Fine-Tuning Next-Generation Genome Editing Tools

Abstract

The availability of genome sequences of numerous organisms and the revolution brought about by genome editing tools (e.g., ZFNs, TALENs, and CRISPR/Cas9 or RGENs) has provided a breakthrough in introducing targeted genetic changes both to explore emergent phenotypes and to introduce new functionalities. However, the wider application of these tools in biology, agriculture, medicine, and biotechnology is limited by off-target mutation effects. In this review, we compare available methods for detecting, measuring, and analyzing off-target mutations. Furthermore, we particularly focus on CRISPR/Cas9 regarding various methods, tweaks, and software tools available to nullify off-target effects. The application of GE tools has significantly influenced medicine, biology, and agriculture for targeted genetic changes to both explore emergent phenotypes and introduce new functionalities with unprecedented control and accuracy. Next-generation GE tools can be optimized to have a method of delivery to cells with high on-target specificity by selecting unique sgRNA or Cas9 variants. Several advanced and optimized methods for unbiased genome-wide profiling of nuclease cleavage sites now provide high-throughput analysis of on- and off-target mutations in single cell-derived clones, humans, animals, and plants. © 2016 Elsevier Ltd