Research progress and application of the CRISPR/Cas9 gene-editing technology based on hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is now a common cause of cancer death,with no obvious change in patient survival over the past few years.Although the traditional therapeutic modalities for HCC patients mainly involved in surgery,chemotherapy,and radiotherapy,which have achieved admirable achievements,challenges are still existed,such as drug resistance and toxicity.The emerging gene therapy of clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9-based(CRISPR/Cas9),as an alternative to traditional treatment methods,has attracted considerable attention for eradicating resistant malignant tumors and regulating multiple crucial events of target gene-editing.Recently,advances in CRISPR/Cas9-based anti-drugs are presented at the intersection of science,such as chemistry,materials science,tumor biology,and genetics.In this review,the principle as well as statues of CRISPR/Cas9 technique were introduced first to show its feasibility.Additionally,the emphasis was placed on the applications of CRISPR/Cas9 technology in therapeutic HCC.Further,a broad overview of non-viral delivery systems for the CRISPR/Cas9-based anti-drugs in HCC treatment was summarized to delineate their design,action mechanisms,and anticancer applications.Finally,the limitations and prospects of current studies were also discussed,and we hope to provide comprehensively theoretical basis for the designing of anti-drugs.

Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR(TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35 S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.

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.

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.

CRISPR–Cas9-mediated promoter editing of FERONIA-Like receptor 13 increases plant growth and disease resistance in rice

Receptor kinases play a pivotal role in detecting environmental signals,and consequently,gene pleiotropy is frequently observed within this family.However,the trade-off in trait expression resulting from gene pleiotropy poses a constraint on the utilization of such genes in agricultural breeding.In this study,we identified the receptor kinase gene FERONIA-Like Receptor 13(FLR13)as a pleiotropic gene influencing plant height,tillering,grain yield,and disease resistance.Using promoter editing,we generated novel alleles(FLR13T5T6-1,FLR13T5T6-2)that confer resistance to rice blast and increase per-plant yield.The knockout of the T5T6 segment alleviates the inhibitory effects of two transcription factors,OsGBP1 and OsWRKY53,on FLR13 expression.In summary,our study presents a promising avenue for enhancing the pivotal attributes of receptor-like kinases through a promoter-editing strategy.

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.

Genome engineering and disease modeling via programmable nucleases for insulin gene therapy;promises of CRISPR/Cas9 technology

Targeted genome editing is a continually evolving technology employing programmable nucleases to specifically change,insert,or remove a genomic sequence of interest.These advanced molecular tools include meganucleases,zinc finger nucleases,transcription activator-like effector nucleases and RNA-guided engineered nucleases(RGENs),which create double-strand breaks at specific target sites in the genome,and repair DNA either by homologous recombination in the presence of donor DNA or via the error-prone non-homologous end-joining mechanism.A recently discovered group of RGENs known as CRISPR/Cas9 gene-editing systems allowed precise genome manipulation revealing a causal association between disease genotype and phenotype,without the need for the reengineering of the specific enzyme when targeting different sequences.CRISPR/Cas9 has been successfully employed as an ex vivo gene-editing tool in embryonic stem cells and patient-derived stem cells to understand pancreatic beta-cell development and function.RNA-guided nucleases also open the way for the generation of novel animal models for diabetes and allow testing the efficiency of various therapeutic approaches in diabetes,as summarized and exemplified in this manuscript.

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.

Studies on the temporal,structural,and interacting features of the clubroot resistance gene Rcr1 using CRISPR/Cas9-based systems

Clubroot disease is a severe threat to Brassica crops globally,particularly in western Canada.Genetic resistance,achieved through pyramiding clubroot resistance(CR)genes with different modes of action,is the most important strategy for managing the disease.However,studies on the CR gene functions are quite limited.In this study,we have conducted investigations into the temporal,structural,and interacting features of a newly cloned CR gene,Rcr1,using CRISPR/Cas9 technology.For temporal functionality,we developed a novel CRISPR/Cas9-based binary vector,pHHIGR-Hsp18.2,to deliver Rcr1 into a susceptible canola line(DH12075)and observed that early expression of Rcr1 is critical for conferring resistance.For structural functionality,several independent mutations in specific domains of Rcr1 resulted in loss-offunction,highlighting their importance for CR phenotype.In the study of the interacting features of Rcr1,a cysteine protease gene and its homologous allele in canola were successfully disrupted via CRISPR/Cas9 as an interacting component with Rcr1 protein,resulting in the conversion from clubroot resistant to susceptible in plants carrying intact Rcr1.These results indicated an indispensable role of these two cysteine proteases in Rcr1-mediated resistance response.This study,the first of its kind,provides valuable insights into the functionality of Rcr1.Further,the new vector p HHIGR-Hsp18.2 demonstrated an inducible feature on the removal of add-on traits,which should be useful for functional genomics and other similar research in brassica crops.

CRISPR/Cas9 technology and its application in horticultural crops

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9(CRISPR/Cas9)system has recently become one popular technology due to its efficiency,precision,and simplicity compared with other genome editing tools such as Zinc Finger Nucleases(ZFNs)and Transcription Activator Like Effector Nucleases(TALENs).Horticultural crops provide energy and health-keeping nutrients to humankind.Genome-editing technology has become widely adopted in horticultural breeding with the increasing demand for high yield and better-quality horticultural crops.Here,we describe the CRISPR/Cas9 system construction,its optimization,including sgRNA promoter,sgRNA design,Cas9 protein promoter,SpCas9 variants and orthologs,and vector delivery methods.We also summarized the application of this technology in horticultural plants for stress responses enhancement,fruit quality improvement,and cultivation traits modification.This detailed review was compiled to help establish comprehensive understanding of the CRISPR/Cas9 systems and provide a reference for further developing this technology to manipulate horticultural plant traits effectively.

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.

CRISPR/Cas9基因编辑技术在羊育种功能基因验证中的应用研究进展

基因编辑技术是一种对目的基因进行特定修饰的技术,被广泛应用于生物育种、疾病模型构建等研究领域。CRISPR/Cas9系统由重复和间隔单元组成的CRISPR阵列和一个Cas基因座位组成,能够对生物体性状进行目的基因定点编辑,具有简单、精确且高效的特点。CRISPR/Cas9基因编辑技术通过引入基因敲除、基因敲入、缺失、点突变和单核苷酸突变等不同形式的修饰广泛应用于畜禽的基因编辑,以研究畜禽关键基因功能,从而加速性状改良。相较于猪、鸡等其他畜禽,CRISPR/Cas9技术对羊的研究相对滞后。文章重点阐述了CRISPR/Cas9基因编辑技术在羊育种功能基因验证中的应用,对其在产肉性状、纤维性状、泌乳性状和人类疾病模型等方面的应用研究进展及前景进行了简要概述,以期为我国羊产业的可持续发展提供参考。

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.

Progress of CRISPR/Cas9 technology in breeding of Brassica napus

Gene editing technology provides important technical basics for the research in plant functional genes and crop genetic improvement.CRISPR/Cas9-mediated gene editing is an effective experimental tool for crop genome directed editing in recent years,which has been widely used in many crops as rice,wheat and other crops.CRISPR/Cas9 system was expected to be a powerful experimental tool in genetic improvement and molecular design breeding of rapeseed.This paper,which based on the development history and the latest research of CRISPR/Cas9-mediated gene editing technology in rapeseed,summarized the progress of CRISPR/Cas9 including plant type improvement,yield traits,quality improvement,disease and stress resistance improvement,yellow seed creation and other utilizes at present.The application scope,development direction and target analysis method of this technology in rape were focused.The problems of CRISPR/cas9 system in rapeseed breeding were analyzed and the improvement strategies were discussed.Finally,views on direction of rapeseed breeding by gene editing were emphasized.

Developing superior alleles of yield genes in rice by artificial mutagenesis using the CRISPR/Cas9 system

Rice yield is an important and complex agronomic trait controlled by multiple genes.In recent decades,dozens of yield-associated genes in rice have been cloned,many of which can increase production in the form of loss or degeneration of function.However,mutations occurring randomly under natural conditions have provided very limited genetic resources for yield increases.In this study,potentially yield-increasing alleles of two genes closely associated with yield were edited artificially.The recently developed CRISPR/Cas9system was used to edit two yield genes:Grain number 1a(Gn1a)and DENSE AND ERECT PANICLE1(DEP1).Several mutants were identified by a target sequence analysis.Phenotypic analysis confirmed one mutant allele of Gn1a and three of DEP1 conferring yield superior to that conferred by other natural high-yield alleles.Our results demonstrate that favorable alleles of the Gnla and DEP1 genes,which are considered key factors in rice yield increases,could be developed by artificial mutagenesis using genome editing technology.

Targeted mutagenesis of amino acid transporter genes for rice quality improvement using the CRISPR/Cas9 system

High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 system. Mutation efficiency varied with genetic background in the T_0 generation, and GPC in the T_1 generation decreased significantly,owing mainly to a reduction in glutelin content. Amylose content was down-regulated significantly in some Osaap6 and all Osaap10 mutants. The increased taste value of these mutants was supported by Rapid Visco Analysis(RVA) profiles, which showed higher peak viscosity and breakdown viscosity and lower setback viscosity than the wild type. There were no significant deficiencies in agronomic traits of the mutants. Targeted mutagenesis of OsAAP6 and OsAAP10, especially OsAAP10, using the CRISPR/Cas9 system can rapidly reduce GPC and improve ECQ of rice, providing a new strategy for the breeding cultivars with desired ECQ.

Generation of tryptophan hydroxylase 2 gene knockout pigs by CRISPR/Cas9-mediated gene targeting

Unbalanced brain serotonin(5-HT) levels have implications in various behavioral abnormalities and neuropsychiatric disorders. The biosynthesis of neuronal 5-HT is regulated by the rate-limiting enzyme, tryptophan hydroxylase-2(TPH2). In the present study, the clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated(Cas) system was used to target the Tph2 gene in Bama mini pig fetal fibroblasts. It was found that CRISPR/Cas9 targeting efficiency could be as high as 61.5%, and the biallelic mutation efficiency reached at38.5%. The biallelic modified colonies were used as donors for somatic cell nuclear transfer(SCNT) and 10 Tph2 targeted piglets were successfully generated. These Tph2 KO piglets were viable and appeared normal at the birth.However, their central 5-HT levels were dramatically reduced, and their survival and growth rates were impaired before weaning. These Tph2 KO pigs are valuable large-animal models for studies of 5-HT deficiency induced behavior abnomality.

Intron-targeted gene insertion in rice using CRISPR/Cas9: A case study of the Pi-ta gene

Intron-targeted gene insertion strategy using CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated Cas9) has been shown to be a potential tool for crop genetic improvement by targeted mutagenesis or gene replacement of an elite allele into widely cultivated rice varieties. The rice blast resistant protein Pi-ta, differs from its susceptible counterpart, pi-ta, by a single amino acid in exon 2. To create new materials resistant to the rice blast disease, we inserted a genomic fragment containing the exon 2 and 3′ untranslated region(3′ UTR) of Pi-ta into intron 1 of pi-ta in rice materials susceptible to rice blast using the intron-targeted insertion strategy. The gene insertion frequency was3.8%. Several novel transgene-free progeny with stably inherited homozygous insert were identified in the T_1 generation, which have been crossed to rice germplasm bearing other resistance gene(R gene) for pyramiding of R genes. This work verified the feasibility of using the genome editing technology in improvement of qualitative agronomic trait in crops.

Applications of CRISPR/Cas9 in retinal degenerative diseases

Gene therapy is a potentially effective treatment for retinal degenerative diseases.Clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated protein 9（Cas9） system has been developed as a new genome-editing tool in ophthalmic studies.Recent advances in researches showed that CRISPR/Cas9 has been applied in generating animal models as well as gene therapy in vivo of retinitis pigmentosa（RP） and leber congenital amaurosis（LCA）.It has also been shown as a potential attempt for clinic by combining with other technologies such as adeno-associated virus（AAV） and induced pluripotent stem cells（i PSCs）.In this review,we highlight the main points of further prospect of using CRISPR/Cas9 in targeting retinal degeneration.We also emphasize the potential applications of this technique in treating retinal degenerative diseases.

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.