Applications and roles of the CRISPR system in genome editing of plants

Genome editing is a valuable tool to target specific DNA sequences for mutagenesis in the genomes of microbes, plants, and animals. Although different genome editing technologies are available, the clustered regularly interspaced short palindromic repeats/Cas9 （CRISPR/ Cas9） system, which utilizes engineered endonucleases to generate a double-stranded DNA break （DSB） in the target DNA region and subsequently stimulates site-specific mutagenesis through DNA repair machineries, is emerging as a powerful genome editing tool for elucidating mecha- nisms of protection from plant viruses, plant disease resistance, and gene functions in basic and applied research. In this review, we provide an overview of recent advances in the CRISPR system associated genome editing in plants by focusing on application of this technology in model plants, crop plants, fruit plants, woody plants and grasses and discuss how genome editing associated with the CRISPR system can provide insights into genome modifications and functional genomics in plants.

Efficient and Specific Modifications of the Drosophila Genome by Means of an Easy TALEN Strategy

Technology development has always been one of the forces driving breakthroughs in biomedical research. Since the time of Thomas Morgan, Drosophilists have, step by step, developed powerful genetic tools for manipulating and functionally dissecting the Drosophila genome, but room for improving these technologies and developing new techniques is still large, especially today as biologists start to study systematically the functional genomics of different model organisms, including humans, in a high-throughput manner. Here, we report, for the first time in Drosophila, a rapid, easy, and highly specific method for modifying the Drosophila genome at a very high efficiency by means of an improved transcription activator-like effector nuclease （TALEN） strategy. We took advantage of the very recently developed ＂unit assembly＂ strategy to assemble two pairs of specific TALENs designed to modify the yellow gene （on the sex chromosome） and a novel autosomal gene. The mRNAs of TALENs were subsequently injected into Drosophila embryos. From 31.2% of the injected Fo fertile flies, we detected inheritable modification involving the yellow gene. The entire process from construction of specific TALENs to detection of inheritable modifications can be accomplished within one month. The potential applications of this TALEN-mediated genome modification method in Drosophila are discussed.

Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System

CRISPR （clustered regularly interspaced short palindromic repeats）/Cas9 system, which is a newly developed technology for targeted genome modification, has been successfully used in a number of species. In this study, we applied this technology to carry out targeted genome modification in maize. A marker gene Zmzb7 was chosen for targeting. The sgRNA-Cas9 construct was transformed into maize protoplasts, and indel （insertion and deletion） mutations could be detected. A mutant seedling with an expected albino phenotype was obtained from screening 120 seedlings generated from 10 callus events. Mutation efficiency in maize heterochromatic regions was also investigated. Twelve sites with different expression levels in maize centromeres or pericentromere regions were selected. The sgRNA- Cas9 constructs were transformed into protoplasts followed by sequencmg the transformed protoplast genomic DNA. The results show that the genes in heterochromatic regions could be targeted by the CRISPR/Cas9 system efficiently, no matter whether they are expressed or not. Meanwhile, off-target mutations were not found in the similar sites having no PAM （protospacer adjacent motif） or having more than two mismatches. Together. our results show that the CRISPR/Cas9 system is a robust and efficient tool for genome modification in both euchromatic and heterochromatic regions in maize.

Characterization and of Ralstonia TAL-Like DNA-Binding Specificities Effectors

Transcription activator-like effectors （TALEs） from Xanthomonas sp. have been used as customizable DNA- binding modules for genome-engineering applications, Ralstonia solanacearum TALE-like proteins （RTLs） exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues （RVDs）, which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts spe- cifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.

TALENs:Customizable Molecular DNA Scissors for Genome Engineering of Plants

Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases（TALENs）,consisting of an engineered specific（TALE） DNA binding domain and a Fok I cleavage domain,are newly developed versatile reagents for genome engineering in different organisms.Because of the simplicity of the DNA recognition code and their modular assembly,TALENs can act as customizable molecular DNA scissors inducing double-strand breaks（DSBs） at given genomic location.Thus,they provide a valuable approach to targeted genome modifications such as mutations, insertions,replacements or chromosome rearrangements.In this article,we review the development of TALENs,and summarize the principles and tools for TALEN-mediated gene targeting in plant cells,as well as current and potential strategies for use in plant research and crop improvement.