Application of CRISPR-Cas9 technology in the screening of tumor resistance genes:A review

Drug resistance is a major problem faced by tumor cell-targeted drugs.Currently,functional gene screening is the most common strategy for screening drug resistance genes.In recent years,Crispr-cas9 gene editing technology has been widely used in the functional studies of tumor-related genes due to their characteristics of accuracy,simplicity and efficiency.The principle of CRISPR-Cas9 Library Screening Technology and its application in functional Gene Screening are reviewed.At the same time,the application prospect of the Crispr-Cas9 technology is forecasted.

Generation and characterization of a novel CYP3A1/2 double knockout rat model using CRISPR-Cas9 system

OBJECTIVE Cytochrome P450(CYP)3A accounts for nearly 30%of total CYP enzymes in human liver and participates in the metabolism of over 50%of clinical drugs.CYP3A also plays an important role in the chemical metabolism,toxicity,and carcinogenicity.New animal models are needed to investigate CYP3A functions.METHODS The CRISPR-Cas9 technology was used to generate Cyp3a1/2 double knockout rat model.The absence of Cyp3a1/2 expression was evaluated through PCR and immunostaining.Metabolic studies of the CYP3A substrates midazolam and nifedipine both in vitro and in vivo were conducted to verify that CYP3A1/2 was functional y inactive in KO rats.In addition,compensatory up-regulation of other P450 genes in Cyp3a1/2 KO rats was detected.RESULTS The Cyp3a1/2 double KO rats were viable and fertile,and had no obvious physiological abnormities.Compared with the wild-type(WT)rat,Cyp3a1/2 expression was completely absent in the liver of the KO rat.In vitro and in vivo metabolic studies of the CYP3A1/2 substrates indicated that CYP3A1/2 was functionally inactive in double KO rats.CONCLUSION The Cyp3a1/2 double KO rat model was successfully generated and characterized.The Cyp3a1/2 KO rats as a novel rodent animal model will be a valuable tool for the study of the physiological and pharmacological roles of CYP3A,and determining whether the absence of CYP3A results in non-CYP mediated metabolism or metabolism by other CYP isoforms.

Genome editing of PAR2 through targeted delivery of CRISPR-Cas9 system for alleviating acute lung inflammation via ERK/NLRP3/IL-1β and NO/iNOS signalling

Excessive and uncontrollable inflammatory responses in alveoli can dramatically exacerbate pulmonary disease progressions through vigorous cytokine releases,immune cell infiltration and protease-driven tissue damages.It is an urgent need to explore potential drug strategies for mitigating lung inflammation.Protease-activated receptor 2(PAR2)as a vital molecular target principally participates in various inflammatory diseases via intracellular signal transduction.However,it has been rarely reported about the role of PAR2 in lung inflammation.This study applied CRISPR-Cas9 system encoding Cas9 and sg RNA(p Cas9-PAR2)for PAR2 knockout and fabricated an anionic human serum albuminbased nanoparticles to deliver p Cas9-PAR2 with superior inflammation-targeting efficiency and stability(TAP/p Cas9-PAR2).TAP/p Cas9-PAR2 robustly facilitated p Cas9-PAR2 to enter and transfect inflammatory cells,eliciting precise gene editing of PAR2 in vitro and in vivo.Importantly,PAR2 deficiency by TAP/p Cas9-PAR2 effectively and safely promoted macrophage polarization,suppressed proinflammatory cytokine releases and alleviated acute lung inflammation,uncovering a novel value of PAR2.It also revealed that PAR2-mediated pulmonary inflammation prevented by TAP/p Cas9-PAR2was mainly dependent on ERK-mediated NLRP3/IL-1β and NO/i NOS signalling.Therefore,this work indicated PAR2 as a novel target for lung inflammation and provided a potential nanodrug strategy for PAR2 deficiency in treating inflammatory diseases.

Applications of CRISPR-Cas9 mediated genome engineering

Targeted mutagenesis based on homologous recombination has been a powerful tool for understanding the mechanisms underlying development, normal physiology, and disease. A recent breakthrough in genome engineering technology based on the class of RNA-guided endonucleases, such as clustered regularly interspaced short palindromic repeats(CRISPR)-associated Cas9, is further revolutionizing biology and medical studies. The simplicity of the CRISPR-Cas9 system has enabled its widespread applications in generating germline animal models, somatic genome engineering, and functional genomic screening and in treating genetic and infectious diseases. This technology will likely be used in all fields of biomedicine, ranging from basic research to human gene therapy.

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.

Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems

The class 2 clustered regularly interspaced short palindromic repeat （CRISPR） systems have been widely used for simultaneous modification of multiple loci in plants. Traditionally, the type II CRISPR-Cas9 or type V CRISPR-Cpfl （also known as Cas12a） system is a two-component transcriptional unit （TCTU） in which the Cas9 or Cpf1 protein is expressed from an RNA polymerase （pol） II promoter, whereas the single guide RNA （sgRNA） is typically expressed from a Pol III promoter, such as U6 or U3 promoter.

Progress and challenges in CRISPR-mediated therapeutic genome editing for monogenic diseases

There are an estimated 10000 monogenic diseases affecting tens of millions of individuals worldwide.The application of CRISPR/Cas genome editing tools to treat monogenic diseases is an emerging strategy with the potential to generate personalized treatment approaches for these patients.CRISPR/Cas-based systems are programmable and sequence-specific genome editing tools with the capacity to generate base pair resolution manipulations to DNA or RNA.The complexity of genomic insults resulting in heritable disease requires patientspecific genome editing strategies with consideration of DNA repair pathways,and CRISPR/Cas systems of different types,species,and those with additional enzymatic capacity and/or delivery methods.In this review we aim to discuss broad and multifaceted therapeutic applications of CRISPR/Cas gene editing systems including in harnessing of homology directed repair,non-homologous end joining,microhomology-mediated end joining,and base editing to permanently correct diverse monogenic diseases.

CRISPR／Cas9系统在现代生物学研究和临床试验中的应用

规律成簇间隔短回文重复结构(clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白9(CRISPR-associated protien 9,Cas9)系统在生物医学研究中被广泛应用于基因编辑及探索基因的功能。这种基因编辑技术越来越多地运用在人类疾病的治疗中,包括巴斯综合征、杜氏肌萎缩症、血友病、地中海贫血和囊性纤维化。CRISPR/Cas 9基因编辑系统可以在体外细胞实验和体内动物实验上修复突变的DNA序列,也能改变CCR5、PD-1/L1、CAR-T等基因序列,用于控制HIV病毒的入侵及提高肿瘤免疫治疗的疗效。该技术还被运用于诱导多能干细胞(induced pluripotent stem cells,iPS)分化,形成某些具有功能的器官用于器官移植。本文综述了CRISPR/Cas 9系统的来源、结构和作用原理,以及其在现代生物学研究和基因治疗领域的应用。

Advances in detecting and reducing off-target effects generated by CRISPR-mediated genome editing

CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors.Currently,poor efficiency and off-target problems have impeded the application of CRISPR systems.The on-target efficiency has been improved in several advanced versions of CRISPR systems,whereas the off-target detection still remains a key challenge.Here,we outline the different versions of CRISPR systems and off-target detection strategies,discuss the merits and limitations of off-target detection methods,and provide potential implications for further gene editing research.

CRISPR-Cas9: A method for establishing rat models of drug metabolism and pharmacokinetics

The 2020 Nobel Prize in Chemistry recognized CRISPR-Cas9,a super-selective and precise gene editing tool.CRISPR-Cas9 has an obvious advantage in editing multiple genes in the same cell,and presents great potential in disease treatment and animal model construction.In recent years,CRISPRCas9 has been used to establish a series of rat models of drug metabolism and pharmacokinetics(DMPK),such as Cyp,Abcb1,Oatp1 b2 gene knockout rats.These new rat models are not only widely used in the study of drug metabolism,chemical toxicity,and carcinogenicity,but also promote the study of DMPK related mechanism,and further strengthen the relationship between drug metabolism and pharmacology/toxicology.This review systematically introduces the advantages and disadvantages of CRISPR-Cas9,summarizes the methods of establishing DMPK rat models,discusses the main challenges in this field,and proposes strategies to overcome these problems.

Gene editing in T cell therapy

The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise. Gene editing holds tremendous potential for further improvements of T cell therapy. Here we review the applications of gene editing in various T cell therapies, focusing on antiviral strategies and cancer immunotherapies, and discuss the challenges and future prospects.

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。

罕见病的基因治疗应用与展望

罕见病发病率极低、但大多病情严重且好发于儿童时期,多由遗传变异所引起。因患者数少、市场需求低,罕见病药物的研发成本极高,有药可治的罕见病仅不足1%。随着基因诊断技术进步,基因治疗凭借其“一次性彻底治愈”的特点,为遗传性罕见病患者带来了希望。本文介绍罕见病的基因治疗现状,论述传统基因递送技术和以CRISPR-Cas 9为代表的基因编辑技术在罕见病中的应用及发展。

Tailoring non-viral delivery vehicles for transporting genome-editing tools

The CRISPR-Cas system, especially the type II CRISPR-Cas9 system from Streptococcuspyogenes, has rapidly emerged as a popular genome editing tool. The development of Cas9 derivatives further expanded the toolbox of CRISPR- Cas9 based genome editing kit. However, therapeutic transla- tion of the CRISPR-Cas9 system in vivo is severely impeded by the absence of an appropriate delivery carrier. The complex- ity and high molecular weight of the CRISPR-Cas9 system, together with the physiological barriers for nucleus targeted cargo transportation have made it a huge challenge for in vivo therapeutic CRISPR-Cas9 delivery. Currently, the main stream carriers for systemic delivery of CRISPR-Cas9 are vi- ral based, such as adeno-associated virus. However, the safety concerns surrounding viral vectors call for the development of non-viral nanocarriers. In this review, we survey the recent advances in the development of non-viral delivery systems for CRISPR-Cas9. Challenges and future directions in this field are also discussed.

Precision Breeding Made Real with CRISPR: Illustration through Genetic Resistance to Pathogens

Since its discovery as a bacterial adaptive immune system and its development for genome editing in eukaryotes,the CRISPR technology has revolutionized plant research and precision crop breeding.The CRISPR toolbox holds great promise in the production of crops with genetic disease resistance to increase agriculture resilience and reduce chemical crop protection with a strong impact on the environment and public health.In this review,we provide an extensive overviewon recent breakthroughs in CRISPR technology,including the newly developed prime editing system that allows precision gene editing in plants.We present how each CRISPR tool can be selected for optimal use in accordance with its specific strengths and limitations,and illustrate how the CRISPR toolbox can foster the development of genetically pathogen-resistant crops for sustainable agriculture.