Yeastriction webservice

Yeastriction is a tool to find CRISPR/Cas9 target sites in various yeast genomes. It first extracts all possible Cas9 target sequences (20 basepairs followed by NGG) from a specified ORF and from its complementary strand. Subsequently, sequences containing 6 or more Ts are discarded as this can terminate transcription (Braglia et al., 2005; Wang et al., 2008). Target sequences are then tested for off-targets (an off-target is defined as a sequence with either the NGG or NAG PAM sequence and 17 or more nucleotides identical to the original 20 bp target sequence (Hsu et al., 2013)) by matching the sequences against the reference genome using Bowtie (version 1) (Langmead et al., 2009). If any off-target is found the original target sequence is discarded. In a next step, the AT content is calculated for the target sequence. Using the RNAfold library (essentially with the parameters --MEA --noLP –temp=30.) (Lorenz et al., 2011) the maximum expected accuracy structure of each RNA molecule is calculated. The target sequence is also searched for the presence of restriction sites based on a default list or a user-defined list. The targets can be ranked based on presence of restriction sites (1 for containing and 0 for lacking a restriction site), AT content (1 having the highest AT-content and 0 for the lowest AT-content) and secondary structure (1 having the lowest amount of pairing nucleotides and 0 for the highest number of nucleotides involved in secondary structures (indicated by brackets)). The range for every parameter is determined per locus and used to normalize the values. Subsequently, the target sequences are ranked by summation of the score for each parameter. These ranking scores should only be used to order the targets from a single locus and not to compare targets for different loci.

Deploy your own CRISPR/Cas9 service

The tool is written in Javascript and based on the MEAN.io stack (MongoDB, Express, AngularJS and Node.js). Running it on your own workstation requires therefore, Node.JS, MongoDB and NPM. You can also run it as a Dokku container on a service like DigitalOcean.

1. First install Dokku.
2. Next you need to install the following plugins:

• dokku-apt
• dokku-mongodb-plugin
• dokku-persistent-storage

3. Push your Yeastriction copy to the dokku server.

4. Modify in /home/dokku/<yourapp> the file ENV to include

export NODE_ENV="production"
export GENOMES_DIR="/genomes/"

5. Edit in the same directory the file PERSISTENT_STORAGE to link the GENOMES_DIR in the container to a directory on the server, e.g.:

/home/<username>/genomes:/genomes

6. Create a MongoDB database for the newly created app: dokku mongodb:create <app>

7. In a webbrowser go your Yeastriction instance and add a user.

8. Using dokku mongodb:console <app> add the 'admin' role to your newly created user.

9. In your genomes directory for every strain you want to include you need a few files:

• STRAIN.tab (contains tab delimited data of all the orfs, including their up and down region)
  The file should contain the following fields (seperated by tabs):

  YAL001C   TFC3       1001    4574    ACTTGTAAAT...
  

  The first field refers to the systematic name, the second to the symbolic name, followed by the start and stop position of the ORF within the sequence followed by the sequence of the ORF including its up and down sequence.

• STRAIN.fsa (the complete genome of the strain, if you've run bowtie, you don't actually need them anymore)

• STRAIN.(rev.)?(1|2).ebwt (files outputed by bowtie using the command bowtie-build -r STRAIN.fsa STRAIN)

10. Next import your strain by going to the URL http://<yoururl>/crispr/import/STRAIN

FAQ

dokku-apt-plugin quits with an error

If the error is something like Err: http://archive.ubuntu.com quantal/main amd64 Packages 404 Not Found, follow the instructions at DigitalOcean.

Is Yeastriction only relevant for finding targets in yeast genomes?

Although Yeastriction is built with Saccharomyces cerevisiae in mind, it can be easily modified to work with other organisms. To do so, alter public/crispr/controllers/crispr.js to match your ORF symbols or systematic names and server/controllers/loci.js to distinguish between symbolic name or systematic name.

More Information

• See our paper:
  Robert Mans, Harmen M. van Rossum, Melanie Wijsman, Antoon Backx, Niels G.A. Kuijpers, Marcel van den Broek, Pascale Daran-Lapujade, Jack T. Pronk, Antonius J.A. van Maris, Jean-Marc G. Daran (2015) CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Research 15. [PubMed] [FEMS Yeast Research]

References

• Braglia P, Percudani R & Dieci G (2005) Sequence context effects on oligo(dT) termination signal recognition by Saccharomyces cerevisiae RNA polymerase III. J Biol Chem 280: 19551–19562.
• Hsu PD, Scott D A, Weinstein J A, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31: 827–832.
• Langmead B, Trapnell C, Pop M & Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10: R25.
• Lorenz R, Bernhart SH, Höner Zu Siederdissen C, Tafer H, Flamm C, Stadler PF & Hofacker IL (2011) ViennaRNA Package 2.0. Algorithms Mol Biol 6: 26.
• Wang Q & Wang L (2008) New methods enabling efficient incorporation of unnatural amino acids in yeast. J Am Chem Soc 130: 6066–6067.

License

The MIT License