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, 112 (10), 2984-9

Rational Design of a split-Cas9 Enzyme Complex

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Rational Design of a split-Cas9 Enzyme Complex

Addison V Wright et al. Proc Natl Acad Sci U S A.

Abstract

Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

Keywords: CRISPR-Cas9; genome engineering; split enzyme.

Conflict of interest statement

Conflict of interest statement: A.V.W., S.H.S. and J.A.D. have filed a related patent.

Figures

Fig. 1.
Fig. 1.
Cas9 can be split into two separate polypeptides that retain the ability to catalyze RNA-guided dsDNA cleavage. (A) Domain organization of WT Cas9 (Top) and split-Cas9 (Bottom), composed of the α-helical lobe and nuclease lobe. Domain junctions are numbered according to Nishimasu et al. (9). BH, bridge helix; PI, PAM-interacting; REC, recognition lobe. The PI domain can be further subdivided into Topo-homology and C-terminal domains (8). (B) Crystal structures of sgRNA/DNA-bound Cas9 (PDB ID code 4OO8) (9) colored according to domain (Left) or by lobe (Right), with the α-helical and nuclease lobes depicted in gray and blue, respectively. Nucleic acids are omitted for clarity. In the observed interface between the lobes (Inset, Left), the dashed line represents a disordered linker spanning residues V713–D718. In the engineered interface (Inset, Right), the dashed line represents a GGS linker connecting E57 to G729, and new N and C termini of the α-helical lobe are shown. (C) SDS polyacrylamide gel electrophoresis (SDS/PAGE) analysis of purified WT Cas9 (159 kDa), the α-helical lobe (77 kDa), and the nuclease lobe (81 kDa). The gel was stained with Coomassie Brilliant Blue. (D) DNA cleavage assay with the indicated Cas9 construct, analyzed by denaturing PAGE. Reactions contained ∼1 nM radiolabeled dsDNA and 100 nM protein–sgRNA complex; split-Cas9 contained a twofold molar excess of the α-helical lobe. Quantified data and kinetic analysis can be found in Fig. S2 and Table S1.
Fig. 2.
Fig. 2.
Split-Cas9 assembly requires the sgRNA. (A) Crystal structure of sgRNA/DNA-bound Cas9 (PDB ID code 4OO8) (9). Cas9 is colored by lobe and shown as a transparent surface, and the sgRNA is colored by motif according to Briner et al. (11), and the DNA is omitted for clarity. (B) Diagrams of full-length and truncated sgRNA variants used in binding experiments; specific motifs of the sgRNA are colored as in A. (C and D) Results from binding experiments using full-length sgRNA (C), and Δhairpins1-2 and Δspacer–nexus sgRNA truncations (D). Radiolabeled RNAs were incubated with increasing concentrations of WT Cas9, individual α-helical and nuclease lobes, or split-Cas9, and the fraction of protein-bound RNA was determined by nitrocellulose filter binding. Equilibrium dissociation constants (Kd) determined from three independent experiments are shown in Table 1. (E) Reference-free class averages from negative-stain EM images of split-Cas9 reconstituted with single-guide RNA (Top Left), WT Cas9 reconstituted with dual-guide RNA (Top Right), and split-Cas9 in the absence of guide RNA (Bottom). For split-Cas9 without sgRNA, several class averages are shown. The width of the boxes corresponds to ∼336 Å. Data with WT Cas9 are adapted from Jinek et al. (8).
Fig. 3.
Fig. 3.
Genomic editing function and selective inactivation of split-Cas9. (A) Schematic of the split-Cas9 RNP nucleofection assay using a full-length EMX1-targeting sgRNA. Illustration and protocol adapted from Lin et al. (13). (B) Analysis of editing efficiencies by nonhomologous end joining (NHEJ) using a T7 endonuclease I assay and agarose-gel electrophoresis. Cells were nucleofected with 100, 30, or 10 pmol of WT or split-Cas9 ribonucleoprotein (RNP) complexes after arrest at mitosis with nocodazole (Sync) or during normal growth (Unsync). Editing efficiencies are shown at the bottom. (C) DNA cleavage time courses using WT and split-Cas9 with either a full-length sgRNA (Top) or the Δhairpins1-2 sgRNA (Bottom). Values were averaged from three independent experiments, and error bars represent the SD. Rate constants can be found in Table S1.

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