Development and Therapeutic Applications of Precise Gene Editing Technology

The advent of gene editing represents one of the most transformative breakthroughs in life science,making genome manipulation more accessible than ever before.While traditional CRISPR/Cas-based gene editing,which involves double-strand DNA breaks(DSBs),excels at gene disruption,it is less effective for accurate gene modification.The limitation arises because DSBs are primarily repaired via non-homologous end joining(NHEJ),which tends to introduce indels at the break site.While homology directed repair(HDR)can achieve precise editing when a donor DNA template is provided,the reliance on DSBs often results in unintended genome damage.HDR is restricted to specific cell cycle phases,limiting its application.Currently,gene editing has evolved to unprecedented levels of precision without relying on DSB and HDR.The development of innovative systems,such as base editing,prime editing,and CRISPR-associated transposases(CASTs),now allow for precise editing ranging from single nucleotides to large DNA fragments.Base editors(BEs)enable the direct conversion of one nucleotide to another,and prime editors(PEs)further expand gene editing capabilities by allowing for the insertion,deletion,or alteration of small DNA fragments.The CAST system,a recent innovation,allows for the precise insertion of large DNA fragments at specific genomic locations.In recent years,the optimization of these precise gene editing tools has led to significant improvements in editing efficiency,specificity,and versatility,with advancements such as the creation of base editors for nucleotide transversions,enhanced prime editing systems for more efficient and precise modifications,and refined CAST systems for targeted large DNA insertions,expanding the range of applications for these tools.Concurrently,these advances are complemented by significant improvements in in vivo delivery methods,which have paved the way for therapeutic application of precise gene editing tools.Effective delivery systems are critical for the success of gene therapies,and recent developments in both viral and non-viral vectors have improved the efficiency and safety of gene editing.For instance,adeno-associated viruses(AAVs)are widely used due to their high transfection efficiency and low immunogenicity,though challenges such as limited cargo capacity and potential for immune responses remain.Non-viral delivery systems,including lipid nanoparticles(LNPs),offer an alternative with lower immunogenicity and higher payload capacity,although their transfection efficiency can be lower.The therapeutic potential of these precise gene editing technologies is vast,particularly in treating genetic disorders.Preclinical studies have demonstrated the effectiveness of base editing in correcting genetic mutations responsible for diseases such as cardiomyopathy,liver disease,and hereditary hearing loss.These technologies promise to treat symptoms and potentially cure the underlying genetic causes of these conditions.Meanwhile,challenges remain,such as optimizing the safety and specificity of gene editing tools,improving delivery systems,and overcoming off-target effects,all of which are critical for their successful application in clinical settings.In summary,the continuous evolution of precise gene editing technologies,combined with advancements in delivery systems,is driving the field toward new therapeutic applications that can potentially transform the treatment of genetic disorders by targeting their root causes.

Development and Application of Prime Editing in Plants

Clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)-mediated genome editing has greatly accelerated progress in plant genetic research and agricultural breeding by enabling targeted genomic modifications.Moreover,the prime editing system,derived from the CRISPR/Cas system,has opened the door for even more precise genome editing.Prime editing has the capability to facilitate all 12 types of base-to-base conversions,as well as desired insertions or deletions of fragments,without inducing double-strand breaks and requiring donor DNA templet.In a short time,prime editing has been rapidly verified as functional in various plants,and can be used in plant genome functional analysis as well as precision breeding of crops.In this review,we summarize the emergence and development of prime editing,highlight recent advances in improving its efficiency in plants,introduce the current applications of prime editing in plants,and look forward to future prospects for utilizing prime editing in genetic improvement and precision molecular breeding.

Prime editing引导植物基因组精确编辑新局面

由于植物细胞内同源重组频率较低、供体传递受限等原因,对植物基因组进行精准编辑十分困难。近期,中国科学院遗传与发育生物学研究所高彩霞团队构建了适用于植物的引导编辑器(plant prime editor,PPE)系统,并在重要作物水稻和小麦中完成了引导编辑。该系统不产生DNA双链断裂,仍可高度准确实现所有可能的12种单碱基替换、多碱基替换及片段缺失插入,从而为植物基因组精确编辑提供了多用途工具。本文介绍了PPE的组成结构和编辑能力,同时也结合其他研究组随后发表的报告综述了植物引导编辑器的优化探索,为合理使用PPEs和继续开展优化工作提供帮助。

PE6c greatly enhances prime editing in transgenic rice plants

Prime editing is a versatile CRISPR/Cas-based precise genome-editing technique for crop breeding.Four new types of prime editors(PEs)named PE6a–d were recently generated using evolved and engineered reverse transcriptase(RT)variants from three different sources.In this study,we tested the editing efficiencies of four PE6 variants and two additional PE6 constructs with double-RT modules in transgenic rice(Oryza sativa)plants.PE6c,with an evolved and engineered RT variant from the yeast Tf1 retrotransposon,yielded the highest prime-editing efficiency.The average fold change in the editing efficiency of PE6c compared with PEmax exceeded 3.5 across 18 agronomically important target sites from 15 genes.We also demonstrated the feasibility of using two RT modules to improve prime-editing efficiency.Our results suggest that PE6c or its derivatives would be an excellent choice for prime editing in monocot plants.In addition,our findings have laid a foundation for prime-editing-based breeding of rice varieties with enhanced agronomically important traits.

Developing an efficient and visible prime editing system to restore tobacco 8-hydroxy-copalyl diphosphate gene for labdane diterpene Z-abienol biosynthesis

Prime editing(PE)is a versatile CRISPR-Cas based precise genome-editing platform widely used to introduce a range of possible base conversions in various organisms.However,no PE systems have been shown to induce heritable mutations in tobacco,nor in any other dicot.In this study,we generated an efficient PE system in tobacco that not only introduced heritable mutations,but also enabled anthocyanin-based reporter selection of transgene-free T_(1) plants.This system was used to confer Zabienol biosynthesis in the allotetraploid tobacco cultivar HHDJY by restoring a G>T conversion in the NtCPS2 gene.High levels of Z-abienol were detected in the leaves of homozygous T_(1) plants at two weeks after topping.This study describes an advance in PE systems and expands genome-editing toolbox in tobacco,even in dicots,for use in basic research and molecular breeding.And restoring biosynthesis of Z-abienol in tobacco might provide an efficient way to obtain Z-abienol in plants.

Recent advances in CRISPR-based genome editing technology and its applications in cardiovascular research

The rapid development of genome editing technology has brought major breakthroughs in the fields of life science and medicine. In recent years, the clustered regularly interspaced short palindromic repeats(CRISPR)-based genome editing toolbox has been greatly expanded, not only with emerging CRISPR-associated protein(Cas) nucleases, but also novel applications through combination with diverse effectors. Recently, transposon-associated programmable RNA-guided genome editing systems have been uncovered, adding myriads of potential new tools to the genome editing toolbox. CRISPR-based genome editing technology has also revolutionized cardiovascular research. Here we first summarize the advances involving newly identified Cas orthologs, engineered variants and novel genome editing systems, and then discuss the applications of the CRISPR-Cas systems in precise genome editing, such as base editing and prime editing. We also highlight recent progress in cardiovascular research using CRISPR-based genome editing technologies, including the generation of genetically modified in vitro and animal models of cardiovascular diseases(CVD) as well as the applications in treating different types of CVD. Finally, the current limitations and future prospects of genome editing technologies are discussed.

引导编辑研究进展及其应用

引导编辑器(prime editor,PE)是继碱基编辑器(base editor,BE)之后新问世的基于CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPR-associated)系统的基因编辑工具,可以在不造成DNA双链断裂的情况下引入碱基替换、插入和删除。PE因其全面的编辑能力,问世即受到全球学者的广泛关注,然而PE表达盒编码较长(>6 kb)、编辑效率较低等问题也亟待研究人员解决。PE的研究方向与BE有许多相似之处,本文首先梳理了学界对PE本身编辑效率和安全性的探索;然后重点介绍了PE效应蛋白、pegRNA和其他细胞因子三个方面对PE的改进手段,以及为方便PE应用而开发的自动化设计工具;最后梳理了PE在动植物以及基因治疗中的应用。方兴未艾的PE领域尽管还难称完善,但在提高编辑效率和改进安全性等方面已取得了许多重要进展。鉴于Cas9、BE等基因编辑工具已广泛应用于遗传病疗法,PE走向遗传病治疗值得期待。

引导编辑技术的研究进展及应用

以CRISPR/Cas9为基础的引导编辑系统是新开发出的一种基因编辑技术,可以精确实现12种碱基的互换、插入和缺失,并且不需要产生双链断裂和引入外源供体DNA。本文从基因编辑的发展、引导编辑系统的原理、特点、优化、脱靶效应、在动植物和基因治疗研究中的应用、引导编辑系统的设计等几方面进行综述,为促进相关领域的科研工作者了解引导编辑系统及进一步利用引导编辑系统在动植物科学基础研究和育种方面的应用提供指导。

Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice

Prime editing(PE)technology enables precise alterations in the genetic code of a genome of interest.PE offers great potential for identifying major agronomically important genes in plants and editing them into superior variants,ideally targeting multiple loci simultaneously to realize the collective effects of the edits.Here,we report the development of a modular assembly-based multiplex PE system in rice and demon-strate its efficacy in editing up to four genes in a single transformation experiment.The duplex PE(DPE)system achieved a co-editing efficiency of 46.1%in the T0 generation,converting TFIIAg5 to xa5 and xa23 to Xa23SW11.The resulting double-mutant lines exhibited robust broad-spectrum resistance against multiple Xanthomonas oryzae pathovar oryzae(Xoo)strains in the T1 generation.In addition,we success-fully edited OsEPSPS1 to an herbicide-tolerant variant and OsSWEET11a to a Xoo-resistant allele,achieving a co-editing rate of 57.14%.Furthermore,with the quadruple PE(QPE)system,we edited four genes—two for herbicide tolerance(OsEPSPS1 and OsALS1)and two for Xoo resistance(TFIIAg5 and OsSWEET11a)—using one construct,with a co-editing efficiency of 43.5%for all four genes in the T0 gen-eration.We performed multiplex PE usingfive more constructs,including two for triplex PE(TPE)and three for QPE,each targeting a different set of genes.The editing rates were dependent on the activity of pegRNA and/or ngRNA.For instance,optimization of ngRNA increased the PE rates for one of the targets(OsSPL13)from 0%to 30%but did not improve editing at another target(OsGS2).Overall,our modular assembly-based system yielded high PE rates and streamlined the cloning of PE reagents,making it feasible for more labs to utilize PE for their editing experiments.Thesefindings have significant implications for advancing gene editing techniques in plants and may pave the way for future agricultural applications.

Efficient and precise genomic deletion in rice using enhanced prime editing

Efficient and precise genomic deletion shows promise for investigating the function of proteins in plant research and enhancing agricultural traits.In this study,we tested the PRIME-Del(PDel)strategy using a pair of prime editing guide RNAs(pegRNAs)that targeted opposite DNA strands and achieved an average deletion efficiency of 55.8%for 60 bp fragment deletions at six endogenous targets.Moreover,as high as 84.2%precise deletion efficiency was obtained for a 2000 bp deletion at the OsGS1 site in transgenic rice plants.To add the bases that were unintentionally deleted between the two nicking sequences,we used the PDel/Syn strategy,which introduced multiple synonymous base mutations in the region that had to be patched in the RT template.The PDel/Syn strategy achieved an average of 58.1%deletion efficiency at six endogenous targets,which was higher than the PDel strategy.The strategies presented in this study contribute to achieving more accurate and flexible deletions in transgenic rice plants.

引导编辑系统研究进展

引导编辑(Prime editing,PE)系统是一种全新的、革命性的基因组编辑策略。该系统由引导编辑器(Prime editor)组成,包括nCas9(H840A)与逆转录酶(Reverse transcriptase,RT)的融合蛋白;以及包含PBS(Primer binding site)序列和RT模板(RT template,RTT)序列的pegRNA(Prime editing guide RNA)两大部分。PE系统可以在双链不断裂的情况下实现所有12种类型的碱基替换及小片段DNA增删,是精准编辑的全新范式。自2019年开发至今不到4年时间,PE系统作为一种通用的技术平台,已广泛应用于医疗、农业等各个领域,产生了一大批新种质资源、基因治疗药物等优秀应用案例。PE作为目前最灵活、最具发展前景的基因组精准编辑新手段,仍旧存在效率偏低、大片段操纵能力不足、系统组分设计复杂(如pegRNA)、安全性未全面评估等问题,仍需要深入研究。本文详细介绍了PE系统的技术原理及限制因素,全面总结了PE系统自开发以来的优化策略及在动植物系统、医疗领域的应用现状,并对PE的发展前景进行了展望。

引导编辑系统的优化及在DNA大片段编辑中的应用

基因编辑技术是指利用人工核酸酶对细胞和个体中特定的基因序列进行插入、替换或删除等编辑修饰。CRISPR/Cas9核酸酶的发现是基因编辑技术发展的一个里程碑,但其编辑产物的精确性和脱靶效应依然是限制其应用的关键因素。近年来以引导编辑技术为代表的衍生性编辑工具因高效且精准而受到广泛关注。该系统能够以不可逆的方式在基因组中靶向引入多种类型的遗传变化,包括12种可能类型的点突变,以及片段的插入和缺失及其组合,而无需DNA双链断裂(DSB)或者供体DNA模板。引导编辑技术结合了CRISPR/Cas9的靶向性和逆转录酶的精准编辑能力,使得编辑产物更加精准。本综述将深入探讨引导编辑技术的发展、优化以及在DNA大片段编辑中的应用。

猪HBB基因中先导编辑效率研究

为了验证先导编辑(prime editing,PE)在猪细胞HBB基因中的编辑效率,并比较其在人细胞与猪细胞中的编辑效率,试验通过生物信息学分析,选择β珠蛋白生成障碍性贫血致病位点-28(A>G)、CD17(A>T)、CD41/42(-TCTT)设计并合成基因编辑质粒,通过脂质体Lipofectamine 2000转染人肾上皮细胞系(HEK293T)和猪肾上皮细胞系(PK-13)并进行药物筛选,提取细胞基因组进行PCR扩增,对PCR扩增产物进行TA克隆后测序,统计各位点的编辑效率;然后通过改进向导RNA骨架序列、添加小分子药物等方法对PK-13中的先导编辑进行优化。结果表明:先导编辑系统PEmax在PK-13中具有编辑作用,猪HBB基因-28(A>G)、CD17(A>T)、CD41/42(-TCTT)位点先导编辑的正确编辑效率分别为9.44%、8.89%和4.76%,较人HBB基因相应位点的正确编辑效率低33.89%、42.22%和26.11%,差异显著(P<0.05)。而经过优化pegRNA序列后猪细胞中正确编辑效率达到了12.78%[CD17(A>T)]、11.67%[CD41/42(-TCTT)],均显著高于pegRNA骨架序列优化前的7.22%和5.33%,是未优化前的1.8~2.2倍,差异显著(P<0.05)。说明先导编辑在猪细胞中具有编辑效率,且可以通过改进pegRNA的策略进一步提高正确编辑效率。

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 strategies have been attempted to optimize both base editor(BE)and prime editor(PE)in order to improve the precise editing efficacy,specificity,and expand the targeting scopes.Here,we summarize the latest development of various BEs and PEs,as well as their applications in plants.Based on these progresses,we recommend the appropriate BEs and PEs for both basic plant research and crop improvement.Moreover,we propose the perspectives for further optimization of these two editors.We envision that both BEs and PEs will become the routine and customized precise gene editing tools for both plant biological research and crop improvement in the near future.

An optimized prime editing system for efficient modification of the pig genome

Prime editing(PE)is a recent gene editing technology that can mediate insertions or deletions and all twelve types of base-tobase conversions.However,its low efficiency hampers the application in creating novel breeds and biomedical models,especially in pigs and other important farm animals.Here,we demonstrate that the pig genome is editable using the PE system,but the editing efficiency was quite low as expected.Therefore,we aimed to enhance PE efficiency by modulating both exogenous PE tools and endogenous pathways in porcine embryonic fibroblasts(PEFs).First,we modified the peg RNA by extending the duplex length and mutating the fourth thymine in a continuous sequence of thymine bases to cytosine,which significantly enhanced PE efficiency by improving the expression of peg RNA and targeted cleavage.Then,we targeted SAMHD1,a deoxynucleoside triphosphate triphosphohydrolase(d NTPase)that impedes the reverse transcription process in retroviruses,and found that treatment with its inhibitor,cephalosporin C zinc salt(CPC),increased PE efficiency up to 29-fold(4-fold on average),presumably by improving the reverse transcription process of Moloney murine leukemia virus reverse transcriptase(M-MLV RT)in the PE system.Moreover,PE efficiency was obviously improved by treatment with a panel of histone deacetylase inhibitors(HDACis).Among the four HDACis tested,panobinostat was the most efficient,with an efficiency up to 122-fold(7-fold on average),partly due to the considerable HDACi-mediated increase in transgene expression.In addition,the synergistic use of the three strategies further enhanced PE efficiency in PEFs.Our study provides novel approaches for optimization of the PE system and broadens the application scope of PE in agriculture and biomedicine.

人机交互理念下CAT+MT+PE师生协同翻译模式探索

随着计算机辅助翻译工具(CAT)和神经网络机器翻译技术(NMT)的迅速发展,基于计算机辅助翻译工具的机器翻译+译后编辑模式(CAT+MT+PE)日益成为语言服务行业的主流翻译模式。本文在总结师生图书翻译项目实践经验的基础上,从人机交互角度提出以在线CAT工具为翻译生产平台的师生协同翻译模式,继而分析了该协同翻译模式的三大优势以及仍需改进和优化的不足之处。研究结论认为,图书翻译应用机器翻译的译后编辑凸显了工具理性和价值理性的融合,符合Web2.0时代人机交互新理念以及"互联网+"时代的翻译生产模式和译后编辑能力培养新模式。

全屏幕编辑软件“PE”简介

PE(Personal Editor)是一个小巧玲珑的全屏幕编辑软件。它适用于IBM-PC及其兼容机。虽然它没有象wordstar那样丰富完善的编辑功能,但是对于编辑小程序或少量数据,阅读或修改某个文件,则有操作方便、使用灵活的特点.PE需占用约60KB的内存容量. PE最明显的特点是具有块操作功能.用PE执行编辑时的复制,删除、转移和查找等操作,比用DoS中的EDLIN命令组简便.例如:用它可以复制或删除任意2行及2列之间的一块文本内容。PE具有灵活的复盖转移功能。用它可复盖一个字符串或一整行内容。

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.

环保践行者 Mercedes-Benz C 250 CDI Blue Efficiency Prime Edition

作为国际车坛中著名的品牌之一,梅赛德斯-奔驰从2008年初就提出了Blue Efficiency（环保高效动力）概念,而随着能源问题和环境污染问题的日益严重,消费者对节能环保车型的关注度也越来越高,为顺应车市发展方向,奔驰推出了一款更节能更环保的C 250 CDI Blue Efficiency领先版车型。

“CAT+MT+PE”模式下的项目管理与翻译实践——以灯塔阅读英汉翻译项目为例

文章利用灯塔阅读提供的翻译项目基于“CAT+MT+PE”模式进行翻译实践,选取高质量文本制作小型双语平行语料库用于翻译研究。同时,使用目前行业中主流CAT软件和平台进行实践,在这一过程中按照该模式完成翻译项目,并从翻译和项目管理的角度将该模式与传统翻译和“CAT+MT+PE”模式项目流程进行对比,旨在研究“CAT+MT+PE”模式在未来翻译领域的发展趋势。