Genome Editing in Rice and Tomato with a Small Un1Cas12f1 Nuclease

The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating prokaryotic and eukaryotic genomes. Despite the robustness and versatility of Cas9 and Cas12a/b nucleases in mammalian cells and plants, their large protein sizes may hinder downstream applications. Therefore, investigating compact CRISPR nucleases will unlock numerous genome editing and delivery challenges that constrain genetic engineering and crop development. In this study, we assessed the archaeal miniature Un1Cas12f1 type-V CRISPR nuclease for genome editing in rice and tomato protoplasts. By adopting the reengineered guide RNA modifications ge4.1 and comparing polymerase II (Pol II) and polymerase III (Pol III) promoters, we demonstrated uncultured archaeon Cas12f1 (Un1Cas12f1) genome editing efficacy in rice and tomato protoplasts. We characterized the protospacer adjacent motif (PAM) requirements and mutation profiles of Un1Cas12f1 in both plant species. Interestingly, we found that Pol III promoters, not Pol II promoters, led to higher genome editing efficiency when they were used to drive guide RNA expression. Unlike in mammalian cells, the engineered Un1Cas12f1-RRA variant did not perform better than the wild-type Un1Cas12f1 nuclease, suggesting continued protein engineering and other innovative approaches are needed to further improve Un1Cas12f1 genome editing in plants. Exploration of a novel compact clustered regularly interspaced short palindromic repeats-Cas system in plants. Testing of uncultured archaeon Cas12f1 in both monocot and dicot plants. Improved genome editing with polymerase III promoters for guide RNA expression. Plant genome editing is a promising breeding technology. Many clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been discovered and engineered as tools for genome editing. Most of the existing CRISPR-Cas nucleases and genome editing systems are based on Cas9 and Cas12a, which are large proteins. The Hypercompact CRISPR-Cas system could enable more innovative delivery approaches. Here, we explored uncultured archaeon Cas12f1 (Un1Cas12f1), a type V-F miniature CRISPR system, for genome editing in plants. We developed an efficient CRISPR-Un1Cas12f1 expression system that is based on an optimized guide RNA scaffold and polymerase III small RNA promoters. We further characterized Un1Cas12f1's sequence preference and editing outcomes in rice and tomato protoplasts by next-generation sequencing. This is a comprehensive study of the Un1Cas12f1 system in plants. Further improvement of Un1Cas12f1 is warranted before it becomes a powerful tool for genome editing in plants.