How do I set the off-target database for CRISPR?

The "Find CRISPR sites" function in Geneious will search for off-target binding sites against a database of sequences. To do this, you will need to create a database of the sequences you wish to test against for off-target binding - this is typically the whole genome of your organism of interest, but can include other sequences, for example, the targeting vector.

To do this, you need to create a new empty folder in Geneious and import the sequences you want to test against in to this folder. As there are a wide variety of genomes researchers may wish to test against; genome sequences can be very large; and new versions of genome assemblies may be released, Geneious does not contain inbuilt copies of any full genome sequences. Genomes can be downloaded directly from NCBI using the "NCBI" folders at the bottom of the Sources panel in Geneious (this is the panel on the left hand side of Geneious). Commonly studied genomes (for example, the human, zebrafish and the rat genomes) can also be downloaded from NCBI using the links found in the "Genomes" folder in the "Sample Documents" in Geneious. Genomes can be also be download from other sources and imported into Geneious using common file formats. This database of off target sequences can be any size and can contain any sequences, although the larger this database is, the longer the search for CRISPR sites will take.

In the "Find CRISPR Sites" settings, click the button next to "Off-target database" and select the folder in Geneious containing the off-target sequences.

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Comments

  • Avatar
    Mihail Sarov

    The CRISPRs design feature was a great addition to Geneious 8, however it is currently painfully slow. Searching a 50-100bp region for guides with the mouse or human genome as off-target references could take 10 or more minutes, orders of magnitude slower than most online services. Searching a bigger region usually slows down to a crawl with fractions of a percent progress per minute and i give up. If the performance of this tool can be optimised it will be very useful indeed, I am afraid the current speed is just not usable. Smaller genomes like worm and fly work much better.

    I have to say it's not a memory problem, I have 6GB RAM allocated to Geneious and they are not used up by the time it slows down. And it's not file access (to a server database) problem either, as I tried the same with locally stored sequences and off target database and it's about as bad.

  • Avatar
    Hilary Miller

    The reason Geneious is slower than online databases is that online CRISPR services generally have a predefined set of genomes available, which are already indexed and stored for searching.  In Geneious, we allow you to fully customize and choose whatever genome you wish, but that means that all the indexing has to be done on the fly so it is much slower to search.  The larger the guide region, the slower it will be as there are more sites to search.  Also bear in mind that the progress bar doesn't run in a linear fashion as some steps take longer than others, so you may find it slows down at one point then speeds up again.  

     

  • Avatar
    Mihail Sarov

    i get all these points plus of course online tools are run on servers much faster than my macbook but still, it's just way too slow. Can't we have the genomes we use all the time indexed once and quick to search the next time?