ssCRISPR

ssCRISPR was created by Austin Rottinghaus.

ssCRISPR can be used to design gRNAs with strain-specific cleavage profiles

This code accompanies the manuscript: Computational design of CRISPR guide RNAs to enable strain-specific control of microbial consortia

Relevant files

Files provided on GitHub: https://github.com/Austin-Rottinghaus/ssCRISPR

1. All_NCBI_Strains.xlsx - Excel sheet containing the strain database used in the program
2. Cas9_gRNA_prediction.py - Standalone script used to predict the efficiency of Cas9 gRNAs using the finalized_model_cas9.sav machine learning model
3. Cas9_gradient boosting.py - Python script used to generate the finalized_model_cas9.sav machine learning model
4. Cas_icon3.ico - Icon used for the ssCRISPR GUI
5. Fastq_analyze_Ecoli.py - Python script used to analyze E. coli next generation sequenceing results
6. Fastq_analyze_pseudo.py - Python script used to analyze Pseudomonas next generation sequenceing results
7. cpf1_gRNA_prediction.py - Standalone script used to predict the efficiency of Cpf1 gRNAs using the finalized_model_cpf1.sav machine learning model
8. cpf1_gradient boosting.py - Python script used to generate the finalized_model_cpf1.sav machine learning model
9. finalized_model_cas9.sav - Machine learning model used to predict the efficiency of Cas9 gRNAs
10. finalized_model_cpf1.sav - Machine learning model used to predict the efficiency of LbCas12a gRNAs
11. ssCRISPR Script.py - Python script containing the GUI and code used to predict gRNAs

Relevant files provided at Mendeley Data: https://data.mendeley.com/datasets/gpgyytwgb5/2

12. ssCRISPR.zip

Tutorials

To use the ssCRISPR program as a stand-alone executable application, perform the following steps:

1. Download the zipped folder, ssCRISPR.zip, from Mendeley Data to your computer; https://data.mendeley.com/datasets/gpgyytwgb5/2
2. Unzip the folder in your desired location
3. Ensure that the following files remain in the same location at all times:
   • Cas_icon3.ico
   • finalized_model_cpf1.sav
   • finalized_model_cas9.sav
   • All_NCBI_Strains.xlsx
   • ssCRISPR.exe
4. Run the ssCRISPR.exe file
5. Fill out the following items in the user interface:
   • Your email address - Note: This is only used to request genome sequences from NCBI. You won't recieve any emails.
   • Whether you are using Cas9, Cpf1, or another Cas protein
   • PAM sequence in A, T, C, and G nucleotides - The program does not accept multi-nucleotide letters, such as R, S, and N.
   • The desired number of nucleotides of specificity
   • Target sequence length
   • PAM orientation
   • List of target strains
   • List of non-target strains
6. Click "Determine gRNAs"
7. When the program finishes running, a text file with the results can be downloaded to your computer by clicking "Download results"

To use the ssCRISPR program in Python, perform the following steps:

1. Download the following files from GitHub to your computer; https://github.com/Austin-Rottinghaus/ssCRISPR
   • Cas_icon3.ico
   • finalized_model_cpf1.sav
   • finalized_model_cas9.sav
   • All_NCBI_Strains.xlsx
   • ssCRISPR.py
2. Ensure that the files remain in the same location at all times:
3. Open the ssCRISPR.py script
4. Ensure that the following function packages are downloaded to your computer: Note, many come with come IDEs such as Anaconda Spyder
   • Bio, re, sys, os, openpyxl, picke, random, seqfold, itertools, math, time
5. Run the script
6. Fill out the following items in the user interface:
   • Your email address - Note: This is only used to request genome sequences from NCBI. You won't recieve any emails.
   • Whether you are using Cas9, Cpf1, or another Cas protein
   • PAM sequence in A, T, C, and G nucleotides - The program does not accept multi-nucleotide letters, such as R, S, and N.
   • The desired number of nucleotides of specificity
   • Target sequence length
   • PAM orientation
   • List of target strains
   • List of non-target strains
7. Click "Determine gRNAs"
8. When the program finishes running, a text file with the results can be downloaded to your computer by clicking "Download results"

Note: User-provided FASTA files can be used as target and non-target strains.

If you want to update the ALL_NCBI_Strains.xlsx file to include the most recent catalogue of sequences, perform the following steps:

1. Go to: https://www.ncbi.nlm.nih.gov/genome/browse#!/prokaryotes/
2. Filter by bacteria and complete genomes
3. Download the resutls table from the search as an excel file
4. Open the downloaded excel file and the All_NCBI_Strains.xlsx file provided with the program
5. Replace the "Organism name," "Strain," and "Replicons" columns in All_NCBI_Strains.xlsx with the same columns from the downloaded excel file
6. Drag the formula in the "Combined" column to apply to all rows with strains