A simple and efficient CRISPR/Cas9 system permits ultra-multiplex genome editing in plants

The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.

CRISPR/Cas9:A powerful tool for crop genome editing

The CRISPR/Cas9 technology is evolved from a type II bacterial immune system and represents a new generation of targeted genome editing technology that can be applied to nearly all organisms. Site-specific modification is achieved by a single guide RNA(usually about 20nucleotides) that is complementary to a target gene or locus and is anchored by a protospaceradjacent motif. Cas9 nuclease then cleaves the targeted DNA to generate double-strand breaks(DSBs), which are subsequently repaired by non-homologous end joining(NHEJ) or homology-directed repair(HDR) mechanisms. NHEJ may introduce indels that cause frame shift mutations and hence the disruption of gene functions. When combined with double or multiplex guide RNA design, NHEJ may also introduce targeted chromosome deletions,whereas HDR can be engineered for target gene correction, gene replacement, and gene knock-in. In this review, we briefly survey the history of the CRISPR/Cas9 system invention and its genome-editing mechanism. We also describe the most recent innovation of the CRISPR/Cas9 technology, particularly the broad applications of modified Cas9 variants, and discuss the potential of this system for targeted genome editing and modification for crop improvement.

A review of the literature on the use of CRISPR/Cas9 gene therapy to treat hepatocellular carcinoma

Noncoding RNAs instruct the Cas9 nuclease to site speifillyl cleave DNA in the CRISPR/Cas9 system.Despite the high incidence of hepatocellular carcinoma(HCC),the patient's outcome is poor.As a result of the emergence of therapeutic resistance in HCC patients,dlinicians have faced difficulties in treating such tumor.In addition,CRISPR/Cas9 screens were used to identify genes that improve the dlinical response of HCC patients.It is the objective of this article to summarize the current understanding of the use of the CRISPR/Cas9 system for the treatment of cancer,with a particular emphasis on HCC as part of the current state of knowledge.Thus,in order to locate recent developments in oncology research,we examined both the Scopus database and the PubMed database.The ability to selectively interfere with gene expression in combinatorial CRISPR/Cas9 screening can lead to the discovery of new effective HCC treatment regimens by combining clinically approved drugs.Drug resistance can be overcome with the help of the CRISPR/Cas9 system.HCC signature genes and resistance to treatment have been uncovered by genome-scale CRISPR activation screening although this method is not without limitations.It has been extensively examined whether CRISPR can be used as a tool for disease research and gene therapy.CRISPR and its applications to tumor research,particularly in HCC,are examined in this study through a review of the literature.

Delivery strategies for CRISPR/Cas genome editing tool for retinal dystrophies:challenges and opportunities

CRISPR/Cas,an adaptive immune system in bacteria,has been adopted as an efficient and precise tool for site-specific gene editing with potential therapeutic opportunities.It has been explored for a variety of applications,including gene modulation,epigenome editing,diagnosis,mRNA editing,etc.It has found applications in retinal dystrophic conditions including progressive cone and cone-rod dystrophies,congenital stationary night blindness,X-linked juvenile retinoschisis,retinitis pigmentosa,age-related macular degeneration,leber’s congenital amaurosis,etc.Most of the therapies for retinal dystrophic conditions work by regressing symptoms instead of reversing the genemutations.CRISPR/Cas9 through indel could impart beneficial effects in the reversal of gene mutations in dystrophic conditions.Recent research has also consolidated on the approaches of using CRISPR systems for retinal dystrophies but their delivery to the posterior part of the eye is a major concern due to high molecular weight,negative charge,and in vivo stability of CRISPR components.Recently,non-viral vectors have gained interest due to their potential in tissue-specific nucleic acid(miRNA/siRNA/CRISPR)delivery.This review highlights the opportunities of retinal dystrophies management using CRISPR/Cas nanomedicine.

Genome-edited rabbits:Unleashing the potential of a promising experimental animal model across diverse diseases

Animal models are extensively used in all aspects of biomedical research,with substantial contributions to our understanding of diseases,the development of pharmaceuticals,and the exploration of gene functions.The field of genome modification in rabbits has progressed slowly.However,recent advancements,particularly in CRISPR/Cas9-related technologies,have catalyzed the successful development of various genome-edited rabbit models to mimic diverse diseases,including cardiovascular disorders,immunodeficiencies,agingrelated ailments,neurological diseases,and ophthalmic pathologies.These models hold great promise in advancing biomedical research due to their closer physiological and biochemical resemblance to humans compared to mice.This review aims to summarize the novel gene-editing approaches currently available for rabbits and present the applications and prospects of such models in biomedicine,underscoring their impact and future potential in translational medicine.

Targeting miRNA by CRISPR/Cas in cancer:advantages and challenges

Clustered regulatory interspaced short palindromic repeats(CRISPR)has changed biomedical research and provided entirely new models to analyze every aspect of biomedical sciences during the last decade.In the study of cancer,the CRISPR/CRISPR-associated protein(Cas)system opens new avenues into issues that were once unknown in our knowledge of the non-coding genome,tumor heterogeneity,and precision medicines.CRISPR/Cas-based geneediting technology now allows for the precise and permanent targeting of mutations and provides an opportunity to target small non-coding RNAs such as microRNAs(miRNAs).However,the development of effective and safe cancer gene editing therapy is highly dependent on proper design to be innocuous to normal cells and prevent introducing other abnormalities.This study aims to highlight the cutting-edge approaches in cancer-gene editing therapy based on the CRISPR/Cas technology to target miRNAs in cancer therapy.Furthermore,we highlight the potential challenges in CRISPR/Cas-mediated miRNA gene editing and offer advanced strategies to overcome them.

Wheat genome editing expedited by efficient transformation techniques:Progress and perspectives

Genome editing is one of the most promising biotechnologies to improve crop performance.Common wheat is a staple food for mankind. In the past few decades both basic and applied research on common wheat has lagged behind other crop species due to its complex,polyploid genome and difficulties in genetic transformation. Recent breakthroughs in wheat transformation permit a revolution in wheat biotechnology. In this review, we summarize recent progress in wheat genetic transformation and its potential for wheat improvement. We then review recent progress in plant genome editing, which is now readily available in wheat. We also discuss measures to further increase transformation efficiency and potential applications of genome editing in wheat. We propose that, together with a high quality reference genome, the time for efficient genetic engineering and functionality studies in common wheat has arrived.

A zwitterionic polymer-inspired material mediated efficient CRISPR-Cas9 gene editing

The typeⅡ prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR/Cas9) adaptive immune system is a cutting-edge genome-editing toolbox.However,its applications are still limited by its inefficient transduction.Herein,we present a novel gene vector,the zwitterionic polymer-inspired material with branched structure (ZEBRA) for efficient CRISPR/Cas9 delivery.Polo-like kinase 1 (PLK1) acts as a master regulator of mitosis and overexpresses in multiple tumor cells.The Cas9 and single guide sgRNA (sgRNA)-encoded plasmid was transduced to knockout Plk1 gene,which was expected to inhibit the expression of PLK1.Our studies demonstrated that ZEBRA enabled to transduce the CRISPR/Cas9 system with large size into the cells efficiently.The transduction with ZEBRA was cell line dependent,which showed~10-fold higher in CD44-positive cancer cell lines compared with CD44-negative ones.Furthermore,ZEBRA induced highlevel expression of Cas9 proteins by the delivery of CRISPR/Cas9 and efficient gene editing of Plk1 gene,and inhibited the tumor cell growth significantly.This zwitterionic polymerinspired material is an effective and targeted gene delivery vector and further studies are required to explore its potential in gene delivery applications.

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

The CRISPR-Cas9 RNA-guided DNA endonuclease has contributed to an explosion of advances in the life sciences that have grown from the ability to edit genomes within living cells.In this Review,we summarize CRISPR-based technologies that enable mammalian genome editing and their various applications.

CRISPR-based genome editing technology and its applications in oil crops

Oil crops,mainly comprised of soybean,rapeseed,groundnut,sunflower and etc.,have provided substantial edible oil and other tremendous nutrients for human beings,as well as valuable biofuels for associated industries.The genetic improvement of significant oil crops and/or domesticating novel high-yielding oil crops are in urgent need to cope with the ever-increasing demand for various oil crop products.CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)-based genome editing technology,born a few years ago,edits stretches of DNA in a targeted and RNA-dependent fashion.The Characteristics of targeted mutagenesis and easy manipulation owned by the technology make it have been applied to many plants and exhibited great potential in the genetic improvement of many important oil crops.In the face of growing need for oil crop products and the rapid developments in CRISPR-based genome editing technology,a critical review regarding the technology and its application in oil crops is badly required to provide references for the better use of this technology to modify the oil crops for higher yield.In this review paper,we briefly described the CRISPR-based genome editing technology and summarized its applications and future prospects in oil crops.

CRISPR/Cas9基因编辑非病毒递送系统

规律间隔成簇短回文重复序列及相关蛋白9(CRISPR/Cas9)系统的基因编辑技术为哺乳细胞基因组的精准修饰与编辑研究提供了高效、快捷的工具,但其化学生物学应用依然面临着CRISPR基因编辑工具Cas9蛋白和gRNA的细胞及活体递送等问题.近年来,研究人员通过开发多种非病毒递送载体,实现了编码CRISPR/Cas9基因编辑工具的DNA和信使RNA(mRNA)以及Cas9/gRNA核糖核蛋白(RNP)复合物的递送,并应用于靶基因的化学修饰与编辑调控.本文主要概述了近期CRISPR/Cas9基因编辑递送的研究进展,并对其化学生物学应用前景进行了展望.

Development of an Agrobacterium-mediated CRISPR/Cas9 system in pea(Pisum sativum L.)

Pea(Pisum sativum L.)is an annual cool-season legume crop.Owing to its role in sustainable agriculture as both a rotation and a cash crop,its global market is expanding and increased production is urgently needed.For both technical and regulatory reasons,neither conventional nor transgenic breeding techniques can keep pace with the demand for increased production.In answer to this challenge,CRISPR/Cas9 genome editing technology has been gaining traction in plant biology and crop breeding in recent years.However,there are currently no reports of the successful application of the CRISPR/Cas9 genome editing technology in pea.We developed a transient transformation system of hairy roots,mediated by Agrobacterium rhizogenes strain K599,to validate the efficiency of a CRISPR/Cas9 system.Further optimization resulted in an efficient vector,PsU6.3-tRNA-PsPDS3-en35S-PsCas9.We used this optimized CRISPR/Cas9 system to edit the pea phytoene desaturase(PsPDS)gene,causing albinism,by Agrobacterium-mediated genetic transformation.This is the first report of successful generation of gene-edited pea plants by this route.

化学调控CRISPR/Cas9基因编辑技术的研究进展

规律间隔成簇短回文重复序列及其相关蛋白9(Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9,CRISPR/Cas9)基因编辑技术作为一项基因工程领域革新式的技术,为癌症、遗传性疾病及感染性疾病等多种重大疾病的治疗提供了极大的帮助.但如何在特定细胞和组织中实现时空调控的精准基因编辑,进而避免脱靶效应,依然是该技术在临床转化领域面临的重要挑战.近年来,通过化学分子和反应实现对CRISPR/Cas9活性的调控已经成为提升这项基因编辑技术效率的重要手段之一.本文综合评述了一些最近报道的化学调控CRISPR/Cas9基因编辑的方法,并对其在临床医学领域的应用前景进行了展望.

Systematic identification of endogenous RNA polymeraseⅢpromoters for efficient RNA guidebased genome editing technologies in maize

Single-guide RNA(sg RNA) is one of the two core components of the CRISPR(clustered regularly interspaced short palindromic repeat)/Cas(CRISPR-associated) genome-editing technology. We established an in vitro Traffic Light Reporter(TLR) system, which is designated as the same colors as traffic lights such as green, red and yellow were produced in cells. The TLR can be readily used in maize mesophyll protoplast for a quick test of promoter activity. The TLR assay indicates the variation in transcription activities of the seven Pol III promoters, from 3.4%(U6-1) to over 21.0%(U6-6). The U6-2 promoter, which was constructed to drive sg RNA expression targeting the Zm Wx1 gene, yielded mutation efficiencies ranging from 48.5% to 97.1%. Based on the reported and unpublished data, the in vitro TLR assay results were confirmed to be a readily system and may be extended to other plant species amenable to efficient genome editing via CRISPR/Cas. Our efforts provide an efficient method of identifying native Pol III-recognized promoters for RNA guide-based genome-editing systems in maize.

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 site- specific 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.

Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR(TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35 S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.

Genome editing technology and application in soybean improvement

Soybean(Glycine max)is a legume crop with great economic value that provides rich protein and oil for human food and animal feed.In order to cope with the ever-increasing need for soybean products and the changing environment,soybean genetic improvement needs to be accelerated.In recent years,the rapid developed genome editing technologies,such as zinc finger nuclease(ZFNs),transcription activator-like effector nucleases(TALENs),and clustered regularly interspaced short palindromic repeats/CRISPR associated protein(CRISPR/Cas),have shown broad application prospects in gene function research and improvement of important agronomic traits in many crops,and has also brought opportunities for soybean breeding.Here we systematically reviewed recent advances in genome editing technology.We also summarized the significances,current applications,challenges and future perspectives in soybean genome editing,which could provide references for exerting the feature and advantage of this technology to better soybean improvement.

PAM-Expanded Streptococcus thermophilus Cas9 C-to-T and C-to-G Base Editors for Programmable Base Editing in Mycobacteria

New therapeutic strategies for the rapid and effective treatment of drug-resistant tuberculosis are highly desirable,and their development can be drastically accelerated by facile genetic manipulation methods in Mycobacterium tuberculosis(M.tuberculosis).Clustered regularly interspaced short palindromic repeat(CRISPR)base editors allow for rapid,robust,and programmed single-base substitutions and gene inactivation,yet no such systems are currently available in M.tuberculosis.By screening distinct CRISPR base editors,we discovered that only the unusual Streptococcus thermophilus CRISPR associated protein 9(St1Cas9)cytosine base editor(CBE)-but not the widely used Streptococcus pyogenes Cas9(SpCas9)or Lachnospiraceae bacterium Cpf1(LbCpf1)CBEs-is active in mycobacteria.Despite the notable C-to-T conversions,a high proportion of undesired byproducts exists with St1Cas9 CBE.We therefore engineered St1Cas9 CBE by means of uracil DNA glycosylase inhibitor(UGI)or uracil DNA glycosylase(UNG)fusion,yielding two new base editors(CTBE and CGBE)capable of C-to-T or C-to-G conversions with dramatically enhanced editing product purity and multiplexed editing capacity in Mycobacterium smegmatis(M.smegmatis).Because wild-type St1Cas9 recognizes a relatively strict protospacer adjacent motif(PAM)sequence for DNA targeting,we engineered a PAM-expanded St1Cas9 variant by means of structureguided protein engineering for the base editors,substantially broadening the targeting scope.We first developed and characterized CTBE and CGBE in M.smegmatis,and then applied CTBE for genome editing in M.tuberculosis.Our approaches significantly reduce the efforts and time needed for precise genetic manipulation and will facilitate functional genomics,antibiotic-resistant mechanism study,and drugtarget exploration in M.tuberculosis and related organisms.

CRISPR/Cas9系统在植物基因组定点编辑中的研究进展

当外源DNA通过转基因技术导入植物细胞后,会以同源重组或非同源重组两种不同的方式整合到基因组中,进而获得相应的目标性状。外源DNA与受体细胞序列相同或相近的位点发生重新组合,从而整合到受体细胞的染色体上称之为同源重组;当发生了DNA双链断裂的细胞为了避免DNA或染色体断裂而造成DNA降解或对生命力的影响,而强行将2个DNA断端彼此连接在一起时则为非同源重组。发生非同源重组的细胞其基因组常出现核苷酸片段的插入和/或缺失以及其他突变等多种情况,使得研究者无法得到精确控制的突变结果;而发生同源重组的细胞基因组序列通常不变,通过加入同源重组的供体DNA,可以实现对基因组的精确修饰和改造。由于在植物中产生自发同源重组的概率很低,对植物基因组进行精确修饰和改造非常困难,位点特异性核酸酶的出现和应用,大大提升了同源重组的效率,使基因组编辑变得更加高效和精确,从而使得对包括植物在内的任何物种进行基因组编辑都将成为可能。锌指核酸酶(ZFN)和TALE核酸酶(TALENs)是能够使DNA的靶位点产生DNA双链断裂进而实现基因组定点编辑的常用系统,但在具体应用中发现这两种系统存在着许多缺陷和不足,如脱靶效应、与基因组进行特异结合与染色体位置及邻近序列有关等,另外技术难度大、构建组装时间长也限制了其应用。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。Ⅱ型CRISPR/Cas系统经过密码子优化等改造后已成为继锌指核酸酶ZFNs和TALENs后的新型高效定点编辑的新技术,具有突变效率高、制作简单、易操作及成本低的特点。目前,该技术成功应用于人类细胞、斑马鱼、小鼠以及细菌的基因组精确编辑,编辑的类型包括基因的定点插入、小片段的缺失、多个位点同时突变、基因定点的indel突变等。目前,CRISPR/Cas系统在植物中的应用还比较有限,但该技术为植物基因工程的发展呈现了美好的前景。文中首先简要介绍了CRISPR/Cas系统的组成和基本原理,进而详细综述了该技术在植物内源基因和外源基因定点编辑中的应用,主要列举了自CRISPR/Cas系统改造成功以来利用该系统对单子叶和双子叶植物进行基因组定点编辑的案例,最后对基因组编辑技术在农业和植物基因工程上的应用进行了展望,希望能够为开展该领域研究的科研工作者提供参考。

茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建

CRISPR/Cas9技术是一门新兴的基因组定点编辑技术,具有操作简单、高效的优点,可轻松实现对目标基因的敲除、替换和定点突变等操作。该技术刚诞生,就受到了全球生命科学领域研究者的关注,不到3年的时间就已经成功应用于多种动、植物当中。然而CRISPR/Cas9技术在茶树中的应用面临载体构建问题,本文以茶树咖啡碱合成酶为例,联合采用常规PCR、Overlapping PCR和Golden Gate Cloning技术,构建了包含茶树咖啡碱合成酶双靶点的CRISPR/Cas9基因编辑载体,为CRISPR/Cas9介导的基因组编辑技术在茶树中的应用奠定了坚实基础。