CRISPR-Cas orthologues and variants: optimizing the repertoire, specificity and delivery of genome engineering tools

Abstract

Robust and cost-effective genome editing in a diverse array of cells and model organisms is now possible thanks to the discovery of the RNA-guided endonucleases of the CRISPR-Cas system. The commonly used Cas9 of Streptococcus pyogenes shows high levels of activity but, depending on the application, has been associated with some shortcomings. Firstly, the enzyme has been shown to cause mutagenesis at genomic sequences resembling the target sequence. Secondly, the stringent requirement for a specific motif adjacent to the selected target site can limit the target range of this enzyme. Lastly, the physical size of Cas9 challenges the efficient delivery of genomic engineering tools based on this enzyme as viral particles for potential therapeutic applications. Related and parallel strategies have been employed to address these issues. Taking advantage of the wealth of structural information that is becoming available for CRISPR-Cas effector proteins, Cas9 has been redesigned by mutagenizing key residues contributing to activity and target recognition. The protein has also been shortened and redesigned into component subunits in an attempt to facilitate its efficient delivery. Furthermore, the CRISPR-Cas toolbox has been expanded by exploring the properties of Cas9 orthologues and other related effector proteins from diverse bacterial species, some of which exhibit different target site specificities and reduced molecular size. It is hoped that the improvements in accuracy, target range and efficiency of delivery will facilitate the therapeutic application of these site-specific nucleases.

Fig. 1

Position of mutagenized residues in SpCas9. a Crystal structure of SpCas9 in complex with sgRNA and target DNA (PDB ID 4OO8). The position of the catalytic residues responsible for the HNH (Asp-10) and the RuvC (His-840) nuclease activity, which are mutated in the D10A and H840A nickases, are shown in black and grey respectively. b Detail of the PAM interaction domain in complex with guide RNA and target DNA showing the position of the key residues mutated in variant Cas9 with altered PAM specificities and how they are mutated. c and d Detail of the target interaction domain in complex with guide RNA and target DNA showing the position of the key residues mutated in SpCas9-HF1 (c) and eSpCas9 (d) and how they are mutated. Residue Arg-1060, mutated in eSpCas9 (1.0) and (1.1), is not annotated in the crystal structure. The 20 bp target DNA is shown in white and the sgRNA is shown in red. (Color figure online)

Fig. 2

Domain structure of CRISPR-Cas effector orthologues. REC recognition domain, NUC Nuclease domain, PI PAM interaction domain, BH bridge helix domain, L1 and L2 linker 1 and 2, WED wedge domain, OBD oligonucleotide-binding domain, LHD looped-out helical domain, UK Unknown function domain, NTD N-terminal domain, PLL phosphate lock loop