/GunnVistaPingry_US

The code for GunnVistaPingry_US's Odigos Project

Primary LanguageHTMLGNU General Public License v3.0GPL-3.0

iGEM_Odigos

Odigos: An Improved CRISPR-Cas9 Effective Guide RNA Predictor

Abstract: CRISPRi is a powerful tool for modulating gene expression in human cells. By designing a gRNA homologous to the target gene of interest, one can achieve targeted knockdown of the specific gene of interest. However, with current methodologies, one has to screen multiple gRNA sequences for efficient targeting while minimizing off-target effects. We present a prediction model for identifying the best gRNA sequence for efficient gene targeting in human cells. Starting with experimental data from knocking down specific genes using several gRNAs in iPS cells, we leverage machine learning to inform better selection of the gRNA. Our tool will be invaluable for designing gene targeting gRNAs and will reveal underlying biochemical principles governing CRISPR efficiency.

This project contains

  • Training and test data from Dr. Perli's collected results in Shinya Yamanaka's Lab at Gladstone Institutes, UCSF
  • Source code to train a new model using RT qRT-PCR data with the pioneering code Horlbeck et al., eLife 2016]
  • Comparative study of Weissman algorithm score, our own project's scores, and Dr. Perli's efficiency measurements of each guide.
  • A tool to generate 10 small guide RNAs using our model and analyzes off-target stringency for genes in host genome.

Dependencies

External command line applications required:

Large genomic data files required:

Recommended HW Configuration:

  • Quad Core 16GB RAM, 100GB Storage Ubuntu 18.x/20.04 LTS

Setup:

Running:

  • It is recommended to use jupyter notebook server to run our software. This package contains the following notebooks.

    • iGEM_CRISPRi_Library_Design.ipynb : To train the model and create igem_v1_estimator that can be used to score and/or generate guideRnas.

    • iGEM_CRISPRi_sgRna_Score_Comparision.ipynb: To do the comparative study of scores of guideRnas across Weissman algorithm, iGEM algorithm and lab scores.

    • iGEM_CRISPRi_Gene_Guide_Selector.ipynb: To generate guides for a gene including offtarget filtering (Currently supports human and can be extended to different hosts)

    • Walk through of above note books also made available with corresponding html extension.

MAC OS X:

  • We observed it is easier to setup these packages and run them using Jupyter Notebook in Anaconda NAVIGATOR
  • Add bioconda,conda-forge to Anaconda default channel to install some of required packages above
  • On Some Mac systems we observed RNAfold giving an error about missing libmpfr.4.dylib. *If required install mpfr using "brew install mpfr" and ensure lib libmpfr.4.dylib is accessible to RNAfold binary.
  • bowtie can be installed from the following location https://sourceforge.net/projects/bowtie-bio/files/bowtie/1.3.0/bowtie-1.3.0-macos-x86_64.zip/download *Please ensure bowtie is add to PATH so that it can be invoked by our modules to run bowtie to do alignment related study.

LIMITATIONS

  • These modules are not supported on Windows systems because few bio modules required by application are not supported on windows.