GuideMaker: Globally design guide RNAs for any CRISPR-Cas system in any small genome

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Authors

• Ravin Poudel, PhD, Department of Microbiology and Cell Science, University of Florida
• Lidimarie Trujillo, Department of Microbiology and Cell Science, University of Florida
• Christopher Reisch, PhD, Department of Microbiology and Cell Science, University of Florida
• Adam R. Rivers, PhD , US Department of Agriculture, Agricultural Research Service

Installation

GuideMaker can be installed from:

1. The Github repository: https://github.com/USDA-ARS-GBRU/GuideMaker

    git clone https://github.com/USDA-ARS-GBRU/GuideMaker.git
    cd GuideMaker
    pip install -e .

    ## check if the installation works
    guidemaker -h

    ## Web app
    streamlit run guidemaker/app.py 

Dependencies

• pybedtools
• NMSLib
• Biopython
• Pandas
• Streamlit for webapp
• altair for plotting

Usage

GuideMaker: Globally design guide RNAs for any CRISPR-Cas system in any small genome

optional arguments:

  -h, --help            show this help message and exit

  --genbank GENBANK [GENBANK ...], -i GENBANK [GENBANK ...]
                        One or more genbank .gbk or gzipped .gbk files for a
                        single genome

  --pamseq PAMSEQ, -p PAMSEQ
                        A short PAM motif to search for, it may use IUPAC
                        ambiguous alphabet
  --outfile OUTFILE, -o OUTFILE
                        The table of PAM sites and data
  --pam_orientation {5prime,3prime}, -r {5prime,3prime}
                        PAM position relative to target: 5prime:
                        [PAM][target], 3prime: [target][PAM]. For example,
                        Cas9 is 3prime
  --guidelength [10-27], -l [10-27]
                        Length of the guide sequence
  --lsr [0-27]          Length of a seed region near the PAM site required to
                        be unique
  --dist [0-5]          Minimum hamming distance from any other potential
                        guide. Default dist is >= 2.
  --before [1-500]      keep guides this far in front of a feature
  --into [1-500]        keep guides this far inside (past the start site)of a
                        feature
  --knum [2-20]         Number of sequences similar to the guide to report
  --controls CONTROLS   Number or random control RNAs to generate
  --threads THREADS     Number of cpu threads to use
  --log LOG             Log file
  --tempdir TEMPDIR     The temp file directory
  --restriction_enzyme_list List of sequence representing restriction enzymes
  --keeptemp            Option to keep intermediate files
  --plot                Option to generate plots
  --config CONFIG       Path to YAML formatted configuration file
  -V, --version         show program's version number and exit

Examples

Use case: Make 20 nucleotide guide sequences for SpCas9 (NGG) in Carsonela ruddii. The length of the seed region near the PAM requred to be unique in each guide is 11 nucleotides.

    guidemaker \
    -i test/test_data/Carsonella_ruddii.gbk \
    -p NGG \
    --pam_orientation 3prime \
    --guidelength 20 \
    --lsr 11 \
    -o OUTDIR \
    --threads 2

Running Web App locally

streamlit run guidemaker/app.py 

API documentation

API documentation for the module can be found here

License information

Guidemaker was created by the United States Department of Agriculture - Agricultural Research Service (USDA-ARS) this software is available under CC0 1.0 Universal (CC0 1.0) Public Domain Dedication