CRISPR-broad
Citation
Alaguraj Veluchamy, Kaian Teles and Wolfgang Fischle. CRISPR-broad: combined design of multitargeting gRNAs and broad, multiplex target finding (Sci Rep. 2023 Nov 12;13(1):19717.).**
https://pubmed.ncbi.nlm.nih.gov/37953351/
Version: 1.1.0
Date: 20-12-2022
Authors: Alaguraj Veluchamy alaguraj.v@gmail.com
The package is developed for the design of gRNA for the targeted epigenome modifications on a broader region.
License: GPL-3
Depends: bwa-0.7.11, Python 3.9.1, biopython 1.78, pandas 1.2.0 pyranges-0.0.95
Installing bwa: git clone https://github.com/lh3/bwa.git; cd bwa; make
Installing dependancies through pip: pip install biopython; pip install pandas; pip install pyranges
Installing CRISPR-broad:
git clone https://github.com/AlagurajVeluchamy/CRISPR-broad.git; cd CRISPR-broad
Description:
CRISPR-broad is a standalone tool that enables user to scan genome for regions that has high frequency of gRNA with user-supplied variation. This open source tool is robust and efficient in finding gRNAs with scores and ranks potential target region.
Input:
CRISPR-broad minimally only requires a genome file in fasta format, PAM sequences in string format "NGG".
QUICK RUN:
If you are planning to split genome into 40 parts in slurm based cluster: srun --time=4:00:00 --mem=100G --nodes=1 -c 40 --pty bash -l
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python crisprbroad.py genomesplit -d /Users/CRISPR -f Inputgenome.fa -t 40 -l 23 -g 50 -p GG
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python crisprbroad.py createindex -f Inputgenome.fa
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python crisprbroad.py maptogenome -d /Users/CRISPR -f Inputgenome.fa -t 40 -nm 5 -nx 10000 -m 2 -g 50 -l 230
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python crisprbroad.py filterhits -d /Users/CRISPR -t 40 -nm 5 -nx 10000
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python crisprbroad.py findwindow -d /Users/CRISPR -t 40 -p GG -w 10000 -l 23 -nm 5 -nw 5
Usage: To list all modules
usage: python crisprbroad.py [-h]
genomesplit, createindex, maptogenome, filterhits, findwindow, findmultiwindow, multisgrna
Module 1: Split genome into candidate gRNA sequences
python crisprbroad.py genomesplit -h
usage: crisprbroad.py genomesplit [-h]
-h, --help show this help message and exit
-f GENOMESPLITFASTA, --genome_fasta GENOMESPLITFASTA
Genome sequence in FASTA format
-q INPUTBED --query_bedfile query in bed format
optional input for finding sgRNA in a given region
-d WORKINGDIRECTORY, --working_directory WORKINGDIRECTORY
Complete path of output directory
-p PAMSEQUENCE, --pam_sequence PAMSEQUENCE
PAM sequence string eg: GG for NGG PAM
-l CANDIDATERNALENGTH, --get_candidaternalength CANDIDATERNALENGTH
Candidate gRNA length
-g GC, --get_gc GC gc content in integer
-t THREADS, --num_threads THREADS
Launch t number of threads in parallel
Module 2: Create index
python crisprbroad.py createindex -h
usage: python crisprbroad.py createindex [-h]
-h, --help show this help message and exit
-f GENOMESPLITFASTA, --genome_fasta GENOMESPLITFASTA
Genome sequence in FASTA format
Module 3: Map gRNA to genome
python crisprbroad.py maptogenome -h
usage: crisprbroad.py maptogenome [-h]
-h, --help show this help message and exit
-f GENOMESPLITFASTA, --genome_fasta GENOMESPLITFASTA
Genome sequence in FASTA format
-d WORKINGDIRECTORY, --working_directory WORKINGDIRECTORY
Complete path of output directory
-m MISMATCH, --num_mismatch MISMATCH
Maximum number of mismatches for Bowtie2 alignment
-t THREADS, --num_threads THREADS
Launch t number of threads in parallel
-nm MINHITS, --get_minhits MINHITS
Minimum number of hits in a window (filter)
-nx MAXHITS, --get_maxmum_total_hits MAXHITS
Maximum total number of hits
-g GC, --get_gc GC gc content in integer
-l CANDIDATERNALENGTH, --get_candidaternalength CANDIDATERNALENGTH
Candidate gRNA length
Module 4:Filter hits for on-target and off-target
python crisprbroad.py filterhits -h
usage: crisprbroad.py filterhits [-h]
-h, --help show this help message and exit
-d WORKINGDIRECTORY, --working_directory WORKINGDIRECTORY
Complete path of output directory
-t THREADS, --num_threads THREADS
Launch t number of threads in parallel
-nm MINHITS, --get_minhits MINHITS
Minimum number of hits in a window (filter)
-nx MAXHITS, --get_maxmum_total_hits MAXHITS
Module 5: Scoring windows and ranking gRNA
python crisprbroad.py findwindow -h
usage: crisprbroad.py findwindow [-h]
-h, --help show this help message and exit
-as AVOIDSCOREOFF, --avoid_scoreoff AVOIDSCOREOFF
Avoid score calculation for off-targets (0 or 1)
-d WORKINGDIRECTORY, --working_directory WORKINGDIRECTORY
Complete path of output directory
-p PAMSEQUENCE, --pam_sequence PAMSEQUENCE
PAM sequence string eg: NGG
-t THREADS, --num_threads THREADS
Launch t number of threads in parallel
-nm MINHITS, --get_minhits MINHITS
Minimum number of hits in a window (filter)
-w WINDOW, --get_window WINDOW
Window size in bp
-l CANDIDATERNALENGTH, --get_candidaternalength CANDIDATERNALENGTH
Candidate gRNA length
-nw WINDOWNUMBERS, --number of window WINDOWNUMBERS
number of windows for targeting
Module 6: gRNA that targets multiple windows
python crisprbroad.py findmultiwindow -h
usage: crisprbroad.py findmultiwindow [-h]
-h, --help show this help message and exit
-f GENOMESPLITFASTA, --genome_fasta GENOMESPLITFASTA
Genome sequence in FASTA format
-d WORKINGDIRECTORY, --working_directory WORKINGDIRECTORY
Complete path of output directory
-p PAMSEQUENCE, --pam_sequence PAMSEQUENCE
PAM sequence string eg: NGG
-t THREADS, --num_threads THREADS
Launch t number of threads in parallel
-nm MINHITS, --get_minhits MINHITS
Minimum number of hits in a window (filter)
-nx MAXHITS, --get_maxmum_total_hits MAXHITS
Maximum total number of hits
-ws WINDOW, --get_window WINDOW
Window size in bp
-sl SLIDINGWINDOWSIZE, --get_slidingwindowlength SLIDINGWINDOWSIZE
sliding window in bp (For consecutive windows: same as window size)
-nw NUMBEROFWINDOW, --get_multiwindow WINDOW
number of target windows
-q INPUTBED --query_bedfile query in bed format
optional input for finding sgRNA in a given region
-l CANDIDATERNALENGTH, --get_candidaternalength CANDIDATERNALENGTH
Candidate gRNA length
Module 7: Finding more than one gRNA that targets multiple windows
python crisprbroad.py multisgrna -h
usage: crisprbroad.py multisgrna [-h]
-h, --help show this help message and exit
-d WORKINGDIRECTORY, --working_directory WORKINGDIRECTORY
Complete path of output directory
-cb CRISPRBROADRESULTFILE, --crisprbroadresultfilt CRISPRBROADRESULTFILE
File obtained as result from module5
-mg SGRNANUMBERS, --numberofgRNA NUMBEROFSGRNA
Number of sgRNA in target window
RESULTS:
**OUTPUT description **
Results ranked list of gRNA and windows with score and dispersion are in tab-limited file: CRISPR_broad_results.xls**
| Column | Column Name | Description |
|---|---|---|
| 1 | Chromosome | Chromosome name of best window for gRNA selection |
| 2 | Start | Start position of best window for gRNA selection |
| 3 | End | End position of best window for gRNA selection |
| 4 | crnaid | Assigned Id for sgRNA (forward/reverse strand; sgRNA number; Start position) |
| 5 | Sequence | sgRNA sequence of user defined length |
| 6 | stringforgroup | Chr-st-end pattern |
| 7 | Hits within Best window | Higher number of hits => Better sgRNA |
| 8 | score | Score assigned (0-2) for sgRNA (Higher the score better is the candidate |
| 9 | stdev | Dispersion/spread from the center of the hits |
| 10 | Finalscore | Sum of score of all hits sgRNA (Higher the score better is the candidate |
| 11 | Total hits | Number of sgRNA hits in whole genome including off-targets |
| 12 | offtargethits | Number of sgRNA hits in outside Best target window based on score |
CIGAR Description:
Example format: ["('I', '+', '12072082', '23M', '0')"] The above is repeated (equal to the number of sgRNA hits)
| CIGAR string | Desction |
|---|---|
| I | chromosome name |
| + | strand |
| 12072082 | start |
| 23M | 23 nucleotide match i,e alignment of sgRNA (23nt length) |
| 0 | Edit distance (Unused) |