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README 

        
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  CasOT - CRISPR/Cas system (Cas9/gRNA) Off-Targeter

******************************************************



CasOT is a genome-wide potential Cas9/gRNA off-target searching tool.



REQUIREMENT

===========

Perl program needs to be installed (http://www.perl.org/get.html).



EXAMPLES

========



All files of examples can be downloaded from 

http://eendb.zfgenetics.org/casot/example.php



Example 0: get a quick help.

----------------------------

    perl casot.pl -h



Example 1: search potential off-targets with default options.

    perl casot.pl -t=example -g=GRCh37



    It searches for potential off-target sites of the targets within the file 

    example in the genome sequence file GRCh37, in a single-gRNA mode, with 

    default PAM type and numbers of mismatches in the seed & non-seed regions 

    allowed (equal to use the options -s=2 -n=255 -p=A).



    The output directory is example-GRCh37-s2/, including a statistic file 

    (_stat) and some result files named by each input target sites in .csv 

    (common seperated version) format (in this example, they are CCR5_#L.csv, 

    CCR5_#R.csv, eGFP_#L.csv, and eGFP_#R.csv).



Example 2: search potential off-targets with some user-specified options.

    perl casot.pl -t=example -g=GRCh37 -e=r73h.gtf -o=tab -s=1 -n=4 -p=B



    It is similar to the above example, while the PAM type allowed is level B 

    (-NGG and -NAG) and only one mismatch allowed in the seed region and two 

    mismatches allowed in the non-seed region. The potential off-target site 

    is checked whether is located in an exon based on the exon-annotation file 

    r37h.gtf.



    The output directory is example-GRCh37-s1n4pB/ and the result files in it 

    are in tabular .txt format.



Example 3: search potential off-targets in the paired-gRNA mode.

    perl casot.pl -t=example -g=GRCh37 -o=tab -m=paired



    It searches for potential off-target sites of the targets in the paired-

    gRNA mode. This is a mode designed for the usage of Cas9-nickase and a 

    pair of gRNAs (see reference of CasOT).



    The output includes the result files of single-gRNA mode, and more files 

    of the paired-gRNA search results for each target pair. (in this example, 

    they are CCR5.txt and eGFP.txt).



Example 4: search potential off-targets in the target-and-off-target mode.

    perl casot.pl -t=example_seq -g=GRCh37 -m=target



    It searches for candidate target site from the input sequence 

    example_seq, and then search for potential off-target sites of them.



    The output also includes a file of candidate target sites (_sites).



OPTIONS

=======



Options -t and -g are required.



General options

---------------

    -h, -help

        Immediately show a quick help.

    -m, -mode

        -- single (default), paired, or target

        Three searching modes are available: the single-gRNA (single), the 

        paired-gRNA (paired), and the target-and-off-target mode (target).

    -t, -target ( *Required* )

        -- Name of a file in FASTA format. 

        File name of target sites (single- or paired-gRNA mode) or sequence 

        to search candidate target sites (target-and-off-target mode). In 

        single- or paired-gRNA mode, all sequences should be ended with -NGG 

        (the PAM) and 21-33 nt in length (18-30-nt protospacer plus the PAM). 

        In paired-gRNA mode, same sequence names should be followed with `_#F' 

        and `_#R' suffixes in pair. In target-and-off-target mode, input 

        sequences should be <1 kb.

    -g, -genome ( *Required* )

        -- Name(s) of one or more files in FASTA format. 

        File names of the genome sequence or other sequences to search for 

        potential off-target sites. Links to download several widely-used 

        genome files are available in the CasOT website. This option can be 

        used more than once for search in multiple genomes, such as:

            -g genome1 -g genome2

    -e, -exon 

        -- Name of the exon annotation file in GTF format.

        File name of the exon annotation of certain genome. Links to several 

        annotation files are available in the CasOT website. If provided, gene 

        IDs and gene symbols will be output if a potential off-target site 

        locates in an exon. The sequence names in the annotation file should 

        be identical to those in the genome sequence file, i.e., `chr1' is not 

        equal to `1'.

    -o, -output 

        -- csv (default) or tab. 

        Output file format. The default .csv (common separated version) file 

        (csv) can be directly opened by most spreadsheet software such as 

        Microsoft Excel. The tabular .txt file (tab) is more readable in text 

        editors and can also be copy-pasted to the spreadsheet software.



Off-target related options for all three modes

----------------------------------------------

    -s, -seed 

        -- 0-6 (default: 2). 

        Maximum number of mismatches allowed in the seed region of potential 

        off-target sites. If this parameter is > 4 (-p=5 or -p=6), a large RAM 

        of the computer is needed.

    -n, -nonseed 

        -- 0-255 (default: 255). 

        Maximum number of mismatches allowed in the non-seed region of 

        potential off-target sites. 

    -p, -pam 

        -- A (default), B, C, or N 

        Allowed level of PAM type. A: -NGG only; B: -NGG and -NAG; C: -NGG, 

        -NAG and -NNGG; N: no limit.



Option for paired-gRNA mode

--------------------------

    -d, -distance 

        -- 0-1000 (default: 100) 

        Maximum distance (i.e., number of nucleotides) allowed between the two 

        potential off-target sequences of an input paired-gRNA.



Options for target-and-off-target mode

--------------------------------------

    -r, -require5g 

        -- yes (default) or no. 

        Allowed type of candidate target sites.

        If the value is no, only -NGG type of PAM is required; if it is yes, a 

        G in the first position is required as well (if T7 promoter is used 

        for gRNA transcription, the first nucleotide in the RNA transcript 

        will be guanine).

    -l, -length 

        -- Two numbers between 18 and 30 connected by a dash (default: 19-20). 

        Allowed range (nt) of protospacer length of the candidate sites.



CONTACT

=======

Website:   http://eendb.zfgenetics.org/casot/

E-mail:    [email protected]



REFERENCE

=========

The manuscript is under review.