An engineered CRISPR–Cas12i tool for efficient multiplexed genome editing

Developing efficient and simplified tools for multiplexed genome editing remains challenging due to limitations in precursor CRISPR RNA (pre-crRNA) processing and reliance on additional RNA-based regulatory components. Cas12i.3, a small RNA-guided nuclease, reportedly lacks pre-crRNA processing ability, restricting its multiplexing capability. Here, we engineered Cas12i.3 by optimizing CRISPR RNA (crRNA) design, codon usage, and exonuclease fusion, generating initial optimized Cas12i (IOCas12i) system. Further rational design and amino acid mutations yielded the highly efficient enhanced optimized Cas12i (EOCas12i) systems, EOCas12i-Combo1 and EOCas12i-Combo2, exhibiting 2.5- to 22.8-fold and 3.0- to 60.0-fold editing efficiencies relative to wild-type Cas12i.3, comparable to Streptococcus pyogenes Cas9 (SpCas9) and Lachnospiraceae bacterium Cas12a (LbCas12a). Additionally, they exhibited high specificity and produced longer insertions and deletions (indels) that may facilitate gene knockout. Notably, both variants enabled efficient multiplexed editing of up to 30 targets using compact crRNA arrays. These advancements position EOCas12i-Combo1 and EOCas12i-Combo2 as promising platforms for multiplexed genome editing applications.