CRISPR-Cas system: Toward a more efficient technology for genome editing and beyond

Genome engineering technology is of great interest for biomedical research that enables scientists to make specific manipulation in the DNA sequence. Early methods for introducing double-stranded DNA breaks relies on protein-based systems. These platforms have enabled fascinating advances, but all are costly and time-consuming to engineer, preventing these from gaining high-throughput applications. The CRISPR-Cas9 system, co-opted from bacteria, has generated considerable excitement in gene targeting. In this review, we describe gene targeting techniques with an emphasis on recent strategies to improve the specificities of CRISPR-Cas systems for nuclease and non-nuclease applications.     

CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production

Cyanobacteria hold promise as a cell factory for producing biofuels and bio-derived chemicals, but genome engineering of cyanobacteria such as Synechococcus elongatus PCC 7942 poses challenges because of their oligoploidy nature and long-term instability of the introduced gene. CRISPR-Cas9 is a newly developed RNA-guided genome editing system, yet its application for cyanobacteria engineering has yet to be reported. Here we demonstrated that CRISPR-Cas9 system can effectively trigger programmable double strand break (DSB) at the chromosome of PCC 7942 and provoke cell death. With the co-transformation of template plasmid harboring the gene cassette and flanking homology arms, CRISPR-Cas9-mediated DSB enabled precise gene integration, ameliorated the homologous recombination efficiency and allowed the use of lower amount of template DNA and shorter homology arms. The CRISPR-Cas9-induced cell death imposed selective pressure and enhanced the chance of concomitant integration of gene cassettes into all chromosomes of PCC 7942, hence accelerating the process of obtaining homogeneous and stable recombinant strains. We further explored the feasibility of engineering cyanobacteria by CRISPR-Cas9-assisted simultaneous glgc knock-out and gltA/ppc knock-in, which improved the succinate titer to 435.0±35.0 μg/L, an ≈11-fold increase when compared with that of the wild-type cells. These data altogether justify the use of CRISPR-Cas9 for genome engineering and manipulation of metabolic pathways in cyanobacteria.     

基于CRISPR/Cas系统的噬菌体基因组编辑

对原核生物获得性免疫系统CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR- associated genes)的研究促进了新一代基因组编辑工具的产生和发展。噬菌体既是原核生物CRISPR阵列(CRISPR array)进化的原动力,又是CRISPR/Cas系统防御的对象。噬菌体功能基因组学研究的速率却落后于发现新噬菌体和测定基因组序列的速率。基于CRISPR/Cas系统的噬菌体基因组编辑,可为噬菌体功能基因组学研究提供新手段。本文评述了基于CRISPR/Cas系统编辑噬菌体基因组的几例开创性研究,并且比较了多种操作程序的异同点和优缺点。同时,进一步构建了联合使用CRISPR/Cas系统与噬菌体重组系统开展噬菌体基因组编辑的新方案,讨论了新方案的潜在局限性,并对如何选择不同方案给予了建议。     

Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference

The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today''s challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications.     

GReEn: a tool for efficient compression of genome resequencing data

Research in the genomic sciences is confronted with the volume of sequencing and resequencing data increasing at a higher pace than that of data storage and communication resources, shifting a significant part of research budgets from the sequencing component of a project to the computational one. Hence, being able to efficiently store sequencing and resequencing data is a problem of paramount importance. In this article, we describe GReEn (Genome Resequencing Encoding), a tool for compressing genome resequencing data using a reference genome sequence. It overcomes some drawbacks of the recently proposed tool GRS, namely, the possibility of compressing sequences that cannot be handled by GRS, faster running times and compression gains of over 100-fold for some sequences. This tool is freely available for non-commercial use at ftp://ftp.ieeta.pt/~ap/codecs/GReEn1.tar.gz.     

构建CRISPR-Cas9介导的耻垢分枝杆菌基因组高效删除系统

【背景】耻垢分枝杆菌具有生长迅速和非致病性的特点,可作为结核分枝杆菌致病机理研究替代菌株和类固醇激素生产的工程菌,但目前耻垢分枝杆菌中缺乏高效率的基因组敲除方法。【目的】基于CRISPR-Cas9介导的定点、高效的DNA切割能力,构建耻垢分枝杆菌染色体DNA片段无痕敲除系统。【方法】构建了包含四环素诱导型启动子驱动的密码子优化的cas9基础载体pCas9101,在双侧同源臂长度约为1 kb条件下选用合适的gRNA表达模块,分别测试了对耻垢分枝杆菌mc2155染色体上的3β-羟基类固醇脱氢酶基因(MSMEG_5228,1 071 bp)和胆固醇降解基因簇(MSMEG_5990-MSMEG_6043,约48kb)敲除效率,使用相同大小的同源臂以经典p2NIL-pGOAL方法进行对照,并计算效率。【结果】使用CRISPR-Cas9方法对耻垢分枝杆菌mc2155的3β-羟基类固醇脱氢酶基因敲除效率为22%,胆固醇降解基因簇敲除效率也达到18%,两者连续敲除效率为4%。但对照p2NIL-pGOAL方法未能获得目标DNA片段敲除的菌株。【结论】本文建立的基于CRISPR-Cas9的耻垢分枝杆菌基因组无痕敲除系统显示出较高的敲除效率,该方法可为耻垢分枝杆菌后续研究提供快速高效的基因组操作方法。     

Harnessing CRISPR-Cas for oomycete genome editing

Oomycetes are a group of microorganisms that include pathogens responsible for devastating diseases in plants and animals worldwide. Despite their importance, the development of genome editing techniques for oomycetes has progressed more slowly than for model microorganisms. Here, we review recent breakthroughs in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technologies that are expanding the genome editing toolbox for oomycetes – from the original Cas9 study to Cas12a editing, ribonucleoprotein (RNP) delivery, and complementation. We also discuss some of the challenges to applying CRISPR-Cas in oomycetes and potential ways to overcome them. Advances in CRISPR-Cas technologies are being used to illuminate the biology of oomycetes, which ultimately can guide the development of tools for managing oomycete diseases.     

A novel compression tool for efficient storage of genome resequencing data

With the advent of DNA sequencing technologies, more and more reference genome sequences are available for many organisms. Analyzing sequence variation and understanding its biological importance are becoming a major research aim. However, how to store and process the huge amount of eukaryotic genome data, such as those of the human, mouse and rice, has become a challenge to biologists. Currently available bioinformatics tools used to compress genome sequence data have some limitations, such as the requirement of the reference single nucleotide polymorphisms (SNPs) map and information on deletions and insertions. Here, we present a novel compression tool for storing and analyzing Genome ReSequencing data, named GRS. GRS is able to process the genome sequence data without the use of the reference SNPs and other sequence variation information and automatically rebuild the individual genome sequence data using the reference genome sequence. When its performance was tested on the first Korean personal genome sequence data set, GRS was able to achieve ～159-fold compression, reducing the size of the data from 2986.8 to 18.8 MB. While being tested against the sequencing data from rice and Arabidopsis thaliana, GRS compressed the 361.0 MB rice genome data to 4.4 MB, and the A. thaliana genome data from 115.1 MB to 6.5 KB. This de novo compression tool is available at http://gmdd.shgmo.org/Computational-Biology/GRS.     

CRISPR-Cas orthologues and variants: optimizing the repertoire,specificity and delivery of genome engineering tools

Mammalian Genome - Robust and cost-effective genome editing in a diverse array of cells and model organisms is now possible thanks to the discovery of the RNA-guided endonucleases of the CRISPR-Cas...     

Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFP^KI) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFP^KI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.     

Cas11 enables genome engineering in human cells with compact CRISPR-Cas3 systems

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Hawkeye: an interactive visual analytics tool for genome assemblies

Genome sequencing remains an inexact science, and genome sequences can contain significant errors if they are not carefully examined. Hawkeye is our new visual analytics tool for genome assemblies, designed to aid in identifying and correcting assembly errors. Users can analyze all levels of an assembly along with summary statistics and assembly metrics, and are guided by a ranking component towards likely mis-assemblies. Hawkeye is freely available and released as part of the open source AMOS project .     

Computer-aided engineering of CRISPR-Cas proteins for enhanced human genome editing

<正>Dear editor.The remarkably diversified CRISPR-Cas systems in nature have provided unlimited valuable resources to develop genome editing tools that are revolutionizing the fields of biotechnology and medicine.However,due to their microbial origin,the activity of most naturally occurring CRISPR-Cas nucleases is relatively poor in mammalian cells (Ran et al.,2015).Thus,improving the mammalian genome editing efficiency becomes the priority for harnessing more CRISPRCas systems for widespre...     

Jenti: an efficient tool for mining complex inbred genealogies

SUMMARY: An efficient tool for mining complex inbred genealogies that identify clusters of individuals sharing the same expected amount of relatedness is described. Additionally it allows for the reconstruction of sub-pedigrees suitable for genetic mapping in a systematic way. AVAILABILITY: http://www.jenti.org.