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Review
, 48 (10), e265

Genome Editing: The Road of CRISPR/Cas9 From Bench to Clinic

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Review

Genome Editing: The Road of CRISPR/Cas9 From Bench to Clinic

Ayman Eid et al. Exp Mol Med.

Abstract

Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9). The CRISPR/Cas9 system's simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. Improvements are urgently needed for various aspects of the CRISPR/Cas9 system, including the system's precision, delivery and control over the outcome of the repair process. Here, we discuss the current status of genome engineering and its implications for the future of biological research and gene therapy.

Figures

Figure 1
Figure 1
Site-specific nucleases (SSNs) generate site-specific genomic double-strand breaks (DSBs). SSNs including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) systems can be used to generate a site-specific DSB. Non-homologous end-joining (NHEJ) or homology-directed repair (HDR) can be used to repair the break. NHEJ is an imprecise repair process and generates indels that can be useful in generating functional knockouts. HDR is a precise repair process and is used mainly in editing the DNA sequence. HDR requires the supply of a repair template to copy information across the break during the repair process. HDR is not efficient in most cellular systems.
Figure 2
Figure 2
Different molecular scissors platforms including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9). (a) ZFNs are hybrid proteins between zinc-finger arrays and the catalytic domain of FokI endonuclease. Each ZF array is capable of binding to three nucleotides in the target sequence. Dimerization of the FokI catalytic domain leads to the formation of double-strand breaks (DSBs). (b) TALENs possess a modular central repeat domain that can be engineered to bind any user-selected sequence. Engineering of the sequences and order of RVDs can confer user-defined sequence specificities. TALENs are hybrid proteins between the TAL effector backbone and the catalytic domain of FokI endonuclease. TALENs require two monomers to bind to the sense and antisense strands, respectively. (c) The CRISPR/Cas9 two-component system is composed of Cas9 endonuclease and the single-guide RNA (sgRNA) molecule. Engineering of 20 nucleotides in the sgRNA can confer user-selected specificity. Cas9 nuclease domains cleave both strands within the target sequence preceding the protospacer-associated motif (PAM) NGG trinucleotide sequence.
Figure 3
Figure 3
Different modalities of non-homologous end-joining (NHEJ) and homology-directed repair (HDR)-mediated repair processes. (a) NHEJ, an error-prone repair mechanism, is capable of generating targeted gene mutagenesis, homologue-specific mutagenesis, deletion of tandem genes and large chromosomal deletions. Example 1 shows targeted mutagenesis by the application of a single-guide RNA (sgRNA1). Example 2 shows the targeting of a duplicate gene using a homolog-specific sgRNA (sgRNA2). Example 3 shows the deletion of tandem genes by the application of a single sgRNA present in the tandem genes (sgRNA3). Example 4 shows the deletion of a chromosomal segment by the application of two sgRNAs (sgRNA2 and sgRNA3). (b) HDR, a precise repair mechanism, is capable of generating gene replacements, gene fusions, gene insertions and gene variants. The HDR process depends on the simultaneous delivery of a repair template and the generation of double-strand breaks (DSBs). Different repair outcomes can be generated by manipulating the repair template and homology arms. Example 1 shows targeted gene replacement in which the entire gene is replaced by a repair template with homology arms for replacement. Example 2 shows that sequences can be fused to the gene by supplying a repair template with homology arms suitable for gene fusions. Example 3 shows targeted gene insertions in which the gene is inserted using a repair template with homology arms to the site of insertion.

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