Cas-OFFinder is OpenCL based, ultrafast and versatile program that searches for potential off-target sites of CRISPR/Cas-derived RNA-guided endonucleases (RGEN).
Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif (PAM) sequences recognized by Cas9, the essential protein com-ponent in RGENs.
Requires an OpenCL device to run properly.
Cas-OFFinder is distributed under new BSD license (3-clauses).
Cas-OFFinder has been tested on the platforms below:
- Microsoft Windows (7 and 8)
- GNU/Linux (CentOS, OpenSUSE, Debian, Ubuntu/Elementary OS)
- Mac OS X (Mavericks)
RGENs use complementary base pairing to recognize target sites.
RGENs consist of two parts.
- Guide RNA, as:
- Dual RNA components comprising sequence-invariant tracrRNA and sequence-variable guide RNA termed crRNA, or,
- Single-chain guide RNA (sgRNA) constructed by linking essential portions of tracrRNA and crRNA
- Cas9 Protein
- A fixed protein component that recognizes the protospacer adjacent motif (PAM) downstream of target DNA sequences corresponding to guide RNA.
PAM sites:
- SpCas9 from Streptococcus pyogenes: 5’-NGG-3’ (to a lesser extent, 5’-NAG-3’)
- StCas9 from Streptococcus thermophilus: 5’-NNAGAAW-3’ (W = A or T)
- NmCas9 from Neisseria meningitidis:5’-NNNNGMTT-3’ (M = A or C)
- SaCas9 from Staphylococcus aureus: 5’-NNGRRT-3’ (R = A or G)
Cas-OFFinder can run with:
cas-offinder {input_filename|-} {G|C|A}[device_id(s)] {output_filename|-}
G stands for using GPU devices, C for using CPUs, and A for using accelerators.
(Optionally, you can set device ID in addition to G/C/A to limit number of devices used by Cas-OFFinder)
The special filename - may be used in place of the input and output filename to read and write
from stdin and stdout, respectively.
A short example may be helpful!
First, download any target organism's chromosome FASTA files. You can find one in below links:
- http://hgdownload.soe.ucsc.edu/downloads.html (UCSC genome sequences library)
- http://ensembl.org/info/data/ftp/index.html (Ensembl sequence library)
Extract all FASTA files in a directory.
For example (human chromosomes, in POSIX environment):
$> wget http://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/chromFa.tar.gz
$> mkdir -p /var/chromosome/human_hg19
$> tar zxf chromFa.tar.gz -C /var/chromosome/human_hg19
$> ls -al /var/chromosome/human_hg19
drwxrwxr-x. 2 user group 4096 2013-10-18 11:49 .
drwxrwxr-x. 16 user group 4096 2013-11-12 12:44 ..
-rw-rw-r--. 1 user group 254235640 2009-03-21 00:58 chr1.fa
-rw-rw-r--. 1 user group 138245449 2009-03-21 01:00 chr10.fa
-rw-rw-r--. 1 user group 137706654 2009-03-21 01:00 chr11.fa
-rw-rw-r--. 1 user group 136528940 2009-03-21 01:01 chr12.fa
-rw-rw-r--. 1 user group 117473283 2009-03-21 01:01 chr13.fa
-rw-rw-r--. 1 user group 109496538 2009-03-21 01:01 chr14.fa
...
Now, download Cas-OFFinder binary here,
https://sourceforge.net/projects/cas-offinder/files/Binaries
and save it to any directory you want.
And just try running it for a short help:
$> ./cas-offinder
Cas-OFFinder v2.4 (Aug 15 2016)
Copyright (c) 2013 Jeongbin Park and Sangsu Bae
Website: http://github.com/snugel/cas-offinder
Usage: cas-offinder {input_file} {C|G|A}[device_id(s)] {output_file}
(C: using CPUs, G: using GPUs, A: using accelerators)
Example input file:
/var/chromosomes/human_hg19
NNNNNNNNNNNNNNNNNNNNNRG
GGCCGACCTGTCGCTGACGCNNN 5
CGCCAGCGTCAGCGACAGGTNNN 5
ACGGCGCCAGCGTCAGCGACNNN 5
GTCGCTGACGCTGGCGCCGTNNN 5
Available device list:
Type: GPU, ID: 0, <GeForce GTX 980> on <NVIDIA CUDA>
Type: GPU, ID: 1, <GeForce GTX 980> on <NVIDIA CUDA>
Also it provides a list of all available OpenCL devices!
On Windows, if you encountered a missing .dll error, you may need to download and install Visual C++ Redistributable Packages for Visual Studio 2013.
Now you should create an input file:
- The first line of the input file gives directory path containing chromosomes FASTA files,
- The second line indicates the desired pattern including PAM site,
- The remaining lines are the query sequences and maximum mismatch numbers, separated by spaces. (The length of the desired pattern and the query sequences should be the same!)
For the pattern and the query sequences, mixed bases are allowed to account for the degeneracy in PAM sequences.
Also, the number of mismatched bases is not limited!
Following codes are supported:
| A | C | G | T |
|---|---|---|---|
| Adenine | Cytosine | Guanine | Thymine |
| R | Y | S | W | K | M |
|---|---|---|---|---|---|
| A or G | C or T | G or C | A or T | G or T | A or C |
| B | D | H | V | N |
|---|---|---|---|---|
| C or G or T | A or G or T | A or C or T | A or C or G | any base |
An example of input file:
/var/chromosomes/human_hg19
NNNNNNNNNNNNNNNNNNNNNRG
GGCCGACCTGTCGCTGACGCNNN 5
CGCCAGCGTCAGCGACAGGTNNN 5
ACGGCGCCAGCGTCAGCGACNNN 5
GTCGCTGACGCTGGCGCCGTNNN 5
...
Save it as 'input.txt'.
Now you can run Cas-OFFinder as following (using GPUs):
$> ./cas-offinder input.txt G out.txt
...
Optionally, you can set the ID of devices to limit the number of devices used by Cas-OFFinder:
$> ./cas-offinder input.txt G1 out.txt
...
You can use commas, or colons for setting range:
$> ./cas-offinder input.txt G0,1 out.txt
or
$> ./cas-offinder input.txt G0:2 out.txt
...
Then output file will be generated :
- The first column is the given query sequence,
- The second column is the FASTA title (if you downloaded it from UCSC or Ensembl, it is usually a chromosome name),
- The third column is the 0-based position of the off-target site (same convention as Bowtie, not 1-based as IGV Viewer and others),
- The fourth column is the actual sequence from the position (mismatched bases noted in lowercase letters),
- The fifth column is the forward strand(+) or reverse strand(-) of the found sequence,
- The last column is the number of the mismatched bases.
out.txt:
GGCCGACCTGTCGCTGACGCNNN chr8 49679 GGgCatCCTGTCGCaGACaCAGG + 5
GGCCGACCTGTCGCTGACGCNNN chr8 517739 GcCCtgCaTGTgGCTGACGCAGG + 5
GGCCGACCTGTCGCTGACGCNNN chr8 599935 tGCCGtCtTcTCcCTGACGCCAG - 5
GGCCGACCTGTCGCTGACGCNNN chr8 5308348 GGCaGgCCTGgCttTGACGCAGG - 5
GGCCGACCTGTCGCTGACGCNNN chr8 9525579 GGCCcAgCTGTtGCTGAtGaAAG + 5
GGCCGACCTGTCGCTGACGCNNN chr8 12657177 GGCCcACCTGTgGCTGcCcaTAG - 5
GGCCGACCTGTCGCTGACGCNNN chr8 12808911 GGCCGACCaGgtGCTccCGCCGG + 5
GGCCGACCTGTCGCTGACGCNNN chr8 21351922 GGCCcACCTGaCtCTGAgGaCAG - 5
GGCCGACCTGTCGCTGACGCNNN chr8 21965064 GGCCGtCCTGcgGCTGctGCAGG - 5
GGCCGACCTGTCGCTGACGCNNN chr8 22409058 GcCCGACCccTCcCcGACGCCAG + 5
...
Cas-OFFinder is mainly designed for CRISPR/Cas9 derived RGENs, however, it is also can be used for searching off-targets of other nucleases, e.g. TALENs(Transcription activator-like effector nucleases) or ZFNs(Zinc-finger nucleases), by specifying pattern sequence as all 'N's.
Example input file for TALENs:
/var/chromosomes/human_hg19
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
TTCTGGAGGTGCCTGAGGCCNNNNNNNNNNNNGAGGCCACCTTTCCAGTCCA 5
TGGCCAATGTGACGCTGACGNNNNNNNNNNNNCTGGAGACTCCAGACTTCCA 5
....
Cas-OFFinder can handle RNA/DNA bulges by using a small wrapper script, found here.
-
Cas-OFFinder requires an OpenCL-enabled device and corresponding runtime API pre-installed to run properly.
OpenCL is supported on various platforms, including many Intel/AMD CPUs and NVidia/AMD graphic cards! Before installing Cas-OFFinder, please check whether your device is an OpenCL-supported one.
Khronos group provides an extensive list of supported devices here:
http://www.khronos.org/conformance/adopters/conformant-products/#opencl
Cas-OFFinder usually runs faster on GPUs than CPUs. If you want to purchase a new graphic card for fast analyzing speed, please check GPU benchmark results below as a reference:
Recently, the OpenCL runtime binaries are already shipped with the device drivers in many cases - so you don't have to install anything to run Cas-OFFinder.
But if it wasn't, you should download and install a proper OpenCL SDK to install runtime APIs. In that case, download an OpenCL SDK among the links below. If you know your device's vendor name, it is enough to install only your vendor's one.
-
Download Cas-OFFinder binary here,
https://sourceforge.net/projects/cas-offinder/files/Binaries
or compile it from its source code (below section).
OpenCL library is required to compile Cas-OFFinder.
To support cross-platform compilation on various operating systems, CMake build system is used (more informations on http://www.cmake.org).
First, download CMake here (http://www.cmake.org/cmake/resources/software.html). If you use Ubuntu linux, you can also install it via apt-get. (apt-get install cmake)
Checkout the source code of Cas-OFFinder with Git client, or download it manually on github website.
In POSIX environment (g++ should be pre-installed), launch terminal and type the following to build Cas-OFFinder:
cmake -G "Unix Makefiles"
make
On Windows (Visual Studio should be pre-installed), launch 'Visual Studio Command Prompt' (You can find it under 'Start menu' - 'Microsoft Visual Studio xxxx' - 'Visual Studio Tools') and type the following (Assuming that the CMake binary is installed in 'C:\Program Files (x86)\CMake 2.8\bin'):
"C:\Program Files (x86)\CMake 2.8\bin\cmake.exe" -G "NMake Makefiles"
nmake
Then cas-offinder binary will be generated. Copy it wherever you want.
-
For reading/parsing FASTA files, the kseq.h library (developed by Heng Li) is used.
The library is distributed under MIT licence.
More informations on: http://lh3lh3.users.sourceforge.net/parsefastq.shtml
-
For supporting Dirent API on Windows environment, Dirent API for Microsoft Visual Studio is used.
More informations on: http://softagalleria.net/dirent.php
The binaries can be downloaded from
https://sourceforge.net/projects/cas-offinder/files/Binaries
And the source code is distributed from
https://github.com/snugel/cas-offinder
- 2.4
- Corrected critical bug (The last 3 bases of 2bit input could be wrong)
- Corrected bug (Segmentation fault if the match occurs at the very first location in chromosome)
- Corrected bug (Cas-OFFinder does not follow symbolic links)
- Now user can limit number of devices used by Cas-OFFinder.
- Now Cas-OFFinder reports the name of platform.
- Now user can set '-' as output file, then the output will be redirected to stdout. All other messages from Cas-OFFinder will be directed to stderr.
- 2.3
- Removed cl.hpp due to lack of C++ binding support in the new OpenCL 2.0 standard.
- Constant arguments are stored in constant or local memory, rather than global memory.
- Added support for 2bit format.
- Removed kseq.h
- Precise running time measurment on POSIX platform.
- 2.2
- Corrected a critical bug (when cas-offinder finds no binding sites in the given genome chunk, it crashes).
- Now Cas-OFFinder reads whole fasta file at once, in order to achieve faster searching speed when it searches in FASTA files which contain many small scaffolds.
- 2.1
- Using atomic operation, reduced computing load on CPU. In our benchmark, the total computation speed increased about twice as fast as before.
- When lowercase sequences are given, convert them uppercase sequences before computation.
- Corrected a bug (mixed bases were shown as lowercases letters, even they had been matched with normal bases).
- Now supports 'accelerators', with 'A' option.
- 1.1
- When Cas-OFFinder is launched without parameters, now it display available device list.
- If the given chromosomes directory does not exist, now it returns an error message.
- Corrected a bug (when Cas-OFFinder couldn't find any OpenCL device it would hang).
- 1.0
- Initial release.
Cas-OFFinder (except dirent.h) is licensed under the new BSD licence.
Copyright (c) 2013, Jeongbin Park and Sangsu Bae All rights reserved.
Redistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met:
-
Redistributions of source code must retain the above copyright notice, this list of conditions and the following disclaimer.
-
Redistributions in binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution.
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Neither the name of the Seoul National University nor the names of its contributors may be used to endorse or promote products derived from this software without specific prior written permission.
THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS "AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE COPYRIGHT OWNERS OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.