CRISPR/Cas9 systems:Delivery technologies and biomedical applications

The emergence of the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)genome-editing system has brought about a significant revolution in the realm of managing human diseases,establishing animal models,and so on.To fully harness the potential of this potent gene-editing tool,ensuring efficient and secure delivery to the target site is paramount.Consequently,developing effective delivery methods for the CRISPR/Cas9 system has become a critical area of research.In this review,we present a comprehensive outline of delivery strategies and discuss their biomedical applications in the CRISPR/Cas9 system.We also provide an indepth analysis of physical,viral vector,and non-viral vector delivery strategies,including plasmid-,mRNA-and protein-based approach.In addition,we illustrate the biomedical applications of the CRISPR/Cas9 system.This review highlights the key factors affecting the delivery process and the current challenges facing the CRISPR/Cas9 system,while also delineating future directions and prospects that could inspire innovative delivery strategies.This review aims to provide new insights and ideas for advancing CRISPR/Cas9-based delivery strategies and to facilitate breakthroughs in biomedical research and therapeutic applications.

Clinical applications of the CRISPR/Cas9 genome-editing system: Delivery options and challenges in precision medicine

CRISPR/Cas9 is an effective gene editing tool with broad applications for the pre-vention or treatment of numerous diseases.It depends on CRiSPR(clustered regularly inter-spaced short palindromic repeats)as a bacterial immune system and plays as a gene editing tool.Due to the higher specificity and efficiency of CRISPR/Cas9 compared to other editing ap-proaches,it has been broadly investigated to treat numerous hereditary and acquired ill-nesses,including cancers,hemolytic diseases,immunodeficiency disorders,cardiovascular diseases,visual maladies,neurodegenerative conditions,and a few X-linked disorders.CRISPR/Cas9 system has been used to treat cancers through a variety of approaches,with sta-ble gene editing techniques.Here,the applications and clinical trials of CRisPR/Cas9 in various illnesses are described.Due to its high precision and efficiency,CRISPR/Cas9 strategies may treat gene-related illnesses by deleting,inserting,modifying,or blocking the expression of specific genes.The most challenging barrier to the in vivo use of CRISPR/Cas9 like off-target effects will be discussed.The use of transfection vehicles for CRISPR/Cas9,including viral vectors(such as an Adeno-associated virus(AAV),and the development of non-viral vectors is also considered.

Construction of a CHO cell line with site‑specific integration to stably express exogenous proteins using the CRISPR–Cas9 technique

Chinese hamster ovary(CHO)cells are widely used in biopharmaceuticals because of their high-density suspension culture,high safety,and high similarity between expressed exogenous proteins and natural proteins.However,the level of exogenous protein expression decreases with increasing culture time;this phenomenon occurs due to the recombination of foreign genes into chromosomes through random integration.The present study integrated the foreign genes into a specific chromosomal site for stable expression based on CRISPR–Cas9 technology.The results showed that the exogenous proteins enhanced green fluorescent protein(EGFP)and human serum albumin(HSA)were successfully integrated into the vicinity of base 1969647 on chromosome NW_003613638.1 of CHO-K1 cells.The obtained positive monoclonal cell lines expressed all the corresponding exogenous proteins after 60 consecutive passages,and no significant differences in expression levels were observed.This study might provide a feasible method to construct a CHO cell line with long-term stable expression of exogenous proteins.

Identification of neutral genome integration sites with high expression and high integration efficiency in Fusarium venenatum TB01

CRISPR/Cas9-mediated homology-directed recombination is an efficient method to express target genes.Based on the above method,providing ideal neutral integration sites can ensure the reliable,stable,and high expression of target genes.In this study,we obtained a fluorescent transformant with neutral integration and high expression of the GFP expression cassette from the constructed GFP expression library and named strain FS.The integration site mapped at 4886 bp upstream of the gene FVRRES_00686 was identified in strain FS based on a Y-shaped adaptor-dependent extension,and the sequence containing 600 bp upstream and downstream of this site was selected as the candidate region for designing sgRNAs(Sites)for CRISPR/Cas9-mediated homology-directed recombination.PCR analysis showed that the integration efficiency of CRISPR/Cas9-mediated integration of target genes in designed sites reached 100%.Further expression stability and applicability analysis revealed that the integration of the target gene into the above designed sites can be stably inherited and expressed and has no negative effect on the growth of F.venenatum TB01.These results indicate the above designed neutral sites have the potential to accelerate the development of F.venenatum TB01 through overexpression of target genes in metabolic engineering.

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein 9(CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs(sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies.Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.

CRISPR/Cas9 and TALE： beyond cut and paste

Nuclease-based genome editing has proven to be a powerful and promising tool for disease modeling and gene therapy. Recent advances in CRISPR/Cas and TALE indicate that they could also be used as a targeted regulator of gene expression, as well as being utilized for illuminating specific chromosomal structures or genomic regions.

Bioinformatics and Functional Analysis of High Oleic Acid-Related Gene GmSAM22 in Soybean [Glycine max (L.) Merr.]

High yield,high quality,stable yield,adaptability to growth period,and modern mechanization are the basic requirements for crops in the 21st century.Soybean oleic acid is a natural unsaturated fatty acid with strong antioxidant properties and stability.Known as a safe fatty acid,it has the ability to successfully prevent cardiovascular and cerebrovascular disorders.Improving the fatty acid composition of soybean seeds,can not only speed up the breeding process of high-quality high-oil and high-oleic soybeans,but also have important significance in human health,and provide the possibility for the development of soybean oil as a new energy source.Hence,the aim of this study was to analyze the high oleic acid elated gene GmSAM22 in soybean.In this research the soybean oleic acid-related gene GmSAM22 was screened out by Genome-wide association analysis,a 662 bp fragment was acquired by specific PCR amplification,and the pMD18T cloning vector was linked by the use of a seamless cloning technique.Bioinformatics analysis of the signal peptide prediction,subcellular localization,protein hydrophobicity,transmembrane region analysis,a phosphorylation site,protein secondary and tertiary structure and protein interaction analysis of the protein encoded by the SAM22 gene was carried out.The plasmid of the gene editing vector is pBK041.The overexpression vector was transformed from pCAMBIA3301 as the base vector,and overexpression vector were designed.Positive plants were obtained by genetic transformation by the pollen tube channel method.Fluorescence quantitative PCR was performed on the T2 generation plants to detect the relative expression levels in different tissues.Southern Blot was used to detect the presence of hybridization signal.Screening genes BAR,35S,and NOS in plants were identified by conventional PCR.10 seeds with high and low oleic acid content were chosen for quantitative PCR identification,and finally,the concentration and morphology of soybean fatty acids were identified by nearfar infrared spectroscopy.On 10 seeds with an upper and lower oleic acid content,a quantitative fluorescence analysis was done.In Southern blot hybridization,the SAM22 gene was integrated into the recipient soybean plant in hands of a sole copy.Fluorescence quantitative PCR appeared that the average relative expression of the SAM22 gene in roots,stems,leaves,and seeds was 1.70,1.67,3.83,and 4.41,respectively.Positive expression seeds had a 4.77%increase in oleic acid content.The level of oleic acid in the altered seeds was reduced by 4.13%when compared to CK,and it was discovered that the GmSAM22 gene could be a regulatory and secondary gene that promotes the conversion of stearic acid to oleic acid in soybean.There has not been a discussion of gene cloning or functional verification.The cloning and genetic transformation of the soybean SAM22 gene can effectively increase the content of oleic acid,which lays a foundation for the study of soybean with high oleic acid.

大豆NIN和NLP基因生物信息学分析及敲除载体构建

为促进大豆NLP家族基因突变大豆材料的获得及大豆NIN和NLP基因功能的研究,本研究对大豆NIN和NLP基因进行生物信息学分析,通过CRISPR/Cas 9基因编辑技术构建基因敲除载体,并且通过大豆毛根转化实验验证靶点的有效性。结果表明:大豆基因组中一共存在4个NIN基因和10个NLP基因家族成员,这些基因都具有RWP-RK和PB1两个保守结构域。亚细胞定位预测表明所有成员都定位在细胞核中,此外GmNIN1b和GmNIN2a还定位于叶绿体。GmNIN1a/b、GmNIN2a/b、GmNLP2a/b及GmNLP3b在根瘤中的表达量相对较高,推测这些基因可能对结瘤过程具有重要的调控功能。成功构建了NLP4和NLP5两个敲除载体,得到可敲除GmNLP4a/b的3个有效靶点和敲除GmNLP5a/b的2个有效靶点。本研究获得了大豆NIN和NLP基因家族的生物信息学依据和创制GmNLP4a/b和GmNLP5a/b基因突变体的技术依据。

大豆phyA下游基因预测和表达分析及敲除载体构建

为了进一步研究大豆光周期调控通路中E3和E4的下游基因,为获得其突变体植株奠定基础,以拟南芥远红光响应受体phyA下游的重要信号传递因子PIF3、LAF1、FHY1和FHL的序列作为参考序列,在Phytozome 12数据库中查找其相似序列,进行序列比对和进化树分析,确定它们在大豆中的同源基因并采用qRT-PCR方法进行组织表达分析,使用CRISPR/Cas 9系统设计同源基因的敲除靶点并通过发根系统验证靶点的有效性。结果显示:AtPIF3在大豆中有6个同源基因,AtLAF1在大豆中有4个同源基因,AtFHL在大豆中有2个同源基因,而AtFHY1在大豆中没有同源基因。4个LAF1基因主要在顶端生长点表达,6个PIF3和2个FHL基因主要在荚中表达。共鉴定出12个有效靶点,能够分别将大豆中6个PIF3、4个LAF1和2个FHL基因成功敲除。研究结果为进一步获得稳定的大豆突变体材料和大豆phyA下游基因的功能研究提供了理论基础。

A NAC transcription factor, NOR-like1, is a new positive regulator of tomato fruit ripening

Ripening of the model fruit tomato(Solanum lycopersicum)is controlled by a transcription factor network including NAC(NAM,ATAF1/2,and CUC2)domain proteins such as No-ripening(NOR),SlNAC1,and SlNAC4,but very little is known about the NAC targets or how they regulate ripening.Here,we conducted a systematic search of fruit-expressed NAC genes and showed that silencing NOR-like1(Solyc07g063420)using virus-induced gene silencing(VIGS)inhibited specific aspects of ripening.Ripening initiation was delayed by 14 days when NOR-like1 function was inactivated by CRISPR/Cas9 and fruits showed obviously reduced ethylene production,retarded softening and chlorophyll loss,and reduced lycopene accumulation.RNA-sequencing profiling and gene promoter analysis suggested that genes involved in ethylene biosynthesis(SlACS2,SlACS4),color formation(SlGgpps2,SlSGR1),and cell wall metabolism(SlPG2a,SlPL,SlCEL2,and SlEXP1)are direct targets of NOR-like1.Electrophoretic mobility shift assays(EMSA),chromatin immunoprecipitation-quantitative PCR(ChIP-qPCR),and dual-luciferase reporter assay(DLR)confirmed that NOR-like1 bound to the promoters of these genes both in vitro and in vivo,and activated their expression.Our findings demonstrate that NOR-like1 is a new positive regulator of tomato fruit ripening,with an important role in the transcriptional regulatory network.

T cells expressing CD5/CD7 bispecific chimeric antigen receptors with fully human heavy-chain-only domains mitigate tumor antigen escape

Bispecific chimeric antigen receptor T-cell(CAR-T)therapies have shown promising results in clinical trials for advanced B-cell malignancies.However,it is challenging to broaden the success of bispecific CAR-T therapies to treat refractory/relapse(r/r)T-cell leukemia/lymphoma because targeting multiple T-cell-expressing antigens leads to exacerbated CAR-T cell fratricide and potential safety concerns.Fully human heavy chain variable(FHVH)antibodies that specifically target CD5 or CD7 were screened and constructed to CD5/CD7 bispecific CARs.A truncated Epidermal growth factor receptor were integrated into CAR constructs to address safety concerns.To tackle the fratricidal issue of CAR-T cells targeting T-cell-pan marker(s),CRISPR/Cas9-based CD5 and CD7 genes knockout were performed before lentiviral transduction of bispecific CARs.Functional comparison between different bispecific CAR structures:tandem CARs and dual CAR were performed in vitro and in vivo to determine the optimal construct suitable for addressing T-cell malignancy antigen escape in clinical setting.Knockout of CD5 and CD7 prevents fratricide of CD5/CD7 bispecific CAR-T cells,and FHVH-derived CD5/CD7 bispecific CAR-T cells demonstrate potent antitumor activity in vitro and in vivo.The fratricide-resistant FHVH-derived CD5/CD7 bispecific CAR-T cells have potent antitumor activity against T-cell malignancies,and tandem CARs are more effective than dual CAR in preventing tumor escape in heterogeneous leukemic cells.The meaningful clinical efficacy and safety of tandem CD5/CD7 CAR-T cells deserve to be explored urgently.

New treatment for osteoarthritis:Gene therapy

Osteoarthritis is a complex degenerative disease that affects the entire joint tissue.Currently,non-surgical treatments for osteoarthritis focus on relieving pain.While end-stage osteoarthritis can be treated with arthroplasty,the health and financial costs associated with surgery have forced the search for alternative non-surgical treatments to delay the progression of osteoarthritis and promote cartilage repair.Unlike traditional treatment,the gene therapy approach allows for long-lasting expression of therapeutic proteins at specific sites.In this review,we summarize the history of gene therapy in osteoarthritis,outlining the common expression vectors(non-viral,viral),the genes delivered(transcription factors,growth factors,inflammation-associated cytokines,non-coding RNAs)and the mode of gene delivery(direct delivery,indirect delivery).We highlight the application and development prospects of the gene editing technology CRISPR/Cas9 in osteoarthritis.Finally,we identify the current problems and possible solutions in the clinical translation of gene therapy for osteoarthritis.

Global analysis of CCT family knockout mutants identifies four genes involved in regulating heading date in rice

Many genes encoding CCT domain-containing proteins regulate flowering time. In rice(Oryza sativa), 41 such genes have been identified, but only a few have been shown to regulate heading date. Here, to test whether and how additional CCT family genes regulate heading date in rice, we classified these genes into five groups based on their diurnal expression patterns. The expression patterns of genes in the same subfamily or in close phylogenetic clades tended to be similar. We generated knockout mutants of the entire gene family via CRISPR/Cas9. The heading dates of knockout mutants of only 4 of 14 genes previously shown to regulate heading date were altered, pointing to functional redundancy of CCT family genes in regulating this trait. Analysis of mutants of four other genes showed that OsCCT22, OsCCT38, and OsCCT41 suppress heading under long-day conditions and promote heading under short-day conditions. OsCCT03 promotes heading under both conditions and upregulates the expression of Hd1 and Ehd1, a phenomenon not previously reported for other such genes. To date, at least 18 CCT domaincontaining genes involved in regulating heading have been identified, providing diverse, flexible gene combinations for generating rice varieties with a given heading date.

Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products

The methylotrophic yeast Pichia pastoris(a.k.a.Komagataella phaffii)is one of the most commonly used hosts for industrial production of recombinant proteins.As a non-conventional yeast,P.pastoris has unique biological characteristics and its expression system has been well developed.With the advances in synthetic biology,more efforts have been devoted to developing P.pastoris into a chassis for the production of various high-value compounds,such as natural products.This review begins with the introduction of synthetic biology tools for the engineering of P.pastoris,including vectors,promoters,and terminators for heterologous gene expression as well as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated System(CRISPR/Cas)for genome editing.This review is then followed by examples of the production of value-added natural products in metabolically engineered P.pastoris strains.Finally,challenges and outlooks in developing P.pastoris as a synthetic biology chassis are prospected.