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PCR primers for off-targets of GTACCGCTGGTGACGCCACT 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.

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_52313548_F Primer3: not found at this Tm N.d. 1.00
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313548_R Primer3: not found at this Tm N.d. 1.00
mm4_exon_Gimap8_chr6_48656340_F Primer3: not found at this Tm N.d. 0.42
mm4_exon_Gimap8_chr6_48656340_R Primer3: not found at this Tm N.d. 0.42
mm4_exon_Syt8_chr7_142440212_F TCGTCGGCAGCGTCCTTCTGGCCAACCCCTACAG 60.0 0.32
mm4_exon_Syt8_chr7_142440212_R GTCTCGTGGGCTCGGGGCCAGAACACGATCCACTT 60.3 0.32
mm4_intergenic_Adad2|Kcng4_chr8_119623367_F TCGTCGGCAGCGTCAGGGAGAGATGCTGAGCTGA 60.0 0.20
mm4_intergenic_Adad2|Kcng4_chr8_119623367_R GTCTCGTGGGCTCGGATCCGAGGCTTTCAGCTCAC 60.1 0.20
mm4_intron_Gm10530_chr1_159635833_F TCGTCGGCAGCGTCCAACTTGACCTTGATGGCCG 59.4 0.18
mm4_intron_Gm10530_chr1_159635833_R GTCTCGTGGGCTCGGGGGAATGGAAAGAGGAGCCC 60.1 0.18
mm4_intron_Cdh13_chr8_118668738_F TCGTCGGCAGCGTCGGTACCTCTGATGACCTTGGG 59.5 0.18
mm4_intron_Cdh13_chr8_118668738_R GTCTCGTGGGCTCGGAGAGGTACCCAGTGAGGTCA 59.2 0.18
mm4_intron_Cdh13_chr8_118668694_F Primer3: not found at this Tm N.d. 0.18
mm4_intron_Cdh13_chr8_118668694_R Primer3: not found at this Tm N.d. 0.18
mm4_intergenic_Grpr|Rnf138rt1_chrX_163694035_F TCGTCGGCAGCGTCTGTCCCTGATGGTGGCTTTC 59.9 0.17
mm4_intergenic_Grpr|Rnf138rt1_chrX_163694035_R GTCTCGTGGGCTCGGACCAATCAGCAGCCAAGGAA 59.8 0.17
mm4_intron_Gpr112_chrX_56893151_F Primer3: not found at this Tm N.d. 0.17
mm4_intron_Gpr112_chrX_56893151_R Primer3: not found at this Tm N.d. 0.17
mm3_intron_Rhoj_chr12_75332811_F Primer3: not found at this Tm N.d. 0.15
mm3_intron_Rhoj_chr12_75332811_R Primer3: not found at this Tm N.d. 0.15
mm3_exon_Agpat3_chr10_78278018_F TCGTCGGCAGCGTCGCAAGCAGGAGCCTTTACCT 60.3 0.10
mm3_exon_Agpat3_chr10_78278018_R GTCTCGTGGGCTCGGGGGGCAGGTGTTCATTCACT 60.2 0.10
mm4_intron_Kdm3b_chr18_34787275_F Primer3: not found at this Tm N.d. 0.09
mm4_intron_Kdm3b_chr18_34787275_R Primer3: not found at this Tm N.d. 0.09
mm4_intergenic_Smyd2|Prox1_chr1_189977583_F Primer3: not found at this Tm N.d. 0.09
mm4_intergenic_Smyd2|Prox1_chr1_189977583_R Primer3: not found at this Tm N.d. 0.09
mm4_intron_Msi2_chr11_88656561_F Primer3: not found at this Tm N.d. 0.08
mm4_intron_Msi2_chr11_88656561_R Primer3: not found at this Tm N.d. 0.08
mm4_exon_Unk_chr11_116056063_F TCGTCGGCAGCGTCTGGCTGCCCTAGAGAAGACT 59.9 0.07
mm4_exon_Unk_chr11_116056063_R GTCTCGTGGGCTCGGGAACCCCAGGACGCCCTTC 62.6 0.07
mm4_intergenic_Commd4|Trcg1_chr9_57224313_F Primer3: not found at this Tm N.d. 0.06
mm4_intergenic_Commd4|Trcg1_chr9_57224313_R Primer3: not found at this Tm N.d. 0.06
mm4_intron_Cdh13_chr8_118668565_F TCGTCGGCAGCGTCTGTCCTCATCTCTGGCTCTGA 59.9 0.05
mm4_intron_Cdh13_chr8_118668565_R GTCTCGTGGGCTCGGCCAGTGATGTCATCAGAGGCA 59.7 0.05
mm4_exon_Zfp536_chr7_37716297_F TCGTCGGCAGCGTCCCACAGCTCAGATGCCTCAT 59.8 0.05
mm4_exon_Zfp536_chr7_37716297_R GTCTCGTGGGCTCGGCTGGGCTTCAGGTCTTCTGG 60.0 0.05
mm4_intron_Fam19a5_chr15_87699827_F Primer3: not found at this Tm N.d. 0.04
mm4_intron_Fam19a5_chr15_87699827_R Primer3: not found at this Tm N.d. 0.04
mm4_intron_Ccdc113_chr8_95543965_F Primer3: not found at this Tm N.d. 0.03
mm4_intron_Ccdc113_chr8_95543965_R Primer3: not found at this Tm N.d. 0.03
mm4_intron_Slc38a1_chr15_96604882_F Primer3: not found at this Tm N.d. 0.03
mm4_intron_Slc38a1_chr15_96604882_R Primer3: not found at this Tm N.d. 0.03
mm4_intron_Cdh13_chr8_118668653_F TCGTCGGCAGCGTCTGCCTCTGATGACATCACTGG 59.7 0.02
mm4_intron_Cdh13_chr8_118668653_R GTCTCGTGGGCTCGGAGAGGTACCCAGTGAGGTCA 59.2 0.02
mm4_intron_Cdh13_chr8_118668631_F Primer3: not found at this Tm N.d. 0.02
mm4_intron_Cdh13_chr8_118668631_R Primer3: not found at this Tm N.d. 0.02
mm4_intron_Cdh13_chr8_118668587_F TCGTCGGCAGCGTCTGTCCTCATCTCTGGCTCTGA 59.9 0.02
mm4_intron_Cdh13_chr8_118668587_R GTCTCGTGGGCTCGGCCAGTGATGTCATCAGAGGCA 59.7 0.02
mm4_intron_Cdh13_chr8_118668543_F TCGTCGGCAGCGTCTGTCCTCATCTCTGGCTCTGA 59.9 0.02
mm4_intron_Cdh13_chr8_118668543_R GTCTCGTGGGCTCGGAGAGGTACCCAGTGAGGTCA 59.2 0.02
mm4_intergenic_Fam173b|Tas2r119_chr15_32036238_F TCGTCGGCAGCGTCAGCCAACGAGGTTACTTTTCCT 59.8 0.02
mm4_intergenic_Fam173b|Tas2r119_chr15_32036238_R GTCTCGTGGGCTCGGCACACAGCTCCTGAGTGTCT 59.3 0.02
mm4_intron_Mmrn2_chr14_34396323_F TCGTCGGCAGCGTCGCCAGGGAGTCAGAGTCATG 59.8 0.00
mm4_intron_Mmrn2_chr14_34396323_R GTCTCGTGGGCTCGGCTGGTACACTGGCTTCTGGG 60.0 0.00
mm4_intron_Dmtn_chr14_70605116_F Primer3: not found at this Tm N.d. 0.00
mm4_intron_Dmtn_chr14_70605116_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.

mm4_exon_Syt8_chr7_142440212 CTTCTGGCCAACCCCTACAGCACTGGGCAGACATGCTGGCCCATGCTCGGCGGCCCATCGCCCAGTGGCACCACCTGCGG
TCCCCCAGGGAAGTGGATCGTGTTCTGGCC
mm4_intergenic_Adad2|Kcng4_chr8_119623367 AGGGAGAGATGCTGAGCTGACAAGGGGTCAGGTCCCCAGTGGCTTCTTCAGCGGTTCCAGAGTGAGCTGAAAGCCTCGGA
T
mm4_intron_Gm10530_chr1_159635833 CAACTTGACCTTGATGGCCGCAGGAAGCCCCTCTGCAGATCCAGAGGGAACTTATAGACTAATTGAATGAATCATGTGGT
TCCTTTCCTCTCCTGCTGGTGACACCACTGGGGATAACGGGCTCCTCTTTCCATTCCC
mm4_intron_Cdh13_chr8_118668738 GGTACCTCTGATGACCTTGGGTACCTCTGATGACATCACTGGGTATCTCTGATGACCTCACTGGGTACCTCTGATGACAT
CACTGGGTAACTCTGATGACCTCACTGGGTATCTCTGATGACCTCACTGGGTACCTCT
mm4_intergenic_Grpr|Rnf138rt1_chrX_163694035 TGTCCCTGATGGTGGCTTTCCTGTTCTATGTGAGCCCCAGTGGCATCACCAGCTCTCCCACTGCTCTTCCTTGGCTGCTG
ATTGGT
mm3_exon_Agpat3_chr10_78278018 GCAAGCAGGAGCCTTTACCTTCTCCTGGTACAGCTTGTGAAGCCACTGGGCGGCGCTGGTCTCATCTGCTGGGATGTCTT
CCAGGGGGAACCTCCTGTAAAGTGTAAAGTGAGTGAATGAACACCTGCCCC
mm4_exon_Unk_chr11_116056063 TGGCTGCCCTAGAGAAGACTTTTGATAACAGCGCAGTGCCCCACCCCAGCAGCGTCACAATCGGTACTGGCTGTGGGTGG
CTGGGACGGCCTGAAGGGCGTCCTGGGGTTC
mm4_intron_Cdh13_chr8_118668565 TGTCCTCATCTCTGGCTCTGATGACCTTACTGGGTACCTCTGATGACTTCACTGGGTACCTCTGATGACCTCACTGGGTA
CCTCTGATGACTTCACTAGGTGCCTCTGATGACATCACTGG
mm4_exon_Zfp536_chr7_37716297 CCACAGCTCAGATGCCTCATAACCGTGAATGAAGTGGTCCTTAGTGGACTCTGCTCGGCTGGTGACTCCACTGGGGCTCC
GGAGGCATGTCTGCTGAATACATATGTTCATCCCACCCAGAAGACCTGAAGCCCAG
mm4_intron_Cdh13_chr8_118668653 TGCCTCTGATGACATCACTGGGTACCTCTGATGACTTCACTGGGTACCTCTGATGACTTCACTGGGTACCTCTGATGACC
TTGGGTACCTCTGATGACATCACTGGGTATCTCTGATGACCTCACTGGGTACCTCT
mm4_intron_Cdh13_chr8_118668587 TGTCCTCATCTCTGGCTCTGATGACCTTACTGGGTACCTCTGATGACTTCACTGGGTACCTCTGATGACCTCACTGGGTA
CCTCTGATGACTTCACTAGGTGCCTCTGATGACATCACTGG
mm4_intron_Cdh13_chr8_118668543 TGTCCTCATCTCTGGCTCTGATGACCTTACTGGGTACCTCTGATGACTTCACTGGGTACCTCTGATGACCTCACTGGGTA
CCTCT
mm4_intergenic_Fam173b|Tas2r119_chr15_32036238 AGCCAACGAGGTTACTTTTCCTTCTTAGTACTGTCTCATTTCAGAAAAAGCTCTTCTAATTACTATCTCTTTAATCCTTT
TTATTTTACTCTTCTAGTGGCGTCATCTGCTGAACAATAGAGACACTCAGGAGCTGTGTG
mm4_intron_Mmrn2_chr14_34396323 GCCAGGGAGTCAGAGTCATGTGAGTAGGGTGGTCAGGCCATTTTCCAGGCCCAGTGGGCTCACCAGAGGCACTGAGCCAC
ACTGCCTTCTTCCTCAGGTATCGAGTGGCCCAGAAGCCAGTGTACCAG

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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