Rapid and efficient gene modification in rice and Brachypodium using TALENs

Journal Article / 2013

Shan, Qiwei; Wang, Yanpeng; Chen, Kunling; Liang, Zhen; Li, Jun; Zhang, Yi; Zhang, Kang; Liu, Jinxing; Voytas, Daniel F.; Zheng, Xuelian; Zhang, Yong; Gao, Caixia

In the past few years, the use of sequence-specific nucleases for efficient targeted mutagenesis has provided plant biologists with a powerful new approach for understanding gene function and developing new traits. These nucleases create DNA double-strand breaks at chromosomal targeted sites that are primarily repaired by the non-homologous end joining (NHEJ) or homologous recombination (HR) pathways. NHEJ is often imprecise and can introduce mutations at target sites resulting in the loss of gene function. In contrast, HR uses a homologous DNA template for repair and can be employed to create site-specific sequence modifications or targeted insertions (Moynahan and Jasin, 2010). In spite of being developed only 3 years ago, TALENs have become the reagent of choice for efficiently modifying eukaryotic genomes in a targeted fashion (Baker, 2012; Liu et al., 2012; Tong et al., 2012). Like ZFNs, TALENs are composed of an engineered array of DNA-binding domains fused with a non-specific FokI nuclease domain (Christian et al., 2010). Two TALEN monomers bind target DNA sequences allowing the FokI domains to dimerize and cleave the sequence between the two recognition sites (Supplemental Figure 1A). Each repeat in the DNA-binding domain of a TALEN recognizes one nucleotide in the target in a largely context-independent fashion, making the engineering of nucleases with new DNA sequence specificities easier and more reliable than ZFNs and meganucleases (Reyon et al., 2012).