Characterization of NiCas12b for In Vivo Genome Editing.

The RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system represents the third family of CRISPR-Cas systems that are harnessed for genome editing. However, only a few nucleases have demonstrated activity in human cells, and their in vivo therapeutic potential remains uncertain. In this study, a green fluorescent protein (GFP)-activation assay is conducted to screen a panel of 15 Cas12b orthologs, and four of them exhibited editing activity in mammalian cells. Particularly noteworthy is the NiCas12b derived from Nitrospira sp., which recognizes a "TTN" protospacer adjacent motif (PAM) and facilitates efficient genome editing in various cell lines. Importantly, NiCas12b also exhibits a high degree of specificity, rendering it suitable for therapeutic applications. As proof of concept, the adeno-associated virus (AAV) is employed to introduce NiCas12b to target the cholesterol regulatory gene proprotein convertase subtilisin/ kexin type 9 (Pcsk9) in the mouse liver. After 4 weeks of injections, an impressive is observed over 16.0% insertion/deletion (indel) efficiency, resulting in a significant reduction in serum cholesterol levels. NiCas12b provides a novel option for both basic research and clinical applications.