CRISPR-Cas systems for editing, regulating and targeting genomes

Abstract

Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

Figure 1. Nuclease-induced genome editing

Nuclease induced double-strand breaks (DSBs) can be repaired by NHEJ or HDR pathways. Imprecise NHEJ-mediated repair can produce variable-length insertion and deletion mutations at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template.

Figure 2. Naturally occurring and engineered CRISPR-Cas9 systems

(a) Naturally occurring CRISPR-Cas systems incorporate foreign DNA sequences into CRISPR arrays, which then produce crRNAs bearing “protospacer” regions that are complementary to the foreign DNA site. crRNAs hybridize to tracrRNAs (also encoded by the CRISPR system) and this pair of RNAs associates with the Cas9 nuclease. crRNA/tracrRNA/Cas9 complexes recognize and cleave foreign DNAs bearing the protospacer sequences. Additional details in the text. (b) and (c) Engineered CRISPR-Cas system consists of a fusion between a crRNA and part of the tracrRNA sequence. This single gRNA then complexes with Cas9 to mediate cleavage of target DNA sites that are complementary to the first (5′) 20 nts of the gRNA and that lie next to a PAM sequence. Additional details in the text.

Figure 3. Cas9-based systems for altering gene sequence or expression

(a) Cas9 nuclease creates double strand breaks at DNA target sites with complementarity to the 5′ end of a gRNA. (b) Cas9 nickase created by mutation of the RuvC nuclease domain with a D10A mutation. This nickase cleaves only the DNA strand that is complementary to and recognized by the gRNA. (c) Cas9 nickase created by mutation of the HNH nuclease domain with a H840A mutation. This nickase cleaves only the DNA strand that does not interact with the sRNA. (d) Paired nickase strategy for improving Cas9 specificity. D10A Cas9 nickase directed by a pair of appropriately oriented gRNAs leads to induction of two nicks that, if introduced simultaneously, would be expected to generate a 5′ overhang. (e) Catalytically inactive or ‘dead’ Cas9 (dCas9) that can be recruited by a gRNA without cleaving the target DNA site. (f) Catalytically inactive dCas9 bearing dual D10A/H840A mutations fused to a heterologous effector domain.

Figure 4. Limitations on targeting range of guide RNAs

(a) Potential target sites of gRNAs expressed from a U6 promoter. Restrictions are imposed by the requirement for a G at the first 5′ nucleotide of the gRNA and by the need for an NGG adjacent to the target site (top). One strategy to work around the requirement for a 5′ G is to append this nucleotide to the beginning of the gRNA (bottom). (b) Potential target sites of gRNAs expressed from a T7 promoter. Restrictions are imposed by the requirement for a GG at the first two nucleotides of the gRNA and by the need for an NGG adjacent to the target site (top). One strategy to work around the requirement for a 5′ GG dinucleotide is to append GG to the beginning of the gRNA (bottom).

Figure 4. Limitations on targeting range of guide RNAs

(a) Potential target sites of gRNAs expressed from a U6 promoter. Restrictions are imposed by the requirement for a G at the first 5′ nucleotide of the gRNA and by the need for an NGG adjacent to the target site (top). One strategy to work around the requirement for a 5′ G is to append this nucleotide to the beginning of the gRNA (bottom). (b) Potential target sites of gRNAs expressed from a T7 promoter. Restrictions are imposed by the requirement for a GG at the first two nucleotides of the gRNA and by the need for an NGG adjacent to the target site (top). One strategy to work around the requirement for a 5′ GG dinucleotide is to append GG to the beginning of the gRNA (bottom).