The DNA inside human cells provides instructions for all of the processes that happen inside the body. Errors in the DNA may lead to cancer, sickle cell disease, cystic fibrosis, Huntington’s disease, or other genetic disorders. Medical researchers are exploring whether it is possible to replace or repair the faulty DNA (an approach known as gene therapy) to reduce the symptoms, or even cure individuals, of these conditions.

Over the last ten years, a new technology known as CRISPR-Cas9 gene editing has proved to be a reliable and efficient way to make small and precise changes to DNA in living cells. First, an enzyme called Cas9 searches for a segment of target DNA segment that matches a template molecule the enzyme carries. Cas9 then cuts the target DNA, which is repaired to match a new customized DNA sequence: this changes the genetic information of the cell.

The Cas9 protein is made of a succession of building blocks called amino acids that create long chains which then fold to form the final three-dimensional shape of the enzyme. A region of Cas9 known as the HNH domain is responsible for cutting the target DNA. However, it remains unclear exactly which amino acids within this domain work together to sever the DNA.

Here, Zuo et al. combined computational and experimental approaches to reveal the three-dimensional structure of the Cas9 enzyme when the HNH domain is poised to cut the target DNA. The findings were used to generate a computational model of Cas9 and this model predicted that the HNH domain relies on a group of three amino acids known collectively as D839-H840-N863 to cleave DNA strands.

This knowledge is useful to understand exactly how Cas9 modifies genetic information. Ultimately, this may help to improve CRISPR-Cas9 technology so it could be safely used in geneediting therapies.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9) has become a gene-editing tool that holds an immense promise for the development of novel therapeutic approaches for human diseases (Cong et al., 2013; Jinek et al., 2012; Knott and Doudna, 2018; Mali et al., 2013). Two, magnesium (Mg²⁺) ion-dependent, nuclease domains (i.e. HNH and RuvC domains) in Cas9 cleave the target DNA strand (tDNA) complementary to the guide region of a dual single-guide RNA (sgRNA) and the non-target DNA strand (ntDNA), respectively (Gasiunas et al., 2012; Jinek et al., 2012). The conformational and mechanistic knowledge of Cas9 activation to achieve DNA cleavage is essential for the rational engineering of Cas9 to possibly ensure minimal off-target effects while retaining high gene-editing efficiency (Chen et al., 2017a; Kleinstiver et al., 2016; Slaymaker et al., 2016; Sternberg et al., 2015).

Several structures of the SpyCas9-sgRNA-DNA ternary complex to depict the HNH nuclease domain in a ‘cleavage-competent’ state have been reported (Huai et al., 2017; Palermo et al., 2018; Palermo et al., 2017; Zuo and Liu, 2017). Notably, the amino acid residue D861 in the HNH domain of SpyCas9 pointed towards the catalytic center in the absolute majority of resolved crystal structures (Anders et al., 2016; Anders et al., 2014; Dong et al., 2017; Hirano et al., 2016; Jiang et al., 2015; Jiang et al., 2016; Jinek et al., 2014; Liu et al., 2019; Nishimasu et al., 2014; Olieric et al., 2016; Yang and Patel, 2017), molecular dynamic simulation models (Palermo et al., 2018; Zuo and Liu, 2017), and cryo-electron microscopy (cryo-EM) structures (Huai et al., 2017; Jiang et al., 2019; Shin et al., 2017) (Figure 1a–1d). Despite lacking experimental evidence, it is generally believed that D861 directly participates in Mg²⁺ chelation and tDNA cleavage (Huai et al., 2017; Palermo et al., 2018; Palermo et al., 2017; Zuo and Liu, 2017). An in silico model (Yoon et al., 2019) that was recently reported also suggested that D861 and N863 are potentially involved in chelating the Mg²⁺ ion at the catalytic center of the HNH domain, although this discovery also remained untested in an experimental setting. In the diverse homologous structures of DNA/RNA nucleases from other species (Yang, 2008; Yang, 2011), however, the residues spatially equivalent to the D861 of SpyCas9 are conserved as an asparagine. The substitution (N62D) of the corresponding asparagine in the active center of bacteriophage T4 Endonuclease VII (T4 Endo VII) has been shown to abrogate its DNA cleavage activity (Biertümpfel et al., 2007). These observations motivated us to examine the potential role of D861 in the HNH domain of Cas9. We mutated D861 to alanine and tested the activity of the D861A variant using an experimental approach based on Cas9-mediated disruption of the egfp gene in EGFP-expressing human cells. Unexpectedly, this Cas9 variant exhibited DNA-cleavage activity level similar to that of the wild-type protein (Figure 1e and Figure 1—figure supplement 1). To further validate our finding, we performed in vitro cleavage assays using either plasmid or oligo DNA as a substrate and observed that the D861 variant retained similar activity as the wild type, given enough reaction time (Figure 1—figure supplement 2 and Figure 1—figure supplement 3). Our experiments thus demonstrate that D861 is not critical for HNH domain-catalyzed tDNA cleavage, unlike what would be expected from the reported Cas9 complex structures (Anders et al., 2014; Huai et al., 2017; Jiang et al., 2016; Palermo et al., 2018; Palermo et al., 2017; Zuo and Liu, 2017). In other words, the previously reported structures of the HNH domain of DNA-bound Cas9 (Huai et al., 2017; Palermo et al., 2018; Palermo et al., 2017; Zuo and Liu, 2017) potentially represent a conformation that is incompetent for tDNA cleavage. Hence, we refer to this cleavage-incompetent conformation with an inward-facing D861 as ‘psuedoactive state’ hereafter. This report aims to unmask the catalytic state of the HNH nuclease domain in Cas9 and explore the underlying mechanism of activation.

Figure 1 with 5 supplements see all

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Figure 1—source data 1

Numeric data for activity quantification of Cas9 and its variants (XLS).

https://doi.org/10.7554/eLife.46500.009

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Intrigued by the above paradoxical findings between the structural and functional experiments, we performed molecular modeling and molecular dynamics (MD) simulations to further investigate the residues that may participate in the catalysis of tDNA cleavage. We first examined the apo-state crystal structure of Cas9 (Jinek et al., 2014). One noticeable feature in this apo-Cas9 structure is that the α-helical element in the HNH domain ββα-metal (ββα-Me) fold appears to pose a unique conformation with N863 pointing toward the catalytic center (Figure 1—figure supplement 4a). This inward orientation of N863 was distinct from the inward orientation of D861 observed in the Cas9 structures co-crystalized with sgRNA and/or DNA (Figure 1a–1d). However, the N-terminal of the α-helical segment is disordered in this apo-Cas9 structure (Figure 1—figure supplement 4a), suggesting a high conformational flexibility around this region. We completed the disordered regions of the apo-Cas9 structure by homology modeling and performed molecular dynamics (MD) simulation with a Mg²⁺ ion placed in the catalytic ββα-Me motif (Figure 1—figure supplement 4b). As a result, the Mg²⁺ ion formed an octahedral coordination with D839, N863, and four water molecules (Figure 1—figure supplement 4c), which closely resembled the Mg²⁺ coordination in the X-ray crystal structure of Actinomyces naeslundii Cas9 (AnaCas9, Figure 1—figure supplement 5a) (Jinek et al., 2014). Encouraged by this finding, we next applied the above Mg²⁺-bound α-helical conformation in our optimized pseudoactive Cas9 complex structure (Zuo and Liu, 2017) (Figure 2a–2b) and performed MD-based refinement on the entire structure (see details in Materials and methods). In our final structure, D839, H840 and N863 on the ββα-Me motif formed the catalytic triad that was poised for cleaving the tDNA. The Mg²⁺ ion was coordinated with the residues D839 and N863 of SpyCas9, a scissile phosphate (the pro-Sp oxygen and leaving O3’ engaged), and two water molecules in a strained geometry (Figure 2d–2e). This structure highly resembled the catalytic configuration present in the X-ray structure of T4 Endo VII/DNA complex (Biertümpfel et al., 2007) (Figure 2c), indicating the N863 of Cas9 could be engaged in the formation of the catalytic center for tDNA cleavage.

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Figure 2—source data 1

Atomic coordinates of active Cas9-gRNA-DNA complex (in PDB format).

https://doi.org/10.7554/eLife.46500.018

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To validate the role of N863 in SpyCas9 function, we examined the gene-editing activities of an N863A variant and a D861A/N863A double-mutant variant using cell-based assays. Both SpyCas9 variants virtually lost the entire gene-editing capability compared to the wild-type protein (Figure 1f and Figure 1—figure supplement 1). Consistently, our in vitro assays also showed an effective loss of tDNA cleavage activity for the N863A variant, essentially producing only nicked products in the plasmid cleavage assay (Figure 1—figure supplement 2) and causing the disappearance of the 23-nucleotide tDNA cleavage product in the oligo assay (Figure 1—figure supplement 3). We note that the DNA nicks produced in the cell-based assay could be efficiently repaired by the cellular machinery, thereby causing the apparent loss of gene-editing capability in the above N863A variants. Together, our results clearly show N863, instead of D861, is the functional catalytic residue. Some studies have also suggested the possibility of N863 in forming the catalytic center of the Cas9 HNH domain (Nishimasu et al., 2014; Yoon et al., 2019). In contrast to the observation from another in silico model that was recently published (Yoon et al., 2019), our computational model indicated that D861 and N863 are unlikely to be simultaneously involved in the coordination of Mg²⁺ ion for cutting the tDNA. Besides N863, the functional significance of H840 (acting as a general base) has been confirmed by experiments (Jinek et al., 2012; Nishimasu et al., 2014). Another putative active residue in the HNH domain is D839, which could be inferred from the structures of homologous nucleases (Yang, 2008), despite lacking experimental data to support its catalytic role. Here, we discovered that the D839A mutation substantially compromised the gene-editing activity of Cas9 (Figure 1f), which is consistent with our simulation studies (Zuo and Liu, 2017) and directly supports the significance of D839 for Cas9 activity. Collectively, our structural and functional data corroborate the essence of D839, H840 and N863 for the cleavage function of the HNH domain.

Our MD simulations also excluded the likelihood of D837 and N854 (at the two ends of the β-sheet of the ββα-Me motif) to function as the catalytic residues previously postulated to be directly engaged in the coordination of the Mg²⁺ ion (Chen and Doudna, 2017b; Huai et al., 2017). The simulations showed that the side chain of N854 is ~10 Å distant from the tDNA, making it unlikely be a residue that directly mediates catalysis. However, an activity assay demonstrated the partial loss of activity in the N854A variant (Nishimasu et al., 2014), suggesting that N854 may play an auxiliary role in catalysis. According to our MD models and the solved Cas9 structures (Figure 1a–1d), N854 may stabilize the activated conformation of the HNH domain by making several intra-molecular hydrogen bonds within the ββα-Me motif. Moreover, our structural analyses revealed that the SpyCas9 N854 is spatially and functionally equivalent to the AnaCas9 N597 and Staphylococcus aureus Cas9 (SauCas9) N571 (Figure 1—figure supplement 5), indicating this stabilization role may exist in multiple Cas9 orthologs (Jinek et al., 2014; Nishimasu et al., 2015).

The D837A Cas9 moderately lost gene-editing activity (Figure 1f). From the solved Cas9 structures and our simulations, D837 may contribute to the structural integrity of the ββα-Me motif, although only one hydrogen bond involving its carboxyl group appear to be established (Figure 1a–1d). It is also possible that D837 may aid in proper positioning of the tDNA relative to the HNH domain (as described below).

The comparison between the active and pseudoactive Cas9-nucleic acid complex structures revealed distinct interactions that stabilize these two conformational states. In the pseudoactive conformation, R864 appeared to stabilize the catalytic core by forming ionic and hydrogen-bonding interactions within the ββα-Me motif (Figure 2a), as observed in various bound forms of Cas9 (Figure 1a–1d), whereas K866 is engaged to E371 to form a salt bridge (Figure 2b). In contrast, the corresponding residue of R864 is surrogated by K866 in the active state (Figure 2d), which is in line with the structures of AnaCas9 (K609) and SauCas9 (K583) (Figure 1—figure supplement 5), while R864, on the other hand, is tethered to a negative pocket lined by E370, E396 and D406 on the REC1 domain (Figure 2e). Further structural and sequence analysis indicated that the arginine residue, which is at an equivalent position of R864 in the pseudoactive state, is highly conserved across the ββα-Me nucleases beyond Cas9 (Figure 2—figure supplement 1), whereas both the Type II-A and II-C Cas9 orthologs feature an invariant lysine residue (Figure 2—figure supplement 2) that is spatially equivalent to K866 in the active state. Therefore, a basic amino acid residue (Lys/Arg) seemed to be required at the catalytic core for the ββα-Me superfamily. Moreover, K862 formed aliphatic-aromatic or cation-π interactions with F405 in the pseudoactive state (Figure 2b), whereas it appeared to interact with D837 and/or the +5 phosphate (relative to the protospacer adjacent motif) in one of our simulations for the active state (Figure 2—figure supplement 3). Despite these differences in the interactions with the REC1 domain, other regions of the HNH domain, especially the helical segment preceding the ββα motif, showed similar patterns of binding to the REC2 domain in the pseudoactive and active states (Figure 2f and Figure 2—figure supplement 4).

The removal of the interfacial Mg²⁺ ion from our models substantially destabilized the pseudoactive and active conformations of the Cas9-nucleic acid complex by ~600 kcal/mol (Supplementary file 1), supporting the indispensable role of the Mg²⁺ ion for Cas9 conformational activation that has been previously demonstrated (Dagdas et al., 2017; Osuka et al., 2018; Zuo and Liu, 2017). The pseudoactive conformation (denoted as N863-OUT hereafter) appeared to be unique to SpyCas9 since the corresponding asparagine residues in other ββα-Me nucleases, regardless of the apo or bound form, typically oriented toward the catalytic center (Figure 1—figure supplement 5 and Figure 2—figure supplement 1). End-state free energy calculations revealed that the N863-OUT SpyCas9 complex is thermodynamically more stable than its catalytically active (N863-IN) form (Supplementary file 1). Long time-scale MD simulations based on the isolated HNH domain of Cas9 also showed that the N863-IN conformation is remarkably flexible and tends to transition to the N863-OUT conformation, but not vice versa (Figure 2—figure supplement 5). These results, at least partially, explain why the N863-OUT SpyCas9 conformation was captured in the majority of experimental structures (Anders et al., 2016; Anders et al., 2014; Hirano et al., 2016; Huai et al., 2017; Jiang et al., 2015; Jiang et al., 2016; Jinek et al., 2014; Nishimasu et al., 2014).

To further enhance the confidence in our MD-derived structure models, QM/MM simulations were performed. The metal centers shown in Figure 2a and d were optimized by DFTB3 QM/MM simulations (Figure 2—figure supplement 6). Compared to our MD simulations, we identified very limited differences from the QM/MD simulations in terms of the active site configuration. Encouragingly, this observation is consistent with our most recent benchmark work (Zuo and Liu, 2018a) demonstrating the good performance of the used nonbonded Mg²⁺ ion model (Liao et al., 2017) in maintaining challenging enzyme metal centers.

Based on the findings reported here and in the previous studies (Dagdas et al., 2017; Osuka et al., 2018; Sternberg et al., 2015; Yang et al., 2018), we present a working model to illustrate how the activation of SpyCas9-mediated DNA cleavage is achieved (Figure 2g). The binding of DNA to Cas9-sgRNA induces the interconversion of the HNH domain between the checkpoint intermediate (I) state and the pre-cleavage (P) state. By bridging a gap between the catalytic center and the opposite scissile phosphate, the ambient Mg²⁺ ion subsequently facilitates a conformational change to transition the HNH domain from the P state to the tDNA docked pseudoactive (D, N863-OUT) or active (D^*, N863-IN) state. Before the cleavage reaction occurs, both states exist and the conformational population of the two states reaches an equilibrium. The lower-energy pseudoactive state has larger conformational population than the active state. Once the irreversible DNA cleavage reaction rapidly occurs, the active state conformational population decreases rapidly. The equilibrium between these two states is lost and the conformational population begins to move from the pseudoactive state to the active state until the reaction is completed. The coexistence of multiple Cas9 ternary complexes has important implications for deciphering the biphasic kinetics of Cas9-catalyzed DNA cleavage that has been shown recently by Raper et al. (2018). Taking their results into considerations, we surmise that the early, fast phase characterizes a direct transition from the precatalytic state to the active state, while the slow transition from the pseudoactive to the active state likely contributes to the late, slow phase. Our conclusion would benefit from structural determination of the activated Cas9 ternary complex with metal ions.

Meanwhile, we note that the possibility cannot be ruled out that the N863-IN conformation may be stably formed when enough Mg²⁺ ions are present in the solvent. In other words, the N863-IN conformation could be induced by Mg²⁺ even without engaging a DNA substrate. To test this hypothesis, we have performed additional simulations on the pre-cleavage Cas9 complex (Jiang et al., 2016) with multiple Mg²⁺ ions positioned around N863, but were unable to observe the trend toward the N863-IN conformation over µs time scales (data not shown). Consistently, the HNH domain in 4UN3 (Figure 1b, right panel) and other crystals (like 5B2R, 5B2T, 5B2S, 5FQ5, 5FW1, 5FW2, 5FW3 and 6AI6) remained in the N863-OUT conformation even though as high as eight Mg²⁺ ions were cocrystalized (Anders et al., 2016; Hirano et al., 2016; Nishimasu et al., 2014; Olieric et al., 2016; Shin et al., 2017). Therefore, the presence of metal ions alone appears to be insufficient for the HNH domain conformational activation. Nevertheless, further experimental and computational research is needed to elucidate the transition process between the two states shown herein as well as the chemical principles defining the requirement for asparagine instead of aspartic acid.

The data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1 and 2.

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Author details

1. Zhicheng Zuo

   1. Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, United States
   2. College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai, China

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2761-5841

2. Ashwini Zolekar

   Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, United States

   Contribution

   Formal analysis, Validation, Investigation

   Competing interests

   No competing interests declared

3. Kesavan Babu

   Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman, United States

   Contribution

   Formal analysis, Validation, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

4. Victor JT Lin

   Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, United States

   Contribution

   Formal analysis, Validation, Investigation

   Competing interests

   No competing interests declared

5. Hamed S Hayatshahi

   Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, United States

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8639-7130

6. Rakhi Rajan

   Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8719-4412

7. Yu-Chieh Wang

   Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, United States

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Writing—original draft, Writing—review and editing

   For correspondence

   yu-chieh.wang@unthsc.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7445-4561

8. Jin Liu

   Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Validation, Investigation, Visualization, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   jin.liu@unthsc.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1067-4063

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