zhangchonglab/crispri-functional-genomics-in-prokaryotes

CRISPRi-functional-genomics-in-prokaryotes

This repository presents the python script collection for CRISPR interference based functional genomics study in prokaryotes, firstly demonstrated in Escherichia coli (for the experiment description and result presentation, see https://doi.org/10.1101/129668). This method enables quantitative measurement of fitness score for thousands of genes in multiple conditions, and it works theoretically in any prokaryotic organism. We demonstrated in our paper (Wang T, Guan C, Guo J, Liu B, Wu Y, Xie Z, Zhang C, Xing X-H (2018) Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance. Nat Commun 9:2475 . doi: 10.1038/s41467-018-04899-x) that CRISPR interference exhibits superior performance over other benchmark methods in microbiology research, such as Tn-seq. Hence, we believe it is potent to be a powerful tool for us to better understand the genomics and physiology of microbes. Considering the need of extensive programming and computation to use this method, which is a major hurdle for experimental biologists with limited programming skills, we develop this software package to facilitate the usage of this method by more people.

The python package basically contains two subpackages, one for the sgRNA (N20) design at either customized manner (given interested gene fasta file) or at genome level (given the gene fasta file encoded by particular genome), while another for the NGS data processing and result visualization after screening experiment. For the details about package usage, see the README file within each subpackage depoisited together with the scripts.

Schematic for the structure of this software package

For any request or question, please contact [email protected] or [email protected].

It is also ok to post your concern directly at this GitHub site. This software package is actively maintained and upgraded.

Wish this tool to benefit your work in microbiology study.

crispri-functional-genomics-in-prokaryotes's Issues

finding gRNAs on a single gene

config_mApple2_gRNA_100919.txt
first of all, nice tool!. I have been trying to use it to automatically design gRNAs for my experiments (I am not running libraries at the moment). I'm doing a control experiment where I want to find gRNAs for a fluorescent protein (mApple2) so that would be just one gene (see attached). I had to modify the code a bit because I am using python 3, however the analysis runs well but I get the following error:

Design process for mApple2 starts.
Copy number: 1
Gene length: 711.0
Command line:./seqmap 5 N20NGGofftarget.fasta NC_022898_PY79.fna seqmapOut.temp.dat /output_all_matches
Loading fasta file N20NGGofftarget.fasta.
56 sequences read, total length is 1288.
analysing probes...totally 56 probes, minimum length = 23, maximum length = 23, set internal key length = 23
importing probes....56 probes imported. 56 are valid.
checking resources...64-bit version.
available memory: 8128MB.
generating reversed probes for searching reverse strand
now 112 probes. 112 are valid.
Split probe into 6 parts.
total 6 copies of probes.
estimated memory usage: 12MB.
Building 6 probe lists......6 probe lists created.
estimated search speed is 302118 bps/sec.
detecting probes
....
average search steps:1.08999 maximum search steps:4
4033459 base pairs processed. average search speed: 694514 bps/sec.
time used: 17.6061 seconds
4 sgRNA detected for mApple2

cut: mApple2_mNeon.gene_statistics.txt: No such file or directory
cut: mApple2_mNeon.sgRNA_statistics.txt: No such file or directory
Traceback (most recent call last):
File "sgRNA_desgin_main.py", line 348, in
main(sys.argv[1])
File "sgRNA_desgin_main.py", line 329, in main
processSgRNASequence('%s.N20.fasta.txt'%(varlist['prefix']),'N20_library.csv','w',False)
File "sgRNA_desgin_main.py", line 265, in processSgRNASequence
with open(N20file,'r') as f:
FileNotFoundError: [Errno 2] No such file or directory: 'mApple2_mNeon.N20.fasta.txt

That means it is not able to create the gRNA fasta file for some reason. Any ideas?

mApple_mNeon.txt