Fulfilling the dream of a perfect genome editing tool.

Abstract

Adaptive nucleic acid-based immune systems of bacteria and archaea provided new tools for genome editing applications and transcription silencing. Despite the rapid success in developing targetable nucleases based on the clustered regularly interspersed short palindromic repeats (CRISPR)-associated (Cas) enzymes, our understanding of how these ribonucleoprotein complexes recognize and process their DNA targets remains rudimentary. A report in PNAS (1) delves into the mechanism of R-loop formation by two very different CRISPR/Cas systems, Cas9 and Cascade (CRISPR-associated complex for antiviral defense). Our ability to tailor the genomes of various organisms has been revolutionized by the development of targetable nucleases with flexible specificity (2). These powerful reagents generate double-strand breaks (DSBs) in the chromosomal DNA with exquisite specificity. Once generated, the DSB is repaired through nonhomologous end-joining or through homologous recombination. The former often leads to gene inactivation, whereas the latter incorporates the desired genetic alterations by using the information from an exogenously supplied template to introduce, correct, or disrupt specific genes. Three types of targetable nucleases are currently in use: zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and the RNA-guided nucleases. Zinc-finger nucleases consist of multiple Zn2+-finger DNA-binding modules selected or engineered to confer specificity for a particular triplet of consecutive base pairs. DNA recognition modules of TALENs are derived from transcription factors found in plant-pathogenic bacteria. Each DNA-binding module of a TALEN specifically interacts with a single base pair, yielding a straightforward recognition code. Two sets of zinc-finger arrays or transcription activator-like effector arrays are engineered to bind the two adjacent DNA sites in a head-to-head orientation, and are fused to the nuclease domains of FokI restriction enzyme. Dimerization activates FokI, allowing it to generate the desired DSB. Requirement for FokI dimerization is an important contributor to cutting specificity, as DSB can only occur when both DNA sequence … [↵][1]1Email: maria-spies{at}uiowa.edu. [1]: #xref-corresp-1-1