SNP CRISPR

SNP-targeted CRISPR sgRNA design pipeline. Online version available at: https://www.flyrnai.org/tools/snp_crispr

Publication: https://fgr.hms.harvard.edu/publications/snp-crispr-web-tool-snp-specific-genome-editing

Prerequisites

• Anaconda or Miniconda for environment management
• Species genome in fasta format

Note: To install without conda, see environment.yml for dependencies

Install

• From the project directory run conda env create -f environment.yml
• Then conda activate snp_crispr
• FlyBase release 6.28 genome fasta file and blast database are included for testing. dm.fasta.zip must be unzipped before running. Remaining installation steps are only necessary for other species
• Download species genome fasta file and put in fasta_files/<species>.fasta
• Run makeblastdb -in fasta_files/<species>.fasta -dbtype nucl -parse_seqids -title <species> -out blast_dbs/<species>
• Species chromosome names -> fasta id mapping files included in fasta_files/<species>_chr_ids.txt

Important Note: <species> name/abbreviation in fasta_files/<species>.fasta, fasta_files/<species>_chr_ids.txt, and blast_dbs/<species> must all match

Species Genome Fasta Download Links

Human - https://www.ncbi.nlm.nih.gov/genome/?term=txid9606
Mouse - https://www.ncbi.nlm.nih.gov/genome/?term=txid10090
Zebrafish - https://www.ncbi.nlm.nih.gov/genome/?term=txid7955
Rat - https://www.ncbi.nlm.nih.gov/genome?term=txid10116

Usage

• Add SNP's or INDEL's of interest to input csv file, see sample_input.csv for format
• Input files in VCF format are also supported (file must have .vcf extension)
• From the project directory run ./snp_crispr.sh <species> <input_file> <PAM> <threads> <-all/-both> <-alt>
• Both -NGG and -NAG PAM sequences supported
• Running with the optional -all argument designs guides where all SNPs are targeted within the 23-mer
• Running with the optional -both argument designs guides where all SNPs are targeted within the 23-mer as well as individually
• Running with the optional -alt argument evaluates the specificity of SNP targeted CRISPRs against a user provided non-reference genome. Before using this option the makeblastdb installation step must be repeated using the non-reference genome fasta file and the non-reference blast db title and output filenames must be <species>_alt
• All arguments are required except -all/-both and -alt. Also note that the order of the arguments must match the provided example
• Design results found in results/designs.csv
• Input variants without any viable designs are logged in results/no_designs.csv
• No viable designs may be found targeting a variant due to the lack of a neighboring PAM sequence, presence of the U6 terminator (TTTT), or the unavailability of a unique sgRNA sequence
• To test the pipeline run ./snp_crispr.sh dm sample_input.csv -NGG 2 and compare the output in results/designs.csv with results/expected_results.csv

Note: The pipeline can be run using any species or genome assembly by adding a new chromosome name to fasta id mapping file in the same format as the examples in fasta_files/<species>_chr_ids.txt and then following the same installation instructions

Multi-Core Performance

• 2 threads recommended on personal computers with limited RAM
• Increasing the number of threads can significantly increase memory usage depending on the genome size
• Setting the threads argument to 2 or more will improve performance when using inputs that span across multiple chromosomes and/or when using the -both argument
• When running on a cluster or other environment with large amounts of memory available, using as many threads as chromosomes in the input will give optimal performance

Example: A test run using 10 threads on an input of 10,000 human snps took ~9 minutes and used ~50 GB of RAM
(Results will vary depending on the machine, input, species, and options used)

Example Commands

• Fly: ./snp_crispr.sh dm dm_snps.csv -NGG 2 -all -alt
• Human: ./snp_crispr.sh hs hs_snps.vcf -NAG 6 -both
• Mouse: ./snp_crispr.sh mm mm_snps.csv -NGG 3

Design Scores

Housden Efficiency Score

• These scores were computed using a position matrix. Detailed information about the input dataset and the algorithm can be found in Housden et al. Sci Signal. 2015 https://www.ncbi.nlm.nih.gov/pubmed/26350902
• Scores range from 1.47-12.32 (higher is better, > 5 recommended)

Off Target Score

• Calculated based on sgRNA sequence blast results.
• Scores range from 0-5441.73 (lower is better, < 1 recommended)

Precomputed Human Clinically Associated SNP Targeted Designs

• Precomputed CRISPR design results provided in clinical_hs_snp_designs.csv
• Clinically associated variant data from Ensembl

Authors

Jonathan Rodiger - jrodiger
Verena Chung - vpchung

License

This project is licensed under the MIT license - see LICENSE for details

Acknowledgements

Perrimon Lab (https://perrimon.med.harvard.edu/)
DRSC (https://fgr.hms.harvard.edu/)