Of note: If you've been using FlashFry before version 1.9, the command-line system has changed slightly.
FlashFry is a fast and flexible command-line tool for characterizing large numbers of potential CRISPR target sequences. FlashFry can be used with any genome, and can run against non-traditional model organisms or transcriptomes. If you're looking to characterize a smaller region or would like a nice web interface we recommend the GT-scan or crispor.org websites.
The easiest way to get started it to try out the quick-start procedure to make sure everything works on your system. If everything looks good, there are few more in-depth tutorials to try out various capacities of FlashFry. Thanks to @drivenbyentropy for the Java implementation of the ViennaRNA energy calculations.
- Quick start
- General options documentation
- Scoring methods
- FAQ
- Database format documentation
- Tutorials - on the wiki
- Citing FlashFry
First, make sure you're running Java version 8 (type java -version
on the command line to see the version). From the UNIX or Mac command line, download the latest release version of the FlashFry jar file:
wget https://github.com/mckennalab/FlashFry/releases/download/1.15/FlashFry-assembly-1.15.jar
Download and then un-gzip the sample data for human chromosome 22:
wget https://raw.githubusercontent.com/aaronmck/FlashFry/master/test_data/quickstart_data.tar.gz
tar xf quickstart_data.tar.gz
Then run the database creation step (this should take a few minutes, it takes ~75 seconds on my laptop):
mkdir tmp
java -Xmx4g -jar FlashFry-assembly-1.15.jar \
index \
--tmpLocation ./tmp \
--database chr22_cas9ngg_database \
--reference chr22.fa.gz \
--enzyme spcas9ngg
Now we discover candidate targets and their potential off-target in the test data (takes a few seconds). Here we're using the EMX1 target with some sequence flanking the target site. This flanking sequnce is needed by on-target scoring metrics to fully evaluate the target's efficiency:
java -Xmx4g -jar FlashFry-assembly-1.15.jar \
discover \
--database chr22_cas9ngg_database \
--fasta EMX1_GAGTCCGAGCAGAAGAAGAAGGG.fasta \
--output EMX1.output
Finally we score the discovered sites (a few seconds):
java -Xmx4g -jar FlashFry-assembly-1.15.jar \
score \
--input EMX1.output \
--output EMX1.output.scored \
--scoringMetrics doench2014ontarget,doench2016cfd,dangerous,hsu2013,minot \
--database chr22_cas9ngg_database
There should now be a set of scored sites in the EMX1.output.scored
. Success! Now check out the documentation and tutorials for more specific details.
FlashFry is published in BMC Biology; if you find it useful please cite:
TY - JOUR
AU - McKenna, Aaron
AU - Shendure, Jay
PY - 2018
DA - 2018/07/05
TI - FlashFry: a fast and flexible tool for large-scale CRISPR target design
JO - BMC Biology
SP - 74
VL - 16
IS - 1
AB - Genome-wide knockout studies, noncoding deletion scans, and other large-scale studies require a simple and lightweight framework that can quickly discover and score thousands of candidate CRISPR guides targeting an arbitrary DNA sequence. While several CRISPR web applications exist, there is a need for a high-throughput tool to rapidly discover and process hundreds of thousands of CRISPR targets.
SN - 1741-7007
UR - https://doi.org/10.1186/s12915-018-0545-0
DO - 10.1186/s12915-018-0545-0
ID - McKenna2018
ER -