CRISPR RNA Array-Guided Multisite Cleavage for Gene Disruption by Cas9 and Cpf1

Area of Science:

• Molecular Biology
• Gene Editing Technologies

Background:

• CRISPR-Cas systems offer precise gene editing capabilities.
• Multisite DNA targeting requires efficient delivery of multiple guide RNAs.

Purpose of the Study:

• To design and evaluate CRISPR RNA arrays (CRISPRay) for simultaneous multisite DNA cleavage.
• To assess the efficacy of CRISPRay with SpCas9 and FnCpf1 nucleases in vitro and in living cells.

Main Methods:

• Fusing four single guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) with uncleavable RNA linkers to create CRISPR RNA arrays.
• Testing CRISPRay's ability to inactivate the firefly luciferase gene in living cells.
• Performing in vitro DNA cleavage assays and DNA sequencing to analyze nuclease activity.

Main Results:

• CRISPRay demonstrated robust inactivation of the firefly luciferase gene.
• sgRNA arrays successfully directed SpCas9 for simultaneous cleavage at four target sites.
• crRNA-array-guided FnCpf1 exhibited nonspecific DNase activity.

Conclusions:

• CRISPRay is a simple and effective approach for multisite-directed gene disruption.
• Optimization of nuclease and CRISPR RNA ratios can further enhance gene interference.
• The study highlights the potential of fused guide RNAs for advanced gene editing applications.