Genome editing of immune cells using CRISPR/Cas9

The ability to read, write, and edit genomic information in living organisms can have a profound impact on research, health, economic, and environmental issues. The CRISPR/Cas system, recently discovered as an adaptive immune system in prokaryotes, has revolutionized the ease and throughput of genome editing in mammalian cells and has proved itself indispensable to the engineering of immune cells and identification of novel immune mechanisms. In this review, we summarize the CRISPR/Cas9 system and the history of its discovery and optimization. We then focus on engineering T cells and other types of immune cells, with emphasis on therapeutic applications. Last, we describe the different modifications of Cas9 and their recent applications in the genome-wide screening of immune cells.     

Genome editing in mammalian cells using the CRISPR type I-D nuclease

Adoption of CRISPR–Cas systems, such as CRISPR–Cas9 and CRISPR–Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense (‘Cascade’: Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR–Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR–Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR–Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.     

基因组编辑技术在猪遗传改良中的应用

核酸酶介导的基因组编辑技术大幅度提高了编辑真核细胞基因组的能力,给生命科学领域带来了革命性地发展,也给猪的遗传改良带来了全新的契机。本文介绍了基因组编辑技术尤其是CRISPR/Cas9系统的发展以及各种天然存在的和人为改造的Cas9变体的作用特点;汇总了利用基因组编辑技术提高猪生产性能,尤其是改善猪肉品质和抵抗病毒感染的研究进展;分析了目前利用基因组编辑技术推进猪遗传改良所面临的挑战;最后,展望了基于基因组编辑技术的猪遗传改良和品种培育的发展趋势。     

In vivo genome editing thrives with diversified CRISPR technologies

Prokaryotic type Ⅱ adaptive immune systems have been developed into the versatile CRISPR technology, which has been widely applied in sitespecific genome editing and has revolutionized biomedical research due to its superior efficiency and flexibility. Recent studies have greatly diversified CRISPR technologies by coupling it with various DNA repair mechanisms and targeting strategies.These new advances have significantly expanded the generation of genetically modified animal models, either by including species in which targeted genetic modification could not be achieved previously, or through introducing complex genetic modifications that take multiple steps and cost years to achieve using traditional methods. Herein, we review the recent developments and applications of CRISPR-based technology in generating various animal models, and discuss the everlasting impact of this new progress on biomedical research.     

Genome editing technologies and their applications in crop improvement

Crop improvement is very essential to meet the increasing global food demands and enhance food nutrition. Conventional crop-breeding methods have certain limitations such as taking lot of time and resources, and causing biosafety concerns. These limitations could be overcome by the recently emerged-genome editing technologies that can precisely modify DNA sequences at the genomic level using sequence-specific nucleases (SSNs). Among the artificially engineered SSNs, the CRISPR/Cas9 is the most recently developed targeted genome modification system and seems to be more efficient, inexpensive, easy, user-friendly and rapidly adopted genome-editing tool. Large-scale genome editing has not only improved the yield and quality but also has enhanced the disease resistance ability in several model and other major crops. Increasing case studies suggest that genome editing is an efficient, precise and powerful technology that can accelerate basic and applied research towards crop improvement. In this review, we briefly overviewed the structure and mechanism of genome editing tools and then emphatically reviewed the advances in the application of genome editing tools for crop improvement, including the most recent case studies with CRISPR/Cpf1 and base-editing technologies. We have also discussed the future prospects towards the improvement of agronomic traits in crops.     

Plant base editing and prime editing: The current status and future perspectives

Precise replacement of an allele with an elite allele controlling an important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement.Base editing and prime editing are two newly developed precision gene editing systems which can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks and donor repair templates,respectively.Since their discoveries,various strate...     

基于CRISPR/Cas9的精准基因编辑技术研究进展

基因编辑技术是一种可以在基因组水平上对DNA序列进行改造的遗传操作技术。基于CRISPR/Cas9系统的精准编辑技术是一个操作方便、应用广泛的基因编辑技术,与传统的CRISPR/Cas9不同,精准基因编辑技术可以在不需要DNA模板的情况下对基因进行定点突变。本文重点介绍了近年来基于CRISPR/Cas9介导的精准基因编辑技术的发展,并深入分析了基因精准编辑技术面临的挑战和机遇。     

Genome editing system CRISPR/CAS9 and peculiarities of its application in monocots

Genome editing is a new methodology for DNA modification that has been developing in recent years. This review compares proposed methods of optimization and development of a modern genome editing system—CRISPR/Cas9—in monocots. Methodical approaches for in silico selecting target sites, designing an expression vector, transferring the vector expression cassette into plant cells, evaluating the results of the editing and nonspecific activity of the system, and obtaining modified plants free of foreign DNA are reviewed. The problem of legislative regulation and the prospects for using this method for commercial purposes are discussed.     

Genome editing: intellectual property and product development in plant biotechnology

Genome editing is a revolutionary technology in molecular biology. While scientists are fascinated with the unlimited possibilities provided by directed and controlled changes in DNA in eukaryotes and have eagerly adopted such tools for their own experiments, an understanding of the intellectual property (IP) implications involved in bringing genome editing-derived products to market is often lacking. Due to the ingenuity of genome editing, the time between new product conception and its actual existence can be relatively short; therefore knowledge about IP of the various genome editing methods is relevant. This point must be regarded in a national framework as patents are instituted nationally. Therefore, when designing scientific work that could lead to a product, it is worthwhile to consider the different methods used for genome editing not only for their scientific merits but also for their compatibility with a speedy and reliable launch into the desired market.     

The Evolving Genome Project: current and future impact.

The National Institutes of Health/Department of Energy Human Genome Project has been funding directed research for only 5 years, and it is understandably difficult to cite important research advances directly attributable to the project. However, the project has been constructive in fostering multidisciplinary group research and an inspiring and synergistic "just do it" attitude in both political and scientific circles, domestically and abroad. This collaborative spirit has spawned large-scale genetic and physical mapping projects, with the most impressive and useful results to date being the dense genetic maps produced by the Généthon, a French organization largely supported by the French muscular dystrophy association. With the genetic and physical map reagents now becoming available, disease-gene cloning is proceeding at an increasingly rapid pace. More important than the predictable acceleration of disease-gene mapping are the unpredictable benefits: Will a dense PCR-based dinucleotide-repeat genetic map open novel alternative approaches to disease-gene isolation? Will it become possible to localize disease genes by simply analyzing unrelated, isolated probands rather than the rarer "extended family"? Proband-based "linkage-disequilibrium cloning" may become possible if adequate density, informativeness, and stability of polymorphic loci are obtained. In addition, "genome exclusion cloning" will be added to the established positional, candidate-gene, and functional-disease-gene-cloning experimental approaches. The anticipated exponential expansion of human genetic disease information over the remainder of the 10-year tenure of the Human Genome Project unveils critical yet unresolved issues for medical education and the practice of medicine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9

Microalgae are promising feedstock for biofuels yet mechanistic probing of their cellular network and industrial strain development have been hindered by lack of genome‐editing tools. Nannochloropsis spp. are emerging model microalgae for scalable oil production and carbon sequestration. Here we established a CRISPR/Cas9‐based precise genome‐editing approach for the industrial oleaginous microalga Nannochloropsis oceanica, using nitrate reductase (NR; g7988) as example. A new screening procedure that compares between restriction enzyme‐digested nested PCR (nPCR) products derived from enzyme‐digested and not‐digested genomic DNA of transformant pools was developed to quickly, yet reliably, detect genome‐engineered mutants. Deep sequencing of nPCR products directly amplified from pooled genomic DNA revealed over an 1% proportion of 5‐bp deletion mutants and a lower frequency of 12‐bp deletion mutants, with both types of editing precisely located at the targeted site. The isolated mutants, in which precise deletion of five bases caused a frameshift in NR translation, grow normally under NH₄Cl but fail to grow under NaNO₃, and thus represent a valuable chassis strain for transgenic‐strain development. This demonstration of CRISPR/Cas9‐based genome editing in industrial microalgae opens many doors for microalgae‐based biotechnological applications.     

CRISPR/Cas9 gene editing in a chicken model: current approaches and applications

Improvements in genome editing technology in birds using primordial germ cells (PGCs) have made the development of innovative era genome-edited avian models possible, including specific chicken...     

Application and future perspective of CRISPR/Cas9 genome editing in fruit crops

Fruit crops, including apple, orange, grape,banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops,including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including d Cas9-base-editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene-free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumerfriendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.     

Precision plant breeding using genome editing technologies

Transgenic Research -     

Genome editing of rodents by electroporation of CRISPR/Cas9 into frozen-warmed pronuclear-stage embryos

Genome edited animals can now be easily produced using the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Traditionally, these animals have been produced by the introduction of endonucleases into pronuclear-stage embryos. Recently, a novel electroporation method, the “Technique for Animal Knockout system by Electroporation (TAKE),” has been established as a simple and highly efficient tool to introduce endonucleases into embryos instead of methods such as microinjection. Use of frozen-warmed pronuclear-stage embryos in this method has further contributed to efficient production of genome edited animals. However, early developmental stage embryos, including pronuclear-stage embryos, especially those of rats, sometimes show low resistance to physical damage by vitrification and introduction of endonucleases during microinjection. In this study, we propose an ethanol-free, slow-freezing method to reduce physical damage to pronuclear-stage embryos followed by the TAKE method. All mouse and rat frozen embryos were survived after electroporation, and 18% and 100% of offspring were edited target gene, respectively. The resulting protocol is an efficient method for producing genome edited animals.