Programmable RNA editing with compact CRISPR–Cas13 systems from uncultivated microbes

Competitive coevolution between microbes and viruses has led to the diversification of CRISPR–Cas defense systems against infectious agents. By analyzing metagenomic terabase datasets, we identified two compact families (775 to 803 amino acids (aa)) of CRISPR–Cas ribonucleases from hypersaline samples, named Cas13X and Cas13Y. We engineered Cas13X.1 (775 aa) for RNA interference experiments in mammalian cell lines. We found Cas13X.1 could tolerate single-nucleotide mismatches in RNA recognition, facilitating prophylactic RNA virus inhibition. Moreover, a minimal RNA base editor, composed of engineered deaminase (385 aa) and truncated Cas13X.1 (445 aa), exhibited robust editing efficiency and high specificity to induce RNA base conversions. Our results suggest that there exist untapped bacterial defense systems in natural microbes that can function efficiently in mammalian cells, and thus potentially are useful for RNA-editing-based research. This work describes the identification of Cas13 proteins from two families by mining public metagenomic data. The newly identified Cas13X.1 shows efficient target knockdown and can be used to degrade SARS-CoV-2 and H1N1 genomes. In addition, the truncated Cas13X.1 offers an advantage in generating mini-RNA base editors.