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Abstract CRISPR–Cas9 (clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9) has been revolutionizing genome engineering, and in-depth understanding of mechanisms governing its DNA discrimination is critical for continuing technology advances. An arginine-rich bridge helix (BH) connecting the nuclease lobe and the recognition lobe, which is conserved across the Cas9 family, exists in a helix–loop–helix conformation in the apo wild-type protein but converts to a long contiguous helix in the Cas9/RNA binary complex. In this work, distances measured with spin labels were utilized to investigate BH’s conformational transitions in the solution state upon single-guide RNA (sgRNA) binding, which is a critical early event preceding DNA binding and cleavage. Analyses show that sgRNA binding drives BH conformational changes in the wild-type SpyCas9 (SpyCas9WT) as well as in two BH-loop variants, SpyCas92Pro and SpyCas92Ala. Each Cas9–sgRNA binary complex, however, exhibits distinct BH features that reveal mutation-specific effects on helical integrity versus side-chain interactions. In addition, the BH conformational variations can be correlated to the observed changes in the mismatch cleavage profiles of the Cas9 variants. The work represents the first use of distances measured by site-directed spin labeling to investigate Cas9 protein conformational changes in the solution state and advances our understanding on the structure–dynamic–function relationship governing DNA target discrimination by Cas9. 

Since mitochondria contribute to tumorigenesis and drug resistance in cancer, mitochondrial genetic engineering promises a new direction for cancer therapy. Here, we report the use of the perimitochondrial enzymatic noncovalent synthesis (ENS) of peptides for delivering genes selectively into the mitochondria of cancer cells for mitochondrial genetic engineering. Specifically, the micelles of peptides bind to the voltage-dependent anion channel (VDAC) on mitochondria for the proteolysis by enterokinase (ENTK), generating perimitochondrial nanofibers in cancer cells. This process, facilitating selective delivery of nucleic acid or gene vectors into mitochondria of cancer cells, enables the mitochondrial transgene expression of CRISPR/Cas9, FUNDC1, p53, and fluorescent proteins. Mechanistic investigation indicates that the interaction of the peptide assemblies with the VDAC and mitochondrial membrane potential are necessary for mitochondria targeting. This local enzymatic control of intermolecular noncovalent interactions enables selective mitochondrial genetic engineering, thus providing a strategy for targeting cancer cells.