In vitro enzymology of Cas9

Abstract

Cas9 is a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNAs with complementarity to the guide RNA. The programmable sequence specificity of Cas9 has been harnessed for genome editing and gene expression control in many organisms. Here, we describe protocols for the heterologous expression and purification of recombinant Cas9 protein and for in vitro transcription of guide RNAs. We describe in vitro reconstitution of the Cas9-guide RNA ribonucleoprotein complex and its use in endonuclease activity assays. The methods outlined here enable mechanistic characterization of the RNA-guided DNA cleavage activity of Cas9 and may assist in further development of the enzyme for genetic engineering applications.

Keywords: Biochemical assay; CRISPR; Endonuclease; Gene targeting; Genome editing; Protein–RNA complex; RNA guided; crRNA; tracrRNA.

Figure 1.1

(A) Schematic representation of the dual-RNA guide structure for programming SpyCas9. Blue: crRNA containing a 20-nt guide sequence and 22-nt invariant sequence. The GG dinucleotide at the 5’ end is appended during in vitro transcription. Red: tracrRNA base-pairing with the invariant sequence of the crRNA. tracrRNA contains three stem-loops (SL1, SL2, and SL3) at its 3’ end. Although SL1 is sufficient for Cas9-mediated cleavage in vitro (Jinek et al., 2012), inclusion of both SL2 and SL3 increases cleavage efficiency by increasing the stability of the Cas9-crRNA-tracrRNA complex (Hsu et al., 2013; Nishimasu et al., 2014). (B) Schematic representation of chimeric single-guide RNA (sgRNA). crRNA- and tracrRNA-derived sequences are connected by a 5’-GAAA-3’ tetraloop linker. (C) Outline of the procedure for sgRNA guide design and preparation by in vitro transcription.

Figure 1.2

(A) sgRNA used in endonuclease activity assays in panels B and C. The sgRNA contains stem-loops SL1 and SL2 but lacks SL3 (total length of sgRNA is 83 nt). (B) Endonuclease activity assay of SpyCas9 using SspI-linearized plasmid (2702 bp). Samples were taken at indicated time points. Cleavage products (2104 and 598 bp) were resolved on a 1% agarose gel and stained with GelRed. The sequence of the target site in the plasmid substrate is shown above the gel image. (C) Endonuclease activity assay of SpyCas9 using a double-stranded oligonucleotide target. The oligonucleotide duplex substrate is shown above the gel image. The cleavage reaction was sampled the indicated time points and analyzed by electrophoresis on a denaturing (7 M urea) polyacrylamide gel. ATTO532 fluorescence was detected using a FLA9500 laser scanner.