1. What data does the tool use to predict the effect of mismatches on sgRNA binding to an off-target site?
The tool leverages experimental data collected by the Zhang laboratory at the Broad Institute (see reference).

2. Where can I find the 5'->3' human genome sequence of the region I want to target?
There are many sources, but we recommend the UCSC human genome browser here. Be sure you are using human genome assembly hg19 (Feb 2009). Enter the chr:pos of the region you would like to target. In the browser window click on "View" in the upper right corner, and scroll down to "DNA". This will take you to a page where you can enter the chr and position location of the region for which you would like to retrieve DNA sequence.

3. I want to optimize CRISPR design across a very large region (>500bp). What should I do?
We find that there is usually a reasonably specific sgRNA sequence available within a ~500bp region. Thus, to reduce the time the tool takes to run, we have capped the input sequence length to 500bp. If you are trying to pick a single best sgRNA across a very large region, we recommend that you simply pick a smaller region, look at your results, and then submit another job with a different region only if no guide sequences of high quality are found. If you are trying to design multiple CRISPRs across a large region to target different sites, we recommend that you submit each one as a separate design job (e.g. break up your input sequence into smaller chunks, one for each target site).

4. I want to design CRISPRs for a non-human genome. Will this tool work?
The current tool is only designed to identify sgRNA sequences and off-target sites in the HUMAN genome, so it will NOT work for CRISPR design in other species. Stay tuned, however, for updates that will enable design for mouse and rat genomes!

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