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.

Lipid nanoparticle-mediated CRISPR/Cas9 gene editing and metabolic engineering for anticancer immunotherapy

Metabolic engineering of the tumor microenvironment has emerged as a new strategy.Lactate dehydrogenase A(LDHA)is a prominent target for metabolic engineering.Here,we designed a cationic lipid nanoparticle formulation for LDHA gene editing.The plasmid DNA delivery efficiency of our lipid nanoparticle formulations was screened by testing the fluorescence of lipid nanoparticles complexed to plasmid DNA encoding green fluorescence protein(GFP).The delivery efficiency was affected by the ratios of three components:a cationic lipid,cholesterol or its derivative,and a fusogenic lipid.The lipid nanoparticle designated formulation F3 was complexed to plasmid DNA co-encoding CRISPR-associated protein 9 and LDHA-specific sgRNA,yielding the lipoplex,pCas9-sgLDHA/F3.The lipoplex including GFP-encoding plasmid DNA provided gene editing in HeLa-GFP cells.Treatment of B16F10 tumor cells with pCas9-sgLDHA/F3 yielded editing of the LDHA gene and increased the pH of the culture medium.pCas9-sgLDHA/F3 treatment activated the interferon-gamma and granzyme production of T cells in culture.In vivo,combining pCas9-sgLDHA/F3 with immune checkpoint-inhibiting anti-PD-L1 antibody provided a synergistic antitumor effect and prolonged the survival of tumor model mice.This study suggests that combining metabolic engineering of the tumor microenvironment with immune checkpoint inhibition could be a valuable antitumor strategy.

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

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

Therapeutic gene editing strategies using CRISPR-Cas9 for theβ-hemoglobinopathies

With advancements in gene editing technologies,our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate,paving the way for scientists and clinicians to uniquely treat a multitude of previously irremediable diseases.CRISPR-Cas9,short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9,is a gene editing platform with the ability to alter the nucleotide sequence of the genome in living cells.This technology is increasing the number and pace at which new gene editing treatments for genetic disorders are moving toward the clinic.Theβ-hemoglobinopathies are a group of monogenic diseases,which despite their high prevalence and chronic debilitating nature,continue to have few therapeutic options available.In this review,we will discuss our existing comprehension of the genetics and current state of treatment forβ-hemoglobinopathies,consider potential genome editing therapeutic strategies,and provide an overview of the current state of clinical trials using CRISPR-Cas9 gene editing.

Harnessing CRISPR-Cas system diversity for gene editing technologies

The discovery and utilization of RNA-guided surveillance complexes,such as CRISPR-Cas9,for sequencespecific DNA or RNA cleavage,has revolutionised the process of gene modification or knockdown.To optimise the use of this technology,an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements,coupled with high cleavage efficacy and specificity.Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.

Traceless photodegradable polymer cocoons for universal protein delivery and light-controlled gene editing

Polymer conjugation was found highly valuable in clinic to improve the bioavailability of protein therapeutics.However,it is still a tremendous challenge to achieve a complete release of original proteins from the conjugated hybrid under external stimulus to recover active proteins in the targeted tissue.Herein,we report a general light-controlled protein delivery methodology by weaving a photodegradable polymer cocoon around proteins,which could reliably protect them from degradation in the dark while efficiently releasing them under illumination without any residual atoms.The surface charge of the polymer shell is easily tunable to facilitate efficient cell uptake.The versatility of this strategy is demonstrated by the delivery of the Cas9/sg RNA complex that realized light-controlled gene editing both in vitro and in vivo,and such repertoire is of particular value in regard to minimizing the off-target toxicity of CRISPR-Cas9-based gene therapy.

When mRNA meets gene editing

The critical challenge of gene therapy lies in delivering gene editing agents.Compared with DNA,while RNA is less stable and more accessible to degrade,it comes with the benefit of lower off-target effects since permanent insertion is not involved.This review focuses on mRNA-based delivery of gene editing agents,highlighting novel mRNA delivery systems.To provide context,a comparison is made between three main gene editing agents:programmable nucleases,base editors,and prime editors.The potential of Cas\pi and transposons is also discussed in this review.Additionally,a summary of four main barriers to mRNAbased in vivo delivery is provided.Furthermore,this review detailedly introduced different delivery systems,both viral(lentivirus)and non-viral vectors(genome editing via oviductal nucleic acids delivery,lipid nanoparticles,polymer-based nanoparticles,viruslike-particles,extracellular vesicles,and migrasome).Each delivery strategy is assessed by comparing its advantages and disadvantages to offer a comprehensive and objective overview of the delivery system.Moreover,we emphasized the vital role of the protein corona as a critical regulator for nanodelivery.Ultimately,we concluded the challenges of mRNA-based gene editing strategies(RNA stability,targeting,potential immunogenicity,cytotoxicity,heterogeneity,and rational design).The purpose of this review is to guide further research and provide a comprehensive analysis of mRNA-based in vivo delivery of gene editing agents in this promising field.

Viral and nonviral nanocarriers for in vivo CRISPR-based gene editing

The continued development of clustered regularly interspaced short palindromic repeats(CRISPR)technology has the potential to greatly impact clinical medicine,particularly for disease diagnosis and treatment.Despite high demand for the in vivo delivery of CRISPR-based therapies,significant challenges persist.These include rapid degradation by enzymes,inefficient disease site targeting,and the risk of undesired off-target outcomes.Nanoparticulate platforms,with their tailorable properties,have been engineered to efficiently package CRISPR payloads in various formats,including as plasmid DNA,mRNA,and ribonucleoprotein complexes,for in vivo delivery.Among them,recombinant adeno-associated viruses,virus-like particles,and lipid nanoparticles have displayed exceptional promise.This review will discuss the development of these and other nanocarriers for in vivo CRISPR-based genome editing.

CRISPR-based gene editing technology and its application in microbial engineering

Gene editing technology involves the modification of a specific target gene to obtain a new function or phenotype.Recent advances in clustered regularly interspaced short palindromic repeats(CRISPR)-Cas-mediated technolo-gies have provided an efficient tool for genetic engineering of cells and organisms.Here,we review the three emerging gene editing tools(ZFNs,TALENs,and CRISPR-Cas)and briefly introduce the principle,classification,and mechanisms of the CRISPR-Cas systems.Strategies for gene editing based on endogenous and exogenous CRISPR-Cas systems,as well as the novel base editor(BE),prime editor(PE),and CRISPR-associated transposase(CAST)technologies,are described in detail.In addition,we summarize recent developments in the application of CRISPR-based gene editing tools for industrial microorganism and probiotics modifications.Finally,the potential challenges and future perspectives of CRISPR-based gene editing tools are discussed.

Treatment of infectious diseases by in vivo gene editing

Gene editing is the specific modification of genome sequences at desired sites using technologies derived from zinc finger nucleases(ZFNs),transcription activator-like effector nucleases(TALENs)and clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR-associated(Cas)nuclease systems.It is a promising tool for the development of new treatment strategies for infectious diseases.Due to its higher editing efficiency and lower off-target effect,gene editing therapy mainly uses CRISPR Cas-derived tools to resist viral and non-viral infections.Here,we reviewed the recent research progress of gene editing in antiviral therapy(human immunodeficiency virus,hepatitis B virus,severe acute respiratory syndrome coronavirus 2,and human papillomavirus)and inhibition of infectious diseases that involve bacteria,fungi and parasites.

Molecular breeding of farm animals through gene editing

The rapid development of biotechnology has facilitated our understanding of the biological functions of candidate genes for important economic traits in farm animals.Molecular breeding by gene editing has greatly revolutionized the breeding of farm animals.Through gene editing and embryo manipulation,breeds with designed economic or disease-resistant traits can be readily generated.Along with this fast progress,the safety assessment of gene-edited farm animals has attracted public and regulatory attention.This review summarizes the research progress of gene editing in farm animals,focusing on performance improvement,disease resistance,bioreactors,animal welfare,and environmental friendliness.The limitations and future development of gene editing technology in farm animal breeding are also discussed.

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

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

Ethical and legal implications of gene editing in plant breeding:a systematic literature review

Biotechnology policies and regulations must be revised and updated to reflect the most recent advances in plantbreeding technology. New Plant Breeding Techniques(NPBT) such as gene editing have been applied to address the myriad of challenges in plant breeding, while the use of NPBT as emerging biotechnological tools raises legal and ethical concerns. This study aims to highlight how gene editing is operationalized in the existing literature and examine the critical issues of ethical and legal issues of gene editing for plant breeding. We carried out a systematic literature review(SLR) to provide the current states of ethical and legal discourses surrounding this topic. We also identified critical research priority areas and policy gaps that must be addressed when designing the future governance of gene editing in plant breeding.

The concept of gene therapy for glaucoma:the dream that has not come true yet

Gene therapies,despite of being a relatively new therapeutic approach,have a potential to become an important alternative to current treatment strategies in glaucoma.Since glaucoma is not considered a single gene disease,the identified goals of gene therapy would be rather to provide neuroprotection of retinal ganglion cells,especially,in intraocular-pressure-independent manner.The most commonly reported type of vector for gene delivery in glaucoma studies is adeno-associated virus serotype 2 that has a high tro pism to retinal ganglion cells,res ulting in long-term expression and low immunogenic profile.The gene thera py studies recruit inducible and genetic animal models of optic neuropathy,like DBA/2J mice model of high-tension glaucoma and the optic nerve crush-model.Reported gene therapy-based neuroprotection of retinal ganglion cells is targeting specific genes translating to growth factors(i.e.,brain derived neurotrophic factor,and its receptor TrkB),regulation of apoptosis and neurodegeneration(i.e.,Bcl-xl,Xiap,FAS system,nicotinamide mononucleotide adenylyl transferase 2,Digit3 and Sarm1),immunomodulation(i.e.,Crry,C3 complement),modulation of neuroinflammation(i.e.,e rythropoietin),reduction of excitotoxicity(i.e.,Com KIlα)and transcription regulation(i.e.,Max,Nrf2).On the other hand,some of gene therapy studies focus on lowering intra ocular pressure,by impacting genes involved in both,decreasing aqueous humor production(i.e.,aquaporin 1),and increasing outflow facility(i.e.,COX2,prostaglandin F2a receptor,RhoA/RhoA kinase signaling pathway,MMP1,Myocilin).The goal of this review is to summarize the current stateof-art and the direction of development of gene therapy strategies for glaucomatous neuropathy.

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.

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.

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

Genome editing tools such as the clustered regularly interspaced short palindromic repeat （CRISPR）-associated system （Cas） have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear （3PN） zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene （HBB）. However, the efficiency of homologous recombination directed repair （HDR） of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene （HBD）, which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.

Highly efficient,genotype-independent transformation and gene editing in watermelon(Citrullus lanatus)using a chimeric ClGRF4-GIF1 gene

Efficient genetic transformation has the potential to advance research and breeding in watermelon(Citrullus lanatus),but regeneration from tissue culture remains challenging.Previous work showed that expressing a fusion of two interacting transcription factors,GROWTH-REGULATING FACTOR4(GRF4)and GRF-INTERACTING FACTOR1(GIF1),improved regeneration in wheat(Triticum aestivum).By overexpressing a chimeric fusion of Cl GRF4 and Cl GIF1,we achieved highly efficient transformation in watermelon.Mutating the mi396 micro RNA target site in Cl GRF further boosted the transformation efficiency up to 67.27%in a genotype-independent manner.Cl GRF4-GIF1 can also be combined with clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)genome editing tools to achieve highly efficient gene editing in watermelon,which we used to successfully create diploid seedless watermelon.This research thus puts forward a powerful transformation tool for future watermelon research and breeding.

Gene editing and its applications in biomedicine

The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats(CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.

A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana

The newly developed CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas(CRISPR-associated) system has emerged as an efficient tool for genome-editing, providing an alternative to classical mutagenesis and transgenic methods to study gene function and improve crop traits. CRISPR/Cas facilitates targeted gene editing through RNA-guided DNA cleavage followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. Here we describe a detailed procedure for our previously developed and highly efficient CRISPR/Cas9 method that allows the generation of heritable-targeted gene mutations andcorrections in Arabidopsis. This protocol describes the strategies and steps for the selection of targets, design of single-guide RNA(sg RNA), vector construction and analysis of transgenic lines. We also offer a method to target two loci simultaneously using vectors containing two different sg RNAs. The principles described in this protocol can be applied to other plant species to generate stably inherited DNA modifications.