Tracking-seq: a universal off-target detection approach for CRISPR-Cas genome editing.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Genome editing technologies like CRISPR-Cas9 are powerful but can cause unintended off-target mutations.
• Accurate detection of these off-target effects is crucial for the safety and efficacy of genome editing applications.

Purpose of the Study:

• To present a detailed protocol for Tracking-seq, a sensitive method for genome-wide off-target detection.
• To enable the identification of on-target and off-target events across diverse genome editing modalities.

Main Methods:

• Tracking-seq leverages replication protein A (RPA) binding to single-stranded DNA (ssDNA) intermediates generated during genome editing.
• The protocol involves cell genome editing, RPA-bound ssDNA extraction, sequencing library construction, and data analysis with the Offtracker tool.

Main Results:

• Tracking-seq demonstrates high sensitivity for detecting off-target effects across various genome editors (Cas9, base editors, prime editors).
• The method is effective even with low cell input and provides high-performance results.

Conclusions:

• Tracking-seq provides a rapid (1-2 weeks) and comprehensive workflow for assessing genome-wide off-target activity.
• This method is a valuable tool for ensuring the safety and precision of genome editing strategies.