Multigene editing: current approaches and beyond.

Area of Science:

• Biomedicine and Biology
• Gene Editing Technologies
• Computational Biology

Background:

• CRISPR/Cas9 multigene editing uses multiple single-guide RNAs (sgRNAs) to edit several genes simultaneously.
• Optimizing sgRNA selection is crucial to minimize off-target editing and its negative consequences.
• Existing methods require computational tools for efficient design and implementation.

Purpose of the Study:

• To review wet-laboratory approaches for CRISPR/Cas9 multigene editing.
• To highlight the need for computational tools in optimizing sgRNA selection.
• To introduce a novel method for sgRNA selection that utilizes off-target sites.

Main Methods:

• Review of current wet-laboratory techniques for multigene editing.
• Development of a 'preference cutting score' to evaluate beneficial off-target sites.
• Prioritization of sgRNAs based on on-target efficiency, off-target site count, and preference cutting score.

Main Results:

• Off-target editing, while unavoidable, can be repurposed as on-target editing sites for other genes.
• The proposed preference cutting score effectively identifies beneficial off-target sites.
• Case studies on cancer-associated genes demonstrate the method's utility.

Conclusions:

• A novel computational approach enhances CRISPR/Cas9 multigene editing by strategically utilizing off-target effects.
• The preference cutting score offers a valuable metric for optimizing sgRNA selection.
• This method holds significant promise for advancing gene editing applications, particularly in cancer research.