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.

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.

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.

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.

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.

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.

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。

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.

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.

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.

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.

Optimized prime editing efficiently generates heritable mutations in maize

Low efficiency is the main obstacle to using prime editing in maize(Zea mays).Recently,prime-editing efficiency was greatly improved in mammalian cells and rice(Oryza sativa)plants by engineering primeediting guide RNAs(pegRNAs),optimizing the prime editor(PE)protein,and manipulating cellular determinants of prime editing.In this study,we tested PEs optimized via these three strategies in maize.We demonstrated that the ePE5max system,composed of PEmax,epegRNAs(pegRNA-evopreQ.1),nicking single guide RNAs(sgRNAs),and MLH1dn,efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS),acetolactate synthase(ALS),or acetyl CoA carboxylase(ACCase)activity.Collectively,we demonstrate that the ePE5max system has sufficient efficiency to generate heritable(homozygous or heterozygous)mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed.

Addition of the T5 exonuclease increases the prime editing efficiency in plants

Prime editing(PE)is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates,but is limited by low editing efficiency.We previously fused the M-MLV reverse transcriptase to the Cas9 nickase,generating the PE2(v1)system,but the editing efficiency of this system is still low.Here we develop different versions of PE2 by adding the 50-to-30 exonuclease at different positions of the nCas9-M-MLV RT fusion protein.PE2(v2),in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions,deletions,and insertions at several genomic sites by 1.7-to 2.9-fold in plant cells compared to PE2(v1).The improved editing efficiency of PE2(v2)is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1,PE-P2,and PPE systems.Using PE2(v2),we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites.The PE efficiency is further improved by combining PE2(v2)and dualpegRNAs.Taken together,the increased genome editing efficiency of PE2(v2)developed in this study may enhance the applications of PE in plants.

Development of Plant Prime-Editing Systems for Precise Genome Editing

Prime-editing systems have the capability to perform efficient and precise genome editing in human cells.In this study,we first developed a plant prime editor 2(pPE2)system and test its activity by generating a targeted mutation on an HPT^(-ATG) reporter in rice.Our results showed that the pPE2 system could induce programmable editing at different genome sites.In transgenic T0 plants,pPE2-generated mutants occurred with 0%–31.3%frequency,suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures.To optimize editing efficiency,guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells.However,at the genomic sites tested in this study,pPE3 systems generated only comparable or even lower editing frequencies.Furthemore,we developed a surrogate pPE2 system by incorporating the HPT^(-ATG) reporter to enrich the prime-edited cells.The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system,presumably due to the enhanced screening efficiency of edited cells.Taken together,our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.

CRISPR base editing and prime editing:DSB and template-free editing systems for bacteria and plants

CRISPR-Cas(Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated)has been extensively exploited as a genetic tool for genome editing.The RNA guided Cas nucleases generate DNA doublestrand break(DSB),triggering cellular repair systems mainly Non-homologous end-joining(NHEJ,imprecise repair)or Homology-directed repair(HDR,precise repair).However,DSB typically leads to unexpected DNA changes and lethality in some organisms.The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools.Hence,in this review,we focus on the non-DSB and template-free genome editing,i.e.,base editing(BE)and prime editing(PE)in bacteria and plants.We first highlight the development of base and prime editors and summarize their studies in bacteria and plants.We then discuss current and future applications of BE/PE in synthetic biology,crop improvement,evolutionary engineering,and metabolic engineering.Lastly,we critically consider the challenges and prospects of BE/PE in PAM specificity,editing efficiency,off-targeting,sequence specification,and editing window.

The CRISPR/Cas9 revolution continues: From base editing to prime editing in plant science

The ability to precisely inactivate or modify genes in model organisms helps us understand the mysteries of life. Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9), a revolutionary technology that could generate targeted mutants, has facilitated notable advances in plant science. Genome editing with CRISPR/Cas9 has gained great popularity and enabled several technical breakthroughs. Herein, we briefly introduce the CRISPR/Cas9, with a focus on the latest breakthroughs in precise genome editing(e.g., base editing and prime editing), and we summarize various platforms that developed to increase the editing efficiency, expand the targeting scope, and improve the specificity of base editing in plants. In addition, we emphasize the recent applications of these technologies to plants. Finally, we predict that CRISPR/Cas9 and CRISPR/Cas9-based genome editing will continue to revolutionize plant science and provide technical support for sustainable agricultural development.

Plant genome editing:CRISPR,base editing,prime editing,and beyond

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system is a fast-growing,genome editing technology that has wide applications in identifying gene functions as well as improving agricultural production and crop breeding.Here,we summarized recent advances in the development and applications of genome editing technologies in plants.We briefly described CRISPR/Cas9 technology and examined the base and prime editing techniques that have been developed from CRISPR technology.Some new prime editing-derived techniques were assessed.

先导编辑的研究进展及应用前景

人类众多遗传性疾病是由于基因组突变所导致,而基于基因编辑技术的基因治疗策略,可从DNA层面对致病突变进行彻底修复,为疾病的治愈提供了新的可能.随着基因编辑技术的不断发展与突破,2019年新型DNA编辑工具先导编辑(PE)成功问世.PE技术并不会引起DNA双链断裂(DSBs),也无需单独引入DNA模板,即可实现基因编辑,可广泛应用于包括点突变及小片段的插入、删除等不同场景.与传统基因编辑工具相比,PE因其高编辑效率及低脱靶率,具备更高的有效性与安全性.本篇综述:(1)详细介绍了先导编辑的工作原理及发展历史.(2)概述了先导编辑系统的改良策略,包括相关酶的改造、工具RNA的结构改进以及递送方式的创新(3)梳理了辅助PE系统设计和分析的网页工具,简化了PE系统的搭建及使用流程.(4)系统总结了PE在疾病模型中的应用案例,并展望了未来的临床应用前景.

Precision Breeding Made Real with CRISPR: Illustration through Genetic Resistance to Pathogens

Since its discovery as a bacterial adaptive immune system and its development for genome editing in eukaryotes,the CRISPR technology has revolutionized plant research and precision crop breeding.The CRISPR toolbox holds great promise in the production of crops with genetic disease resistance to increase agriculture resilience and reduce chemical crop protection with a strong impact on the environment and public health.In this review,we provide an extensive overviewon recent breakthroughs in CRISPR technology,including the newly developed prime editing system that allows precision gene editing in plants.We present how each CRISPR tool can be selected for optimal use in accordance with its specific strengths and limitations,and illustrate how the CRISPR toolbox can foster the development of genetically pathogen-resistant crops for sustainable agriculture.