Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States

Abstract

High-resolution Cas9 structures have yet to reveal catalytic conformations due to HNH nuclease domain positioning away from the cleavage site. Nme1Cas9 and Nme2Cas9 are compact nucleases for in vivo genome editing. Here, we report structures of meningococcal Cas9 homologs in complex with sgRNA, dsDNA, or the AcrIIC3 anti-CRISPR protein. DNA-bound structures represent an early step of target recognition, a later HNH pre-catalytic state, the HNH catalytic state, and a cleaved-target-DNA-bound state. In the HNH catalytic state of Nme1Cas9, the active site is seen poised at the scissile phosphodiester linkage of the target strand, providing a high-resolution view of the active conformation. The HNH active conformation activates the RuvC domain. Our structures explain how Nme1Cas9 and Nme2Cas9 read distinct PAM sequences and how AcrIIC3 inhibits Nme1Cas9 activity. These structures provide insights into Cas9 domain rearrangements, guide-target engagement, cleavage mechanism, and anti-CRISPR inhibition, facilitating the optimization of these genome-editing platforms.

Keywords: AcrIIC3; HNH domain; Nme1Cas9; Nme2Cas9; PAM; anti-CRISPR; catalytic state; genome editing; sgRNA.

Figure 1.. The overall structure of Nme1Cas9 in complex with truncated sgRNA

A. Domain organization of Nme1Cas9. B. Schematic representation of the 135-nt sgRNA used for crystallization. The repeat:anti-repeat region is highlighted in green background. The stem-loops 1 and 2 are highlighted in pink and grey background, respectively. The black and blue dashed-line boxes indicate the truncated nts of the 122-nt and 102-nt sgRNAs, respectively. The cartoon diagram represents the state of Nme1Cas9 in this structure. C. Crystal structure of the Nme1Cas9-sgRNA binary complex. Individual Nme1Cas9 domains are colored according to the scheme in Figure 1A. D. DNA cleavage by wild-type and mutant Nme1Cas9 using linearized plasmid DNA containing a target sequence fully complementary to the sgRNA, and various sgRNAs containing different truncations. E. Top: schematic representation of the partially double-stranded DNA target with its TS complementary to guide nts 17-24 (seed only). Bottom: crystal structure of the seed-only Nme1Cas9-sgRNA-dsDNA complex. F. Conformational changes of Nme1Cas9 before (C) and after (E) PAM recognition and seed pairing. See also Figures S1, S2, and Table S1.

Figure 2.. Overall structure of the HNH-inactivated, fully complementary Nme1Cas9-sgRNA-dsDNA complex

A. Schematic diagram of the sgRNA and DNA target used for co-crystallization. Guide RNA, TS DNA and NTS DNA are in orange, red and black, respectively. B. Cartoon and surface representation of Nme1Cas9 His588Ala in complex with sgRNA and a fully complementary DNA duplex bearing a 5'-N₄GATT-3' PAM sequence. C. Contacts between Nme1Cas9 residues and the guide-TS heteroduplex in the catalytically poised structure. Residues are color-coded by domain as in Figures 1A and 2B. D. Zoomed-in view of the PAM-binding region in Nme1Cas9. E. DNA endonuclease activity of wild-type (WT) and mutant Nme1Cas9 using a linearized plasmid DNA containing a target sequence fully complementary to the sgRNA shown in (A). See also Figure S3 and Table S1.

Figure 3.. HNH catalytic conformation

A. The interaction between the HNH domain in the catalytic state and the TS. B. Close-up view of the catalytic site of the HNH domain and the TS cleavage site (the phosphate between TS 3 and 4). The Mg²⁺ is shown as a magenta sphere. The distances between the Mg²⁺ and the coordinated atoms are 2.1-2.3 Å. C. The interaction of Linker L1 and the RNA-DNA duplex. D-E Structural comparison of the HNH domain in the seed-matched (D) and fully paired (E) states showing that the formation of the RNA-DNA heteroduplex drives the conformational change of the HNH domain. F-G. The DNA cleavage assay using linear plasmid DNA. H. The schematic representation of the Cy3- and Cy5- labeled short DNA (TS and NTS, respectively). I-J. DNA cleavage of the Cy3- and Cy5-labeled DNA with Nme1Cas9 mutants at 25°C. See also Figure S3.

Figure 4.. The crystal structure of Nme1Cas9-sgRNA in complex with the cleaved, partial-duplex DNA

A. Overall structure of the product-bound wild-type Nme1Cas9. B. Close-up view of the catalytic site of the HNH domain and the RNA-DNA duplex. The 3'-product of the cleaved DNA is in light magenta (left panel). The Fo-Fc electron density omit map of nts 2-5 in the target strand (contoured at 3.0 σ) is shown. C. Interactions between Nme1Cas9 and the RNA-DNA duplex. The two fragments of broken DNA are in red and light magenta. D. Structural comparison of the Linker L1 and HNH domain in the HNH catalytic state (in grey) and the post-cleavage state. E. Conformational change of Nme1Cas9 upon TS cleavage. F. RNA-DNA duplex comparison in the HNH catalytic state (in grey) and the product-bound state showing the shift of the cleaved TS. The colors in the product-bound state are identical to those used in (A). See also Table S1.

Figure 5.. The crystal structure of the Nme2Cas9-sgRNA-dsDNA complex, and requirements for Nme1Cas9/Nme2Cas9/sgRNA residues during genome editing

A. The overall structure of the Nme2Cas9-sgRNA-dsDNA complex. B. Detailed view of the interaction between the PI domain of Nme2Cas9 and the 5'-N₄CC-3' PAM sequence. C. Editing efficiency of wild-type and mutant Nme1Cas9 at two genomic sites in HEK293T cells. D. Nme1Cas9 editing efficiency of wild-type and mutant sgRNA at two genomic sites in HEK293T cells. E. Editing efficiency of wild-type and mutant Nme2Cas9 at two genomic sites in HEK293T cells. Editing efficiency values are mean ± s.d. from three biological replicates. See also Figures S4, S5 and Table S2.

Figure 6.. Structures of Nme1Cas9-sgRNA in complex with AcrIIC3 or with both AcrIIC3 and dsDNA

A. AcrIIC3 binds to Nme1Cas9 in the apo, sgRNA-bound, and sgRNA-dsDNA-bound states. B. Architecture of the Nme1Cas9-sgRNA in complex with AcrIIC3, showing that two AcrIIC3 monomers fasten two Nme1Cas9-sgRNA complexes together. C. Architecture of the Nme1Cas9-sgRNA in complex with AcrIIC3 and dsDNA, showing that AcrIIC3 does not fully prevent target DNA binding. See also Figure S6 and Table S3.

Figure 7.. Two AcrIIC3 monomers interact with the HNH and REC2 domains from two Nme1Cas9 complexes, inhibiting the HNH domain from rotating to the scissile site

A. AcrIIC3 interacts with the HNH and REC2 domains from two Nme1Cas9 complexes. B-D. The detailed interactions between AcrIIC3 and the HNH domain in Nme1Cas9.1 (B), Linker L1 in Nme1Cas9.1 (C), and the REC2 domain in Nme1Cas9.2 (D). E. The HNH domain in the AcrIIC3-bound Nme1Cas9 complex is located distant from the cleavage site (highlighted by a magenta sphere) on the target strand. The RuvC and PI domains are omitted for clarity. F. Schematic representations of interactions that enable AcrIIC3 inhibition of DNA cleavage by Nme1Cas9 and Nme2Cas9. See also Figure S7.