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PCR primers for off-targets of GGTCGCCGTCGACACGGCGC 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.

If a primer was not found, the reason is usually that the region around the off-target is too repetitive. To avoid unspecific primers, all repeats are masked for the primer design (not for off-target search). If you think that we should change the parameters here or should use different primer3 settings, please let us know.

Maximum amplicon length:     Primer Tm:

NamePrimer Sequence Tm CFD Score
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313703_F TCGTCGGCAGCGTCCGCATCACAAGGTGACCCTA 59.7 1.00
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313703_R GTCTCGTGGGCTCGGCTCCTAATCCCAGGCTCCCT 60.1 1.00
mm4_exon_Lce1l_chr3_92850244_F Primer3: not found at this Tm N.d. 0.47
mm4_exon_Lce1l_chr3_92850244_R Primer3: not found at this Tm N.d. 0.47
mm4_exon_6330416G13Rik_chr4_63584499_F TCGTCGGCAGCGTCCAGGGGAGAGGAGGAGTCAA 59.9 0.33
mm4_exon_6330416G13Rik_chr4_63584499_R GTCTCGTGGGCTCGGGACAGTCTGAGGCCAACAGG 60.3 0.33
mm4_exon_Tmem126a_chr7_90457134_F TCGTCGGCAGCGTCACAGCTACTCACCATTCGGC 60.1 0.17
mm4_exon_Tmem126a_chr7_90457134_R GTCTCGTGGGCTCGGATGGAGCTCAGACGACCCTA 59.7 0.17
mm4_intergenic_Gm26497|Piezo1_chr8_122539483_F Primer3: not found at this Tm N.d. 0.13
mm4_intergenic_Gm26497|Piezo1_chr8_122539483_R Primer3: not found at this Tm N.d. 0.13
mm4_exon_Hoxa2_chr6_52164435_F TCGTCGGCAGCGTCCATCCAGGGATACTCAGGCG 59.6 0.12
mm4_exon_Hoxa2_chr6_52164435_R GTCTCGTGGGCTCGGAGACCATTCCCAGCCTGAAC 59.6 0.12
mm4_intron_Cradd_chr10_95323928_F TCGTCGGCAGCGTCACGGTAAATGAAGCCGCAGA 60.0 0.09
mm4_intron_Cradd_chr10_95323928_R GTCTCGTGGGCTCGGTGACTCCATCTTCTCTCGCTG 59.2 0.09
mm4_intergenic_AI854517|Mir9-3_chr7_79505203_F TCGTCGGCAGCGTCTCTGTCGGTCCCCTCTGG 59.9 0.08
mm4_intergenic_AI854517|Mir9-3_chr7_79505203_R GTCTCGTGGGCTCGGGAGAAACGGGCCTCCCATTC 60.7 0.08
mm3_exon_Nrf1_chr6_30048065_F TCGTCGGCAGCGTCGCAGGAGGCTGCGAGGAG 62.5 0.07
mm3_exon_Nrf1_chr6_30048065_R GTCTCGTGGGCTCGGGGTAGGGCACTCACCTCAGA 60.6 0.07
mm4_intron_Grin2a_chr16_9896440_F Primer3: not found at this Tm N.d. 0.07
mm4_intron_Grin2a_chr16_9896440_R Primer3: not found at this Tm N.d. 0.07
mm4_intergenic_Pax2|Fam178a_chr19_44838348_F TCGTCGGCAGCGTCTGTCCTCGCACTGGTAAACC 59.9 0.06
mm4_intergenic_Pax2|Fam178a_chr19_44838348_R GTCTCGTGGGCTCGGTCCAGGGGAGCTTAGGTCAA 59.8 0.06
mm4_exon_Ttc32_chr12_9030094_F TCGTCGGCAGCGTCGGTAGCTGTGGTTCCGCTT 60.0 0.02
mm4_exon_Ttc32_chr12_9030094_R GTCTCGTGGGCTCGGCAAACTCGCCCCTGGAGAAG 60.6 0.02
mm4_intron_Eif4e_chr3_138527731_F TCGTCGGCAGCGTCGAGAGAGCCGGAGAGGGAG 60.5 0.02
mm4_intron_Eif4e_chr3_138527731_R GTCTCGTGGGCTCGGTCCTCCGCAGCTCCAGAG 60.7 0.02
mm3_exon_Slc27a4_chr2_29812307_F TCGTCGGCAGCGTCCACACCTGACTGGCTTCCTC 60.3 0.01
mm3_exon_Slc27a4_chr2_29812307_R GTCTCGTGGGCTCGGTTGTTGTTGGCACCCTGGTT 60.6 0.01
mm4_exon_Pced1a/Vps16_chr2_130423790_F TCGTCGGCAGCGTCGGGAAGAAGGGTCACTTGGG 59.9 0.01
mm4_exon_Pced1a/Vps16_chr2_130423790_R GTCTCGTGGGCTCGGAGAGACTTGGTAACTGGCGC 60.0 0.01
mm4_exon_Cdh22_chr2_165142081_F TCGTCGGCAGCGTCTGCATGGCAGGACAGCAG 60.3 0.00
mm4_exon_Cdh22_chr2_165142081_R GTCTCGTGGGCTCGGCTACTGGAGGTGCAGGAGGA 60.3 0.00
mm4_intron_Txlng_chrX_162828632_F Primer3: not found at this Tm N.d. 0.00
mm4_intron_Txlng_chrX_162828632_R Primer3: not found at this Tm N.d. 0.00

Off-target amplicon sequences with primers

These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.

ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313703 CGCATCACAAGGTGACCCTAGCTCCCCACTGCCATCTCCGCGGTCGCCGTCGACACGGCGCTGGGGCTACCCGGCGCCTG
CCTTGTCGCCTTAGCTCCTCTTCTCAGCCAAGATCCCAGGGAGCCTGGGATTAGGAG
mm4_exon_6330416G13Rik_chr4_63584499 CAGGGGAGAGGAGGAGTCAAAAAGCCGATGCCCGTCAACACGGCGCTGGAAAAGAAGACCCTGTTGGCCTCAGACTGTC
mm4_exon_Tmem126a_chr7_90457134 ACAGCTACTCACCATTCGGCAGAATGAAGGCAGCTAATGGCAGCCTCCCCAGCCGCCGGTGCTTGCCCGGGTCCTCCCGC
GCCGAGTCTACCGCAACCTTCGCTAGGGTCGTCTGAGCTCCAT
mm4_exon_Hoxa2_chr6_52164435 CATCCAGGGATACTCAGGCGGCTGCAGGGCGCCGGCAGGCACCGGGCTGCCGCGACTGCCCGCGGGGCTCGACTTGGGGC
GGCCGCCAACGCCAGCGCCGTGGCGAGGGTGACTGCCCGGGTTCAGGCTGGGAATGGTCT
mm4_intron_Cradd_chr10_95323928 ACGGTAAATGAAGCCGCAGACTGTCCTCTCGGAGGAAACCGACCCCCGGCACCGCCACCCGAACCCCGGTCCAGCCCCCG
GGAGAGCCGCGCAGGGGCGATGTCGCCACGGCGCGGGGACAGCGAGAGAAGATGGAGTCA
mm4_intergenic_AI854517|Mir9-3_chr7_79505203 TCTGTCGGTCCCCTCTGGCTCTCCGCGTGCGCCCGGGGCTGCGGGAGCGGAGTCCGCTCAGGCCCCCGCGCCGGGTCGGC
GGCGGCACGTGGGGCCCACAGCGCGGCAGCGGTGCCGGCGAATGGGAGGCCCGTTTCTC
mm3_exon_Nrf1_chr6_30048065 GCAGGAGGCTGCGAGGAGCCGGCGCGGTCGCAGTCTCCACGGCGCAGGCCCACGGTAGCGCAGCCGCTCTGAGGTGAGTG
CCCTACC
mm4_intergenic_Pax2|Fam178a_chr19_44838348 TGTCCTCGCACTGGTAAACCCTCGATAAGTCAAGGATCCAGCTCCGTTTCCACTGCGACCTCCTCTAAATTCTTGACCTA
AGCTCCCCTGGA
mm4_exon_Ttc32_chr12_9030094 GGTAGCTGTGGTTCCGCTTCCACCCGGGTGAGGCGGTCATGGCCTTGCGGCCAGGCCGAGAGGGCGGAGAGAGCTCGGCC
GCCCTGGCCACGGCGCAGGCTCGCTTCTCCAGGGGCGAGTTTG
mm4_intron_Eif4e_chr3_138527731 GAGAGAGCCGGAGAGGGAGCCCGAGGCAGCGGCGCCTCGCCGTCGCCGGCGACCCGCCGCCGGGGGTCCTCGTGCGGGCC
GCGCTCTGGAGCTGCGGAGGA
mm3_exon_Slc27a4_chr2_29812307 CACACCTGACTGGCTTCCTCGTCCCTAGGTCAGCCAGGCCAGCTGGTGGGTCGCATCATCCAGCAGGACCCTCTGCGCCG
TTTCGACGGGTACCTCAACCAGGGTGCCAACAACAA
mm4_exon_Pced1a/Vps16_chr2_130423790 GGGAAGAAGGGTCACTTGGGCCGTAGTCTTAGACTACAGCGCTTCGCGCGCGCAGAGACGCCTTTGCCCTGCCACGTGTC
AACGGCGGCCCCGCGCGTGCGCATTCGTCCGCTCGCGCCAGTTACCAAGTCTCT
mm4_exon_Cdh22_chr2_165142081 TGCATGGCAGGACAGCAGGCAGCAGGCGTGGCCCCGGGCGGGGGTCTCACCGGACGGGCCGGTTGGCGGCGTCGGGGTCC
CGCGCCGTCACCACGCCGACCAGGGAGCCCACCTGCGCGTCCTCCTGCACCTCCAGTAG

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_mN1FkRligAThyqKv3eg3.txt --name MY_EXPERIMENT

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