¹H, ¹³C, ¹⁵ N backbone resonance assignment of the recognition lobe subdomain 3 (Rec3) from Streptococcus pyogenes CRISPR-Cas9

Abstract

Rec3 is a subdomain of the recognition (Rec) lobe within CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated protein Cas9 that is involved in nucleic acid binding and is critical to HNH endonuclease activation. Here, we report the backbone resonance assignments of an engineered construct of the Rec3 subdomain from Streptococcus pyogenes Cas9. We also analyze backbone chemical shift data to predict secondary structure and an overall fold that is consistent with that of Rec3 from the full-length S. pyogenes Cas9 protein.

Biological context

CRISPR-associated protein 9 (Cas9) is a component of the bacterial immune system now widely used as an innovative genome-editing tool by harnessing its endonuclease function for applications in bioengineering and medicine (Jinek et al. 2012; Charpentier and Doudna 2013; Charpentier and Marraffini 2014; Wang et al. 2016; Hsu et al. 2014). The Cas9 machine is a large, multidomain enzyme that associates with a guide RNA, consisting of either a CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) pair (Deltcheva et al. 2011; Jinek et al. 2012) or a synthetic single-guide RNA (sgRNA), to target and site-specifically cleave DNA (Jiang and Doudna 2017). The Cas9 protein itself is a 160 kDa polypeptide chain comprised of a Photo-spacer Adjacent Motif (PAM) interacting domain (PI) for recognition of the target DNA sequence, an alpha-helical recognition (Rec) lobe that mediates nucleic acid binding, and two endonuclease domains, HNH and RuvC, that cleave the double-stranded DNA in a concerted fashion (Jiang and Doudna 2017). The proposed allosteric network that transmits signals throughout Cas9 as the recognition, binding, and subsequent cleavage events occur is not well understood, though molecular simulations have suggested a continuous allosteric pathway from the PI domain through the Rec lobe to the HNH domain (Oakes et al. 2016; Palermo et al. 2017; East et al. 2020). Despite being poised as a precision genome editing technology for human diseases (Maeder and Gersbach 2016; Strong and Musunuru 2017; Xiong et al. 2016; Martinez-Lage et al. 2018; Tian et al. 2019), a greater understanding of the Cas9 structure is necessary to mitigate its known off-target effects (Zhang et al. 2015) and improve control over the Cas9 machinery. Solution NMR is widely employed to study dynamic mechanisms of proteins (Lisi and Loria 2016a, b), and is now being applied to Cas9 with the purpose of detecting structural fluctuations that underlie its function.

Full-length Cas9, however, is a difficult system to study by NMR due to its size and low thermostability that precludes the collection of data at elevated temperatures to improve spectral quality (Arbogast et al. 2015). Thus, an alternative approach to studying this system is required, and we recently engineered a construct of the HNH domain (15.4 kDa) to characterize site-specific dynamics that span the nuclease with NMR and molecular simulations (Belato et al. 2019; East et al. 2020). Utilizing this “per domain” technique to map the allosteric network in Cas9 by NMR, we have engineered a construct of Rec3 (25.7 kDa, Fig. 1), a subdomain of the Rec lobe. Rec3, which forms a pocket with HNH where the RNA:DNA hybrid resides, undergoes a significant conformational rearrangement upon RNA binding (Jiang et al. 2015). Previous computational studies suggested that Rec3 was critical for HNH activation, based on high motional correlation between these domains that indicated an inter-dependence of their conformational dynamics (Chen et al. 2017; Palermo et al. 2016, 2018). Here, we report the NMR resonance assignments for the amide backbone (H^N, N^H, C_α, C_β, C_O) of Rec3 as a foundational step to study the structure and dynamics of this subdomain, and to probe the contribution of Rec3 to the larger allosteric mechanism that regulates Cas9 function. Importantly, we also demonstrate good agreement of the secondary structure and overall fold between our construct and Rec3 within full-length Cas9 (Fig. 2).

Fig. 1.

¹H-¹⁵ N TROSY HSQC NMR spectrum of Rec3 from S. pyogenes Cas9 collected at 25 °C and 600 MHz. The inset shows assigned residues within the crowded center of the spectrum

Fig. 2.

a Differences in C_α, of C_β, and C_O chemical shifts from random coil as a metric for Rec3 secondary structure. Positive values indicate alpha helical secondary structure and negative values denote beta sheet secondary structure. b CS23D2.0 model of the Rec3 structure based on NMR chemical shifts (cyan) overlaid with Rec3 from full-length Cas9 (black, PDB: 4OO8)

Methods and experiments

Sample preparation

The Rec3 sequence from S. pyogenes Cas9 comprising residues 497–713 was cloned into the pET28a vector with an N-terminal His₆-tag and a TEV protease cleavage site. The plasmid was then transformed into BL21 (DE3) cells (New England Biolabs). Isotopically labeled samples were grown in deuterated M9 minimal media containing CaCl₂, MgSO₄, and MEM vitamins, and supplemented with ¹⁵ N ammonium chloride and ¹³C glucose (1.0 g/liter and 2.0 g/liter, respectively; Cambridge Isotope Laboratories). Glucose supplements had the chemical formula HO¹³CH₂(¹³CHOH)₄¹³CHO, retaining protons on the sugar. Cells were grown at 37 °C to an OD₆₀₀ of 0.8 – 1.0, and then induced with 1 mM IPTG. Following an overnight incubation at 20 °C, the cells were harvested by centrifugation and stored at − 80 °C. For purification of Rec3, the cells were resuspended in a buffer of 20 mM sodium phosphate, 300 mM NaCl, and 5 mM imidazole at pH 8.0, containing a mini EDTA-free protease inhibitor cocktail tablet (Sigma Aldrich), and then lysed by sonication. Cell debris was removed from the lysate by centrifugation. Rec3 was purified from the supernatant by Ni–NTA affinity chromatography and eluted with the same buffer containing 250 mM imidazole. The N-terminal His-tag was cleaved by incubation of Rec3 with TEV protease for 4 h while being dialyzed against a buffer of 20 mM sodium phosphate and 300 mM NaCl at pH 8.0. The His-tag and TEV protease were subsequently removed by Ni–NTA chromatography. Purified Rec3 was dialyzed into a buffer containing 20 mM sodium phosphate, 50 mM KCl, 1 mM EDTA, and 6% ²H₂O at pH 7.5, and concentrated to 650 mM for NMR experiments.

NMR experiments

NMR experiments for backbone resonance assignment were collected on a Bruker Avance NEO 600 MHz spectrometer at 25 °C equipped with pulsed field gradients and a triple resonance cryoprobe. The backbone assignments were completed using a TROSY HSQC and the following TROSY triple resonance experiments (Pervushin et al. 1997): HNCA, HN(CO)CA, HN(CA)CB, HN(COCA)CB, HN(CA)CO, and HNCO (Salzmann et al. 1998). All NMR spectra were processed with NMRPipe (Delaglio et al. 1995) and analyzed in Sparky (Lee et al. 2015). Three-dimensional correlations and resonance assignments were determined in CARA (Keller 2005). Backbone chemical shift data were used in comparison to random coil chemical shifts to estimate the Rec3 secondary structure and a model of the Rec3 construct was developed with the CS23D2.0 server (Wishart et al 2008). CS23D2.0 employs a suite of chemical shift analysis programs and known structures with homologous sequences to generate a 3D model. 4UN4B from PPT-DB (PDB: 4UN4), a full length Cas9 X-ray structure, was utilized in the modeling of the Rec3 construct. For comparison, Fig. 2b over-lays the CS23D2.0 generated structure with an alternative Rec3 structure (PDB: 4OO8) from the full-length Cas9 protein to assess the agreement of the CS23D2.0 model. Many X-ray crystal structures of Cas9 lack electron density in some regions of Rec3, thus we used PDB entry 4OO8 to generate the most complete alignment with visible structural elements.

Assignment and data deposition

The assigned ¹H-¹⁵ N TROSY HSQC NMR spectrum for Rec3 is shown in Fig. 1. 92% of the backbone HN resonances have been assigned, as well as 92% of C_α, 91% of C_β, and 90% of C_O. Missing assignments are relatively dispersed in the sequence, with one cluster (residues 38–42) found in a flexible loop region in the protein core. Figure 2 estimates the secondary structure of the construct and shows good agreement with Rec3 within the full-length Cas9 protein. A list of the ¹H, ¹³C, and ¹⁵ N chemical shifts has been deposited into the BioMagResBank under accession number 50389.