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PCR primers for off-targets of AGGCGGCACTAGGCTCGGAC 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.

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
mm0_exon_Pop5_chr5_115238001_F TCGTCGGCAGCGTCGAGCGTGGAGATTGACAGGG 60.4 1.00
mm0_exon_Pop5_chr5_115238001_R GTCTCGTGGGCTCGGTTACCTGTGCTTGAACCGCA 60.1 1.00
mm4_intergenic_Gm22618|Gm22976_chr5_50969965_F Primer3: not found at this Tm N.d. 0.47
mm4_intergenic_Gm22618|Gm22976_chr5_50969965_R Primer3: not found at this Tm N.d. 0.47
mm4_intergenic_Foxc1|Gmds_chr13_31815946_F TCGTCGGCAGCGTCATACATTCCCTTGCCCGTGG 60.1 0.47
mm4_intergenic_Foxc1|Gmds_chr13_31815946_R GTCTCGTGGGCTCGGTAAACCCAGGCTCCACCAAC 59.8 0.47
mm4_intergenic_Gm22251|Zfp521_chr18_13489322_F Primer3: not found at this Tm N.d. 0.42
mm4_intergenic_Gm22251|Zfp521_chr18_13489322_R Primer3: not found at this Tm N.d. 0.42
mm4_exon_Pde4a_chr9_21165855_F TCGTCGGCAGCGTCCACTGAGGCCAGGAGCTC 59.4 0.41
mm4_exon_Pde4a_chr9_21165855_R GTCTCGTGGGCTCGGCTCCCGCTCTGTCTTGTGG 60.0 0.41
mm4_intron_Rfx4_chr10_84778750_F Primer3: not found at this Tm N.d. 0.21
mm4_intron_Rfx4_chr10_84778750_R Primer3: not found at this Tm N.d. 0.21
mm4_intergenic_Grb14|Cobll1_chr2_65028920_F Primer3: not found at this Tm N.d. 0.16
mm4_intergenic_Grb14|Cobll1_chr2_65028920_R Primer3: not found at this Tm N.d. 0.16
mm4_intron_Thsd7a_chr6_12444035_F TCGTCGGCAGCGTCTGCAAGAAAACCAGTGCTGA 58.2 0.15
mm4_intron_Thsd7a_chr6_12444035_R GTCTCGTGGGCTCGGGGCACATTGAACGCCATCTG 60.1 0.15
mm4_intergenic_Bin3|Bin3/Ccar2_chr14_70135410_F TCGTCGGCAGCGTCGAGTGCAGACCAGTATGGGG 59.8 0.12
mm4_intergenic_Bin3|Bin3/Ccar2_chr14_70135410_R GTCTCGTGGGCTCGGGATGAACAGCGAGAGGAGGG 59.8 0.12
mm3_intergenic_Gm24637|Dcaf6_chr1_165459876_F TCGTCGGCAGCGTCAACTTTGGCGTTCAACGTGG 59.9 0.12
mm3_intergenic_Gm24637|Dcaf6_chr1_165459876_R GTCTCGTGGGCTCGGCTCAAGCTGGGCTTCCCTC 60.0 0.12
mm4_intron_Arhgap22_chr14_33277725_F Primer3: not found at this Tm N.d. 0.11
mm4_intron_Arhgap22_chr14_33277725_R Primer3: not found at this Tm N.d. 0.11
mm4_intergenic_Gm26603|Fmnl2_chr2_52898731_F TCGTCGGCAGCGTCGAGGCTGAAGTCTGTCTGGG 59.7 0.08
mm4_intergenic_Gm26603|Fmnl2_chr2_52898731_R GTCTCGTGGGCTCGGAGTAATTCAACAGGAATGTGCTAAA 57.2 0.08
mm4_exon_Fndc3b_chr3_27710405_F TCGTCGGCAGCGTCGTCGTCCTCTGCTTCCCG 59.8 0.05
mm4_exon_Fndc3b_chr3_27710405_R GTCTCGTGGGCTCGGTCCTCCCTCCCCGTCCTG 62.0 0.05
mm4_intergenic_Gm26468|Rpsa-ps10_chr3_149399220_F TCGTCGGCAGCGTCAGTTCCTTGGGGTAGCTCCT 59.8 0.05
mm4_intergenic_Gm26468|Rpsa-ps10_chr3_149399220_R GTCTCGTGGGCTCGGTCTCTCCAGGAAGCCCCTAC 60.0 0.05
mm4_intergenic_Gm24556|2210019I11Rik_chr5_147218695_F TCGTCGGCAGCGTCGGACTGTCTCACCAGAGCTG 59.7 0.04
mm4_intergenic_Gm24556|2210019I11Rik_chr5_147218695_R GTCTCGTGGGCTCGGGGTTGGAGAGCTTCCTGGAC 60.0 0.04
mm4_exon_Gnb1l_chr16_18564907_F TCGTCGGCAGCGTCAGGCCAGATAGAAGACCAGGA 59.7 0.03
mm4_exon_Gnb1l_chr16_18564907_R GTCTCGTGGGCTCGGAGTTTGTGGCCTGTCGAGAG 59.9 0.03
mm4_intron_4931429P17Rik_chr13_47975402_F Primer3: not found at this Tm N.d. 0.03
mm4_intron_4931429P17Rik_chr13_47975402_R Primer3: not found at this Tm N.d. 0.03
mm4_exon_Cacybp_chr1_160212862_F TCGTCGGCAGCGTCGAGACGGCTCTGGAACCTTC 60.1 0.02
mm4_exon_Cacybp_chr1_160212862_R GTCTCGTGGGCTCGGCTCGCGAGAATTGAGCGTTG 59.9 0.02
mm4_intergenic_Rnu5g|Parp16_chr9_65205625_F TCGTCGGCAGCGTCGCAACTGCGTGAGGCTTAAC 60.1 0.02
mm4_intergenic_Rnu5g|Parp16_chr9_65205625_R GTCTCGTGGGCTCGGCCCTCTTCAGCTCAGCACAA 59.9 0.02
mm4_intergenic_Hspbp1|Brsk1_chr7_4685256_F Primer3: not found at this Tm N.d. 0.02
mm4_intergenic_Hspbp1|Brsk1_chr7_4685256_R Primer3: not found at this Tm N.d. 0.02
mm4_intron_Zfp143_chr7_110061900_F TCGTCGGCAGCGTCGCTGCTTATGCTGGTCCTCA 60.1 0.01
mm4_intron_Zfp143_chr7_110061900_R GTCTCGTGGGCTCGGTAAACTCCGATTCTGCCCGG 59.8 0.01
mm4_exon_Dmxl2_chr9_54376302_F TCGTCGGCAGCGTCCCTAGCCATGGCAGAGATGG 59.9 0.01
mm4_exon_Dmxl2_chr9_54376302_R GTCTCGTGGGCTCGGACATGCAACTGTTCTCCACA 57.6 0.01
mm4_exon_Vps9d1_chr8_123246985_F Primer3: not found at this Tm N.d. 0.01
mm4_exon_Vps9d1_chr8_123246985_R Primer3: not found at this Tm N.d. 0.01
mm4_intergenic_6430628N08Rik|Dmbx1_chr4_115908133_F TCGTCGGCAGCGTCGGTGGCTGATGTGAGGGAAA 59.9 0.00
mm4_intergenic_6430628N08Rik|Dmbx1_chr4_115908133_R GTCTCGTGGGCTCGGGGTTGGCTGGAAACTGGAGA 59.8 0.00
mm4_intron_Ptch1_chr13_63512776_F TCGTCGGCAGCGTCGGACGGGGACACAATTCACT 59.9 0.00
mm4_intron_Ptch1_chr13_63512776_R GTCTCGTGGGCTCGGGCTCCAAAACTCTGGCTTGC 60.0 0.00
mm4_intron_Ssu2_chr6_112385435_F Primer3: not found at this Tm N.d. 0.00
mm4_intron_Ssu2_chr6_112385435_R Primer3: not found at this Tm N.d. 0.00
mm4_intergenic_Gm13198|Ccdc3/Gm26776_chr2_5185813_F Primer3: not found at this Tm N.d. 0.00
mm4_intergenic_Gm13198|Ccdc3/Gm26776_chr2_5185813_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.

mm0_exon_Pop5_chr5_115238001 GAGCGTGGAGATTGACAGGGCACGCTGCCTGTCCGAGCCTAGTGCCGCCTTCCTCCCGCATCCGGCGCGGACGCCATGGT
GCGGTTCAAGCACAGGTAA
mm4_intergenic_Foxc1|Gmds_chr13_31815946 ATACATTCCCTTGCCCGTGGCCCGCCACTGCAGTCCGACCTCGGGCTGCTCTGCTTCCTTTCTGGCCAGTCTGAGTCCAG
TGCCTCCTTTGGAGCAGTCTGGTTGGTGGAGCCTGGGTTTA
mm4_exon_Pde4a_chr9_21165855 CACTGAGGCCAGGAGCTCCCGTGGGTCCCCAAGCACCTCGGCCGTGGCGCCACTGAGCTCGGACAGGCCAGGGGACGTTT
TTGCCCCCGGGCACCCCACAAGACAGAGCGGGAG
mm4_intron_Thsd7a_chr6_12444035 TGCAAGAAAACCAGTGCTGATATACAATGGGGGAAGGCACTAGGCTCTGACTGGAGCAGATGGCGTTCAATGTGCC
mm4_intergenic_Bin3|Bin3/Ccar2_chr14_70135410 GAGTGCAGACCAGTATGGGGGTGGCAGCCAGAAAGAGGGTAAAACCCAGTCGGAGCCCAGTGTCTCCTGCTGCTGGGTCC
AATTGGGATGGCATGCAGCAGGCTCCTGCCCTCCTCTCGCTGTTCATC
mm3_intergenic_Gm24637|Dcaf6_chr1_165459876 AACTTTGGCGTTCAACGTGGCATCTCCGTAAGGCTCGCGGAGTCTTCTCCGGAGTCCCAGCCCACGAGCGACCCCAACAC
CTTTCCGGGCTTAGTGCCGCCTCCCCACGGCCCGGCTCTCCGAGGGAAGCCCAGCTTGAG
mm4_intergenic_Gm26603|Fmnl2_chr2_52898731 GAGGCTGAAGTCTGTCTGGGCTAAATATTGAGACACTGTCTCCAAAGTCTAGGTATGAGTTACTATTTGTGAGTTTAACC
CGTTCCGTTCCTAGTGCCTCCTTTTTAGCACATTCCTGTTGAATTACT
mm4_exon_Fndc3b_chr3_27710405 GTCGTCCTCTGCTTCCCGCCGCGCCCCGCTCCCCCGCCCGAGCCTGCAGCCGCCTGGCTCGCCGAGTCCAGCCGCGGCCG
CCGGTTCCCGCCCCCAGCCCGGGGGGAGCCCAGGGGGCAGGACGGGGAGGGAGGA
mm4_intergenic_Gm26468|Rpsa-ps10_chr3_149399220 AGTTCCTTGGGGTAGCTCCTATTTTGATAAGATGAAGCCAAGGGGCCACTAGGCTTGCACAGGGAGGTTAGGCTATAGCT
TCTCCTCTGTAGGGGCTTCCTGGAGAGA
mm4_intergenic_Gm24556|2210019I11Rik_chr5_147218695 GGACTGTCTCACCAGAGCTGATCCTTGTAGCAGGAGAGGCGGCTGAAGGCTCGGCCAGGATTCAGGAGATCAAGAGTGTC
AGAGGACTGGATCCTCAAATGGAGAGCAGTGCAATATGTCCAGGAAGCTCTCCAACC
mm4_exon_Gnb1l_chr16_18564907 AGGCCAGATAGAAGACCAGGAAAGGAGGCTCTAGCCTCTGACAGGATAAGGTAAAGGGATAGCTTGTCTTTCAGTGAACC
TGCAGGAGGCAAGGGAAAGCACTCTCGACAGGCCACAAACT
mm4_exon_Cacybp_chr1_160212862 GAGACGGCTCTGGAACCTTCGCAAGGCTCCACCGGAGCCTCCCAAGGCCCCGCCCCTCCGAGTCTAGTCCCGCCCCCAGG
CTGGTTGCCTAGCAACGCTCAATTCTCGCGAG
mm4_intergenic_Rnu5g|Parp16_chr9_65205625 GCAACTGCGTGAGGCTTAACAAGTACACGGTCCTGCTTTGCTGCTGCCTTCAGCATGTTCTTAGTAACTTAGTCAGCACT
AGGATGGGACTGGCACCTCGTGACCGCTAAGCAAGCGCTTTGTGCTGAGCTGAAGAGGG
mm4_intron_Zfp143_chr7_110061900 GCTGCTTATGCTGGTCCTCAGGCCTGGCCGATCCTACGGCCGCCTTCGTAGCGCAGCCCGGGCAGAATCGGAGTTTA
mm4_exon_Dmxl2_chr9_54376302 CCTAGCCATGGCAGAGATGGAGGTGGGTGTGGCTGGCTTGAGGAATGGGAGGCTGCACCAGGCTGGTACAGGGTTGTAGT
GGAACCACGGTAATCAATATCATCTGAACTGTGGAGAACAGTTGCATGT
mm4_intergenic_6430628N08Rik|Dmbx1_chr4_115908133 GGTGGCTGATGTGAGGGAAAGTAAGATAAGGGCCAGGCCAAGCCAAGTCCCGCCTGCATATCTCCAGTTTCCAGCCAACC
mm4_intron_Ptch1_chr13_63512776 GGACGGGGACACAATTCACTGGCCAGCCCTTCTCTCCTTCAAGGAAGGCTGCTCTAGCCTGGGACTGGAATACACATTTC
CTGTAAACATGGTGGGGGCCTCAGGCAAGCCAGAGTTTTGGAGC

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_ozJbN0W08qlgEBn3RvZK.txt --name MY_EXPERIMENT

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