In Vitro Reconstitution and Crystallization of Cas9 Endonuclease Bound to a Guide RNA and a DNA Target

Abstract

The programmable RNA-guided DNA cleavage activity of the bacterial CRISPR-associated endonuclease Cas9 is the basis of genome editing applications in numerous model organisms and cell types. In a binary complex with a dual crRNA:tracrRNA guide or single-molecule guide RNA, Cas9 targets double-stranded DNAs harboring sequences complementary to a 20-nucleotide segment in the guide RNA. Recent structural studies of the enzyme have uncovered the molecular mechanism of RNA-guided DNA recognition. Here, we provide protocols for electrophoretic mobility shift and fluorescence-detection size exclusion chromatography assays used to probe DNA binding by Cas9 that allowed us to reconstitute and crystallize the enzyme in a ternary complex with a guide RNA and a bona fide target DNA. The procedures can be used for further mechanistic investigations of the Cas9 endonuclease family and are potentially applicable to other multicomponent protein-nucleic acid complexes.

Keywords: CRISPR-Cas; Cas9; Double-strand DNA break; EMSA; Endonuclease; Fluorescence detection; Genome editing; HPLC; Macromolecular complexes; Protein–RNA interactions.

Figure 1

(A) Schematic workflow of the electrophoretic mobility shift assay (EMSA) to probe for ternary complex formation. In a first step, apo-dCas9 and guide RNA are preincubated to form a binary complex. Addition of annealed duplex substrate leads to the assembly of ternary dCas9-guide RNA-target DNA complex. (B) Nucleic acid components used for the EMSA shown in panel (C). sgRNA is shown in orange with the 5’-terminal GG dinucleotide originating from in vitro transcription using T7 RNA polymerase marked in gray. The complementary and noncomplementary DNA strands are colored in blue and black, respectively. The noncomplementary strand is labeled at its 3’ end with an ATTO532 fluorophore. The PAM sequence and its complement are marked with a green box. (C) Target DNA duplex (50 nM) was titrated with increasing concentrations (0, 10, 50, 100, 250, and 1000 nM) of in vitro reconstituted dCas9-sgRNA complex. Binding reactions were analyzed using a native 8% polyacrylamide gel and visualized by detection of ATTO532 fluorescence using a laser scanner. The lower and upper bands represent the unbound and complex-bound DNA fractions, respectively.

Figure 2

(A and B) Schematic representation of two target DNA variants used in fluorescence-detection size exclusion chromatography (FSEC). The complementary DNA strand (blue) is identical in both DNA targets. The 3’-Cy3-labeled noncomplementary DNA strands (black) differ in length (panel A: target DNA 1, 15 nt; panel B: target DNA 2, 31 nt) to form either a partially or fully double-stranded target substrate. The PAM sequence and its complement are marked with a green box. The sgRNA is colored grey. (C-I) FSEC chromatograms for apo-dCas9 (C), sgRNA (D), binary dCas9-sgRNA complex (E), the ternary dCas9–sgRNA–target DNA 1 complex (F) ternary dCas9–sgRNA–target DNA 2 complex (G), target DNA 1 alone (H), and target DNA 2 alone (I). The measured absorbances at 260 and 280 nm are shown with red and blue lines, respectively. The yellow line depicts the Cy3 fluorescence signal. For the ternary complexes, 20 μl of the labeled peak fractions were analyzed on a native polyacrylamide gel and detected by the Cy3 fluorescence (F and G). (J) Summary of elution volumes from FSEC experiments in (C-I).

Figure 3

(A) Schematic representation of sgRNA and target DNA sequences in the dCas9-sgRNA-target DNA complex used for crystallization screening. The color coding is as in Fig. 2A. (B and C) Two examples of hits from initial crystallization screenings using 1.8 mg ml^-1 ternary complex containing dCas9, sgRNA, and target DNA 1 (see panel A). The crystal screenings were set up at 20 °C and incubated either at 4 or 20 °C. Five out of 19 hits grew at 4 °C. Four out of 19 hits were found in KSCN, Tris-acetate, and polyethylene glycols of different chain length (PEG1500, PEG3350, and PEG4000). Thirteen out of 19 hits were found in Li₂SO₄, PEG4000, and different buffers spanning the pH range from pH 6.0 to 10.0. Crystals in (B) grew from 0.1 M Tris-acetate, pH 8.5, 0.15 M KSCN, 18% PEG3350. Crystals in (C) grew from 0.02 M CAPS, pH 10.0, 0.2 M Li₂SO₄, 15% PEG4000. (D) Optimized crystal after iterative rounds of seeding. The crystallization was performed at 20 °C with 1.5 mg ml^-1 ternary complex in 20 mM HEPES, pH 7.5, 250 mM KCl, 5 mM MgCl₂. The crystal was grown from 0.1 M Tris-acetate, pH 8.5, 0.3 M KSCN, 16% PEG3350. (E) Ternary complex crystal harvested in a 200–300 μm nylon loop. (F) Front and rear views of dCas9-sgRNA-target DNA 1 complex shown in cartoon representation.