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.

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.

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.

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.

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 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.

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.

Construction and Functional Analysis of CRISPR/Cas9 Vector of FAD2 Gene Family in Soybean

Soybean oleic acid content is one of the important indexes to evaluate the quality of soybean oil.In the synthesis pathway of soybean fatty acids,the FAD2 gene family is the key gene that regulates the production of linoleic acid from soybean oleic acid.In this study,CRISPR/Cas9 gene editing technology was used to regulate FAD2 gene expression.Firstly,the CRISPR/Cas9 single knockout vectors GmFAD2-1B and GmFAD2-2C and double knockout vectors GmFAD2-2A-3 were constructed.Then,the three vectors were transferred into the recipient soybean variety Jinong 38 by Agrobacterium-mediated cotyledon node transformation,and the mutant plants were obtained.Functional analysis and comparison of the mutant plants of the T2 and T3 generations were carried out.The results showed that there was no significant difference in agronomic traits between the CRISPR/Cas9 single and double knockout vectors and the untransformed CRISPR/Cas9 receptor varieties.The oleic acid content of the plants that knocked out the CRISPR/Cas9 double gene vector was significantly higher than that of the single gene vector.

Gene Repair of iPSC Line with GARS (G294R) Mutation of CMT2D Disease by CRISPR/Cas9

Objective Charcot-Marie-Tooth disease(CMT)severely affects patient activity,and may cause disability.However,no clinical treatment is available to reverse the disease course.The combination of CRISPR/Cas9 and iPSCs may have therapeutic potential against nervous diseases,such as CMT.Methods In the present study,the skin fibroblasts of CMT type 2D(CMT2D)patients with the c.880G>A heterozygous nucleotide mutation in the GARS gene were reprogrammed into iPSCs using three plasmids(pCXLE-hSK,pCXLE-hUL and pCXLE-hOCT3/4-shp5-F).Then,CRISPR/Cas9 technology was used to repair the mutated gene sites at the iPSC level.Results An iPSC line derived from the GARS(G294R)family with fibular atrophy was successfully induced,and the mutated gene loci were repaired at the iPSC level using CRISPR/Cas9 technology.These findings lay the foundation for future research on drug screening and cell therapy.Conclusion iPSCs can differentiate into different cell types,and originate from autologous cells.Therefore,they are promising for the development of autologous cell therapies for degenerative diseases.The combination of CRISPR/Cas9 and iPSCs may open a new avenue for the treatment of nervous diseases,such as CMT.

利用CRISPR/Cas-9技术创制水稻温敏核不育系

水稻光温敏核不育系的选育是两系法杂交稻选育的关键环节。温敏不育基因tms5在生产上应用最为广泛,通过CRISPR/Cas9基因组编辑技术突变可育水稻品种的TMS5基因,可以快速选育水稻温敏型两系不育系。利用CRISPR/Cas-9技术创制水稻温敏核不育系的试验结果表明:通过构建CRISPR/Cas-9基因编辑载体TMS502,转化优良中间材料GH89获得10株T0代转基因苗,有6株产生了插入或缺失突变,其中纯合突变株tms5-1和双等位突变株tms5-4在T0代就表现出温敏不育特征;T1代非转基因单株中共有4种纯合突变基因型,突变类型与T0代检测结果一致,并未产生新的突变;T2代tms5不育系材料的温敏不育起点温度约为24℃,符合水稻两系不育系选育要求。研究结果证明了利用基因编辑技术培育水稻两系不育系的有效性,所创制的不育系可供进一步选育利用。

利用CRISPR/Cas 9技术构建大肠杆菌aroA基因的敲除系统及其初步应用

旨在利用CRISPR/Cas 9新型基因编辑技术构建大肠杆菌aroA基因的敲除系统,并分析其对不同大肠杆菌aroA基因敲除、修复的差异。首先构建靶向aroA基因的CRISPR/Cas 9载体;随后人工设计同源修复供体基因序列,并亚克隆到CRISPR/Cas 9载体中,构建成完整的CRISPR/Cas 9基因敲除体系;将该系统分别应用到大肠杆菌DH10B、DH5α和JM109细胞中,PCR鉴定筛选到的阳性菌株,并回收其扩增条带进行克隆、测序。CRISPR/Cas 9载体的酶切与测序结果均正确,表明载体构建成功;PCR鉴定结果显示,该系统对三种大肠杆菌均能进行aroA基因的有效敲除,敲除效率为46%~58%;测序结果进一步证实目的基因敲除成功。本试验成功构建大肠杆菌aroA基因CRISPR/Cas 9敲除系统,为进一步研究致病菌aroA基因功能及开发减毒大肠杆菌疫苗提供新型、有效的基因敲除工具。

Targeting miRNA by CRISPR/Cas in cancer:advantages and challenges

Clustered regulatory interspaced short palindromic repeats(CRISPR)has changed biomedical research and provided entirely new models to analyze every aspect of biomedical sciences during the last decade.In the study of cancer,the CRISPR/CRISPR-associated protein(Cas)system opens new avenues into issues that were once unknown in our knowledge of the non-coding genome,tumor heterogeneity,and precision medicines.CRISPR/Cas-based geneediting technology now allows for the precise and permanent targeting of mutations and provides an opportunity to target small non-coding RNAs such as microRNAs(miRNAs).However,the development of effective and safe cancer gene editing therapy is highly dependent on proper design to be innocuous to normal cells and prevent introducing other abnormalities.This study aims to highlight the cutting-edge approaches in cancer-gene editing therapy based on the CRISPR/Cas technology to target miRNAs in cancer therapy.Furthermore,we highlight the potential challenges in CRISPR/Cas-mediated miRNA gene editing and offer advanced strategies to overcome them.

CRISPR/Cas9技术在卵巢癌诊疗及其转移相关机制研究中的进展

卵巢癌是常见的妇科恶性肿瘤之一,其治疗手段以手术为主,放、化疗作为卵巢癌术后的辅助治疗;晚期卵巢癌尤其容易发生远处转移,所以卵巢癌转移的诊疗也是我们卵巢癌临床治疗的重点。CRISPR/Cas9系统介导的基因组编辑技术系统相比传统的基因编辑技术来说可以更快获得基因敲除突变体,有望通过CRISPR/Cas9来解析卵巢癌的发生及转移机制,从而加速卵巢癌的研究。本文简要的介绍了CRISPR/Cas9技术以及近年来CRISPR/Cas9技术在卵巢癌的治疗及晚期发生、发展转移的应用现状及展望。

CRISPR/Cas 9基因编辑技术降低贵州禾株高研究

为获得株高降低的贵州禾水稻新种质,以贵州禾高秆糯稻材料黎平杂边禾为研究材料,根据CRISPR/Cas 9靶位点设计原则设计水稻OsGA20ox2基因的特异性靶点sgRNA序列,构建水稻OsGA20ox2基因的CRISPR/Cas 9定点突变载体,利用农杆菌介导的愈伤转化法遗传转化黎平杂边禾,通过潮霉素筛选,以及PCR分子检测,获得16株转基因植株。对筛选获得的转基因植株基因进行测序,获得7株OsGA20ox2基因靶位点被编辑的突变转基因植株。研究表明,T 1代突变体植株株高比黎平杂边禾降低了29.6%。

CRISPR/Cas 9介导的基质Gla蛋白基因敲除对破骨细胞分化的影响

目的利用CRISPR/Cas 9基因编辑技术,构建基质Gla蛋白(MGP)基因敲除的RAW264.7巨噬细胞系,明确MGP基因在破骨细胞分化过程中的作用。方法利用在线软件分析并筛选出3个针对MGP基因外显子区的单链向导RNA(gRNA),人工合成gRNA寡核苷酸序列,并将其插入线性化的LentiCRISPRv2质粒中,构建成LentiCRISPRv2 MGP gRNA重组质粒。将重组质粒与psPAX2、VSVG包装质粒一同转染293 T细胞,包装并收集重组慢病毒,将其感染RAW264.7细胞。经嘌呤霉素筛选得到稳定RAW264.7细胞系,实时荧光定量多聚核苷酸链式反应(qPCR)、蛋白质印迹法(Western Blot)验证MGP mRNA及蛋白表达水平。qPCR检测破骨细胞分化标志分子的表达情况。结果成功构建了LentiCRISPRv2 MGP gRNA重组质粒,成功包装了含有MGP gRNA的重组慢病毒,筛选得到了稳定低表达MGP的RAW264.7细胞系。qPCR及蛋白质印迹法检测显示,其MGP mRNA、蛋白表达显著性下调(P<0.05)。qPCR结果显示,最具有代表性的LentiCRISPRv2 MGP gRNA1细胞破骨标志分子Itgb3(2.29±0.17)、Acp5(2.86±0.15)、Ctsk(2.07±0.13)的mRNA水平均显著上调(P<0.05)。结论利用CRISPR/Cas 9基因编辑技术成功构建了MGP基因敲除的RAW264.7细胞系,敲除MGP后RAW264.7细胞破骨分化能力增强。

利用CRISPR/Cas 9基因编辑技术提高贵州禾产量研究

以贵州禾糯稻黎平杂边禾为研究材料,设计水稻产量控制关键基因OsCKX2的CRISPR/Cas 9编辑特异性靶点序列,构建水稻OsCKX2基因的CRISPR/Cas 9定点编辑载体,并利用农杆菌介导法导入黎平杂边禾,通过潮霉素筛选及PCR分子检测,获得20个独立的转基因植株。对筛选获得的转基因T0代植株基因进行测序比对,获得7株OsCKX2基因靶位点被编辑的突变转基因植株。对纯合的突变体T1代植株农艺性状进行分析发现,与野生型黎平杂边禾相比,突变体的穗粒数增加16.67%,单株产量增加25.03%。本研究获得了产量增加的黎平杂边禾基因编辑的新种质。

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.