CRISPRimmunity: the tool for CRISPR-associated Important Molecular events and Modulators Used in geNome edIting Tool identifYing
The CRISPR-Cas system is a highly adaptive and RNA-guided immune system found in bacteria and archaea that has applications as a genome editing tool and is a valuable system for studying co-evolutionary dynamics of bacteriophage interactions. This repository presents CRISPRimmunity, a new tool designed for Acr prediction, identification of novel class 2 CRISPR-Cas loci, and dissection of CRISPR-associated important molecular events. CRISPRimmunity is built on a series of CRISPR-oriented databases that offer a comprehensive co-evolutionary perspective of the CRISPR-Cas and anti-CRISPR protein systems. CRISPRimmunity also provide a web server, offering a well-designed graphical interface, a detailed tutorial, and multi-faceted information, making it easy to use and facilitating future experimental design and further data mining. The web server is accessible without registration at http://www.microbiome-bigdata.com/CRISPRimmunity/index/home.
The source code is written by python3. In addition, several tools have been applied in CRISPRimmunity.
First, please check for the following packages and install them as needed:
- Python packages：numpy，Biopython，sklearn，Levenshtein，re，lxml，requests，shutil，pandas，numpy，scipy，matplotlib，codecs，multiprocessing
- R packages：treeio，ggtree，ggrepel
Second, please check for the following tools and install them as needed:
- Prokka in https://github.com/tseemann/prokka
- diamond in https://github.com/bbuchfink/diamond
- BLAST+ in https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/
- HMMER in http://hmmer.org/download.html
- cd-hit in https://www.bioinformatics.org/cd-hit
- pilercr in https://www.drive5.com/pilercr/
- CRISPRCasFinder in https://github.com/dcouvin/CRISPRCasFinder
- CRISPRidentify in https://github.com/BackofenLab/CRISPRidentify
- AcRanker-master in https://github.com/amina01/AcRanker
- mafft in https://mafft.cbrc.jp/alignment/software/
- clustalo in http://www.clustal.org/omega/
- mview in https://github.com/desmid/mview
- FastTree in http://www.microbesonline.org/fasttree/
- step1: Clone the repository of CRISPRimmunity from Github
git clone https://github.com/HIT-ImmunologyLab/CRISPRimmunity.git- step2: Download CRISPRimmunity database and data for standalone from CRISPRimmunity webserver and check MD5
Due to the large size of the database file (~4G), we recommend using -c (continue) and -b (background) parameters of wget to avoid the data loss caused by network outages, and checking MD5 to verify the integrity of the downloaded files.
## md5 checksum: XXX
### Download Database
wget -c -b http://www.microbiome-bigdata.com/CRISPRimmunity/static/files/download/database.tar.gz
### Check md5 (e2ac0981ab07b4529d857c5c778f30e6 )
md5sum database.tar.gz
### Download Data
wget -c -b http://www.microbiome-bigdata.com/CRISPRimmunity/static/files/download/data.tar.gz
### Check md5 (6f577bbc661276e597b237f80870f2b0)
md5sum data.tar.gz- step3: Unzip the database and data file to specified subdirectory under CRISPRimmunity installation directory
### Unzip the database file
tar -zxvf database.tar.gz
cp -r <download_database_path>/database <CRISPRimmunity_path>/CRISPRimmunity
### Unzip the database file
tar -zxvf data.tar.gz
cp -r <download_data_path>/data <CRISPRimmunity_path>/CRISPRimmunityWhen you organize the whole files well,the corresponding directory structure are displayed as shown below.
- step4: Create soft links to dependent tools in the software directory
### Create soft links
ln -s <path>/prokka <CRISPRimmunity_path>/software/prokka
ln -s <path>/diamond <CRISPRimmunity_path>/software/diamond
ln -s <path>/blastn <CRISPRimmunity_path>/software/blastn
ln -s <path>/blastdbcmd <CRISPRimmunity_path>/software/blastdbcmd
ln -s <path>/rpsblast <CRISPRimmunity_path>/software/rpsblast
ln -s <path>/prodigal <CRISPRimmunity_path>/software/prodigal
ln -s <path>/hmmscan <CRISPRimmunity_path>/software/hmmscan
ln -s <path>/nhmmscan <CRISPRimmunity_path>/software/nhmmscan
ln -s <path>/CRISPRCasFinder.pl <CRISPRimmunity_path>/software/CRISPRCasFinder.pl
ln -s <path>/CRT1.2-CLI.jar <CRISPRimmunity_path>/software/CRT1.2-CLI.jar
ln -s <path>/pilercr <CRISPRimmunity_path>/software/pilercr
ln -s <path>/clustalo <CRISPRimmunity_path>/software/clustalo
ln -s <path>/mafft <CRISPRimmunity_path>/software/mafft
ln -s <path>/mview <CRISPRimmunity_path>/software/mview
ln -s <path>/FastTree <CRISPRimmunity_path>/software/FastTree
ln -s <path>/acranker.py <CRISPRimmunity_path>/software/acranker.py
### Copy CRISPRidentify to software directory
cp -r <path>/CRISPRidentify <CRISPRimmunity_path>/softwareCRISPRimmunity is an comprehensive tool for Acr prediction, identification of novel class 2 CRISPR-Cas loci, and dissection of CRISPR-associated important molecular events.
General:
--input <file name> : Genome file path: FASTA or GenBank file
--output <folder name> : Output folder in which results will be stored
--annotation <x> : function selection: effector,anti,self-targeting,prophage,MGE-targeting
--prog_dir <x> : directory of CRISPRimmunity
Acr prediction module:
--anti_flag <x> : flag for predicting homologs for known or novel Acrs, values: novel, known
--anti_identity <x> : minimum identity to report an alignment with konwn Acrs, default:0.4 [0-1]
--anti_coverage <x> : minimum coverage to report an alignment with konwn Acrs, default:0.7 [0-1]
--anti_up_num <x> : maximum protein number separated from HTH domain-containing proteins, default:3
--anti_prot_size <x> : maximum protein size for Acr (aa), default:400
Important molecular events annotation module:
Detection and classification of CRISPR-Cas event:
--att_spacer_num <x> : minimum spacer of the CRISPR array, default:2
--att_min_repeat_length <x>: minimum repeat length of the CRISPR array, default:18
--att_max_repeat_length <x>: maximum repeat length of the CRISPR array, default:45
--range <x> : length of the sequence of interest in the vicinity of the CRISPR array (bp), default:20000
--neighbor <x> : number of the protein of interest in the vicinity of the CRISPR array, default:10
--repeat_identity <x> : minimum identity to report an alignment with konwn type repeats, default:0.9 [0-1]
--repeat_coverage <x> : minimum coverage to report an alignment with konwn type repeats, default:0.9 [0-1]
Detection of self-targeting event:
--mismatch <x> : maximum mismatch to report an alignment with self-genome for self-targeting , default:2
--cov <x> : minimum coverage to report an alignment with self-genome for self-targeting, default:1
Detection of prophage event:
--prophage_thread_num <x> : number of threads (CPUs) to use in the prophage detection, default:3
Detection of MGE-targeting event:
--phage_mismatch <x> : maximum mismatch to report an alignment with MGE database for MGE-targeting, default:2
--phage_coverage <x> : minimum coverage to report an alignment with MGE database for MGE-targeting, default:0.7
Novel class 2 CRISPR-Cas loci identification module:
--att_protein_size <x> : minimum protein size for novel class 2 effector candidate, default:500
--att_neighbor_distance <x>: maximum protein number of interest in the vicinity of the CRISPR array for mining novel class 2 effector candidate, default:5
--homo_identity <x> : minimum identity to report an alignment with novel class 2 effector candidate, , default:0.3
--homo_coverage <x> : minimum coverage to report an alignment with novel class 2 effector candidate, default:0.6- Identification of novel class 2 effector protein:
python <path>/predict_crispr_cas_self_targeting.py --input <genome file path> --output <outdir> --annotation 'effector' --prog_dir <program directory>- Acr prediction:
python <path>/predict_crispr_cas_self_targeting.py --input <genome file path> --output <outdir> --annotation 'anti,self-targeting,prophage' --anti_flag novel --prog_dir <program directory>- Annotation of important molecular events:
python <path>/predict_crispr_cas_self_targeting.py --input <genome file path> --output <outdir> --annotation 'self-targeting,MGE-targeting,prophage,anti' --prog_dir <program directory>| File Name | Description | Module |
|---|---|---|
| crispr_cas_self-targeting_statis_result.txt | Overview of the result of CRISPR-associated event, including CRISPR array detection, Cas protein annotation, repeat type annotation, self-targeting detection. | IME, ACR, EFF |
| anti_statis_result.txt | Overview of the result of predicted Acrs. | IME, ACR |
| .merge_spc_blastn_fasta_add_ cov_filter_mismatch_2_cov_1_hit_not_in_array |
Detail of the result of sequence alignment between spacer and self-genome. | IME, ACR, EFF |
| merge_crispr_array.txt_merge.spc | Overview of the result of CRISPR array detection. | IME, ACR, EFF |
| merge_crispr_array.txt | Detail of the result of CRISPR array detection. | IME, ACR, EFF |
| _merge.csp | Sequence of the spacers. | IME, ACR, EFF |
| self-targeting/spacer_target_hit_result.txt | Detail of the result of self-targeting detection. | IME, ACR |
| self-targeting/*.*_multialign | Detail of the result of spacer-protospacer alignment. | IME, ACR |
| self-targeting/*.*_fa | Detail of the result of spacer-protospacer sequence. | IME, ACR |
| prophage/complete_prophage_summary.txt | Overview of the result of prophage detection. | IME, ACR |
| prophage/complete_prophage_detail.txt | Detail of the result of prophage region. | IME, ACR |
| prophage/complete_prophage_protein.faa | Protein sequence of prophage region. | IME, ACR |
| prophage/complete_prophage_nucl.fa | Nucleotide sequence of prophage region. | IME, ACR |
| interacting_phage/spacer_blastn_phage_filter.txt | Overview of the result of MGE-targeting detection. | IME, ACR |
| anti/acr_candidate_info_integrated_three_methods.txt | Overview of the predicted ant. | IME, ACR |
| novel_effector_protein/filter_repeat_homo_ protein_crispr_filter_result.txt |
Overview of the identified novel class 2 effector proteins | EFF |
| novel_effector_protein/effector_tree.png | Phylogenetic tree of novel class 2 effector candidates and known class 2 effector proteins | EFF |
IME：important molecular events detection module ACR：Acr prediction module EFF：novel class 2 effector proteins identification module
In the CRISPRimmunity package, there is a directory named "example" which includes input sequence and result for examples of three modules. "Armatimonadetes-bacterium-isolate-ATM2-J3.gb" is used for identifying novel class 2 effector proteins. "Staphylococcus-schleiferi-strain-5909-02.gb" is used for Acr prediction and annotation of important molecular events, respectively.
## run the example
python example_run.py(1) Result for identifying novel class 2 effector proteins
(2) Result for Acr prediction and annotation of important molecular events
This project exists thanks to all the people who contribute.
This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.