The ability to precisely modify endogenous genes can significantly facilitate biological studies and disease treatment, and the clustered regularly interspaced short palindromic repeats (CRISPR) systems have the potential to be powerful tools for genome engineering. However, the target specificity of CRISPR systems is largely unknown. Here we demonstrate that CRISPR/Cas9 systems targeting the human hemoglobin β and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin δ and C-C chemokine receptor type 2 genes, respectively, causing gross chromosomal deletions. The guide strands of the CRISPR/Cas9 systems were designed to have a range of mismatches with the sequences of potential off-target sites. Off-target analysis was performed using the T7 endonuclease I mutation detection assay and Sanger sequencing. We found that the repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions and point mutations. Therefore, CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites, including those with mismatches to the 12-bases proximal to the guide strand protospacer-adjacent motif.

Jasmonic acid (JA) is a natural hormone regulator involved in development, responses against wounding and pathogen attack. Upon perception of pathogens, JA is synthesized and mediates a signaling cascade initiating various defense responses. Traditionally, necrotrophic fungi have been shown to be the primary activators of JAdependent defenses through the JA-receptor, COI1. Conversely, plants infected with biotrophic fungi have classically been associated with suppressing JA-mediated responses. However, recent evidence has shown that certain biotrophic fungal species also trigger activation of JA-mediated responses and mutants deficient in JA signaling show an increase in susceptibility to certain biotrophic fungal pathogens. These findings suggest a new role for JA in defense against fungal biotrophs. This review will focus on recent research advancing our knowledge of JA-dependant responses involved in defense against both biotrophic and necrotrophic fungi.

Transcription activator-like effector nucleases (TALENs) have become a powerful tool for genome editing due to the simple code linking the amino acid sequences of their DNA-binding domains to TALEN nucleotide targets. While the initial TALEN-design guidelines are very useful, user-friendly tools defining optimal TALEN designs for robust genome editing need to be developed. Here we evaluated existing guidelines and developed new design guidelines for TALENs based on 205 TALENs tested, and established the scoring algorithm for predicting TALEN activity (SAPTA) as a new online design tool. For any input gene of interest, SAPTA gives a ranked list of potential TALEN target sites, facilitating the selection of optimal TALEN pairs based on predicted activity. SAPTA-based TALEN designs increased the average intracellular TALEN monomer activity by >3-fold, and resulted in an average endogenous gene-modification frequency of 39% for TALENs containing the repeat variable di-residue NK that favors specificity rather than activity. It is expected that SAPTA will become a useful and flexible tool for designing highly active TALENs for genome-editing applications. SAPTA can be accessed via the website at http://baolab.bme.gatech.edu/Research/BioinformaticTools/TAL_targeter.html.