BiooTools

Latest updates. 28 Jul 15
1. sgRNAcas9_3.0.5.zip  (Standalone installer package)

sgRNAcas9 can design sgRNA for any species!

It's a command-line tool, which can run on the Windows, Mac OS X and Linux system.

This software requires the pre-install Perl on the computer. See the README.txt file for detailed instruction.

Manually download and install Perl for your computer (http://www.perl.org/).

Sequence Format Converter (FASTA file): http://www.simgene.com/ReadSeqor http://sequenceconversion.bugaco.com/converter/biology/sequences/

Ensembl Genome Browser, manually download human, mouse, rat, pig etc. genome DNA FASTA files from FTP Download (http://asia.ensembl.org/info/data/ftp/index.html?redirect=no) website.

Usage example(for windows system):

(1) sense strand searching mode
perl sgRNAcas9_3.0.5.pl -i hEMX1_example.txt -x 20 -l 40 -m 80 -g genome_example.fa -o s -t s -v w -n 5

(2) anti-sense strand searching mode
perl sgRNAcas9_3.0.5.pl -i hEMX1_example.txt -x 20 -l 40 -m 80 -g genome_example.fa -o a -t s -v w -n 5

(3) both strand searching mode
perl sgRNAcas9_3.0.5.pl -i hEMX1_example.txt -x 20 -l 40 -m 80 -g genome_example.fa -o b -t s -v w -n 5

(4) paired-gRNA searching mode
perl sgRNAcas9_3.0.5.pl -i hEMX1_example.txt -x 20 -l 40 -m 80 -g genome_example.fa -o b -t p -v w -n 5 -s 5 -e 35

 

Usage example(for linxu system):

Using the chmod command to change the access mode of  Seqmap program(in the "Seqmap" file).

chmod +x seqmap-1.0.12-linux

chmod +x seqmap-1.0.12-linux-64


(1) sense strand searching mode
perl sgRNAcas9_3.0.5.pl -i hEMX1_example.txt -x 20 -l 40 -m 80 -g genome_example.fa -o s -t s -v l -n 5

(2) anti-sense strand searching mode
perl sgRNAcas9_3.0.5.pl -i hEMX1_example.txt -x 20 -l 40 -m 80 -g genome_example.fa -o a -t s -v l -n 5

(3) both strand searching mode
perl sgRNAcas9_3.0.5.pl -i hEMX1_example.txt -x 20 -l 40 -m 80 -g genome_example.fa -o b -t s -v l -n 5

(4) paired-gRNA searching mode
perl sgRNAcas9_3.0.5.pl -i hEMX1_example.txt -x 20 -l 40 -m 80 -g genome_example.fa -o b -t p -v l -n 5 -s 5 -e 35

 

 

2. sgRNAcas9_Java  (GUI)

Manually download and install Java (alsoknown as Java Runtime Environment or JRE) for your computer (http://www.java.com/zh_CN/).

Manually download and install Perl for your computer (http://www.perl.org/).



3. There are 23 columns in the sgRNAcas9 output file. Their meanings are:

field                                                     meaning

sgRID                   ID of the CRISRP target site

Start                     start sites  

End                      End sites

CRISPR_target_sequence    CRISRP target sequence(5'-3')    

GC%                    GC content

OT                        annotation of off-targets with No.of mismatches <=5

0M(on-/off-)          on target site, if 0M >1, Repeat gene? or off-target sites with 0 bp mismatch

1M                        off-target sites with 1 bp mismatch

2M                        off-target sites with 2 bp mismatches

3M                        off-target sites with 3 bp mismatches

4M                        off-target sites with 4 bp mismatches

5M                        off-target sites with 5 bp mismatches

No.OT                   total number of off-target sites

POT                      annotation of off-targets with perfect match to the 12-nt seed region of thesgRNA (No.of mismatches <=5)

0M(on-/off-)          perfect match to the 12-nt seed region of the sgRNA, outer segments with 0mismatch

1M                        perfect match to the 12-nt seed region of the sgRNA, outer segments with 1mismatch

2M                        perfect match to the 12-nt seed region of the sgRNA, outer segments with 2mismatches

3M                        perfect match to the 12-nt seed region of the sgRNA, outer segments with 3mismatches

4M                        perfect match to the 12-nt seed region of the sgRNA, outer segments with 4mismatches

5M                        perfect match to the 12-nt seed region of the sgRNA, outer segments with 5mismatches

No.POT                 total number of off-targets sites with perfect match to the 12-nt seed regionof the sgRNA  

Risk_evaluation     evaluation of risk

   

Risk:

Discard > High_risk > moderate_risk> low_risk > repeat_sites_or_bad ? > Best