Split-tracrRNA as an efficient tracrRNA system with an improved potential of scalability

Area of Science:

• Molecular Biology
• Biotechnology
• Gene Editing

Background:

• Standard CRISPR-Cas9 systems rely on long tracrRNAs, limiting their scalability and cost-effectiveness due to chemical synthesis challenges.
• Shorter RNA guides are desirable for improving the accessibility and affordability of CRISPR-Cas9 technology.

Purpose of the Study:

• To engineer shortened, cost-effective RNA guides for CRISPR-Cas9 systems by developing a split-tracrRNA approach.
• To identify optimal nicking positions within the tracrRNA sequence to maintain gene editing functionality.

Main Methods:

• Development of a split-tracrRNA system by nicking the 67-mer tracrRNA (tracrRNA(67)).
• Evaluation of gene editing efficiency and specificity using cellular assays and in vitro DNA cleavage assays.
• Assessment of binding affinity to Cas9 and reaction kinetics using binding assays and single-molecule experiments.

Main Results:

• The position of the nick in tracrRNA is critical for maintaining its activity.
• TracrRNA(41 + 23), nicked in stem loop 2, demonstrated comparable gene editing efficiency and specificity to the full-length tracrRNA(67).
• Removal of the stem loop 2 loop was feasible without compromising performance when the stem duplex was stabilized by high GC content.

Conclusions:

• Efficient split-tracrRNAs can be engineered by maintaining similar binding affinity to Cas9 and reaction kinetics as the full-length tracrRNA(67).
• The split-tracrRNA system offers a promising strategy for enhancing the scalability and cost-effectiveness of CRISPR-Cas9 RNA guides.