Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells.

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• CRISPR-Cas9 systems, including Staphylococcus aureus Cas9 (SaCas9), are powerful tools for genome editing.
• Understanding and predicting off-target activity is crucial for the safe and effective therapeutic application of SaCas9 in the human genome.

Purpose of the Study:

• To develop and validate a high-throughput screening approach for measuring SaCas9 genome editing variation.
• To assess the impact of single guide RNA (sgRNA) spacer length and target mismatches on SaCas9 activity and specificity.
• To create a predictive model for SaCas9 off-target sites.

Main Methods:

• A high-throughput screening approach using a barcoded pairwise library of 88,692 sgRNAs paired with matched or mismatched target sites in a synthetic cassette.
• Incorporation of randomized barcodes for accurate molecule identification and downstream analysis, mitigating oligonucleotide synthesis errors.
• Development of an SaCas9 specificity model based on the generated dataset.

Main Results:

• SaCas9 sgRNAs with 21-mer or 22-mer spacer sequences demonstrated generally higher activity.
• Shorter, highly efficient 20-mer spacers exhibited reduced tolerance to target mismatches.
• The developed SaCas9 specificity model effectively ranked potential off-target sites.

Conclusions:

• The barcoded pairwise library screen provides a scalable and high-fidelity framework for investigating CRISPR enzyme properties and nucleic acid interactions.
• This methodology enhances the understanding of SaCas9 off-target activity, paving the way for improved genome editing safety.
• The findings contribute to the development of more precise and reliable SaCas9-based therapeutic strategies.