The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5′ coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

Transcription activator-like (TAL) effectors of Xanthomonas oryzae pv. oryzae (Xoo) contribute to pathogen virulence by transcriptionally activating specific rice disease-susceptibility (S) genes 1, 2. TAL effector nucleases (TALENs)-fusion proteins derived from the DNA recognition repeats of native or customized TAL effectors and the DNA cleavage domains of FokI 3, 4, 5-have been used to create site-specific gene modifications in plant cells 6, 7 , yeast 8 , animals 9, 10, 11, 12 and even human pluripotent cells 13. Here, we exploit TALEN technology to edit a specific S gene in rice to thwart the virulence strategy of X. oryzae and thereby engineer heritable genome modifications for resistance to bacterial blight, a devastating disease in a crop that feeds half of the world's population. We targeted the rice bacterial blight susceptibility gene Os11N3 (also called OsSWEET14) for TALEN-based disruption. This rice gene encodes a member of the SWEET sucrose-efflux transporter family and is hijacked by X. oryzae pv. oryzae, using its endogenous TAL effectors AvrXa7 or PthXo3, to activate the gene and thus divert sugars from the plant cell so as to satisfy the pathogen's nutritional needs and enhance its persistence 2, 14. The Os11N3 promoter contains an effector-binding element (EBE) for AvrXa7, overlapping with another EBE for PthXo3 and with the TATA box (Figure 1a and Supplementary Figure 1). We deployed two pairs of designer TALENs (pair 1 and pair 2) independently to induce mutations in these overlapping EBEs of the Os11N3 promoter and thus to interfere with the virulence function of AvrXa7 and PthXo3, but not the developmental function of Os11N3 (Supplementary Figure 1 and Supplementary Note). The TALE repetitive regions used for nuclease fusions included the native AvrXa7 and three designer TALE repetitive regions custom synthesized using a modular

The Cas9/sgRNA of the CRISPR/Cas system has emerged as a robust technology for targeted gene editing in various organisms, including plants, where Cas9/sgRNA-mediated small deletions/insertions at single cleavage sites have been reported in transient and stable transformations, although genetic transmission of edits has been reported only in Arabidopsis and rice. Large chromosomal excision between two remote nuclease-targeted loci has been reported only in a few non-plant species. Here we report in rice Cas9/sgRNA-induced large chromosomal segment deletions, the inheritance of genome edits in multiple generations and construction of a set of facile vectors for high-efficiency, multiplex gene targeting. Four sugar efflux transporter genes were modified in rice at high efficiency; the most efficient system yielding 87–100% editing in T0 transgenic plants, all with di-allelic edits. Furthermore, genetic crosses segregating Cas9/sgRNA transgenes away from edited genes yielded several genome-edited but transgene-free rice plants. We also demonstrated proof-of-efficiency of Cas9/sgRNAs in producing large chromosomal deletions (115–245 kb) involving three different clusters of genes in rice protoplasts and verification of deletions of two clusters in regenerated T0 generation plants. Together, these data demonstrate the power of our Cas9/sgRNA platform for targeted gene/genome editing in rice and other crops, enabling both basic research and agricultural applications.

DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination.