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LICENSE

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+MIT License
+
+Copyright (c) 2023 gprezza
+
+Permission is hereby granted, free of charge, to any person obtaining a copy
+of this software and associated documentation files (the "Software"), to deal
+in the Software without restriction, including without limitation the rights
+to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
+copies of the Software, and to permit persons to whom the Software is
+furnished to do so, subject to the following conditions:
+
+The above copyright notice and this permission notice shall be included in all
+copies or substantial portions of the Software.
+
+THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
+IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
+FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE
+AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
+LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM,
+OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE
+SOFTWARE.

README.md

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+# CRISPRi_designer
+Tools to design gRNA/array pools targeting a list of genes. 
+
+## Dependencies
+Create a conda environment from the environment.yml file:
+```
+conda env create -f environment.yml
+```
+## get_sequences_from_gff
+The first step in the pipeline is to obtain a fasta file with the sequences of the genes that are to be targeted. These sequences must contain the promoter. The file can be created from a gff annotation with the get_sequences_from_gff.py script.
+
+### Usage
+```
+usage: get_sequences_from_gff.py [-h] [--outname OUTNAME]
+                                 [--promoter PROMOTER] [--genetype GENETYPE]
+                                 [--attribute ATTRIBUTE] [--name NAME]
+                                 gff_file fasta_file
+
+Extract gene sequences from gff and genome files.
+
+positional arguments:
+  gff_file              Input annotation (gff) file with sequences of the
+                        target genes.
+  fasta_file            Input genome file.
+
+optional arguments:
+  -h, --help            show this help message and exit
+  --outname OUTNAME, -o OUTNAME
+                        Base name of the output files. The .fasta extension
+                        will be appended to this. (Default: target_genes.
+  --promoter PROMOTER, -p PROMOTER
+                        Length before the gene start to be considered as the
+                        promoter. (Default: 50)
+  --genetype GENETYPE, -t GENETYPE
+                        Consider only genes of this type (Third column of the
+                        gff file).
+  --attribute ATTRIBUTE, -a ATTRIBUTE
+                        Consider only genes that have this attribute (ninth
+                        column of the gff file).E.g. if you want to filter for
+                        the attribute "sRNA_type=Intergenic"
+                        writesRNA_type=Intergenic.
+  --name NAME, -n NAME  Take gene name from this attribute of the gff
+                        attribute column (ninth column of the gff file)
+```
+### Input files
+* A gff annotation (*gff_file*) in the standard format (tab-separated, no header, columns: seqname,feature,start,end,score,strand,frame,attribute).
+* A (multi)fasta file (*fasta_file*) containing the genome file (chromosome(s) and plasmid(s), if present).
+
+### Output
+* A _basename_coordinates.txt_ file containing the coordinates of each gene (promoter included).
+* A _basename_sequences.fasta_ file containing the genes (promoter included).
+_basename_ is defined by the --outname option.
+
+## design_CRISPRi_gRNAs.py
+This script designs the gRNA/array library.
+
+### Usage
+
+```
+usage: design_CRISPRi_gRNAs.py [-h] [--processors PROCESSORS]
+                               [--in_name IN_NAME] [--coordinates COORDINATES]
+                               [--folding FOLDING] [--left LEFT]
+                               [--right RIGHT] [--arrays] [--non_targeting]
+                               [--PAM PAM] [--spacer_length SPACER_LENGTH]
+                               [--promoter_length PROMOTER_LENGTH]
+                               [--mutants MUTANTS] --reference REFERENCE
+
+Designs spacers targeting the input genes.
+
+optional arguments:
+  -h, --help            show this help message and exit
+  --processors PROCESSORS, -p PROCESSORS
+                        Number of processors (Default: 1).
+  --in_name IN_NAME, -i IN_NAME
+                        Input base name of the fasta file with sequences of
+                        the target genes.(Default: target_genes.)
+  --coordinates COORDINATES, -c COORDINATES
+                        File with coordinates of the target genes. (Default: "
+                        --in_name + _coordinates.txt".)
+  --folding FOLDING, -f FOLDING
+                        Minimal accepted correct folding probability of the
+                        arrays. Scale: 0-1. (Default: 0.2).
+  --left LEFT, -lo LEFT
+                        Left overhang for cloning mature spacers. (Default:
+                        atctttgcagtaatttctactgttgtagat).
+  --right RIGHT, -ro RIGHT
+                        Right overhang for cloning mature spacers. (Default:
+                        ccggcttatcggtcagtttcacctgattta).
+  --arrays, -a          The script attempts to design one array per target
+                        gene if this flag is present.
+  --non_targeting, -nt  The script designs nontargeting gRNAs if this flag is
+                        present.
+  --PAM PAM, -pam PAM   PAM sequence. (Default: TTV).
+  --spacer_length SPACER_LENGTH, -sl SPACER_LENGTH
+                        Length of each spacer. No more than 26 if the -a flag
+                        is used. (Default: 20).
+  --promoter_length PROMOTER_LENGTH, -pl PROMOTER_LENGTH
+                        Length of the promoter region. (Default: 50).
+  --mutants MUTANTS, -m MUTANTS
+                        Number of spacers (including one array if using the -a
+                        flag) to be designed per targeted gene. (Default: 4).
+  --reference REFERENCE, -r REFERENCE
+                        Folder containing the genome sequence(s).
+```
+### Input
+* The two files generated by _get_sequences_from_gff_, which are indicated by *in_name* (and *--coordinates*, if the names are different).
+
+### Output
+The following files are generated by the script:
+* _constructs_mature.txt_: fasta file with all gRNAs + homology arms. Non-targeting gRNAs are also included if the _-nt_ option is used.
+* _mature_oligo_pool2.txt_: text file with all gRNAs + homology arms. Like the previous file, but without sequence ID.
+* _nontargeting_oligo_pool2.txt_: only if the _-nt_ option is used. Text file with the non-targeting gRNAs + homology arms.
+* _constructs_array.txt_: only if the _-a_ option is used. Fasta file with all arrays.
+* _array_oligo_pool_n.txt_: only if the _-a_ option is used. n Text files with the sequences of the oligos needed to construct the array via CRATES. One file is generated per each pool to be ordered.