#GuideMaker

Globally design gRNAs for any CRISPR-Cas system in any small genome

Authors

• Lidimarie Trujillo, Department of Microbiology and Cell Science, University of Florida
• Ravin Poudel, PhD, 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

Introduction

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/guiemaker.git

Dependencies

Following are the required softwares/programs.

• pybedtools
• NMSLib
• Biopython
• Pandas

Usage

GuideMaker: globally design guide RNAs for any CRISPR-Cas system in any 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
  --strand {forward,reverse,both}, -s {forward,reverse,both}
                        Strand of DNA to search
  --lcp [0-27]          Length of the guide closest to the PAM required to be
                        unique
  --dist [1-5]          Minimum hamming distance from any other potential
                        guide
  --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]         how many sequences similar to the guide to report
  --threads THREADS     The number of cpu threads to use
  --log LOG             Log file
  --tempdir TEMPDIR     The temp file directory
  -V, --version         show program's version number and exit

Examples

Use case: Retrieving target sequence for a given PAM motif in the forward and reverse strands, where length of guide sequence is 20 base pair. 12 base pair close to PAM motif is conserved ,i.e. unique and the full sequence has a hamming distance of more than 2. Here the number of used threads is 2 Return a table of pam sites and associated data, at in current folder.

    guidemaker -i sample.gbk -p NGG --targetlength 20 --strand both \
    --lcp 10 --dist 2 --outfile out.txt \
    --log logfile.txt --threads 2

API documentation

API documentation for the module can be found here

License information

As a work of the United State Governemnt this software is available under CC0 1.0 Universal (CC0 1.0) Public Domain Dedication