An engineered CRISPR-Cas12i tool for efficient multiplexed genome editing

Area of Science:

• Molecular Biology
• Genome Engineering
• Biotechnology

Background:

• Multiplexed genome editing tools face challenges in precursor CRISPR RNA (pre-crRNA) processing and require additional regulatory components.
• The Cas12i.3 nuclease lacks pre-crRNA processing ability, limiting its multiplexing potential.

Purpose of the Study:

• To engineer a highly efficient and simplified multiplexed genome editing system based on Cas12i.3.
• To overcome the limitations of wild-type Cas12i.3 for broad genome editing applications.

Main Methods:

• Optimized CRISPR RNA (crRNA) design, codon usage, and exonuclease fusion to create initial optimized Cas12i (IOCas12i).
• Employed rational design and amino acid mutations to develop enhanced optimized Cas12i (EOCas12i) systems (EOCas12i-Combo1 and EOCas12i-Combo2).
• Evaluated editing efficiency, specificity, and multiplexing capability using up to 30 target crRNA arrays.

Main Results:

• Engineered EOCas12i systems demonstrated significantly enhanced editing efficiencies (2.5- to 60.0-fold) compared to wild-type Cas12i.3.
• Achieved editing efficiencies comparable to established systems like Streptococcus pyogenes Cas9 (SpCas9) and Lachnospiraceae bacterium Cas12a (LbCas12a).
• Demonstrated efficient multiplexed editing of up to 30 targets using compact crRNA arrays, producing longer indels beneficial for gene knockout.

Conclusions:

• The developed EOCas12i-Combo1 and EOCas12i-Combo2 systems represent significant advancements in multiplexed genome editing.
• These engineered nucleases offer a simplified and highly efficient platform for diverse genome editing applications.
• The enhanced Cas12i variants overcome previous limitations, paving the way for broader use in genetic research and engineering.