NSCCN/konezumiaid：命令行工具，用于基于 Target-AID 自动设计多重 KO 小鼠实验所需的 gRNA。

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|ranscript 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.

Recommended: whether the gRNA is recommended or not.
Target amino acid: the amino acid that the gRNA targets.
Exon　index: the index of the exon where the gRNA is located.

[!NOTE] KOnezumi-AID ignores the minor version of the RefSeq ID (e.g., NM_111111.2 → NM_111111) because the refFlat file does not support version numbers.

Batch processing

KOnezumi-AID can process multiple search queries at once using the batch command. For this purpose, a CSV or Excel file containing gene symbols or transcript names is required.

konezumiaid batch <-f|--file> <gene symbols or transcripts list CSV or Excel file>

[!NOTE] The CSV or Excel file must follow the format below.

The file should consist of a single column.

No header row is required. Data should start from the first row.

Each row should contain only one gene or transcript.

e.g. CSV file
Xkr4
Rp1
Sox17
e.g. Excel file | | |—| | Xkr4 | | Rp1 | | Sox17 |

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

Version check

$ konezumiaid <-v|--version>