WARNING: Cas-OFFinder 3 is not production ready yet, it is known that the result can be different from that of Cas-OFFinder 2. For production use please use the latest Cas-OFFinder 2 instead.

Cas-OFFinder

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 component 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)

Download

Cas-OFFinder binaries are available at: https://github.com/snugel/cas-offinder/releases

For further information about installation, check out below Installation section.

CRISPR/Cas-derived RNA-guided endonucleases (RGEN)

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)

Usage

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 IDs in addition to G, C, A to limit number of devices used by Cas-OFFinder. A range of device IDs can be specified by using comma or colon to separate the IDs. See examples below.

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 3.0.0 beta (Jan 24 2021)

Copyright (c) 2021 Jeongbin Park and Sangsu Bae
Website: http://github.com/snugel/cas-offinder

Usage: cas-offinder {input_filename|-} {C|G|A}[device_id(s)] {output_filename|-}
(C: using CPUs, G: using GPUs, A: using accelerators)

Example input file (DNA bulge size 2, RNA bulge size 1):
/var/chromosomes/human_hg38
NNNNNNNNNNNNNNNNNNNNNRG 2 1
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 2015, 2017 and 2019.

Now you should create an input file:

The first line is a directory containing FASTA or 2BIT files,
The second line indicates the desired pattern including PAM site and optional DNA or RNA bulge sizes, separated by spaces,
The remaining lines are the query sequences and maximum mismatch numbers, separated by spaces,
Optionally, you can specify an ID for each query sequence, which will be included in the output (Id column).

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 an input file:

/var/chromosomes/human_hg38
NNNNNNNNNNNNNNNNNNNNNRG 2 1
GGCCGACCTGTCGCTGACGCNNN 3 Seq1
CGCCAGCGTCAGCGACAGGTNNN 3 Seq2
...

Save it as input.txt.

Now you can run Cas-OFFinder as following (using GPUs):

$ ./cas-offinder input.txt G output.txt

Optionally, you can set the ID of devices to select a specific device used by Cas-OFFinder. For example, to use the second GPU device (0-based):

$ ./cas-offinder input.txt G1 output.txt

You can use commas or colons to select range of devices:

$ ./cas-offinder input.txt G0,1 output.txt

$ ./cas-offinder input.txt G0:2 output.txt

Then the TAB separated output file will be generated with the following columns:

The first column is the sequence id, by default line numbers (0-based), or given IDs from the input file,
The second column is the type of bulge, the value is one of X, DNA, or RNA,
The third column is the given query sequence (including gaps in case of DNA bulge),
The fourth column is the actual sequence at the location (including gaps in case of RNA bulge; mismatched bases are noted in lowercase letters),
The fifth column is the sequence name (if you downloaded it from UCSC or Ensembl, it is usually a chromosome name),
The sixth column is the 0-based location of the off-target site (same convention as Bowtie, not 1-based as IGV Viewer and others),
The seventh column is the direction of sequence, either forward (+) or reverse (-) strand of the found sequence,
The eighth column is the number of the mismatched bases,
The last column is the size of bulge.

output.txt will be (shown here as a table for convenience):

Id	Bulge type	crRNA	DNA	Chromosome	Location	Direction	Mismatches	Bulge Size
Seq2	DNA	`CGCCAGCGTCAGCGACAGG--TNNN`	`CcCCAGtGTCAGCcACAGGGCTCAG`	chr1	34978273	-	3	2
Seq2	DNA	`CGC--CAGCGTCAGCGACAGGTNNN`	`CaCTCCAGCcTCAGCGACAGGcAAG`	chr1	18173251	+	3	2
Seq2	DNA	`CG--CCAGCGTCAGCGACAGGTNNN`	`CaCTCCAGCcTCAGCGACAGGcAAG`	chr1	18173251	+	3	2
Seq2	DNA	`C--GCCAGCGTCAGCGACAGGTNNN`	`CACtCCAGCcTCAGCGACAGGcAAG`	chr1	18173251	+	3	2
Seq1	DNA	`GGCCGACC--TGTCGCTGACGCNNN`	`GGCCcAgCTCTGTCGCTGACGgGAG`	chr1	40979785	+	3	2
Seq1	DNA	`GGCCGACCTGTCGCTGA--CGCNNN`	`GGCCGtCCTGTtGCTGAGACtCGGG`	chr1	17408102	-	3	2
Seq1	DNA	`GGCCGACCTGTCGCTG--ACGCNNN`	`GGCCGtCCTGTtGCTGAGACtCGGG`	chr1	17408102	-	3	2
Seq1	DNA	`GGCCGACCTGTCGCT--GACGCNNN`	`GGCCGtCCTGTtGCTGAGACtCGGG`	chr1	17408102	-	3	2
…
Seq2	X	`CGCCAGCGTCAGCGACAGGTNNN`	`CtCCAGCcTCAGCGACAGGcAAG`	chr1	18173253	+	3	0
Seq2	RNA	`CGNCCCAGCGTCAGCGACAGGTNNN`	`C-CCCCAGtGTCActGACAGGTGGG`	chr1	5502832	-	3	1
Seq2	RNA	`CGNCCCAGCGTCAGCGACAGGTNNN`	`t-CtCCAGCcTCAGCGACAGGcAAG`	chr1	18173254	+	3	1
Seq2	RNA	`CGCCGCAGCGTCAGCGACAGGTNNN`	`CG-CGCAGCGaCAGgGAgAGGTGAG`	chr1	1273663	-	3	1
Seq2	RNA	`CGCCGCAGCGTCAGCGACAGGTNNN`	`CGC-GCAGCGaCAGgGAgAGGTGAG`	chr1	1273663	-	3	1
Seq2	RNA	`CGCCAGCGTCGCAGCGACAGGTNNN`	`CGCCtGCG-CGgAGCtACAGGTGAG`	chr1	18888560	+	3	1
Seq2	RNA	`CGCCAGCGTCGCAGCGACAGGTNNN`	`aGCCAGCt-CtCAGCGACAGcTGAG`	chr1	91631879	+	3	1
Seq2	RNA	`CGCCAGCGTCGTAGCGACAGGTNNN`	`CGCCtGCGg-GgAGCtACAGGTGAG`	chr1	18888560	+	3	1
Seq2	RNA	`CGCCAGCGTCAGCGGCACAGGTNNN`	`CcCCAGaGTCAGC-GCACAGaTGGG`	chr1	4006295	-	3	1
Seq2	RNA	`CGCCAGCGTCAGCGACGAAGGTNNN`	`tGCCAGCGgCAGCGA-GAAGtTTAG`	chr1	8462729	+	3	1
…

Advanced Usage

Cas-OFFinder is mainly designed for CRISPR/Cas9 derived RGENs, however, it can also 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_hg38
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
TTCTGGAGGTGCCTGAGGCCNNNNNNNNNNNNGAGGCCACCTTTCCAGTCCA 5
TGGCCAATGTGACGCTGACGNNNNNNNNNNNNCTGGAGACTCCAGACTTCCA 5
....

Installation

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:
- https://compubench.com/result.jsp
- http://www.luxrender.net/luxmark/top/top20/Room/GPU/1
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://github.com/snugel/cas-offinder/releases

or compile it from its source code (below section).

Compile

The OpenCL library is required to compile Cas-OFFinder.

The OpenMP library is optional, for faster processing of 2BIT files.

To support cross-platform compilation on various operating systems, the CMake build system is used. See http://www.cmake.org for more information.

If you use Ubuntu linux, you can also install it via apt-get, apt-get install cmake.

If you use macOS and homebrew, you can also install it via brew, brew install cmake.

Otherwise, download CMake here.

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’.)

Assuming the CMake binary is installed in C:\Program Files (x86)\CMake 3.19\bin, type the following:

  "C:\Program Files (x86)\CMake 3.19\bin\cmake.exe" -G "NMake Makefiles" .
  nmake

Then cas-offinder binary will be generated. Copy it wherever you want.

If you have difficulty compiling cas-offinder, cmake can output system information to a file. Please open a GitHub issue and attach the file.

  $ cmake --system-information cmake-sys-info.log

Module reference

For reading/parsing FASTA files, the kseq.h library (developed by Heng Li) is used.

The library is distributed under MIT licence.

More information at: http://lh3lh3.users.sourceforge.net/parsefastq.shtml
For supporting Dirent API on Windows environment, Dirent API for Microsoft Visual Studio is used.

More information at: http://softagalleria.net/dirent.php

Download & Source

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

Publication

Cas-OFFinder is discussed in a paper published in the journal Bioinformatics, Volume 30, Issue 10, 15 May 2014, Pages 1473–1475, https://doi.org/10.1093/bioinformatics/btu048

Changelog

3.0.0
- Native support of DNA/RNA bulges
- Update output format to display bulge information
- Code cleanup / Better library handling by CMake (@richardkmichael)
2.4.1
- Corrected warnings, code cleanups, and document updates (@richardkmichael)
- Follow symlinks (@richardkmichael)
- Allow stdin for input (@richardkmichael)
- Tagging of input sequences (#28)
- Github Actions based CI & automatic release
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.

License

Cas-OFFinder (except dirent.h) is licensed under the new BSD licence.

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.
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.