Optimized TAL effector nucleases (TALENs) for use in treatment of sickle cell disease

TAL effector nucleases (TALENs) represent a new class of artificial nucleases capable of cleaving long, specific target DNA sequences in vivo and are powerful tools for genome editing with potential therapeutic applications. Here we report a pair of custom-designed TALENs for targeted genetic correction of the sickle cell disease mutation in human cells, which represents an example of engineered TALENs capable of recognizing and cleaving a human disease-associated gene. By using a yeast reporter system, a systematic study was carried out to optimize TALEN architecture for maximal in vivo cleavage efficiency. In contrast to the previous reports, the engineered TALENs were capable of recognizing and cleaving target binding sites preceded by A, C or G. More importantly, the optimized TALENs efficiently cleaved a target sequence within the human β-globin (HBB) gene associated with sickle cell disease and increased the efficiency of targeted gene repair by >1000-fold in human cells. In addition, these TALENs showed no detectable cytotoxicity. These results demonstrate the potential of optimized TALENs as a powerful genome editing tool for therapeutic applications.     

Applications of CRISPR/Cas9 technology for targeted mutagenesis,gene replacement and stacking of genes in higher plants

Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described.     

CRISPR/Cas系统：RNA靶向的基因组定向编辑新技术

CRISPR/Cas系统广泛存在于细菌及古生菌中, 是机体长期进化形成的RNA指导的降解入侵病毒或噬菌体DNA的适应性免疫系统。对Ⅱ型CRISPR/Cas系统的改造使其成为继锌指核酸酶(ZFNs)和TALE核酸酶(TALENs)以来的另一种对基因组进行高效定点修饰的新技术, 与ZFNs和TALENs相比, CRISPR/Cas系统更简单, 并且更容易操作。文章重点介绍了Ⅱ型CRISPR/Cas系统的基本结构、作用原理及这一技术在基因组定点修饰中的应用, 剖析了该技术可能存在的问题, 展望了CRISPR/Cas系统的应用前景, 为开展这一领域的研究工作提供参考。     

红枣活性成分及其生物活性研究进展

红枣中的活性成分以多糖、黄酮类、环核苷酸类、多酚类、五环三萜类、生物碱为主,具有抗氧化、免疫调节及抗肿瘤、保护肝脏、降血糖、抗炎等多种生物活性。本文综述了红枣中活性成分及生物活性的研究进展,并对红枣产业的发展进行展望,为红枣中活性成分的开发与利用提供科学依据。     

Agrobacterium-mediated delivery of CRISPR/Cas reagents for genome editing in plants enters an era of ternary vector systems

Lack of appropriate methods for delivery of genome-editing reagents is a major barrier to CRISPR/Cas-mediated genome editing in plants. Agrobacterium-mediated genetic transformation(AMGT) is the preferred method of CRISPR/Cas reagent delivery,and researchers have recently made great improvements to this process. In this article, we review the development of AMGT and AMGT-based delivery of CRISPR/Cas reagents. We give an overview of the development of AMGT vectors including binary vector, superbinary vector, dual binary vector, and ternary vector systems. We also review the progress in Agrobacterium genomics and Agrobacterium genetic engineering for optimal strains. We focus in particular on the ternary vector system and the resources we developed. In summary, it is our opinion that Agrobacterium-mediated CRISPR/Cas genome editing in plants is entering an era of ternary vector systems, which are often integrated with morphogenic regulators. The new vectors described in this article are available from Addgene and/or MolecularCloud for sharing with academic investigators for noncommercial research.     

Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease 9(CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructed one vector targeting eight agronomic genes in rice using the CRISPR/Cas9 multiplex genome editing system. By subsequent genetic transformation and DNA sequencing, we found that the eight target genes have high mutation efficiencies in the T_0 generation. Both heterozygous and homozygous mutations of all editing genes were obtained in T_0 plants. In addition, homozygous sextuple, septuple, and octuple mutants were identified. As the abundant genotypes in T_0 transgenic plants, various phenotypes related to the editing genes were observed. The findings demonstrate the potential of the CRISPR/Cas9 system for rapid introduction of genetic diversity during crop breeding.     

Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome

The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.     

利用rBE3和rBE4系统进行水稻基因单碱基编辑的特异性和遗传稳定性分析

对基于rBE3(Rice base editor)和rBE4碱基编辑系统创制获得Os SERK1(D428N)和pi-ta(S918F)等基因的单碱基编辑突变体材料进行编辑特异性和遗传稳定性分析,旨在全面了解和更好地利用该碱基编辑系统。首先对Os SERK1(D428N)和pi-ta(S918F)sg RNA的潜在脱靶位点进行预测,并对T0代材料中的各脱靶位点进行PCR扩增和Sanger测序检测;同时对该两个基因的突变体材料自交获得的T1代植株的靶位点序列和外源T-DNA分离进行检测。结果显示各T0代材料均未检测到潜在脱靶位点发生单碱基编辑;此外,Os SERK1(D428N)和Os08g07774含有相同的sg RNA位点,且两个位点均能发生单碱基编辑;rBE3或rBE4系统介导产生的碱基编辑可稳定遗传至T1代,并在T1代可获得无外源T-DNA的纯合突变体。上述结果表明由rBE3或rBE4介导的碱基编辑具有较高的特异性,可进行多位点编辑,引入的碱基替换可稳定遗传至后代。