CRISPOR

Contents:

PCR Primers
Amplicon sequences
Crispresso support

PCR primers for off-targets of CTCGTCGTCGACAACGGCTC 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.

Name	Primer Sequence	Tm	CFD Score
ontarget_mm0__chr7 5.53 Mbp_chr7_5529613_F	`TCGTCGGCAGCGTCCACGATGGAGGGGAAGACG`	59.8	1.00
ontarget_mm0__chr7 5.53 Mbp_chr7_5529613_R	`GTCTCGTGGGCTCGGGCTATTCTCGCAGCTCACCA`	60.1	1.00
mm3__chr1 223.86 Mbp_chr1_223864807_F	`TCGTCGGCAGCGTCCATGGAAGGGAAGATGGCCC`	60.4	0.87
mm3__chr1 223.86 Mbp_chr1_223864807_R	`GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT`	60.1	0.87
mm2__chr5 77.78 Mbp_chr5_77784907_F	`TCGTCGGCAGCGTCCACCAGCTCACCATGGATGA`	59.7	0.87
mm2__chr5 77.78 Mbp_chr5_77784907_R	`GTCTCGTGGGCTCGGAACGATGGAGGGGAAGACTG`	59.0	0.87
mm2__chr22 15.72 Mbp_chr22_15721507_F	`TCGTCGGCAGCGTCACTGAATGAGCTCACCATGGA`	59.0	0.87
mm2__chr22 15.72 Mbp_chr22_15721507_R	`GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC`	60.1	0.87
mm2__chr2 131.63 Mbp_chr2_131626159_F	`TCGTCGGCAGCGTCACCATGGATGATGATACCGCT`	59.0	0.87
mm2__chr2 131.63 Mbp_chr2_131626159_R	`GTCTCGTGGGCTCGGCCTCCATCATGCCCTGGTG`	60.1	0.87
mm2__chr2 131.26 Mbp_chr2_131263576_F	`TCGTCGGCAGCGTCTGGATGATGATACCGCCGTG`	59.9	0.87
mm2__chr2 131.26 Mbp_chr2_131263576_R	`GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC`	60.1	0.87
mm2__chr2 130.66 Mbp_chr2_130656770_F	`TCGTCGGCAGCGTCACCATGGATGATGATACCGCT`	59.0	0.87
mm2__chr2 130.66 Mbp_chr2_130656770_R	`GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC`	60.1	0.87
mm2__chr2 130.46 Mbp_chr2_130463899_F	`Primer3: not found at this Tm`	N.d.	0.87
mm2__chr2 130.46 Mbp_chr2_130463899_R	`Primer3: not found at this Tm`	N.d.	0.87
mm2__chr2 130.08 Mbp_chr2_130075327_F	`TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC`	60.1	0.87
mm2__chr2 130.08 Mbp_chr2_130075327_R	`GTCTCGTGGGCTCGGACCATGGATGATGATACCGCT`	59.0	0.87
mm2__chr14 19.40 Mbp_chr14_19402864_F	`TCGTCGGCAGCGTCGATGGAAGGGAAGACAGCCC`	60.1	0.87
mm2__chr14 19.40 Mbp_chr14_19402864_R	`GTCTCGTGGGCTCGGACTGAATGAGCTCACCATGGA`	59.0	0.87
mm2__chr14 19.00 Mbp_chr14_18998888_F	`TCGTCGGCAGCGTCACTGAATGAGCTCACCATGGA`	59.0	0.87
mm2__chr14 19.00 Mbp_chr14_18998888_R	`GTCTCGTGGGCTCGGGATGGAAGGGAAGACAGCCC`	60.1	0.87
mm1__chr9 6.83 Mbp_chr9_6834476_F	`TCGTCGGCAGCGTCCCGCTACCAGCTCACCATAG`	59.9	0.85
mm1__chr9 6.83 Mbp_chr9_6834476_R	`GTCTCGTGGGCTCGGACACGATGAAGGGGAAGACG`	59.7	0.85
mm3__chr22 41.08 Mbp_chr22_41075195_F	`TCGTCGGCAGCGTCGATGGAAGGGAAGATGGCCC`	60.1	0.81
mm3__chr22 41.08 Mbp_chr22_41075195_R	`GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT`	60.1	0.81
mm3__chr9 100.73 Mbp_chr9_100731260_F	`TCGTCGGCAGCGTCGCCAGCTCACCATGGATGAT`	60.1	0.60
mm3__chr9 100.73 Mbp_chr9_100731260_R	`GTCTCGTGGGCTCGGCTAGGGAGCTCCACGATGGA`	60.4	0.60
mm4__chr3 169.77 Mbp_chr3_169769476_F	`Primer3: not found at this Tm`	N.d.	0.22
mm4__chr3 169.77 Mbp_chr3_169769476_R	`Primer3: not found at this Tm`	N.d.	0.22
mm3__chrX 46.29 Mbp_chrX_46289084_F	`TCGTCGGCAGCGTCTGGAGGGGAAGACAACTGGG`	61.1	0.11
mm3__chrX 46.29 Mbp_chrX_46289084_R	`GTCTCGTGGGCTCGGCTCCATCACCAGTCCACACC`	60.0	0.11
mm4__chr15 34.79 Mbp_chr15_34794758_F	`TCGTCGGCAGCGTCGGGAAGTTTACCTGGTGCCG`	60.9	0.10
mm4__chr15 34.79 Mbp_chr15_34794758_R	`GTCTCGTGGGCTCGGATGTGTGACGACGAGGAGAC`	59.4	0.10
mm4__chr2 16.08 Mbp_chr2_16083760_F	`TCGTCGGCAGCGTCAGTTAGAAGCCCGCAAGCAT`	60.0	0.09
mm4__chr2 16.08 Mbp_chr2_16083760_R	`GTCTCGTGGGCTCGGAAGAGGACAAGGTGCTGGTG`	59.8	0.09
mm4__chr4 140.15 Mbp_chr4_140154394_F	`TCGTCGGCAGCGTCCAACCACACTACCGCCCC`	60.3	0.09
mm4__chr4 140.15 Mbp_chr4_140154394_R	`GTCTCGTGGGCTCGGAAGTTGATTTATTTCTGAGATCCCC`	57.1	0.09
mm2__chr18 62.44 Mbp_chr18_62442029_F	`TCGTCGGCAGCGTCGCCAGCTCACCATGGATGAT`	60.1	0.06
mm2__chr18 62.44 Mbp_chr18_62442029_R	`GTCTCGTGGGCTCGGATGATGGAGAGGAAGACGGC`	59.2	0.06
mm4__chrX 53.14 Mbp_chrX_53143869_F	`TCGTCGGCAGCGTCGATGGAGGGGAACACAGCTC`	60.1	0.05
mm4__chrX 53.14 Mbp_chrX_53143869_R	`GTCTCGTGGGCTCGGTGCTCTTCCACAGGTTGCAA`	60.1	0.05
mm4__chr16 50.06 Mbp_chr16_50059187_F	`TCGTCGGCAGCGTCGAGGGGAACATGGTTCAGGG`	60.0	0.05
mm4__chr16 50.06 Mbp_chr16_50059187_R	`GTCTCGTGGGCTCGGACTTCTCCACGATGGCGATG`	60.1	0.05
mm2__chr5 131.66 Mbp_chr5_131660071_F	`TCGTCGGCAGCGTCATTATGGAGGGGAAGACGGC`	59.2	0.04
mm2__chr5 131.66 Mbp_chr5_131660071_R	`GTCTCGTGGGCTCGGGCCACCAGTTCACCATGGAT`	60.3	0.04
mm2__chr15 43.99 Mbp_chr15_43990140_F	`TCGTCGGCAGCGTCAATGAAGGGGAAGATGGCCC`	59.7	0.04
mm2__chr15 43.99 Mbp_chr15_43990140_R	`GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT`	60.1	0.04
mm4__chr7 289.59 Kbp_chr7_289586_F	`Primer3: not found at this Tm`	N.d.	0.04
mm4__chr7 289.59 Kbp_chr7_289586_R	`Primer3: not found at this Tm`	N.d.	0.04
mm4__chr5 80.30 Mbp_chr5_80302803_F	`TCGTCGGCAGCGTCATGATGAAGGGGAAGGCAGC`	60.1	0.03
mm4__chr5 80.30 Mbp_chr5_80302803_R	`GTCTCGTGGGCTCGGAGCTCACCATGCATGACGAT`	59.8	0.03
mm4__chr17 81.51 Mbp_chr17_81512310_F	`TCGTCGGCAGCGTCCCATCCACTCACCTGGTGTC`	60.0	0.02
mm4__chr17 81.51 Mbp_chr17_81512310_R	`GTCTCGTGGGCTCGGATGGAAGAAGAGATCGCCGC`	60.2	0.02
mm4__chr5 98.34 Mbp_chr5_98338764_F	`TCGTCGGCAGCGTCGCAGCTAGCTCACCATGGAT`	59.8	0.02
mm4__chr5 98.34 Mbp_chr5_98338764_R	`GTCTCGTGGGCTCGGGCATTGTCACCTGTGAAGCC`	59.7	0.02
mm4__chr6 46.20 Mbp_chr6_46204998_F	`TCGTCGGCAGCGTCATGGAAGAAGAGATCCCCGC`	59.2	0.02
mm4__chr6 46.20 Mbp_chr6_46204998_R	`GTCTCGTGGGCTCGGATATGGCTCGGAGGGCATTG`	59.9	0.02
mm4__chr1 229.43 Mbp_chr1_229433065_F	`TCGTCGGCAGCGTCAAGCCGGCTTTCACCAGG`	60.2	0.01
mm4__chr1 229.43 Mbp_chr1_229433065_R	`GTCTCGTGGGCTCGGGCTGAGACTTCTGCGCTGAT`	60.4	0.01
mm4__chr1 218.76 Mbp_chr1_218759728_F	`TCGTCGGCAGCGTCCAGCAGTCCCAGACTTGAGT`	59.3	0.01
mm4__chr1 218.76 Mbp_chr1_218759728_R	`GTCTCGTGGGCTCGGCCCTGGCTAGACCCACCATA`	60.4	0.01
mm2__chr6 88.28 Mbp_chr6_88276942_F	`TCGTCGGCAGCGTCCCCACAATGGAGGGGAAGAC`	60.0	0.00
mm2__chr6 88.28 Mbp_chr6_88276942_R	`GTCTCGTGGGCTCGGGCCAGCTCACCATGGATGAT`	60.1	0.00

Off-target amplicon sequences

Primers underlined, off-targets in bold.

ontarget_mm0__chr7 5.53 Mbp_chr7_5529613	`CACGATGGAGGGGAAGACGGCCCGGGGGGCATCGTCGCCCGCGAAGCCGGCCTTGCACATGCCGGAGCCGTTGTCGACGA CGAGCGCGGCGATATCATCATCCATGGTGAGCTGCGAGAATAGC`
mm3__chr1 223.86 Mbp_chr1_223864807	`CATGGAAGGGAAGATGGCCCAGGGGTCATCATCCCCTGTGAAGCCAGCCTTGCACATGCCGGAGCTGTTGTCAATGACGA GCGCCGCGATATCATCATCCATGGTGAGCTGGC`
mm2__chr5 77.78 Mbp_chr5_77784907	`CACCAGCTCACCATGGATGATGATATCACCGCGCTCGTCATTGACAACGGCTCCGGCATGTGCAAGGCCAGCTTCACGGG CGACAATGCCGCCCGGGCAGTCTTCCCCTCCATCGTT`
mm2__chr22 15.72 Mbp_chr22_15721507	`ACTGAATGAGCTCACCATGGATGATGATACTGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTG CAGGTGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC`
mm2__chr2 131.63 Mbp_chr2_131626159	`ACCATGGATGATGATACCGCTGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTGCGGGCGACGATGC CCCCCAGGCTGTCTTCCCTTCTATCGTGGGGCGCCCCAGGCACCAGGGCATGATGGAGG`
mm2__chr2 131.26 Mbp_chr2_131263576	`TGGATGATGATACCGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTGCGGGCGACGATGCCCCC CGGGCTGTCTTCCCTTCCATC`
mm2__chr2 130.66 Mbp_chr2_130656770	`ACCATGGATGATGATACCGCTGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTGCGGGCGACGATGC CCCCCGGGCTGTCTTCCCTTCCATC`
mm2__chr2 130.08 Mbp_chr2_130075327	`GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCGCCCGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA GCACAGCGGTATCATCATCCATGGT`
mm2__chr14 19.40 Mbp_chr14_19402864	`GATGGAAGGGAAGACAGCCCGGGGGGCATCGTCACCTGCAAAGCCGGCCTTGCACATGCCAGAGCCGTTGTCAATGACGA GCACGGCAGTATCATCATCCATGGTGAGCTCATTCAGT`
mm2__chr14 19.00 Mbp_chr14_18998888	`ACTGAATGAGCTCACCATGGATGATGATACTGCCGTGCTCGTCATTGACAACGGCTCTGGCATGTGCAAGGCCGGCTTTG CAGGTGACGATGCCCCCCGGGCTGTCTTCCCTTCCATC`
mm1__chr9 6.83 Mbp_chr9_6834476	`CCGCTACCAGCTCACCATAGATGATGATATTGCTACGCCCGTCGTCGACAACGGCTCCGGCATGTGGAAGGCTGGCTTCG CAGGTGACGATGACCCCCGGCCGTCTTCCCCTTCATCGTGT`
mm3__chr22 41.08 Mbp_chr22_41075195	`GATGGAAGGGAAGATGGCCCAGGGGACATCGTCACCCGCGAAGCCGGCCTTGCACATGCCAGAGCTGTTGTCAACAACGA GCGCAGCAATATCATCATCCATGGTGAGCTGGC`
mm3__chr9 100.73 Mbp_chr9_100731260	`GCCAGCTCACCATGGATGATGATATCGCCCCACTTGTCATTGACAACGGCTCAGGCATGTGCAAGGCCGGCTTTGCAGGT GATAATGTCCCCCTGGGCCATCTTCCCCCTCCATCGTGGAGCTCCCTAG`
mm3__chrX 46.29 Mbp_chrX_46289084	`TGGAGGGGAAGACAACTGGGGGGCATCATCGCCCATGAAGGCAGCCTTGCACATGCCAGAGCCTTTGTTGAAGACGAGAG CAGTGATATCATCATTCATGGTGATCTGGCAGTGGGTGTGGACTGGTGATGGAG`
mm4__chr15 34.79 Mbp_chr15_34794758	`GGGAAGTTTACCTGGTGCCGCGGGCGGCCCACGATGGACGGGAAGACAGCGCGGGGCGCGTCATCGCCCGCAAAGCCGGC CTTCACCAGCCCAGAGCCGTTGTCGCACACCAGGGCGGTGGTCTCCTCGTCGTCACACAT`
mm4__chr2 16.08 Mbp_chr2_16083760	`AGTTAGAAGCCCGCAAGCATTTGCCGAGGGTAGTGCGGGCATCTCAGGACTCCATCCTCCCCTCCTCGTGGACAGCGGTT CCGGGAGCCCCAACTCACTGGCTGGGGACTTGCCTCCCACACCAGCACCTTGTCCTCTT`
mm4__chr4 140.15 Mbp_chr4_140154394	`CAACCACACTACCGCCCCGCCGAGAAGCCCAGAGCCGTCGGCGACGGAGAGAAGAAAGATTCTGGGGGGATCTCAGAAAT AAATCAACTT`
mm2__chr18 62.44 Mbp_chr18_62442029	`GCCAGCTCACCATGGATGATGATACTGTCGCCCTCGTCATCGACAATGGCTCTGGCATGTGCAGGGCTGGCTTCGCAGGC GACAGTGCTCTCCAGGCCGTCTTCCTCTCCATCAT`
mm4__chrX 53.14 Mbp_chrX_53143869	`GATGGAGGGGAACACAGCTCAGGGGCCATCCATCCCCAGCAAAGCCAGCTTTGCACATGCCAGAGCCATTGTCAATGATG AGCGCGGCGATCTCTATTTCCATTGCAACCTGTGGAAGAGCA`
mm4__chr16 50.06 Mbp_chr16_50059187	`GAGGGGAACATGGTTCAGGGGGCATCGTCCCTAGCAAAGCCAGCTTTGCACCTGCCAGAGCCATTGTCAATGATGAGCAC GATGATCTCTTCTTCCATCGCCATCGTGGAGAAGT`
mm2__chr5 131.66 Mbp_chr5_131660071	`ATTATGGAGGGGAAGACGGCCCGGGGGCATCGTTGCCCGTGAAGCAGACCTTCCACATGCCAGAGCCATTGTCGACGACA AGCACAGCAATATCATTATCCATGGTGAACTGGTGGC`
mm2__chr15 43.99 Mbp_chr15_43990140	`AATGAAGGGGAAGATGGCCCGGGGTGGGCATCGTCACCTGCAAAGCTGGCCTTGCACATGCCAGAGCCATTGTCGACGAC AAGCATGGTGATATCATCATCCATGGTGAGCTGGC`
mm4__chr5 80.30 Mbp_chr5_80302803	`ATGATGAAGGGGAAGGCAGCCCGGGGGACACCATCGCTCGCAAAGCTAGCCTTGCATATGCCAGAGCCATTGTTGAATAC GAGAGCAGTGATATCGTCATGCATGGTGAGCT`
mm4__chr17 81.51 Mbp_chr17_81512310	`CCATCCACTCACCTGGTGTCTGGGGCGCCCGACGATGGAAGGAAACACGGCTCGGGGAGCGTCGTCCCCAGCAAAACCAG CTTTGCACATGCCGGAGCCATTGTCAATGACCAGCGCGGCGATCTCTTCTTCCAT`
mm4__chr5 98.34 Mbp_chr5_98338764	`GCAGCTAGCTCACCATGGATGATGATATCAGCACGCTCCTCATCAACAAGGGCTCTGGCATGTGCAAGGCCGGCTTCACA GGTGACAATGC`
mm4__chr6 46.20 Mbp_chr6_46204998	`ATGGAAGAAGAGATCCCCGCGCTCTTCATTGACAATGGCTCCGGCATGTGAAAAGCAGCTTTGCTGGGACAATGCCCTCC GAGCCATAT`
mm4__chr1 229.43 Mbp_chr1_229433065	`AAGCCGGCTTTCACCAGGCCGGAGCCATTGTCGCACACGAGGGCGGTGGTCTCGTCTTCGTCGCACATTGTGTCTAGTTT CTGCAAGGACAGGGAGACCGCGAAGAGGCGCGGTGCATCAGCGCAGAAGTCTCAGC`
mm4__chr1 218.76 Mbp_chr1_218759728	`CAGCAGTCCCAGACTTGAGTAAGTGAAAATAGAGGTGGGACTCTGTTTCATTTTCAGGTAAAATCCCAAAAGACAACAAG TATCCTCCTTGTCCACAATGGCTCAGGAGGAGTAAGTATCTATGGTGGGTCTAGCCAGGG`
mm2__chr6 88.28 Mbp_chr6_88276942	`CCCACAATGGAGGGGAAGACGGCCTGGGGAGCCTTGCGCATGCTGGAGCAGTTGTCGACGACGACGAGCGCGGTGATAGC ATCATCCATGGTGAGCTGGC`

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

Version 4.7 - Documentation - Contact us - Downloads/local installation - Paper - License