ExsgRNA: reduce off-target efficiency by on-target mismatched sgRNA

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• CRISPR/Cas9 is a powerful gene editing tool.
• Existing sgRNA design tools overlook on-target mismatches, potentially leading to off-target mutations.
• On-target mismatches may offer a strategy for enhanced sgRNA design.

Purpose of the Study:

• To develop a novel method for generating sgRNAs that include on-target mismatches.
• To evaluate the efficacy and specificity of sgRNAs with on-target mismatches.
• To create a user-friendly web server for designing such sgRNAs.

Main Methods:

• A seq2seq-attention model with a feedback-loop architecture was trained to generate sgRNAs with on-target mismatches.
• Dual-luciferase reporter assays were used to assess sgRNA activity.
• Off-target sites were analyzed to evaluate specificity.

Main Results:

• Generated sgRNAs with three on-target mismatches retained approximately 80% of the activity of perfect-matched sgRNAs.
• The use of sgRNAs with on-target mismatches significantly reduced the number of off-target sites.
• A web server, ExsgRNA, was developed for designing optimal sgRNAs.

Conclusions:

• Incorporating on-target mismatches into sgRNA design is a viable strategy for improving CRISPR/Cas9 specificity.
• The ExsgRNA tool provides an accessible platform for designing more precise gene editing tools.
• This approach balances on-target activity with reduced off-target effects for enhanced genome editing.