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Description:

KOnezumiAID 0.3.1

KOnezumi-AID
KOnezumi-AID is the command-line tool to automate the gRNA design for multiplex KO mouse using Target-AID
Installation
Prerequisits

Python 3.9 or later
Unix-like environment (Linux, macOS, WSL2, etc.)

Installation🔨
From Bioconda (Recommended)
conda install -c conda-forge -c bioconda konezumiaid
From PyPI:
pip install KOnezumiAID
Required Packages (Not needed if installed via Bioconda)

bedtools
Follow the official instruction

bowtie
Follow the official instruction

Input data set (e.g. Mus musculus mm39)
Locus information
refFlat.txt.gz from UCSC
genomic sequence
mm39.fa.gz from UCSC
Download scripts (bash)
mkdir -p data
curl https://hgdownload.soe.ucsc.edu/goldenPath/mm39/database/refFlat.txt.gz |
gzip -dc > data/refFlat.txt
curl https://hgdownload.soe.ucsc.edu/goldenPath/mm39/bigZips/mm39.fa.gz |
gzip -dc > data/mm39.fa

Usage
KOnezumi-AID's output

gRNAs to generate PTC (premature termination codon)
gRNAs to disrupt splice acceptor site
gRNAs to disrupt splice donor site

KOnezumi-AID provides these gRNAs in standerd output and CSV format.
The CSV file is located in data/output directory and named as <gene symbol/transcript name>_ptc_gRNA.csv or <gene symbol/transcript name>_splice_gRNA.csv.
Create data set for KOnezumi-AID
konezumiaid preprocess <your refFlat.txt Path> <your mm39.fa Path>
Example
konezumiaid preprocess data/refFlat.txt data/mm39.fa
Search candidate by gene symbol or transcript name (Refseq id)
KOnezumi-AID accepts a gene symbol or a transcript name.
konezumiaid <-n | --name> <gene symbol | transcript name>
Search by gene symbol
You can obtain the gRNAs that are present in all transcript variants.

[!NOTE]
If the gene has one transcript, the result is the same as searching by the transcript name

Search by transcript name
You can obtain the transcript's gRNAs and access more information about the gRNAs.

in_start_150bp: The gRNA is located in the first 150bp of the transcript or not.
in_50bp_from_LEJ: The gRNA is located in the 50bp from the last exon junction or not.
exon_index: the index of the exon where the gRNA is located.

Examples
Search candidate by the gene symbol (gene symbol with multiple transcripts)
$konezumiaid -n Rp1
Processing NM_001370921...
Processing NM_001195662...
Processing NM_011283...
List of gRNAs to generate PTC (premature termination codon)
Target sequence (20mer + PAM) Target amino acid link to CRISPRdirect
0 ACAGTTTGGCGGCGTTCGGGTGG Q https://crispr.dbcls.jp/?userseq=ACAGTTTGGCGGCGTTCGGGTGG&pam=NGG&db=mm39
1 ACGACACAGCATCACCAGGCTGG R https://crispr.dbcls.jp/?userseq=ACGACACAGCATCACCAGGCTGG&pam=NGG&db=mm39
2 ACAGGTTATGCAGTGTCCTGTGG Q https://crispr.dbcls.jp/?userseq=ACAGGTTATGCAGTGTCCTGTGG&pam=NGG&db=mm39
3 CCAGGGCCGAGGGCGCCTGCGGG W https://crispr.dbcls.jp/?userseq=CCAGGGCCGAGGGCGCCTGCGGG&pam=NGG&db=mm39
4 GCCAGGGCCGAGGGCGCCTGCGG W https://crispr.dbcls.jp/?userseq=GCCAGGGCCGAGGGCGCCTGCGG&pam=NGG&db=mm39
List of gRNAs to disrupt splice acceptor site
No gRNA found.
List of gRNAs to disrupt splice donor site
No gRNA found.

Search candidate by the gene symbol (gene symbol with single transcript)
$konezumiaid -n Mafa
List of gRNAs to generate PTC (premature termination codon)
Target sequence (20mer + PAM) Recommended Target amino acid link to CRISPRdirect
0 CTCAGGCCGGGGGCGCCCCGGGG True 87Q https://crispr.dbcls.jp/?userseq=CTCAGGCCGGGGGCGCCCCGGGG&pam=NGG&db=mm39
1 GCTCAGGCCGGGGGCGCCCCGGG True 87Q https://crispr.dbcls.jp/?userseq=GCTCAGGCCGGGGGCGCCCCGGG&pam=NGG&db=mm39
2 CCAGCACCACCTGAACCCCGAGG True 121Q https://crispr.dbcls.jp/?userseq=CCAGCACCACCTGAACCCCGAGG&pam=NGG&db=mm39
3 GGTCAGAGCTTCGCGGGCGGCGG True 167Q https://crispr.dbcls.jp/?userseq=GGTCAGAGCTTCGCGGGCGGCGG&pam=NGG&db=mm39
List of gRNAs to disrupt splice acceptor site
No gRNA found.
List of gRNAs to disrupt splice donor site
No gRNA found.

Search candidate by the transcript name
$ konezumiaid -n NM_001370921
List of gRNAs to generate PTC (premature termination codon)
Target sequence (20mer + PAM) Recommended Target amino acid link to CRISPRdirect
0 ACAGTTTGGCGGCGTTCGGGTGG False 46Q https://crispr.dbcls.jp/?userseq=ACAGTTTGGCGGCGTTCGGGTGG&pam=NGG&db=mm39
1 ACGACACAGCATCACCAGGCTGG True 88R https://crispr.dbcls.jp/?userseq=ACGACACAGCATCACCAGGCTGG&pam=NGG&db=mm39
2 ACAGGTTATGCAGTGTCCTGTGG True 192Q https://crispr.dbcls.jp/?userseq=ACAGGTTATGCAGTGTCCTGTGG&pam=NGG&db=mm39
3 ACAACCTGTCCTTCCAGGTAAGG True 389Q https://crispr.dbcls.jp/?userseq=ACAACCTGTCCTTCCAGGTAAGG&pam=NGG&db=mm39
4 ACCAATCAGAACAATCCCACTGG True 698Q https://crispr.dbcls.jp/?userseq=ACCAATCAGAACAATCCCACTGG&pam=NGG&db=mm39
5 ACGAATGTATCTGAGGATTAAGG True 723R https://crispr.dbcls.jp/?userseq=ACGAATGTATCTGAGGATTAAGG&pam=NGG&db=mm39
6 TCAGGCCAATGTCACATTGTGGG True 861Q https://crispr.dbcls.jp/?userseq=TCAGGCCAATGTCACATTGTGGG&pam=NGG&db=mm39
7 CTCAGGCCAATGTCACATTGTGG True 861Q https://crispr.dbcls.jp/?userseq=CTCAGGCCAATGTCACATTGTGG&pam=NGG&db=mm39
8 CCAGGGCCGAGGGCGCCTGCGGG True 126W https://crispr.dbcls.jp/?userseq=CCAGGGCCGAGGGCGCCTGCGGG&pam=NGG&db=mm39
9 GCCAGGGCCGAGGGCGCCTGCGG True 126W https://crispr.dbcls.jp/?userseq=GCCAGGGCCGAGGGCGCCTGCGG&pam=NGG&db=mm39
10 TCCAGTGGGATTGTTCTGATTGG True 704W https://crispr.dbcls.jp/?userseq=TCCAGTGGGATTGTTCTGATTGG&pam=NGG&db=mm39
11 CCAGTACTGGGATTTGTCACTGG True 1052W https://crispr.dbcls.jp/?userseq=CCAGTACTGGGATTTGTCACTGG&pam=NGG&db=mm39
List of gRNAs to disrupt splice acceptor site
Target sequence (20mer + PAM) Exon index link to CRISPRdirect
0 ACCTGGGATTGAAAGGAACAAGG 20 https://crispr.dbcls.jp/?userseq=ACCTGGGATTGAAAGGAACAAGG&pam=NGG&db=mm39
1 TCTGTTGGAGAAAAGCCCCATGG 22 https://crispr.dbcls.jp/?userseq=TCTGTTGGAGAAAAGCCCCATGG&pam=NGG&db=mm39
2 ACCTGAAGAAAATGGAAAACAGG 23 https://crispr.dbcls.jp/?userseq=ACCTGAAGAAAATGGAAAACAGG&pam=NGG&db=mm39
List of gRNAs to disrupt splice donor site
Target sequence (20mer + PAM) Exon index link to CRISPRdirect
0 TACCTTGCCCAAGTCCATCATGG 8 https://crispr.dbcls.jp/?userseq=TACCTTGCCCAAGTCCATCATGG&pam=NGG&db=mm39
1 TTACCTCTCACAGGTGAAGATGG 22 https://crispr.dbcls.jp/?userseq=TTACCTCTCACAGGTGAAGATGG&pam=NGG&db=mm39