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.

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.

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.

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.

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.

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.

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.

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.

Base editing:a brief review and a practical example

Genome editing has undergone rapid development in recent years,yielding new approaches to make precise changes in genes.In this review,we discuss the development of various adenine and cytosine base-editing technologies,which share the ability to make specific base changes at specific sites in the genome.We also describe multiple applications of base editing in vitro and in vivo.Finally,as a practical example,we demonstrate the use of a cytosine base editor and an adenine base editor in human cells to introduce and then correct a prevalent mutation responsible for hereditary tyrosinemia type 1.

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.

Reflections on Amendments to the Journal's Editing Standards

A 10-year-old unified editing standard was recently abolished in the field of humanities and social science journals. This is because the standard failed in showing equal respect to disciplinary differences, in keeping with Chinese culturaltraditions, and in gaining acceptance among journal editors. As such, amendment efforts to the National Standard should be undertaken patiently and in a practical manner to achieve agreement within this publishing field.

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.

A new gain-of-function OsGS2/GRF4 allele generated by CRISPR/Cas9 genome editing increases rice grain size and yield

Grain size is one of the most important factors affecting rice grain quality and yield,and attracts great attention from molecular biologists and breeders.In this study,we engineered a CRISPR/Cas9 system targeting the miR396 recognition site of the rice GS2 gene,which encodes growth-regulating factor 4(OsGRF4)and regulates multiple agronomic traits including grain size,grain quality,nitrogen use efficiency,abiotic stress response,and seed shattering.In contrast to most previous genome editing efforts in which indel mutations were chosen to obtain null mutants,a mutant named GS2^(E) carrying an in-frame 6-bp deletion and 1-bp substitution within the miR396-targeted sequence was identified.GS2^(E) plants showed increased expression of GS2 in consistent with impaired repression by miR396.As expected,the gain-of-function GS2^(E) mutant exhibited multiple beneficial traits including increased grain size and yield and bigger grain length/width ratio.Thousand grain weight and grain yield per plant of GS2^(E) plants were increased by 23.5%and 10.4%,respectively.These improved traits were passed to hybrids in a semidominant way,suggesting that the new GS2^(E) allele has great potential in rice improvement.Taken together,we report new GS2 germplasm and describe a novel gene-editing strategy that can be widely employed to improve grain size and yield in rice.This trait-improvement strategy could be applied to other genes containing miRNA target sites,in particular the conserved miR396-GRF/GIF module that governs plant growth,development and environmental response.

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.