Cas9 as a versatile tool for engineering biology

Abstract

RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have dramatically transformed our ability to edit the genomes of diverse organisms. We believe tools and techniques based on Cas9, a single unifying factor capable of colocalizing RNA, DNA and protein, will grant unprecedented control over cellular organization, regulation and behavior. Here we describe the Cas9 targeting methodology, detail current and prospective engineering advances and suggest potential applications ranging from basic science to the clinic.

Figure 1

Functioning of the type II CRISPR-Cas systems in bacteria. Phase 1: in the immunization phase, the CRISPR system stores the molecular signature of a previous infection by integrating fragments of invading phage or plasmid DNA into the CRISPR locus as ‘spacers’. Phase 2: in the immunity phase, the bacterium uses this stored information to defend against invading pathogens by transcribing the locus and processing the resulting transcript to produce CRISPR RNAs (crRNAs) that guide effector nucleases to locate and cleave nucleic acids complementary to the spacer. First, tracrRNAs hybridize to repeat regions of the pre-crRNA. Second, endogenous RNase III cleaves the hybridized crRNA-tracrRNA, and a second event removes the 5′ end of the spacer, yielding mature crRNAs that remain associated with the tracrRNA and Cas9. The complex cleaves complementary ‘protospacer’ sequences only if a PAM sequence is present.

Figure 2

Cas9-sgRNA targeting complexes. (a) The basic S. pyogenes Cas9-sgRNA RNA-guided nuclease complex for eukaryotic genome engineering. Target recognition and cleavage require protospacer sequence complementary to the spacer and presence of the appropriate NGG PAM sequence at the 3′ of the protospacer. (b) Cas9 enables programmable localization of dsDNA, RNA and proteins. Proteins can be targeted to any dsDNA sequence by simply fusing them to Cas9_{nuclease-null}, and additional RNA can be tethered to sgRNA termini without compromising Cas9 binding. Attaching RNA binding sites can in turn recruit RNA-binding proteins or directly recruit other RNAs via sequence hybridization. Finally, Cas9 can theoretically bring together any two dsDNA regions by employing sgRNA-Cas9 ‘staples’ that bind to the targeted loci and to one another. Consequently, Cas9 can in principle bring together any fusion proteins, any natural or fusion RNAs and/or any dsDNA locus to any other dsDNA sequence of interest. (c) The diverse potential applications of Cas9 range from targeted genome editing (via simplex and multiplex double-strand breaks and nicks) to targeted genome regulation (via tethering of epigenetic effector domains to either the Cas9 or sgRNA, and via competition with endogenous DNA binding factors) and possibly programmable genome reorganization and visualization. Cas9 might also be engineered to function as an RNA-guided recombinase, and via RNA tethers could serve as a scaffold for the assembly of multiprotein and nucleic acid complexes.

Figure 3

Platform for multiplex biological screens. To modulate multiple genomic sites, sgRNA libraries can be generated and delivered into target cells that also express orthogonal Cas9 effectors (nucleases, activators and/or repressors). This format enables multiplex ex vivo and in vivo genetic screens via targeted genome editing and/ or regulation.

Figure 4

Cas9 therapeutics. Potential Cas9-mediated therapeutic approaches include targeted genome editing to correct genetic disorders and targeted genome regulation to modify endogenous protein levels (top). Technical hurdles and potential routes to achieve these objectives are listed (bottom).