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Guide sequence: GGGAGGCCAGGAGGAGGAGC CGG
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 gggaggccaggaggaggagc, the following primers should be ordered for cloning into the BsaI-digested plasmid
DR274 generated by the Joung lab.
Name | Primer Sequence |
guideRna553fwT7sense |
TAGgggaggccaggaggaggagc |
guideRna553fwT7antisense |
AAACgctcctcctcctggcctcc |
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 |
guideRNA553fwT7crTarget |
GAAATTAATACGACTCACTATAgggaggccaggaggaggagcGTTTTAGAGCTAGAAATAGCAAG |
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 |
guideRNA553fwGeneArtFw |
TACGACTCACTATAGgggaggccaggaggaggagc |
guideRNA553fwGeneArtRev |
TTCTAGCTCTAAAACgctcctcctcctggcctccc |
U6 expression from an Addgene plasmid
The guide sequence gggaggccaggaggaggagc 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:
Name | Primer Sequence |
guideRNA553fwU6senseMLM3636 |
ACACCgggaggccaggaggaggagcG |
guideRNA553fwU6antisenseMLM3636 |
AAAACgctcctcctcctggcctcccG |
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 |
batchOligo553fw |
GGAAAGGACGAAACACCGgggaggccaggaggaggagcGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC |
Summary of main cloning/expression primers
guideRna553fwT7sense |
TAGgggaggccaggaggaggagc |
guideRna553fwT7antisense |
AAACgctcctcctcctggcctcc |
guideRNA553fwT7crTarget |
GAAATTAATACGACTCACTATAgggaggccaggaggaggagcGTTTTAGAGCTAGAAATAGCAAG |
guideRNAallT7common |
AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC |
guideRNA553fwU6senseMLM3636 |
ACACCgggaggccaggaggaggagcG |
guideRNA553fwU6antisenseMLM3636 |
AAAACgctcctcctcctggcctcccG |
guideRNA553fwGeneArtFw |
TACGACTCACTATAGgggaggccaggaggaggagc |
guideRNA553fwGeneArtRev |
TTCTAGCTCTAAAACgctcctcctcctggcctccc |
PCR to amplify the on-target site
Use these primers to amplify a genomic fragment around the on-target site:
OntargetGuideRna553fwLeft |
AACCGCCAGAGAAGATGGTG |
Tm 60.036 |
OntargetGuideRna553fwRight |
GAAGGGAACTGTGGGTGGAG |
Tm 59.963 |
Genome fragment with validation primers (underlined) and guide sequence (yellow)
Genomic sequence chr6:52314029-52314052 including primers, genomic forward strand:
AACCGCCAGAGAAGATGGTGCCCCACGGTTGCCTGAGCCCGCGAGCCGGCCCTCCGACTTCCCGGGAGCGTGGCGGGGGA
GGCCAGGAGGAGGAGCCGGTCGATGGACTAGCAGGCAGTGCTGCAGGGCTGGGCGCCGAGCCACGGTCTGCTGGAGCGGC
CATGCTTGGCCCGGGACCCCCAGTCCCCTCCGCGGACAGCCTCTCTGGCCAAGGGCAACCTAGTAGCTCAGACACCGAAT
CGGATTTCTATGAAGAAATCGAGGTGAGCTGCACCCCAGACTGCGCCACCGGGAACGCCGAGTACCAGCACAGCAAAGGT
AGCCACCTTGCCCCTCCGCTCCCCGGTCCGCCCACTCCACCCACAGTTCCCTTC
AACCGCCAGAGAAGATGGTGCCCCACGGTTGCCTGAGCCCGCGAGCCGGCCCTCCGACTTCCCGGGAGCGTGGCGGGGGA
GGCCAGGAGGAGGAGCCGGTCGATGGACTAGCAGGCAGTGCTGCAGGGCTGGGCGCCGAGCCACGGTCTGCTGGAGCGGC
CATGCTTGGCCCGGGACCCCCAGTCCCCTCCGCGGACAGCCTCTCTGGCCAAGGGCAACCTAGTAGCTCAGACACCGAAT
CGGATTTCTATGAAGAAATCGAGGTGAGCTGCACCCCAGACTGCGCCACCGGGAACGCCGAGTACCAGCACAGCAAAGGT
AGCCACCTTGCCCCTCCGCTCCCCGGTCCGCCCACTCCACCCACAGTTCCCTTC
Sequence length: 373
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 |
NlaIV/BmiI/PspN4I/BspLI | GGNNCC |
GGGAGGCCAGGAGGAGGAGCCGG
|
Life Technologies, NEB, SibEnzyme, Vivantis |
MspI/HpaII/HapII/BsiSI | CCGG |
GGGAGGCCAGGAGGAGGAGCCGG
|
EURx, Life Technologies, Minotech, Molecular Biology Resources, NEB, Nippon Gene, Promega, SibEnzyme, Sigma, SinaClon BioScience, Takara, Vivantis |
TaqI | TCGA |
GGGAGGCCAGGAGGAGGAGCCGG
|
EURx, Life Technologies, Minotech, Molecular Biology Resources, NEB, Nippon Gene, Promega, Roche, SibEnzyme, Sigma, Takara, Vivantis |
BseRI | GAGGAG |
GGGAGGCCAGGAGGAGGAGCCGG
|
NEB |
LpnPI | CCDG |
GGGAGGCCAGGAGGAGGAGCCGG
|
NEB |
Bsh1285I/BsiEI/BstMCI | CGRYCG |
GGGAGGCCAGGAGGAGGAGCCGG
|
Life Technologies, NEB, SibEnzyme, Vivantis |
Cfr10I/Bse118I/BssAI/BsrFI | RCCGGY |
GGGAGGCCAGGAGGAGGAGCCGG
|
Life Technologies, Minotech, NEB, SibEnzyme, Takara, Vivantis |
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: NlaIV/BmiI/PspN4I/BspLI, Site: GGNNCC, Restriction fragment lengths: 16bp, 69bp, 34bp, 35bp, -5bp, 195bp
AACCGCCAGAGAAGATGGTGCCCCACGGTTGCCTGAGCCCGCGAGCCGGCCCTCCGACTTCCCGGGAGCGTGGCGGGGGA
GGCCAGGAGGAGGAGCCGGTCGATGGACTAGCAGGCAGTGCTGCAGGGCTGGGCGCCGAGCCACGGTCTGCTGGAGCGGC
CATGCTTGGCCCGGGACCCCCAGTCCCCTCCGCGGACAGCCTCTCTGGCCAAGGGCAACCTAGTAGCTCAGACACCGAAT
CGGATTTCTATGAAGAAATCGAGGTGAGCTGCACCCCAGACTGCGCCACCGGGAACGCCGAGTACCAGCACAGCAAAGGT
AGCCACCTTGCCCCTCCGCTCCCCGGTCCGCCCACTCCACCCACAGTTCCCTTC
Enzyme: MspI/HpaII/HapII/BsiSI, Site: CCGG, Restriction fragment lengths: 45bp, 12bp, 30bp, 71bp, 114bp, 50bp, 28bp
AACCGCCAGAGAAGATGGTGCCCCACGGTTGCCTGAGCCCGCGAGCCGGCCCTCCGACTTCCCGGGAGCGTGGCGGGGGA
GGCCAGGAGGAGGAGCCGGTCGATGGACTAGCAGGCAGTGCTGCAGGGCTGGGCGCCGAGCCACGGTCTGCTGGAGCGGC
CATGCTTGGCCCGGGACCCCCAGTCCCCTCCGCGGACAGCCTCTCTGGCCAAGGGCAACCTAGTAGCTCAGACACCGAAT
CGGATTTCTATGAAGAAATCGAGGTGAGCTGCACCCCAGACTGCGCCACCGGGAACGCCGAGTACCAGCACAGCAAAGGT
AGCCACCTTGCCCCTCCGCTCCCCGGTCCGCCCACTCCACCCACAGTTCCCTTC
Enzyme: TaqI, Site: TCGA, Restriction fragment lengths: 99bp, 155bp, 112bp
AACCGCCAGAGAAGATGGTGCCCCACGGTTGCCTGAGCCCGCGAGCCGGCCCTCCGACTTCCCGGGAGCGTGGCGGGGGA
GGCCAGGAGGAGGAGCCGGTCGATGGACTAGCAGGCAGTGCTGCAGGGCTGGGCGCCGAGCCACGGTCTGCTGGAGCGGC
CATGCTTGGCCCGGGACCCCCAGTCCCCTCCGCGGACAGCCTCTCTGGCCAAGGGCAACCTAGTAGCTCAGACACCGAAT
CGGATTTCTATGAAGAAATCGAGGTGAGCTGCACCCCAGACTGCGCCACCGGGAACGCCGAGTACCAGCACAGCAAAGGT
AGCCACCTTGCCCCTCCGCTCCCCGGTCCGCCCACTCCACCCACAGTTCCCTTC
Enzyme: BseRI, Site: GAGGAG, Restriction fragment lengths: 86bp, -3bp, 279bp
AACCGCCAGAGAAGATGGTGCCCCACGGTTGCCTGAGCCCGCGAGCCGGCCCTCCGACTTCCCGGGAGCGTGGCGGGGGA
GGCCAGGAGGAGGAGCCGGTCGATGGACTAGCAGGCAGTGCTGCAGGGCTGGGCGCCGAGCCACGGTCTGCTGGAGCGGC
CATGCTTGGCCCGGGACCCCCAGTCCCCTCCGCGGACAGCCTCTCTGGCCAAGGGCAACCTAGTAGCTCAGACACCGAAT
CGGATTTCTATGAAGAAATCGAGGTGAGCTGCACCCCAGACTGCGCCACCGGGAACGCCGAGTACCAGCACAGCAAAGGT
AGCCACCTTGCCCCTCCGCTCCCCGGTCCGCCCACTCCACCCACAGTTCCCTTC
Enzyme: LpnPI, Site: CCDG, Restriction fragment lengths: 5bp, 22bp, 10bp, 12bp, 17bp, 9bp, 71bp, 5bp, 92bp, 9bp, 12bp, 34bp, 28bp
AACCGCCAGAGAAGATGGTGCCCCACGGTTGCCTGAGCCCGCGAGCCGGCCCTCCGACTTCCCGGGAGCGTGGCGGGGGA
GGCCAGGAGGAGGAGCCGGTCGATGGACTAGCAGGCAGTGCTGCAGGGCTGGGCGCCGAGCCACGGTCTGCTGGAGCGGC
CATGCTTGGCCCGGGACCCCCAGTCCCCTCCGCGGACAGCCTCTCTGGCCAAGGGCAACCTAGTAGCTCAGACACCGAAT
CGGATTTCTATGAAGAAATCGAGGTGAGCTGCACCCCAGACTGCGCCACCGGGAACGCCGAGTACCAGCACAGCAAAGGT
AGCCACCTTGCCCCTCCGCTCCCCGGTCCGCCCACTCCACCCACAGTTCCCTTC
Enzyme: Bsh1285I/BsiEI/BstMCI, Site: CGRYCG, Restriction fragment lengths: 96bp, 272bp
AACCGCCAGAGAAGATGGTGCCCCACGGTTGCCTGAGCCCGCGAGCCGGCCCTCCGACTTCCCGGGAGCGTGGCGGGGGA
GGCCAGGAGGAGGAGCCGGTCGATGGACTAGCAGGCAGTGCTGCAGGGCTGGGCGCCGAGCCACGGTCTGCTGGAGCGGC
CATGCTTGGCCCGGGACCCCCAGTCCCCTCCGCGGACAGCCTCTCTGGCCAAGGGCAACCTAGTAGCTCAGACACCGAAT
CGGATTTCTATGAAGAAATCGAGGTGAGCTGCACCCCAGACTGCGCCACCGGGAACGCCGAGTACCAGCACAGCAAAGGT
AGCCACCTTGCCCCTCCGCTCCCCGGTCCGCCCACTCCACCCACAGTTCCCTTC
Enzyme: Cfr10I/Bse118I/BssAI/BsrFI, Site: RCCGGY, Restriction fragment lengths: 44bp, 44bp, 274bp
AACCGCCAGAGAAGATGGTGCCCCACGGTTGCCTGAGCCCGCGAGCCGGCCCTCCGACTTCCCGGGAGCGTGGCGGGGGA
GGCCAGGAGGAGGAGCCGGTCGATGGACTAGCAGGCAGTGCTGCAGGGCTGGGCGCCGAGCCACGGTCTGCTGGAGCGGC
CATGCTTGGCCCGGGACCCCCAGTCCCCTCCGCGGACAGCCTCTCTGGCCAAGGGCAACCTAGTAGCTCAGACACCGAAT
CGGATTTCTATGAAGAAATCGAGGTGAGCTGCACCCCAGACTGCGCCACCGGGAACGCCGAGTACCAGCACAGCAAAGGT
AGCCACCTTGCCCCTCCGCTCCCCGGTCCGCCCACTCCACCCACAGTTCCCTTC
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:
GTG: 0.010084
CAG: 0.011433
CGG: 0.011937
CGT: 0.013445
GAG: 0.015514
CTG: 0.016230
CGA: 0.028812
ACG: 0.036168
CAT: 0.036735
ATT: 0.040000
GCA: 0.042191
GCT: 0.043744
CCC: 0.072250
ATG: 0.113263
TCC: 0.132557
TAA: 0.151099
TAT: 0.167411
TGG: 0.228006
GGT: 0.279104
TTC: 0.301491
TAC: 0.389668
GGG: 0.465918
AAT: 0.517453
ATA: 0.551961
CTC: 0.598916
GCC: 0.619595
CAC: 0.712138
AAG: 0.730214
ACA: 0.799483
TGA: 1.032148
GTC: 1.045309
GAC: 1.250916
TGT: 1.547813
AAA: 2.655363
ACC: 3.215632
ATC: 3.671306
CGC: 4.048454
GGA: 6.571779
AGT: 9.955140
TGC: 10.992422
GGC: 13.476135
AAC: 16.083439
AGG: 35.666189
AGA: 70.068891
AGC: 361.886929
Saturating mutagenesis using all guides
Oligonucleotides of all guides for pooled cloning into a lentiviral vector can be downloaded from the Saturating mutagenesis page.