Multi-gene engineering in plants with RNA-guided Cas9 nuclease

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Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos

Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrp^y11 mutant embryos or cardia bifida phenotypes in fau^tm236a mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.     

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering.     

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases

Dear Editor, Recently, zinc finger nuclease, transcrip- tion activator-like effector nuclease, and RNA-guided Cas9 endonuciease （Cas9） have emerged as powerful means for genome editing （Conklin, 2013; Gaj eta[., 2013）. These nucleases are efficient in gen- erating double-strand breaks in the genome that can be repaired by error-prone nonho- mologous end joining leading to a functional knockout （KO） of the targeted gene or used to integrate a DNA sequence at a specific locus through homologous recombination.     

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。     

Combinatorial complexity of pathway analysis in metabolic networks

Elementary flux mode analysis is a promising approach for a pathway-oriented perspective of metabolic networks. However, in larger networks it is hampered by the combinatorial explosion of possible routes. In this work we give some estimations on the combinatorial complexity including theoretical upper bounds for the number of elementary flux modes in a network of a given size. In a case study, we computed the elementary modes in the central metabolism of Escherichia coli while utilizing four different substrates. Interestingly, although the number of modes occurring in this complex network can exceed half a million, it is still far below the upper bound. Hence, to a certain extent, pathway analysis of central catabolism is feasible to assess network properties such as flexibility and functionality.     

Combinatorial pathway optimization for streamlined metabolic engineering

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Mosaicism in CRISPR/Cas9-mediated genome editing

The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, using this technology for generating gene-edited animals involves a number of obstacles. One such obstacle is mosaicism, which is common in founder animals. This is especially the case when the CRISPR/Cas9 system is used in embryos. Here we review the pros and cons of mosaic mutations of gene-edited animals caused by using the CRISPR/Cas9 system in embryos. Furthermore, we will discuss the mechanisms underlying mosaic mutations resulting from the CRISPR/Cas9 system, as well as the possible strategies for reducing mosaicism. By developing ways to overcome mosaic mutations when using CRISPR/Cas9, genotyping for germline gene disruptions should become more reliable. This achievement will pave the way for using the CRISPR technology in the research and clinical applications where mosaicism is an issue.     

Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs

Recombineering, the use of endogenous homologous recombination systems to recombine DNA in vivo, is a commonly used technique for genome editing in microbes. Recombineering has not yet been developed for animals, where non-homology-based mechanisms have been thought to dominate DNA repair. Here, we demonstrate, using Caenorhabditis elegans, that linear DNAs with short homologies (∼35 bases) engage in a highly efficient gene conversion mechanism. Linear DNA repair templates with homology to only one side of a double-strand break (DSB) initiate repair efficiently, and short overlaps between templates support template switching. We demonstrate the use of single-stranded, bridging oligonucleotides (ssODNs) to target PCR fragments for repair of DSBs induced by CRISPR/Cas9 on chromosomes. Based on these findings, we develop recombineering strategies for precise genome editing that expand the utility of ssODNs and eliminate in vitro cloning steps for template construction. We apply these methods to the generation of GFP knock-in alleles and gene replacements without co-integrated markers. We conclude that, like microbes, metazoans possess robust homology-dependent repair mechanisms that can be harnessed for recombineering and genome editing.     

DNA-free genome editing with preassembled CRISPR/Cas9 ribonucleoproteins in plants

Transgenic Research - Processes of traditional trait development in plants depend on genetic variations derived from spontaneous mutation or artificial random mutagenesis. Limited availability of...     

Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation,target binding and cleavage

CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9''s ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9''s off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex.     

Editing the genome of hiPSC with CRISPR/Cas9: disease models

Mammalian Genome - The advent of human-induced pluripotent stem cell (hiPSC) technology has provided a unique opportunity to establish cellular models of disease from individual patients, and to...     

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.     

CRISPR/Cas9基因编辑在三维基因组研究中的应用

CRISPR/Cas9系统在基因编辑方面具有巨大优势,能够低成本、可编程、方便快捷地用于动物、植物以及微生物的基因组靶向编辑和功能改造。三维基因组学是近年来兴起的一门研究染色质高级结构动态调控及基因组生物学功能的交叉学科。在三维基因组研究中,通常采用对DNA片段进行基因编辑以模拟基因组结构性变异,标记特定DNA片段,进而研究调控元件对于基因调控、细胞分化、组织发生、器官形成、个体发育的影响,最终阐明三维基因组的组装调控机制和生物学功能。因此,CRISPR及其衍生技术为研究三维基因组提供了极好的遗传学工具。本文主要综述了CRISPR片段编辑及其衍生技术在三维基因组调控与功能研究中的应用,以期为后续研究工作提供理论参考以及新的研究思路。