Package: aceofbases
ACEofBASEsis a tool to determine sites to be editted with the CRISPR/Cas9 technology
in a input sequence and predict its potential off-target sites. The online version ofACEofBASEsis available athttp://aceofbases.cos.uni-heidelberg.de/This is a command line version ofACEofBASEsthat is designed mainly to allow search
of large volume of sequences and higher flexibility.If you use this tool for your scientific work, please cite it as:
Cornean, A., Gierten, J., Welz, B., Mateo, J.L., Thumberger, T. and Wittbrodt, J.
Stemmer, M., Thumberger, T., del Sol Keyer, M., Wittbrodt, J. and Mateo, J.L.Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction.eLife (2022).RequirementsACEofBASEsis implemented in Python and it requires a version 3.5 or above.In addition we relay on the short read aligner Bowtie 1 to identify the
off-target sites. Bowtie can be downloaded from this sitehttp://bowtie-bio.sourceforge.net/index.shtmlin binary format for the main
platforms.
You need to create an indexed version of the genome sequence of your
target species. This can be done with the tool bowtie-build included in the
Bowtie installation. For that you simply need a fasta file containing the genome
sequence. To get the index you can do something like:$ bowtie-build -r -f <your-fasta-file> <index-name>The previous command will create the index files in the current folder.To handle 2bit files and, optionally, gene and exon annotations we use the python librarybx-python.The exon and gene files contain basically the coordinates of those elements inbed format, which are the first
three columns of the file. The exon file can contain two more columns with the
ID and name of the corresponding gene.
You can generate easily such kind of files for you target organism using the
scriptgff2bedFilesincluded in this package. As the name
of this script suggests, you only need a GFF file with the annotation.
Additionally, you can also useEnsembl Biomart,
if your species is available there, to generate files complying with these
requirements.In case of difficulties with these files contact us and we can provide you the
files you need or help to generate them on your own.InstallPlease, refer to the fileINSTALL.md.UsageAfter a successful installation you should have the mainACEofBASEsexecutable,
together with the script to generate the gene/exons files, ready to be used.
You can runACEofBASEswith the -h flag to get a detailed list of the available
parameters. For instance:$ aceofbases -hAt minimum it is necessary to specify the input (multi)fasta file (--input), the
Bowtie index (--index) and the 2bit file (--twobit). In this caseACEofBASEsassumes that the Bowtie and blat executables can be found in thePATHsystem variable,
there are not gene and exon files to use and the rest of parameters will take default values.
Notice that the index parameter to specify here refers to the name of the
index, without any file extension, together with the path, if necessary.A command for a typical run will look something like this:$ aceofbases --input <query.fasta> --index <path/index-name> --twobit <file.2bit> --output <output-folder>The result of the run will be three files for each sequence in the input query
file. These files will have extension .fasta, .xls and html, containing,
respectively, the sequence of the target sites, their detailed information either
as tab separated file that can be open with any spreadsheet program or the html file to be open
with any web browser. The name of the output file(s) will be taken from the
name of the sequences in the input fasta file.Generating Exon/Gene filesFor any species you have to work with it is very likely that there is an
annotation file in GFF format. From any of these files you can generate
the files thatACEofBASEsneeds to annotate the off-target sites.
The scriptgff2bedFilesexpects as first argument the input file inGFF version 3format.
Files in this format can be usually found with their corresponding assemblies
in the web sites NCBI or Ensembl.
With the input file downloaded, it doesn't need to be uncompressed if it is in
gz format, specify it as first argument to the script followed by
the prefix you prefer for the output files.$ gff2bedFiles <input-gff> <prefix>The result will be two files named<prefix>_exons.bed.gzand<prefix>_genes.bed.gz.
These files are compressed, to save space, and can be passed directly toACEofBASEs.Docker imageACEofBASEsis also available as a Docker image athttps://hub.docker.com/r/juanlmateo/aceofbasesThis image contains everything ready to useACEofBASEs.
Simply download the image with this commanddocker pull juanlmateo/aceofbases:latestWith this image you can run the commandsaceofbasesandgff2bedFiles, but also
you can run Bowtie to create the index of your target species or faToTwoBit to create the 2bit file.Below you have an example that shows how to get CRISPR/Cas candidates for a
sequence using the yeast as target species. This example shows all the steps,
from creating the Bowtie index, the exon and gene files to the generation of
the final output.# downloading the genome of the target species in fasta forma
wget ftp://ftp.ensembl.org/pub/release-105/fasta/saccharomyces_cerevisiae/dna/Saccharomyces_cerevisiae.R64-1-1.dna.toplevel.fa.gz
# building the bowtie index from the fasta file
docker run -v `pwd`:/data/ aceofbases bowtie-build -r -f Saccharomyces_cerevisiae.R64-1-1.dna.toplevel.fa.gz saccharomyces_cerevisiae
# downloading the annotation of this assembly in GFF format
wget ftp://ftp.ensembl.org/pub/release-105/gff3/saccharomyces_cerevisiae/Saccharomyces_cerevisiae.R64-1-1.105.gff3.gz
# generating the exon and gene files
docker run -v `pwd`:/data/ aceofbases gff2bedFiles Saccharomyces_cerevisiae.R64-1-1.105.gff3.gz saccharomyces_cerevisiae
# generating the 2bit file
ocker run -v `pwd`:/data/ aceofbases faToTwoBit Saccharomyces_cerevisiae.R64-1-1.dna.toplevel.fa.gz yeast.2bit
# defining the input sequence(s)
echo -e ">YDL194W\nATGGATCCTAATAGTAACAGTTCTAGCGAAACATTACGCCAAGAGAAACAGGGTTTCCTA" > test.fa
# running ACEofBASEs
docker run -v `pwd`:/data/ aceofbases aceofbases --input test.fa --index saccharomyces_cerevisiae --twobit yeast.2bit --exons saccharomyces_cerevisiae_exons.bed.gz --genes saccharomyces_cerevisiae_genes.bed.gz