Using CRISPOR with gen_sgRNAs.py

CRISPOR is a scoring functionality adapted from the tool described in this publication. We incorporated its scoring functionality to enable users to easily use predicted specificity of guides when designing their experiments. We did not include efficiency predictions because ExcisionFinder is intended to be used to identify genomic excisions, and efficiency scores describe the likelihood of a cut to disturb its locus via indels.

Setting up a Conda environmnet

For the CRISPOR element of gen_sgRNAs.py to work, you need to install the relevant dependencies. We encompassed these in an Anaconda environment file, crispor.yml, located in the scripts/ directory of ExcisionFinder. To install this environment, navigate to the scripts/ directory and use the following command:

conda env create -f crispor.yml

Downloads

Additionally, you need the relevant genome files for the reference genome you're using. If you make a directory in your project directory titled genomes, you can download all needed files using a CRISPOR script as such:

bash ExcisionFinder/scripts/crispor/downloadGenome.sh hg19 genomes

which would download necessary files for human reference hg19 to your genomes directory.

After you download the genome files, use the tool twoBitToFa to generate a fasta file from the 2bit file. Then, delete the "segments" file, as this makes CRISPOR glitch.

Directory

You have to be in your genomes directory in order to run ExcisionFinder with CRISPOR, although this is something we're looking to fix in the future. This is so the tool can find the genomes correctly. For example, if you downloaded hg38 to a directory titled genomes, such that the directory structure is genomes/hg38/hg38/hg38.fa, you should be in the genomes/ directory when you run the gen_sgRNAs.py script.

Additional outputs

When you use the --crispor command with gen_sgRNAs.py, it will output a file titled crispor_error.txt. This simply contains any errors CRISPOR may have outputted. You don't need to worry about this file unless no output is generated by gen_sgRNAs.py, in which case it can be used to debug.