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4. Advanced usage

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Noonanav edited this page Nov 15, 2021 · 6 revisions

Overview

This page provides an example of a common advanced usage of sgRNAble:

  • Genome-wide sgRNA library design for genome-wide screening in a user-defined host.

Setup

This advanced usage requires the identification of all open reading frames (ORFs) in the host genome. This will be accomplished here using the Prodigal protein-coding gene prediction tool1 (see GitHub repo here). Installation instruction can be found through the GitHub repo, or Prodigal can be installed in an Anaconda enviroment using the commands below:

conda create --name Prodigal
conda activate Prodigal
conda install -c bioconda prodigal
conda deactivate

Table of Contents

Genome-wide sgRNA libary design

The following example describes the usage of sgRNAble to design a library of sgRNAs targeting all chromosomal genes in an E. coli MG1655 host.

Setup

Navigate to a working directory in which sgRNAble can be run. Place input files in sub-directory named /data (this directory structure is not necessary and relative or absolute paths can be used to select input or direct output anywhere on machine).

Input Files

This example requires the installation of Progidal, as described above, and the following file that be downloaded from the sgRNAble GitHub repository in the sgRNAble/tests/data/ directory:

  • ecoli_genome.fasta - host genome

Commands

1) Using Prodigal to identify ORFs in the host genome

If Prodigal was installed following the instructions above, activate the Prodigal Anaconda environment using:

conda activate Prodigal

The following command will identify protein coding ORFs in the E. coli MG1655 host genome (ecoli_genome.fasta) and output a .fasta file (ecoli_target_ORFs.fasta) containing the nucleotide sequences of all the open-reading frames predicted by Prodigal. This will serve as the TARGET_SEQUENCE input file for sgRNAble.

prodigal -i ./data/ecoli_genome.fasta -d ./data/ecoli_target_ORFs.fasta

Check you /data directory for the ecoli_target_ORFs.fasta output file.

2) Using sgRNAble to design sgRNAs for all ORFs in the host genome

The following command will identify and rank 10 sgRNAs targeting each predicted ORF in the E. coli MG1655 host and provide output in a directory named ecoli_library_output. sgRNAble will use 4 threads and a miximum of 2GB of RAM.

sgrnable -t ./data/ecoli_target_ORFs.fasta \
         -g ./data/ecoli_genome.fasta \
         -o ./ecoli_library_output \
         -th 4 \
         -m 2

Optional arguments can be used to specify additional sgRNA design criteria.

Citation

  1. Hyatt, D., Chen, GL., LoCascio, P.F. et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11, 119 (2010). https://doi.org/10.1186/1471-2105-11-119

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