAGCGACCGCACCTCGCCCTACGACCACATGCTGCCCGGGGCCGAGCATTTCGCGGAGTACGCA
GGCCGCCTGGGCGTGGGCGCGGCCACCCACGTCGTGATCTAC
mm4__chr19 5.04 Mbp_chr19_5041583 GTACTTTCCCAGTCCCTGCCGCTCGTCTTCCTGGCACTGTGGAGCCTCGAGTCCTCAGCCCGCCAGGCCACCTGCTGGAT
TCGGCTCCAACCCGGTCCTGACTTGGGCTCAG
mm4__chr17 43.88 Mbp_chr17_43879955 GCTGTAACCCTGACCTTCCCGCTCTGAGTCTTTCGGCCGCCGGGAGGTGTCTAGGGGGATGGGGGTAGGTTGGACCAAAT
GGGCAGGGGCAGGTTACAGTGCCTCAGA
mm4__chr5 138.93 Mbp_chr5_138927927 ACTGTCAGTGCTGGCATTGTGTTTTGTGCCACCGAGTCCATCGGCCTCCAGGACCTCACACCGACATGCTGTCTGGTCCC
CTGCGTGAGCGTCCACTGTTCTCAC
mm4__chr7 56.05 Mbp_chr7_56052230 CGGAGAACAAAGCGGCTCTAGAGCGCGGCGTTCCCCGCCCTCTGGGGCTTCCAGGCACCCTGAGGACTCGGCACAATCTC
TGCGGGGACGGGGTGGGCACTACACCTGTCCTCCATCCTGCTGGACATAACTAGCCCC
mm4__chr19 49.89 Mbp_chr19_49888611 GACAGCAGCCTGGAGATCCTGCCCGAGGAGGAGGACGAGGGCGCCGACTCCTAACCCCGCCAGGCAGCCTCGTTCTGCAC
AGGCACTTTAGCCCGAGCCAGG
mm4__chr19 57.32 Mbp_chr19_57317580 ACACACGTTGAGCACCACTAACTGGTTAGCCCCCATCCTCCTCCCCGCGGCCGCCGCCTAGTCCTCAAGCCGCTCGGTCT
CCGCGGGGCCGGAGGCCGCTCCCACCCCCGCGGGCGCTTCAAGGGGCCTGAGC
mm4__chr19 6.21 Mbp_chr19_6212209 CTGTTGGCGCTAGTCTGAGTAGAGCCCGAGAGCAGACTGGTGGCTCCCGGGCGCCCTGAGGACTCGCTGCCCTTTGTAGT
GTTGGCTGACCCCCCCGGGAGCCCCGGTAGGAGGCGGCGGCATCCTTGGAACGGCAAAG
mm4__chr18 51.20 Mbp_chr18_51196566 TTCTCTCGTCTCCCCACCCACGCCCGGGGCCATGCCCAGCTGGACCTCCCCACCCCTGCACTCACCCTGGCGGCCGACGA
TCTCGGCGACGTGCTCGGAGCTGGGCACCGGGACGCACTCGGTGGTGTTGACGCTCTTTC
mm4__chr4 76.42 Mbp_chr4_76421362 CATCCGAGCGTCGAAGTCCCCACCACCGCCCGGGCGCCCTAAGGAGTGGGGGTGTACCCAGCCCGGAGTCAAATCCTGGA
GCCCTGCCCCAGAAACTACCCTG
mm4__chr19 30.32 Mbp_chr19_30322473 TCCACTGTGCTTGTCCATCCTGCTTCACAAACCCATCTCTGTTTCTGCGGGACTTGGTCCTGTCGCCCTAAGGACTCTGC
AGGCGCCTTCTTGGGAGGGCTGCGTGTGTTACATGCTTGC
mm4__chr17 81.46 Mbp_chr17_81461124 CACCCATATTTGGCAACGGCTTCCGCGCCGACTCCTTCAGCAGCCTGGCCAGCTCCTACGCGCCCTTCGTCGGGGGGACC
GGGCCGGGCCTCCCCAGGGGAGCCCACAAGCTGCTGCGGGCTAAGAAGGCCGAGAGGGTG
mm4__chr7 1.36 Mbp_chr7_1360765 TTGGGGAGAATGCAGGTTCCCCTGGGGCACCCCCCATCAAAGGGCAGAGAGCTGGGGCCAGGCCAGAGCCACCCTCACCG
GGCCGCCCAAGGGCTCAGCGGCACCGCTGTGAAGTGGGCATGGGC
mm4__chr9 127.94 Mbp_chr9_127944879 ACGATCTTCTCCACGATGGCATCCGCATCGATCCGCGTGTCGTCCACCCAGGTCGGGTGGCTCTCCACCGAGTCCTTCTT
CCGCCTGGGTCAGGTCGTGGGGGGACAAGTCACAACGGTCAGGGCAGTAACAG

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.