Documentation

Platform Overview

MultiEdit PTG Designer 1.0 utilizes polycistronic tRNA-gRNA (PTG)-based multiplex editing system, where the PTG assembly is efficiently processed into individual guide RNAs (gRNAs) that direct Cas9 to edit multiple chromosomal targets simultaneously.

The tool automates the design of gRNA spacer-specific primers with 4-bp overlap for Golden Gate assembly. Each module is designed from a template containing 77 bp tRNA sequence followed by 86 bp optimized gRNA scaffold sequence, enabling seamless assembly without extra nucleotides.

Technical Methodology

Assembly Process

• Golden Gate Assembly: Uses BsaI and FokI restriction enzymes to create compatible 4-bp overhangs
• Primer Design: Forward and reverse primers anchor the first and last 12 nucleotides of the 20 bp spacer
• Module Construction: Each module contains tRNA sequence + gRNA scaffold + spacer sequence
• Terminal Assembly: FokI sites enable cloning into Cas9 editor plasmids

Template Sequences

• Complete Template: 247 nucleotides total
• gRNA Scaffold: 86 nucleotides (optimized for Cas9)
• tRNA Sequence: 77 nucleotides (processing signal)
• Compatibility: Validated for monocot and dicot plants

Output Specifications

• Primer sequences for each module (5' to 3' orientation)
• Individual tRNA-gRNA hybrid module sequences
• Complete PTG assembly sequence
• Laboratory-ready CSV export format

Requirements & Best Practices

Input Requirements

• Spacer sequences: exactly 20 base pairs (A, T, G, C only)
• Maximum 6 spacers per assembly
• Automatic sequence validation

Recommended Workflow

• Verify spacer sequences target intended genomic locations
• Check for potential off-target effects using appropriate tools
• Generate and review all primer sequences before ordering
• Export results for laboratory records and team collaboration

Development & Validation

This platform was developed by researchers at ICAR-IARI and ICAR-IASRI. The methodology is based on established molecular biology protocols and has been wet lab validated for seamless assembly of multiple PTG modules.

The tool is designed for both research and practical applications in plant genome editing, supporting efficient multiplex CRISPR-Cas9 experiments.