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Guide sequence: CCCGTGCTTGAACCGCACCA TGG

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

NamePrimer Sequence
guideRna53rvT7sense TAGGCCCGTGCTTGAACCGCACCA
guideRna53rvT7antisense AAACTGGTGCGGTTCAAGCACGGG

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:

NamePrimer Sequence
guideRNA53rvT7crTarget GAAATTAATACGACTCACTATAGCCCGTGCTTGAACCGCACCAGTTTTAGAGCTAGAAATAGCAAG
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:

NamePrimer Sequence
guideRNA53rvGeneArtFw TACGACTCACTATAGCCCGTGCTTGAACCGCACCA
guideRNA53rvGeneArtRev TTCTAGCTCTAAAACTGGTGCGGTTCAAGCACGGG

U6 expression from an Addgene plasmid

The guide sequence CCCGTGCTTGAACCGCACCA 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 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:
NamePrimer Sequence
gN20-guideRNA53rvU6senseMLM3636 ACACCGCCCGTGCTTGAACCGCACCAG
gN20-guideRNA53rvU6antisenseMLM3636 AAAACTGGTGCGGTTCAAGCACGGGCG

Primers for gN19 guides:
Kim et al 2020. showed that changing the first nucleotide to 'G' is slightly more efficient.

NamePrimer Sequence
gN19-gN20-guideRNA53rvU6senseMLM3636 ACACCGCCGTGCTTGAACCGCACCAG
gN19-gN20-guideRNA53rvU6antisenseMLM3636 AAAACTGGTGCGGTTCAAGCACGGCG

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.
NameOligonucleotide Sequence
batchOligo53rv GGAAAGGACGAAACACCGCCCGTGCTTGAACCGCACCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC

Summary of main cloning/expression primers

guideRNA53rvGeneArtFw TACGACTCACTATAGCCCGTGCTTGAACCGCACCA
guideRNA53rvGeneArtRev TTCTAGCTCTAAAACTGGTGCGGTTCAAGCACGGG
gN20-guideRNA53rvU6senseMLM3636 ACACCGCCCGTGCTTGAACCGCACCAG
gN20-guideRNA53rvU6antisenseMLM3636 AAAACTGGTGCGGTTCAAGCACGGGCG
guideRna53rvT7sense TAGGCCCGTGCTTGAACCGCACCA
guideRna53rvT7antisense AAACTGGTGCGGTTCAAGCACGGG
guideRNA53rvT7crTarget GAAATTAATACGACTCACTATAGCCCGTGCTTGAACCGCACCAGTTTTAGAGCTAGAAATAGCAAG
guideRNAallT7common AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC
gN19-gN20-guideRNA53rvU6senseMLM3636 ACACCGCCGTGCTTGAACCGCACCAG
gN19-gN20-guideRNA53rvU6antisenseMLM3636 AAAACTGGTGCGGTTCAAGCACGGCG

PCR to amplify the on-target site

Not found in genome, cannot design primer

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

Click here to list mutated guides sorted by off-targetactivity

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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CRISPR/Cas9 Guide Designer for chordate vertebrate ecdysozoans lophotrochozoans protostomes spongi corals plants butterflies metazoans genomes fruitflies insects nematodes mammals.