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.

Germline Gene-Editing Creates Enhanced Livestock-Technical and Especially Ethical Issues Challenge Its Use in Humans

Using clustered regularly interspaced short palindromic repeats(CRISPR)-based molecular tools,scientists are engineering-as they are also doing with plants.-animals with advantageous traits,like disease resistance and improved food yield.While these innovative techniques could one day be applied in humans,technical hurdles and ethical concerns likely place this possibility far in the future,The enhancements rely on germline gene editing,which alters the genes in a way that passes the changes on to offspring.Ger m-line gene editing differs from the somatic cell gene editing used in the highly promising new treatment recently approved for the human disease sickle cell anemia.

Development of an Agrobacterium-mediated CRISPR/Cas9 gene editing system in jute(Corchorus capsularis)

Jute(Corchorus capsularis L.)is the second most important natural plant fiber source after cotton.However,developing an efficient gene editing system for jute remains a challenge.In this study,the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4,an elite jute variety widely cultivated in China.The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation(BiFC)assays.The CHLOROPLASTOS ALTERADOS 1(CcCLA1)gene,which is involved in the development of chloroplasts,was targeted for editing at two sites in Meifeng 4.Based on this hairy root transformation,the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and-GhU6.9 promoters,respectively,to assess the efficiency of gene editing.Results indicated the 50.0%(GhU6.7)and 38.5%(GhU6.9)editing events in the target 2 alleles(gRNA2),but no mutation was detected in the target 1 allele(gRNA1)in transgenic-positive hairy roots.CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation.Two CcCLA1 mutants were albinic,with a gene editing efficiency of 5.3%.These findings confirm that the CRISPR/Cas9 system,incorporating promoter GhU6.7,can be used as a gene editing tool for jute.

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.

Engineering high amylose and resistant starch in maize by CRISPR/Cas9-mediated editing of starch branching enzymes

To improve the amylose content(AC)and resistant starch content(RSC)of maize kernel starch,we employed the CRISPR/Cas9 system to create mutants of starch branching enzyme I(SBEI)and starch branching enzyme IIb(SBEIIb).A frameshift mutation in SBEI(E1,a nucleotide insertion in exon 6)led to plants with higher RSC(1.07%),lower hundred-kernel weight(HKW,24.71±0.14 g),and lower plant height(PH,218.50±9.42 cm)compared to the wild type(WT).Like the WT,E1 kernel starch had irregular,polygonal shapes with sharp edges.A frameshift mutation in SBEIIb(E2,a four-nucleotide deletion in exon 8)led to higher AC(53.48%)and higher RSC(26.93%)than that for the WT.E2 kernel starch was significantly different from the WT regarding granule morphology,chain length distribution pattern,X-ray diffraction pattern,and thermal characteristics;the starch granules were more irregular in shape and comprised typical B-type crystals.Mutating SBEI and SBEIIb(E12)had a synergistic effect on RSC,HKW,PH,starch properties,and starch biosynthesis-associated gene expression.SBEIIa,SS1,SSIIa,SSIIIa,and SSIIIb were upregulated in E12 endosperm compared to WT endosperm.This study lays the foundation for rapidly improving the starch properties of elite maize lines.

Metabolic engineering and genome editing strategies for enhanced lipid production in microalgae

Depleting global petroleum reserves and skyrocketing prices coupled with succinct supply have been a grave concern,which needs alternative sources to conventional fuels.Oleaginous microalgae have been explored for enhanced lipid production,leading towards biodiesel production.These microalgae have short life cycles,require less labor,and space,and are easy to scale up.Triacylglycerol,the primary source of lipids needed to produce biodiesel,is accumulated by most microalgae.The article focuses on different types of oleaginous microalgae,which can be used as a feedstock to produce biodiesel.Lipid biosynthesis in microalgae occurs through fatty acid synthesis and TAG synthesis approaches.In-depth discussions are held regarding other efficient methods for enhancing fatty acid and TAG synthesis,regulating TAG biosynthesis bypass methods,blocking competing pathways,multigene approach,and genome editing.The most potential targets for gene transformation are hypothesized to be a malic enzyme and diacylglycerol acyltransferase while lowering phosphoenolpyruvate carboxylase activity is reported to be advantageous for lipid synthesis.

Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa

Alfalfa(Medicago sativa.L.)is a globally significant autotetraploid legume forage crop.However,despite its importance,establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge.In this study,we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa,a variety widely cultivated in Northeast China.Subsequently,we created a single transcript CRISPR/Cas9(CRISPR_2.0)toolkit,incorporating multiplex gRNAs,designed for gene editing in Gongnong 1.Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter,a widely employed polymeraseⅡconstitutive promoter known for strong transgene expression in dicots.To assess the toolkit’s efficiency,we targeted PALM1,a gene associated with a recognizable multifoliate phenotype.Utilizing the CRISPR_2.0 toolkit,we directed PALM1 editing at two sites in the wild-type Gongnong 1.Results indicated a 35.1%occurrence of editing events all in target 2 alleles,while no mutations were detected at target 1 in the transgenic-positive lines.To explore more efficient sgRNAs,we developed a rapid,reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation,incorporating the visible reporter MtLAP1.This screening system demonstrated that most purple visible hairy roots underwent gene editing.Notably,sgRNA3,with an 83.0%editing efficiency,was selected using the visible hairy root system.As anticipated,tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype.These palm1 lines demonstrated an average crude protein yield increase of 21.5%compared to trifoliolate alfalfa.Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.

New insights into ATR inhibition in muscle invasive bladder cancer:The role of apolipoprotein B mRNA editing catalytic subunit 3B

Background:Apolipoprotein B mRNA editing catalytic polypeptide(APOBEC),an endogenous mutator,induces DNA damage and activates the ataxia telangiectasia and Rad3-related(ATR)-checkpoint kinase 1(Chk1)pathway.Although cisplatin-based therapy is the mainstay for muscle-invasive bladder cancer(MIBC),it has a poor survival rate.Therefore,this study aimed to evaluate the efficacy of an ATR inhibitor combined with cisplatin in the treatment of APOBEC catalytic subunit 3B(APOBEC3B)expressing MIBC.Methods:Immunohistochemical staining was performed to analyze an association between APOBEC3B and ATR in patients with MIBC.The APOBEC3B expression in MIBC cell lines was assessed using real-time polymerase chain reaction and western blot analysis.Western blot analysis was performed to confirm differences in phosphorylated Chk1(pChk1)expression according to the APOBEC3B expression.Cell viability and apoptosis analyses were performed to examine the anti-tumor activity of ATR inhibitors combined with cisplatin.Results:There was a significant association between APOBEC3B and ATR expression in the tumor tissues obtained from patients with MIBC.Cells with higher APOBEC3B expression showed higher pChk1 expression than cells expressing low APOBEC3B levels.Combination treatment of ATR inhibitor and cisplatin inhibited cell growth in MIBC cells with a higher APOBEC3B expression.Compared to cisplatin single treatment,combination treatment induced more apoptotic cell death in the cells with higher APOBEC3B expression.Conclusion:Our study shows that APOBEC3B’s higher expression status can enhance the sensitivity of MIBC to cisplatin upon ATR inhibition.This result provides new insight into appropriate patient selection for the effective application of ATR inhibitors in MIBC.

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。

Devised Guidelines of Rapid Post-Editing in Machine Translation Output of Mark on Some Aspects

One of uses of machine translation(MT),is helping readers to read for the gist of a foreign text through a draft transla tion produced by MT engines.Rapid post-editing,as Jeffrey Allen defines it as a"strictly minimal editing on texts in order to re move blatant and significant errors without considering stylistic issues",can help present the reader with a roughly comprehensi ble translation as quickly as possible.The purpose of this article is on a proposed set of rapid post-editing guidelines for Biblical Chinese-English MT,with its application on editing the English MT version of Chapter one of Mark(马尔谷福音) of the Chi nese Catholic Bible(天主教思高本圣经) as an example.

Genome editing opens a new era of genetic improvement in polyploid crops

Sequence-specific nucleases(SSN) that generate double-stranded DNA breaks(DSBs) in genes of interest are the key to site-specific genome editing in plants. Genome editing has developed into one method of reducing undesirable traits in crops by the induction of knockout mutations. Different SSN-mediated genome-editing systems, including LAGLIDADG homing endonucleases or meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats, are emerging as robust tools for introducing functional mutations in polyploid crops including citrus, wheat, cotton, soybean, rapeseed, potato, grapes, Camelina sativa,dandelion, and tobacco. The approach utilizes knowledge of biological mechanisms for targeted induction of DSBs and their error-prone repair, allowing highly specific changes at designated genome loci. In this review, we briefly describe genome-editing technologies and their application to genetic improvement of polyploid crops.

Genome-wide identification of RNA editing in seven porcine tissues by matched DNA and RNA high-throughput sequencing

Background: RNA editing is a co/posttranscriptional modification mechanism that increases the diversity of transcripts, with potential functional consequences. The advent of next-generation sequencing technologies has enabled the identification of RNA edits at unprecedented throughput and resolution. However, our knowledge of RNA editing in swine is still limited.Results: Here, we utilized RES-Scanner to identify RNA editing sites in the brain, subcutaneous fat, heart, liver,muscle, lung and ovary in three 180-day-old Large White gilts based on matched strand-specific RNA sequencing and whole-genome resequencing datasets. In total, we identified 74863 editing sites, and 92.1% of these sites caused adenosine-to-guanosine(A-to-G) conversion. Most A-to-G sites were located in noncoding regions and generally had low editing levels. In total, 151 A-to-G sites were detected in coding regions(CDS), including 94 sites that could lead to nonsynonymous amino acid changes. We provide further evidence supporting a previous observation that pig transcriptomes are highly editable at PRE-1 elements. The number of A-to-G editing sites ranged from 4155(muscle) to 25001(brain) across the seven tissues. The expression levels of the ADAR enzymes could explain some but not all of this variation across tissues. The functional analysis of the genes with tissuespecific editing sites in each tissue revealed that RNA editing might play important roles in tissue function.Specifically, more pathways showed significant enrichment in the fat and liver than in other tissues, while no pathway was enriched in the muscle.Conclusions: This study identified a total of 74863 nonredundant RNA editing sites in seven tissues and revealed the potential importance of RNA editing in tissue function. Our findings largely extend the porcine editome and enhance our understanding of RNA editing in swine.

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.

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.

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.

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.

Primordial germ cell-mediated transgenesis and genome editing in birds

Transgenesis and genome editing in birds are based on a unique germline transmission system using primordial germ cells(PGCs), which is quite different from the mammalian transgenic and genome editing system. PGCs are progenitor cells of gametes that can deliver genetic information to the next generation. Since avian PGCs were first discovered in nineteenth century, there have been numerous efforts to reveal their origin, specification, and unique migration pattern, and to improve germline transmission efficiency. Recent advances in the isolation and in vitro culture of avian PGCs with genetic manipulation and genome editing tools enable the development of valuable avian models that were unavailable before. However, many challenges remain in the production of transgenic and genome-edited birds,including the precise control of germline transmission, introduction of exogenous genes, and genome editing in PGCs.Therefore, establishing reliable germline-competent PGCs and applying precise genome editing systems are critical current issues in the production of avian models. Here, we introduce a historical overview of avian PGCs and their application, including improved techniques and methodologies in the production of transgenic and genome-edited birds, and we discuss the future potential applications of transgenic and genome-edited birds to provide opportunities and benefits for humans.

Genome-wide profiling of RNA editing sites in sheep

Background: The widely observed RNA-DNA differences(RDDs) have been found to be due to nucleotide alteration by RNA editing. Canonical RNA editing(i.e., A-to-I and C-to-U editing) mediated by the adenosine deaminases acting on RNA(ADAR) family and apolipoprotein B mRNA editing catalytic polypeptide-like(APOBEC)family during the transcriptional process is considered common and essential for the development of an individual.To date, an increasing number of RNA editing sites have been reported in human, rodents, and some farm animals;however, genome-wide detection of RNA editing events in sheep has not been reported. The aim of this study was to identify RNA editing events in sheep by comparing the RNA-seq and DNA-seq data from three biological replicates of the kidney and spleen tissues.Results: A total of 607 and 994 common edited sites within the three biological replicates were identified in the ovine kidney and spleen, respectively. Many of the RDDs were specific to an individual. The RNA editing-related genes identified in the present study might be evolved for specific biological functions in sheep, such as structural constituent of the cytoskeleton and microtubule-based processes. Furthermore, the edited sites found in the ovine BLCAP and NEIL1 genes are in line with those in previous reports on the porcine and human homologs, suggesting the existence of evolutionarily conserved RNA editing sites and they may play an important role in the structure and function of genes.Conclusions: Our study is the first to investigate RNA editing events in sheep. We screened out 607 and 994 RNA editing sites in three biological replicates of the ovine kidney and spleen and annotated 164 and 247 genes in the kidney and spleen, respectively. The gene function and conservation analysis of these RNA editing-related genes suggest that RNA editing is associated with important gene function in sheep. The putative functionally important RNA editing sites reported in the present study will help future studies on the relationship between these edited sites and the genetic traits in sheep.

A Review and Prospects on Collaborative Ontology Editing Tools

Building ontology is a fundamental but also hard work. Collaborative ontology editing tools can make ontology development more efficiently. In this paper, the important features of collaborative ontology development were analyzed, and several tools such as AGROVOC Concept Server Workbench （ACSW）, Collaborative Prot6g6 and WebProt6g6 were studied. Besides, some comparisons among them from several aspects were made and some prospects for the further improvement of these tools were given. Finally, we show it is a good way to build agricultural ontology with these tools collaboratively and simultaneously.

Crop genome editing: A way to breeding by design

Increasing population and consumption in our planet is placing unprecedented challenges on agriculture for meeting food security and sustainability needs[1].Meanwhile,the adaptation of modern agricultural techniques[2]is central to minimize extensive losses due to abiotic stresses[3]under global climate change.