Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9

Abstract

The past decade has been one of rapid innovation in genome-editing technology. The opportunity now exists for investigators to manipulate virtually any gene in a diverse range of cell types and organisms with targeted nucleases designed with sequence-specific DNA-binding domains. The rapid development of the field has allowed for highly efficient, precise, and now cost-effective means by which to generate human and animal models of disease using these technologies. This review will outline the recent development of genome-editing technology, culminating with the use of CRISPR-Cas9 to generate novel mammalian models of disease. While the road to using this same technology for treatment of human disease is long, the pace of innovation over the past five years and early successes in model systems build anticipation for this prospect.

Figure 3. Binding specificity of CRISPR-Cas9.

(A) With the most commonly used CRISPR-Cas9 system, a guide RNA recognizes and hybridizes a 20-bp protospacer in the genome. The DSB occurs at a site 3-bp upstream of the PAM sequence. (B) “Nickase” CRISPR-Cas9 bind to flanking DNA sequences and generate single-strand nicks that are the equivalent of a DSB. (C) Fusion proteins of catalytically dead CRISPR-Cas9 and FokI nuclease domains bind to flanking DNA sequences and position their FokI domains such that they dimerize and generate a DSB between binding sites.

Figure 2. Binding specificity of ZFNs and TALENs.

(A) The variable length ZFN DNA–binding domains bind to flanking DNA sequences and position their FokI nuclease domains such that they dimerize and generate a DSB between the binding sites. (B) Heterodimeric binding of TALENS, which like ZFNs, bind regions of variable length to generate DSB between binding sites.

Figure 1. Repair of DSBs.

With the creation of each DSB, two DNA repair processes proceed in concert. HDR results in high-fidelity repair using a template strand. If desired, an exogenous oligonucleotide sequence can be introduced to achieve site-specific mutagenesis. NHEJ yields WT clones as well as clones with frameshift/indel mutations through its inherently more error-prone mechanism of repair.