Linking CRISPR-Cas9 double-strand break profiles to gene editing precision with BreakTag

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas9 gene editing relies on DNA cleavage, but the determinants of blunt versus staggered double-strand breaks (DSBs) are not fully understood.
• Understanding Cas9 incision types is crucial for predicting and controlling gene editing outcomes.

Purpose of the Study:

• To develop and apply a novel method, BreakTag, for profiling Cas9-induced DSBs.
• To identify the factors influencing the type of Cas9 DNA incisions.
• To correlate Cas9 incision types with DNA repair outcomes.

Main Methods:

• Development of BreakTag, a versatile method for profiling Cas9-induced DSBs.
• Assessment of SpCas9 cleavage at over 150,000 endogenous sites using ~3,500 single-guide RNAs.
• Application of machine learning models to analyze determinants of Cas9 incision and repair outcomes.

Main Results:

• Approximately 35% of SpCas9-induced DSBs were found to be staggered.
• DNA:gRNA complementarity and engineered Cas9 variants influence DSB incision type.
• Cas9 incision patterns are dependent on protospacer sequence and impacted by human genetic variation.
• Staggered Cas9 breaks correlate with precise, templated single-nucleotide insertions.

Conclusions:

• The type of Cas9 DNA cut is influenced by sequence context, genetic variation, and Cas9 variants.
• BreakTag provides a method to profile Cas9-induced DSBs and their determinants.
• Scission-based guide RNA design can leverage staggered breaks for precise gene correction, including single-nucleotide deletions.