AltEx-BE 1.0.9

automatically design sgRNA for exon skipping with many base editors

AltEx-BE: Alternate Exon Skipping by Base Editing

• AltEx-BE: Alternate Exon Skipping by Base Editing
  • Overview
  • Key Features
  • Workflow Diagram
  • Installation
  • Required dataset
  • Usage
    • Graphical User Interface (UI)
    • Command-Line Interface (CLI)
      • 1. Using a Preset Editor:
      • 2. Input Base Editor Information in the Command Line:
      • 3. Input a CSV/TSV/TXT File Containing Information about Your Base Editors:
  • List of command line options
  • Format of AltEx-BE output
• License

Overview

AltEx-BE is a command-line bioinformatics tool that designs sgRNAs (single guide RNAs) to induce targeted exon skipping using Base Editing technology.

Manipulating alternative splicing is key to understanding diseases like cancer and neurodegenerative disorders, but designing the right tools for the job is a major bottleneck. The manual process of identifying targetable exons, designing sgRNAs for specific base editors, and assessing off-target risks is complex, tedious, and slows down critical research.

AltEx-BE is a powerful command-line tool built to automate this entire workflow. It intelligently parses transcript data to find the best exon targets, designs candidates for a multitude of base editors, and evaluates their off-target risk to provide a ranked list of high-confidence sgRNAs.

By transforming a complex, multi-step design process into a single command, AltEx-BE bridges the gap between your scientific question and a successful wet lab experiment, significantly accelerating research into splicing-related diseases and therapies.

Key Features

• 🧬 Automated Target Exon Annotation:

  • Automatically parses transcript structures from refFlat files to identify and classify potential targets for exon skipping. This includes Skipped Exons (SE) and exons with Alternative 3'/5' Splice Sites (A3SS/A5SS), eliminating the need for tedious manual searches.

• ⚙️ Universal Base Editor Compatibility:

  • Supports virtually any ABE or CBE. You can use built-in presets or define any custom editor by specifying its PAM sequence and editing window, allowing immediate use of the latest editors from new publications.
  • AltEx-BE can design sgRNAs for multiple Base-Editors in one run

• 🚀 Streamlined End-to-End Workflow:

  • Seamlessly moves from data input to candidate selection. The design command generates sgRNAs, while the visualize command creates comprehensive reports to help you evaluate and rank the best candidates for your experiment.

Workflow Diagram

Here is a simplified diagram illustrating the workflow of AltEx-BE:

Installation

To get started with AltEx-BE, clone the repository and install the required dependencies.

# 1. install via bioconda
conda install -c conda-forge -c bioconda altex-be

# 2. install via pypi
pip install AltEx-BE

[!CAUTION] AltEx-BE required python 3.10~ 3.12 So when you face to some install errors, please suspect version conflict.

Required dataset

To use AltEx-BE, you should prepare 2 input files in your computer

• refFlat file or gtf file of your interest species
  • refflat file contains Refseq infomations: explanation of refFlat format is here
  • you can download refflat files from UCSC goldenpath: refflat files of mm39 is here
  • also you can use GTF file as a input
    • If you use GTF, AltEx-BE automatically converts GTF into refflat format and generate them into output directory
• Fasta files contain all chromosome sequence of your interest species
  • you can download Fasta file also from UCSC goldenpath
  • please comfirm your .fa files contain all of chromosome. if not, AltEx-BE process will fail
• (optional) CSV or TXT or TSV contain the gene symbols or Refseq IDs
  • AltEx-BE is avalilable for many genes. When you want to design sgRNAs for many genes, You can input gene list via --gene-file option.
  • The input file should only have 1 column with gene symbols or refseq IDs (No need the header row)

[!NOTE] Point of Gene and RefseqID input

• When providing a gene symbol (e.g., MYGENE), AltEx-BE will analyze all known transcripts of that gene to identify alternative splicing events.
• When providing a RefSeq ID (e.g., NM_0012345), AltEx-BE will automatically identify the corresponding gene and analyze all of its transcripts. This ensures a comprehensive analysis even when starting from a single transcript identifier.

Usage

AltEx-BE can be run via a graphical user interface (UI) or directly from the command line.

Graphical User Interface (UI)

For users who prefer a graphical interface, AltEx-BE includes a web-based UI built with Streamlit. It allows you to configure and run the pipeline without using the command line.

To launch the UI, run:

altex-be --ui

The UI helps you:

• 📥 Select input files (FASTA, transcript annotation)
• ⚙️ Configure all options for the analysis
• ▶️ Run the AltEx-BE pipeline locally
• 📝 Monitor execution logs in real time
• 📊 Preview and browse output files

Command-Line Interface (CLI)

For command-line usage, AltEx-BE is operated via the altex-be command. Here are a few examples:

1. Using a Preset Editor:

• By default, AltEx-BE design sgRNAs for below 6 Base Editing Tools

[!NOTE] Preset Base Editors:

base_editor_name	pam_sequence	editing_window_start	editing_window_end	base_editor_type
target_aid_ngg	NGG	17	19	cbe
be4max_ngg	NGG	12	17	cbe
abe8e_ngg	NGG	12	17	abe
target_aid_ng	NG	17	19	cbe
be4max_ng	NG	12	17	cbe
abe8e_ng	NG	12	17	abe

altex-be \
    --refflat-path /path/to/your/refFlat.txt \
    --fasta-path /path/to/your/genome.fa \
    --output-dir /path/to/output_directory \
    --gene-symbols MYGENE \
    --assembly-name hg38

2. Input Base Editor Information in the Command Line:

altex-be \
    --refflat-path /path/to/your/refFlat.txt \
    --fasta-path /path/to/your/genome.fa \
    --output-dir /path/to/output_directory \
    --gene-symbols MYGENE \
    --assembly-name hg38 \
    --be-name target-aid \
    --be-type cbe \
    --be-pam NGG \
    --be-start 17 \
    --be-end 19

[!CAUTION] --be-start and --be-end specify the editing window of your base editor. The location of the editing window is counted from the base next to the PAM (1-indexed).

3. Input a CSV/TSV/TXT File Containing Information about Your Base Editors:

You can provide a file containing the information for one or more base editors. This is useful when you want to design sgRNAs for multiple editors at once.

[!CAUTION] The input file should have the following columns: base_editor_name, pam_sequence, editing_window_start, editing_window_end, base_editor_type.

altex-be \
    --refflat-path /path/to/your/refFlat.txt \
    --fasta-path /path/to/your/genome.fa \
    --output-dir /path/to/output_directory \
    --gene-symbols MYGENE \
    --assembly-name hg38 \
    --be-files /path/to/your/base_editor_info.csv

List of command line options

Short Option	Long Option	Argument	Explanation
-h	--help		Show the help message and exit.
-v	--version		Show the version of Altex BE.
	--ui		Launch the Streamlit web UI for AltEx-BE.
-r	--refflat-path	FILE	(Mutually Required -r or -g) Path to the refFlat file.
-g	--gtf-path	FILE	(Mutually Required with -r or -g) Path to the GTF file.
-f	--fasta-path	FILE	(Required) Path to the FASTA file.
-o	--output-dir	DIR	(Required) Directory for the output files.
	--gene-symbols	SYMBOL [SYMBOL ...]	A space-separated list of gene symbols of interest.
	--refseq-ids	ID [ID ...]	A space-separated list of RefSeq IDs of interest.
	--gene-file	FILE	Path to a CSV or TXT file contain your interest gene symbols/RefseqIDs
	--run-all_genes	store true	when user input this option, AltEx-BE design sgRNAs for all genes
-a	--assembly-name	ASSEMBLY	(Required) The name of the genome assembly to use (e.g., hg38, mm39).
-n	--be-name	NAME	The name of the base editor to use.
-p	--be-pam	SEQUENCE	The PAM sequence for the base editor.
-s	--be-start	INTEGER	The start of the editing window for the base editor (1-indexed from the base next to the PAM).
-e	--be-end	INTEGER	The end of the editing window for the base editor (1-indexed from the base next to the PAM).
-t	--be-type	TYPE	The type of base editor (ABE or CBE).
	--be-files	FILE	Path to a CSV or TXT file containing information about one or more base editors.

Format of AltEx-BE output

altex-be makes 2 output files in Path/To/YourOutput/ directory which you specified in --output-dir command

• Summary sgRNA table (.csv)
  • this table contain imformation of sgRNAs designed and ranked by AltEx-BE

- Meaning of each column is :

column name	meaning	remark
geneName	gene symbol of target gene
chrom	location of target gene
strand	strand of target gene
exonstart, exonend, exonlength	general information of target exon
coding	whether target gene is protein coding or non coding gene
frame	mod3 of the length of target exon	0 = in-frame or 1,2 = out-frame
exon_position	relative location of target exon in target gene	"first" or "internal" or "last"
uuid	the unique id for each sgRNAs	changes in every run
exon_intron_boundary+-25bp_sequence	sequence around SA or SD
sgrna_sequence	sgRNA sequence	Thymine is not replaced by Uracil
sgrna_target_pos_in_seq	position of target A or C in sgRNA	relative location in sgrna
sgrna_overlap_between_cds_and_editing_window	number of overlapping bases with editing window
sgrna_unintended_edited_base_count	number of possible being edited bases (A or C) in cds
sgrna_start/end_in_genome	location of sgrna
site type	target splicing site of sgRNA	acceptor or donor
sgrna_strand	strand of sgRNA
base_editor_name/pam_sequence/window_start or end / base editor type	infomation of BE to design sgRNA
crispr_direct_url	link to CRISPR direct
pam+20bp exact match	pam+20bp (23-mer) exact match in all chromosome
pam+12bp exact match	pam+12bp (12-mer) exact match in all chromosome
sgrna_priority	ranking of sgRNA for each target exon	ranked by off-target specificity and GC content

sgRNA Prioritization

To facilitate the selection of optimal sgRNAs for experimental validation, AltEx-BE ranks sgRNAs for each target splice site based on predicted off-target binding specificity. The tool prioritizes sgRNAs primarily by the number of exact 20-nucleotide matches (PAM + 20bp) across the genome, selecting those with the fewest potential off-target sites. For sgRNAs with equivalent off-target profiles, GC content is considered as a secondary criterion, favoring sgRNAs within the optimal range of 40-60%. In rare cases where multiple sgRNAs remain equivalent, extended off-target matches (PAM + 12bp) and the number of editable bases within the CDS region are used as final tiebreakers.

• BED file for UCSC custom track (.bed)
  • this bed file can use as a UCSC custom tracks, you can input that bed file into this webpage

- colored box (red, blue) is sgRNA sequences. red means sgRNAs for abe, blue means sgRNAs for cbe. - score columns in bed file means offtarget count of 20bp+PAM - when you assign bed file, you should choose correct assembly name in above website

License

• Please see LICENSE.md