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Guide sequence: CCGTCTTCCCCTCCATCGTG 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 CCGTCTTCCCCTCCATCGTG, the following primers should be ordered for cloning into the BsaI-digested plasmid DR274 generated by the Joung lab.

NamePrimer Sequence
guideRna233fwT7sense TAGGCCGTCTTCCCCTCCATCGTG
guideRna233fwT7antisense AAACCACGATGGAGGGGAAGACGG

T7 in vitro expression from overlapping oligonucleotides

Template for in vitro synthesis of guide RNA with T7 RNA polymerase can be prepared by annealing and primer extension of the following primers:

NamePrimer Sequence
guideRNA233fwT7crTarget GAAATTAATACGACTCACTATAGCCGTCTTCCCCTCCATCGTGGTTTTAGAGCTAGAAATAGCAAG
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.

U6 expression from an Addgene plasmid

The guide sequence CCGTCTTCCCCTCCATCGTG does not contain the motif TTTT, which terminates RNA polymerase, so it can be transcribed in mammalian cells.

Select your Addgene plasmid:

To clone the guide into MLM3636 (Joung lab), use these primers:
Note: Efficient transcription from the U6 promoter requires a 5' G. This G has been added in the sequence below, it is underlined. For a full discussion about G- prefixing, see the discussion of G-prefixing under overlapping oligonucleotides. Do not use G- prefixing with the high-fidelity Cas9 variants HF1 and eSpCas9 1.1

NamePrimer Sequence
guideRNA233fwU6senseMLM3636 ACACCGCCGTCTTCCCCTCCATCGTGG
guideRNA233fwU6antisenseMLM3636 AAAACCACGATGGAGGGGAAGACGGCG
The plasmid has to be digested with: BsmBI
Click here to download the cloning protocol for MLM3636 (Joung lab)

Direct PCR for C. intestinalis

Only usable at the moment in Ciona intestinalis (alias Ciona robusta). DNA construct is assembled during the PCR reaction; expression cassettes are generated with One-Step Overlap PCR (OSO-PCR) Gandhi et al., Dev Bio 2016 (preprint) following this protocol. The resulting unpurified PCR product can be directly electroporated into Ciona eggs.
NamePrimer Sequence
.233fw.sgF gCGTCTTCCCCTCCATCGTGgtttaagagctatgctggaaacag
.233fw.U6R CACGATGGAGGGGAAGACGcatctataccatcggatgccttc

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.
NameOligonucleotide Sequence
batchOligo233fw GGAAAGGACGAAACACCGCCGTCTTCCCCTCCATCGTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC

Summary of main cloning/expression primers

guideRna233fwT7sense TAGGCCGTCTTCCCCTCCATCGTG
guideRna233fwT7antisense AAACCACGATGGAGGGGAAGACGG
guideRNA233fwT7crTarget GAAATTAATACGACTCACTATAGCCGTCTTCCCCTCCATCGTGGTTTTAGAGCTAGAAATAGCAAG
guideRNAallT7common AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC
guideRNA233fwU6senseMLM3636 ACACCGCCGTCTTCCCCTCCATCGTGG
guideRNA233fwU6antisenseMLM3636 AAAACCACGATGGAGGGGAAGACGGCG

PCR to amplify the on-target site

Warning: Found multiple perfect matches for this guide sequence in the genome. For the PCR, we are using the on-target match in the input sequence chr7:5529550-5529572 (gene: chr7 5.53 Mbp), but this guide will not be specific. Is this a polyploid organism? Try selecting another guide sequence or email crispor@tefor.net to discuss your strategy or modifications to this software.

Use these primers to amplify a genomic fragment around the on-target site:
OntargetGuideRna233fwLeft TCGATGGGGTACTTCAGGGT Tm 59.958
OntargetGuideRna233fwRight GCTATTCTCGCAGCTCACCA Tm 60.179

Genome fragment with validation primers (underlined) and guide sequence (yellow)

Maximum amplicon length:     Primer Tm:

Your guide sequence is on the reverse strand relative to the genome sequence, so it is reverse complemented in the sequence below.

Genomic sequence chr7:5529549-5529572 including primers, genomic forward strand:
TCGATGGGGTACTTCAGGGTGAGGATGCCTCTCTTGCTCTGGGCCTCGTCGCCCACATAGGAATCCTTCTGACCCATGCC
CACCATCACGCCCTGGGAAGGAAAGGACAAGAAGCCCTGAGCACGGGCGCAGCCCCCACCCCGGAAACCGGGAGGCTCCT
GTGCAGAGAAAGCGCCCTTGCCTCCCGCCCGCTCCCGGGGCTGCCCCACCCAGCCAGCTCCCCTACCTGGTGCCTGGGGC
GCCCCACGATGGAGGGGAAGACGGCCCGGGGGGCATCGTCGCCCGCGAAGCCGGCCTTGCACATGCCGGAGCCGTTGTCG
ACGACGAGCGCGGCGATATCATCATCCATGGTGAGCTGCGAGAATAGC


TCGATGGGGTACTTCAGGGTGAGGATGCCTCTCTTGCTCTGGGCCTCGTCGCCCACATAGGAATCCTTCTGACCCATGCC
CACCATCACGCCCTGGGAAGGAAAGGACAAGAAGCCCTGAGCACGGGCGCAGCCCCCACCCCGGAAACCGGGAGGCTCCT
GTGCAGAGAAAGCGCCCTTGCCTCCCGCCCGCTCCCGGGGCTGCCCCACCCAGCCAGCTCCCCTACCTGGTGCCTGGGGC
GCCCCACGATGGAGGGGAAGACGGCCCGGGGGGCATCGTCGCCCGCGAAGCCGGCCTTGCACATGCCGGAGCCGTTGTCG
ACGACGAGCGCGGCGATATCATCATCCATGGTGAGCTGCGAGAATAGC

Sequence length: 367

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.

EnzymePatternGuide with Restriction SiteSuppliers
BslI/BseLI/Bsc4I/AfiICCNNNNNNNGG CCGTCTTCCCCTCCATCGTGGGG Life Technologies, NEB, SibEnzyme, 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: BslI/BseLI/Bsc4I/AfiI, Site: CCNNNNNNNGG, Restriction fragment lengths: 90bp, 14bp, 7bp, -4bp, 36bp, -10bp, 20bp, 11bp, 116bp
TCGATGGGGTACTTCAGGGTGAGGATGCCTCTCTTGCTCTGGGCCTCGTCGCCCACATAGGAATCCTTCTGACCCATGCC
CACCATCACGCCCTGGGAAGGAAAGGACAAGAAGCCCTGAGCACGGGCGCAGCCCCCACCCCGGAAACCGGGAGGCTCCT
GTGCAGAGAAAGCGCCCTTGCCTCCCGCCCGCTCCCGGGGCTGCCCCACCCAGCCAGCTCCCCTACCTGGTGCCTGGGGC
GCCCCACGATGGAGGGGAAGACGGCCCGGGGGGCATCGTCGCCCGCGAAGCCGGCCTTGCACATGCCGGAGCCGTTGTCG
ACGACGAGCGCGGCGATATCATCATCCATGGTGAGCTGCGAGAATAGC


PCR to amplify off-target sites

Primers for all off-targets can be downloaded from the Off-target PCR page.

Saturating mutagenesis using all guides

Oligonucleotides of all guides for pooled cloning into a lentiviral vector can be downloaded from the Saturating mutagenesis page.


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