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Guide sequence: CAGCGGGGTGAGCGCGGCCC GGG
Contents:
Cloning and expression of guide RNA
T7 in vitro expression from a plasmid
To produce guide RNA by in vitro transcription with T7 RNA polymerase, the guide RNA sequence can be cloned into a variety of plasmids (see
AddGene website).
For the guide sequence CAGCGGGGTGAGCGCGGCCC, the following primers should be ordered for cloning into the BsaI-digested plasmid
DR274 generated by the Joung lab.
Name | Primer Sequence |
guideRna108rvT7sense |
TAGGCAGCGGGGTGAGCGCGGCCC |
guideRna108rvT7antisense |
AAACGGGCCGCGCTCACCCCGCTG |
T7 in vitro expression from overlapping oligonucleotides
For spCas9, template for
in vitro synthesis of guide RNA with T7 RNA polymerase can be prepared by annealing and primer extension of the following primers:
Name | Primer Sequence |
guideRNA108rvT7crTarget |
GAAATTAATACGACTCACTATAGCAGCGGGGTGAGCGCGGCCCGTTTTAGAGCTAGAAATAGCAAG |
guideRNAallT7common (constant primer used for all guide RNAs) |
AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC |
T7 RNA polymerase starts transcription most efficiently if the first two nucleotides to be transcribed are GG. A common recommendation is to add the prefix GG- if our guide does not start with G (5'-N20-(NGG)-3'), to add G- if your guide starts with a single G (5'-GN19-(NGG)-3') and to not add anything if your guide starts with GG already (5'-GGN18-(NGG)-3').
One protocol for template preparation from oligonucleotides and in-vitro transcription can be found in Bassett et al. Cell Rep 2013. We also provide our own optimized protocol for T7 guide expression.
Gagnon et al. PLoS ONE 2014 prefixed guides with GG to ensure high efficiency in vitro transcription by T7 RNA polymerase. It has been shown by other authors that the 5' nucleotides of the guide have little or no role in target specificity and it is therefore generally accepted that prefixing guides with GG should not affect activity.
However, in our lab, we found that in vitro transcription with T7 RNA polymerase is efficient enough when the sequence starts with a single G rather than with GG. This took some optimization of the reaction conditions including using large amounts of template DNA and running reactions overnight. Click here to download our optimized protocol for T7 guide expression.
Do not use G-prefixing with high-fidelity Cas9 Variants like HF1 and eSpCas9 1.1 when this adds a mismatch in the genome as the efficiency will most likely be very low.
T7 in vitro expression with the GeneArt kit
Use these two primers for the Invitrogen GeneArt kit:
Name | Primer Sequence |
guideRNA108rvGeneArtFw |
TACGACTCACTATAGCAGCGGGGTGAGCGCGGCCC |
guideRNA108rvGeneArtRev |
TTCTAGCTCTAAAACGGGCCGCGCTCACCCCGCTG |
U6 expression from an Addgene plasmid
The guide sequence CAGCGGGGTGAGCGCGGCCC does not contain the motif TTTT, which terminates RNA polymerase, so it can be transcribed in mammalian cells.
To clone the guide into MLM3636 (Joung lab), use these primers:
Note: Efficient transcription from the U6 promoter requires a 5' G. This is not the case for this guide. Several options are possible, you can either add an additional G- prefix to the N20 guide sequence, called gN20 guides here, or replace the first with a G and create a gN19 guide. For users of HF1 and eSpCas9: G- prefixing with the high-fidelity variants may reduce efficiency, as it introduces a mismatch.
Primers for gN20 guides:
Name | Primer Sequence |
gN20-guideRNA108rvU6senseMLM3636 |
ACACCGCAGCGGGGTGAGCGCGGCCCG |
gN20-guideRNA108rvU6antisenseMLM3636 |
AAAACGGGCCGCGCTCACCCCGCTGCG |
Primers for gN19 guides:
Kim et al 2020. showed that changing the first nucleotide to 'G' is slightly more efficient.
Name | Primer Sequence |
gN19-gN20-guideRNA108rvU6senseMLM3636 |
ACACCGAGCGGGGTGAGCGCGGCCCG |
gN19-gN20-guideRNA108rvU6antisenseMLM3636 |
AAAACGGGCCGCGCTCACCCCGCTCG |
The plasmid has to be digested with: BsmBI
Click here to download the cloning protocol for MLM3636 (Joung lab)
Lentiviral vectors: cloning with Gibson assembly
Order the following oligonucleotide to clone with Gibson assembly into the vector
pLentiGuide-puro. See the
protocol by Matt Canver.
To clone with restriction enzymes into this vector, see the section
U6 expression from an AddGene plasmid and choose pLentiGuide-puro from the list of AddGene plasmids.
If you use lentiviral vectors, you may be interested in our tools for
saturating mutagenesis and for
gene knockout libraries.
Name | Oligonucleotide Sequence |
batchOligo108rv |
GGAAAGGACGAAACACCGCAGCGGGGTGAGCGCGGCCCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC |
Summary of main cloning/expression primers
guideRNA108rvGeneArtFw |
TACGACTCACTATAGCAGCGGGGTGAGCGCGGCCC |
guideRNA108rvGeneArtRev |
TTCTAGCTCTAAAACGGGCCGCGCTCACCCCGCTG |
gN20-guideRNA108rvU6senseMLM3636 |
ACACCGCAGCGGGGTGAGCGCGGCCCG |
gN20-guideRNA108rvU6antisenseMLM3636 |
AAAACGGGCCGCGCTCACCCCGCTGCG |
guideRna108rvT7sense |
TAGGCAGCGGGGTGAGCGCGGCCC |
guideRna108rvT7antisense |
AAACGGGCCGCGCTCACCCCGCTG |
guideRNA108rvT7crTarget |
GAAATTAATACGACTCACTATAGCAGCGGGGTGAGCGCGGCCCGTTTTAGAGCTAGAAATAGCAAG |
guideRNAallT7common |
AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC |
gN19-gN20-guideRNA108rvU6senseMLM3636 |
ACACCGAGCGGGGTGAGCGCGGCCCG |
gN19-gN20-guideRNA108rvU6antisenseMLM3636 |
AAAACGGGCCGCGCTCACCCCGCTCG |
PCR to amplify the on-target site
Use these primers to amplify a genomic fragment around the on-target site:
OntargetGuideRna108rvLeft |
TGCGGTTCAAGCACAGGTAA |
Tm 60.179 |
OntargetGuideRna108rvRight |
CTGTTCAGTTTTCGACCCGC |
Tm 59.765 |
Genome fragment with validation primers (underlined) and guide sequence (yellow)
Your guide sequence is on the reverse strand relative to the genome sequence, so it is reverse complemented in the sequence below.
Genomic sequence chr5:115238102-115238125 including primers, genomic forward strand:
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Sequence length: 351
Method: Primer3.2 with default settings, target length 250-400 bp,
Restriction Sites for PCR product validation
Cas9 induces mutations, usually 3bp 5' of the PAM site.
If a mutation is induced, then it is very likely that one of the following enzymes no longer cuts your PCR product amplified from the mutant sequence.
For each restriction enzyme, the guide sequence with the restriction site underlined is shown below.
Enzyme | Pattern | Guide with Restriction Site | Suppliers |
BshFI/BsuRI/HaeIII/BspANI/AoxI/BsnI | GGCC |
CAGCGGGGTGAGCGCGGCCCGGG
|
EURx, Life Technologies, Minotech, Molecular Biology Resources, NEB, Nippon Gene, Promega, Roche, SibEnzyme, Sigma, Takara, Toyobo, Vivantis |
BmeT110I/Eco88I/AvaI/BsiHKCI/Ama87I/BsoBI | CYCGRG |
CAGCGGGGTGAGCGCGGCCCGGG
|
EURx, Life Technologies, Molecular Biology Resources, NEB, Nippon Gene, SibEnzyme, Takara, Vivantis |
TauI | GCSGC |
CAGCGGGGTGAGCGCGGCCCGGG
|
Life Technologies |
MwoI/HpyF10VI/BstMWI | GCNNNNNNNGC |
CAGCGGGGTGAGCGCGGCCCGGG
|
Life Technologies, NEB, SibEnzyme |
NciI/BpuMI/AsuC2I/BcnI | CCSGG |
CAGCGGGGTGAGCGCGGCCCGGG, CAGCGGGGTGAGCGCGGCCCGGG
|
Life Technologies, NEB, Nippon Gene, Promega, SibEnzyme, Takara, Vivantis |
MspI/HpaII/HapII/BsiSI | CCGG |
CAGCGGGGTGAGCGCGGCCCGGG
|
EURx, Life Technologies, Minotech, Molecular Biology Resources, NEB, Nippon Gene, Promega, SibEnzyme, Sigma, SinaClon BioScience, Takara, Vivantis |
BseDI/BsaJI/BssECI | CCNNGG |
CAGCGGGGTGAGCGCGGCCCGGG
|
Life Technologies, NEB, SibEnzyme |
PspPI/BmgT120I/Sau96I/Cfr13I/AspS9I | GGNCC |
CAGCGGGGTGAGCGCGGCCCGGG
|
Life Technologies, Minotech, NEB, Nippon Gene, SibEnzyme, Takara, Vivantis |
BspFNI/AccII/BstUI/Bsh1236I/MvnI/BstFNI | CGCG |
CAGCGGGGTGAGCGCGGCCCGGG
|
Life Technologies, NEB, Nippon Gene, Roche, SibEnzyme, Takara, Vivantis |
LpnPI | CCDG |
CAGCGGGGTGAGCGCGGCCCGGG
|
NEB |
StyD4I/Bme1390I/BstSCI/BmrFI/ScrFI/MspR9I | CCNGG |
CAGCGGGGTGAGCGCGGCCCGGG, CAGCGGGGTGAGCGCGGCCCGGG
|
Life Technologies, NEB, Nippon Gene, SibEnzyme, Vivantis |
Cfr9I/SmaI/TspMI/XmaI | CCCGGG |
CAGCGGGGTGAGCGCGGCCCGGG
|
EURx, Life Technologies, Minotech, Molecular Biology Resources, NEB, Nippon Gene, Promega, Roche, SibEnzyme, Sigma, SinaClon BioScience, Takara, Toyobo, Vivantis |
Fsp4HI/PkrI/Fnu4HI/BlsI/BisI/GluI/SatI | GCNGC |
CAGCGGGGTGAGCGCGGCCCGGG
|
Life Technologies, NEB, SibEnzyme |
All restriction enzyme sites on the amplicon sequence
Restriction sites are shown in yellow, the guide sequence is highlighted in bold. Use this schema to check if the sites are unique enough to give separate bands on a gel:
Enzyme: BshFI/BsuRI/HaeIII/BspANI/AoxI/BsnI, Site: GGCC, Restriction fragment lengths: 53bp, 129bp, 82bp, 1bp, 71bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Enzyme: BmeT110I/Eco88I/AvaI/BsiHKCI/Ama87I/BsoBI, Site: CYCGRG, Restriction fragment lengths: 49bp, 118bp, 173bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Enzyme: TauI, Site: GCSGC, Restriction fragment lengths: 54bp, 15bp, 117bp, -2bp, 125bp, 18bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Enzyme: MwoI/HpyF10VI/BstMWI, Site: GCNNNNNNNGC, Restriction fragment lengths: 1bp, 13bp, 0bp, -2bp, 3bp, -2bp, 40bp, 10bp, -7bp, 32bp, 8bp, 17bp, 3bp, -6bp, 88bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Enzyme: NciI/BpuMI/AsuC2I/BcnI, Site: CCSGG, Restriction fragment lengths: 49bp, -4bp, 118bp, -4bp, 95bp, 73bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Enzyme: MspI/HpaII/HapII/BsiSI, Site: CCGG, Restriction fragment lengths: 50bp, 120bp, 97bp, 73bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Enzyme: BseDI/BsaJI/BssECI, Site: CCNNGG, Restriction fragment lengths: 49bp, 49bp, 63bp, 30bp, 48bp, 83bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Enzyme: PspPI/BmgT120I/Sau96I/Cfr13I/AspS9I, Site: GGNCC, Restriction fragment lengths: 52bp, 99bp, 24bp, 82bp, 75bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Enzyme: BspFNI/AccII/BstUI/Bsh1236I/MvnI/BstFNI, Site: CGCG, Restriction fragment lengths: 33bp, 7bp, 8bp, 51bp, -2bp, 22bp, 17bp, 90bp, 30bp, 60bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Enzyme: LpnPI, Site: CCDG, Restriction fragment lengths: 50bp, 75bp, 41bp, 25bp, 56bp, 8bp, 0bp, 69bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Enzyme: StyD4I/Bme1390I/BstSCI/BmrFI/ScrFI/MspR9I, Site: CCNGG, Restriction fragment lengths: 49bp, -4bp, 74bp, 39bp, -4bp, 84bp, 6bp, 73bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Enzyme: Cfr9I/SmaI/TspMI/XmaI, Site: CCCGGG, Restriction fragment lengths: 49bp, 118bp, 173bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
Enzyme: Fsp4HI/PkrI/Fnu4HI/BlsI/BisI/GluI/SatI, Site: GCNGC, Restriction fragment lengths: 20bp, 29bp, 15bp, 37bp, 75bp, -2bp, 30bp, 90bp, 18bp
TGCGGTTCAAGCACAGGTAAGCAGCGCTGTCCCCGCGCCGTCCCCGCGACCCGGGCCGCGCTCACCCCGCTGTCGCCGCA
GGTACCTTCTGTGCGAGCTGGTGTCCGAGGACGCGCGCTGCCGTTTGAGCCTGGACGACCGCGTGCTGGGCGGGCTGGTC
CGCGACACGATCGCCCGGGTGCACGGGGCCTTCGGCGCCGCCGCCTGCTCCGTGGGCTTTGCAGGTACGAGGGCGCTGCG
GATGCAGTGCGGGGCGCGCTTTGCCAGGGCGAGGCCCGGCCAGTGTGTCGCGTTTTGTACGATAGTGGAGGGTTCTAGAA
AATCGGAGTGCGGCGGGTCGAAAACTGAACAG
PCR to amplify off-target sites
Primers for all off-targets can be downloaded from the Off-target PCR page.
BETA: Guide mutations to minimize on-target activity
Sums of CFD scores for all possible mutations of the last three nucleotides of the guide:
ACA: 0.000000
CGG: 0.000000
CTT: 0.012530
ACC: 0.019692
TCC: 0.084839
CCG: 0.173801
CAC: 0.203154
CGC: 0.291573
CCA: 0.306220
GCC: 0.341512
CCT: 0.400179
CTC: 0.510507
CCC: 8.411031
Saturating mutagenesis using all guides
Oligonucleotides of all guides for pooled cloning into a lentiviral vector can be downloaded from the Saturating mutagenesis page.