A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants

CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high- efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/ Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edi- ted 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homol- ogous end-joining mechanism followed by homologous recombination-based repair. We also obtained uni- form biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mu- tations in To rice and T1Arabidopsis plants by simultaneous targeting of multiple （up to eight） members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.

IBM1-dependent H3K9 demethylation enables self-silencing of an exogenous silencer for the non-cell autonomous silencing of an endogenous target gene

RNA silencing(RNAi)is a nucleotide sequence-specific process that results in blockage of gene expression(Baulcombe,2004,2005;Gunter Meister,2004;Vaucheret,2006;Chinnusamy and Zhu,2009;Heo and Kim,2009;Matzke et al.,2009;Simon and Meyers,2011).In plants,post-transcriptional gene silencing(PTGS)occurs in the cytoplasm that is induced by small RNAs(sRNAs),which are the products of double-stranded RNAs(dsRNAs)processed by Dicer-like ribonucleases and achieve specificity through base pairing with targeted RNA sequences(Carmell and Hannon,2004;Gunter Meister,2004;Gasciolli et al.,2005).In an alternative pathway occurring in the nucleus,sRNAs corresponding to promoter sequences direct the silencing machinery to block the transcription of homologous promoters(transcriptional gene silencing,TGS).This process requires 24-nt sRNAs for de novo DNA methylation,a process known as RNA-directed DNA methylation(RdDM)(James P.Jackson and Jacobsen,2002;Matzke and Birchler,2005;Matzke et al.,2007;Zilberman et al.,2007;Wierzbicki et al.,200&Zilberman,2008).Histone modifications also play an important role in the establishment and maintenance of DNA methylation(Zuzana Jasencakova,2003;Ooi et al.,2007;Cedar and Bergman,2009;Law and Jacobsen,2010).In plants,it has been shown that the transcription of exogenous transgene transcribing inverted-repeat(exo-/R)sequences produces dsRNAs,triggering exo-//?PTGS that is negatively autoregulated through methylation spreading/transitive silencing.This transitive silencing「einforces the self-silencing of exo-//?and leads to reduced exo-//?PTGS and exo-/R-derived sRNA production.exo-/R-derived sRNAs function as mobile signals to trigger sRNA-mediated non-cell autonomous silencing of an endogenous homologous target gene(endo-gene)(Dong et al.,2011).It remains unknown whether histone modifications play a role in the exo-IR-triggered endo-gene silencing.

Research progresses of artificial nucleic acid cleavage agents

Artificial nucleic acid cleavage agents have attracted close attention because they play important roles in biochemistry and molecular biology. According to the cleavage mechanism of nucleic acid, they are divided into three types, namely free radical, phosphodiester bond hydrolysis and elimination cleavage agents. In this review, a series of cleavage agents, including the site- and sequence-specific ones, are illustrated, and some suggestions for the future researches in this field are also put forward.

Evidence for oxidative damage to prion protein in prion diseases

In prion diseases the irreversible protein structural transformation process is completed in the brains of mammals within a few months, the uniformly generated infectivity displays extraordinary resistance to inactivation, suggesting that a vital energy source is required for the production of infectious particles. Considering the high oxygen-respiration rate in the brains, prion protein oxidative damage can be the crucial factor. Both theoretical consideration of the nature of protein radical reactions and a large body of previously unraveled feature of scrapie and prion diseases have provided multiple distinct lines of compelling evidence which persuasively support a suggestion that the infectious agents may be prion (free) radicals produced from protein oxidative damage. This paper describes that scrapie prions are most likely formed from prion radicals and oxidative species-mediated sequence-specific cross-linking of benign prion proteins.

In-depth cDNA Library Sequencing Provides Quantitative Gene Expression Profiling in Cancer Biomarker Discovery

Quantitative gene expression analysis plays an important role in identifying differentially expressed genes in various pathological states, gene expression regulation and co-regulation, shedding light on gene functions. Although microarray is widely used as a powerful tool in this regard, it is suboptimal quantitatively and unable to detect unknown gene variants. Here we demonstrated effective detection of differential expression and co-regulation of certain genes by expressed sequence tag analysis using a selected subset of cDNA libraries. We discussed the issues of sequencing depth and library preparation, and propose that increased sequencing depth and improved preparation procedures may allow detection of many expression features for less abundant gene variants. With the reduction of sequencing cost and the emerging of new generation sequencing technology, in-depth sequencing of cDNA pools or libraries may represent a better and powerful tool in gene expression profiling and cancer biomarker detection. We also propose using sequence-specific subtraction to remove hundreds of the most abundant housekeeping genes to increase sequencing depth without affecting relative expression ratio of other genes, as transcripts from as few as 300 most abundantly expressed genes constitute about 20% of the total transcriptome. In-depth sequencing also represents a unique advantage of detecting unknown forms of transcripts, such as alternative splicing variants, fusion genes, and regulatory RNAs, as well as detecting mutations and polymorphisms that may play important roles in disease pathogenesis.