Abstract 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.