High-accuracy crRNA array assembly strategy for multiplex CRISPR.

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Systems Biology

Background:

• Multiplex CRISPR enables simultaneous targeting of multiple genetic loci for complex cellular network analysis.
• Existing CRISPR array assembly methods lack convenience, accuracy, and efficiency.
• Emerging Cas nucleases and CRISPR arrays have expanded multiplex CRISPR applications in vitro and in vivo.

Purpose of the Study:

• To develop a streamlined, accurate, cost- and time-saving strategy for CRISPR array assembly.
• To facilitate the convenient implementation of multiplex CRISPR for DNA and RNA targets.
• To enable dissection of complex cellular networks and advance multi-target gene therapy.

Main Methods:

• Developed a novel strategy for CRISPR array assembly.
• Efficiently assembled 12 CRISPR RNAs (crRNAs) for AsCas12a and 15 crRNAs for RfxCas13d in a single reaction.
• Investigated CRISPR array expression patterns driven by Pol II versus Pol III promoters and developed improved methods for long CRISPR array expression.

Main Results:

• Demonstrated a highly accurate, cost- and time-saving CRISPR array assembly strategy.
• Successfully assembled large numbers of crRNAs in a single reaction for both DNA (AsCas12a) and RNA (RfxCas13d) targeting.
• Observed distinct expression patterns for CRISPR arrays driven by Pol II and Pol III promoters, allowing for controlled CRISPR intensity distribution.

Conclusions:

• The novel strategy provides a flexible and powerful tool for multiplex CRISPR implementation.
• This approach significantly facilitates the dissection of complex biological networks.
• The developed method is crucial for advancing multi-target gene therapy and understanding cellular functions.