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1、CRISPR:UPDATES ON THE GROWING IMPORTANCE OF THIS TECHNOLOGYCAS INSIGHTSCRISPR(clustered regularly interspaced short palindromic repeats)and CRISPR-associated proteins(Cas)are naturally occurring mechanisms in bacteria and archaea that work as a defense mechanism against viral infection that have sin

2、ce been harnessed to enable precise and efficient genome editing in a variety of organisms.16 This technology has revolutionized the field of genetic engineering and therapeutic development,1,79 offering unprecedented opportunities to advance our understanding of genetic diseases,develop novel thera

3、pies,and potentially cure previously intractable conditions.1 However,it is important to recognize its broader potential in medical diagnosis,synthetic biology,agriculture and food,and environmental bioremediation.For example,CRISPR/Cas has been harnessed for plant genome editing17,18 to develop cro

4、ps that are high-yielding and resistant to abiotic and biotic stresses,and around 13%of CRISPR-related documents are associated with the application of CRISPR in plants and agricultural use-according to our search in the CAS Content CollectionTM.In this report,we examine data from the CAS Content Co

5、llection,the largest human-compiled multi-disciplinary database of published documents and substances,to highlight the latest advances and developments in CRISPR technology,with a focus on its potential in medicine.IntroductionCRISPR INSIGHT REPORT|3From nature to the lab:How does the CRISPR/Cas sys

6、tem work?In bacteria,the CRISPR/Cas system works by capturing snippets of DNA from invading viruses and storing them.When the same virus attacks again,the bacteria can quickly produce segments of a molecule called ribonucleic acid(RNA)to target the viral DNA.This RNA guides Cas proteins to the compl

7、ementary viral DNA,where they can act like molecular scissors,cutting the DNA and neutralizing the threat.3,10This natural mechanism has been adapted for use in gene editing.1,11 The most commonly used system,CRISPR/Cas9,involves a guide RNA(gRNA)that matches the target DNA sequence and the Cas9 enz

8、yme,which cuts the DNA at a specific desired location.This break in the DNA can then be repaired by the cells natural repair mechanisms,allowing for the insertion,deletion,or modification of genes.12,13 Figure 1 summarizes both the role of Cas9 in nature and its use as a genome-editing tool.Since it

9、s adaptation for gene editing,CRISPR/Cas technology has rapidly advanced,and researchers have developed various modifications of the original CRISPR/Cas9 system to improve specificity,efficiency,and versatility.The approval of the first CRISPR-Cas9-based gene editing therapy,Casgevy,approved by the

10、U.S.Food and Drug Administration(FDA)for the treatment of patients with transfusion-dependent-thalassemia,marked a significant milestone in CRISPR technology development.The same therapy has also been approved in Europe and the U.K.with the additional indication of sickle cell disease.1416Figure 1.C

11、RISPR/Cas9 system in nature(left)versus its adaptation for genome editing(right).S,Spacer;R,Repeats.OutcomeTarget DNA cutTarget searchCas9&gRNAproducedtracrRNAcas9RRSTargetPhage genomeDNA destroyedProkaryotic cell(immune system)Eukaryotic cell(genome editing)Custom sgRNAdesigned by scientistDelivery

12、 of genome editing toolsChromatinNucleusDNA editedAmong the key milestones for CRISPR(Figure 2),understanding of CRISPR-mediated mechanisms has significantly improved.Firstly,the natural CRISPR defense mechanism that protects bacteria was defined in three basic stages(Figure 3A):adaptation(spacer ac

13、quisition),CRISPR RNA(crRNA)maturation,and target interference.2830 Another breakthrough was the synthesis of single guide RNA(sgRNA)in the lab which combines the role of crRNA and trans-activating crRNA(tracrRNA)into a single molecule by fusing them together with a linker.26,31 The mechanism of CRI

14、SPR/Cas-9 genome editing contains three steps:recognition,cleavage,and repair,as depicted in Figure 3B.32 Figure 2.Timeline of significant events for CRISPR technology.The CRISPR timeline1987 Presence of CRISPR first reported in the Escherichia coli genome by Nakata et al.19Mid 2000s Functionality a

15、nd importance of CRISPR first described in prokaryotes wherein the CRISPR system is a key component of their adaptive immunity.23252020 Doudna and Charpentier awarded the Nobel Prize in Chemistry.272002 Cas discovered by Jansen et al.20Further identification in both Gram-positive and Gram-negative b

16、acteria,as well as archaea,led to the obvious questions regarding the relevance of CRISPR to these organisms.21,222012 Jennifer Doudna and EmmanuelleCharpentier propose that the bacterial CRISPR-Cas9 system could be used as a programmable toolkit for genome editing in humans and other animal species

17、.262023 First CRISPR-Cas9-based gene editing therapy,Casgevy,approved by the FDA for the treatment of patients with transfusion-dependent-thalassemia.14-16 CRISPR INSIGHT REPORT|5Figure 3.Mechanisms of CRISPR/Cas9.(A)CRISPR/Cas9 adaptive immunity.The CRISPR/Cas systems are composed of a Cas operon a

18、nd a CRISPR array,which comprises identical repeat sequences that are interspersed by phase-derived spacers.The CRISPR/Cas-mediated adaptive immunity consists of three stages:adaptation,maturation,and interference.(B)CRISPR/Cas9-mediated genome engineering.The synthetic sgRNA guides Cas9 in introduc

19、ing a double-strand break in the target DNA.The double-strand break is repaired by DNA repair mechanisms.Figures were created with www.BioR and adapted from Jiang and Doudna(Jiang F,Doudna,2017.Annu.Rev.Biophys.46:50529).crRNA,CRISPR RNA;PAM,protospacer adjacent motif;sgRNA,single guide RNA;tracrRNA

20、,trans-activating crRNA.sgRNACas9sgRNA(crRNA+tracrRNA)Double stranded break(DSB)Endogenous DNA repairs at theDSB siteHomology directed repairKnocked-in DNAsequence of interest(Precise gene modification)InsertionDonor DNADeletionSubstitution(Random mutations causedisruption of gene function)Nonhomolo

21、gous end joiningPAM5335353355Spacer acquisitioncrRNA biogenesisTarget interferenceLocusCas operonTranscriptionSpacersRepeatsPre-crRNAcrRNASite-specific cleavageCas9Cas9PAMcrRNAtracrRNAComplementary foreign DNAcrRNA maturation(RNase III processing)IntegrationInvasive viral DNAProtospacerCas1-Cas2CRIS

22、PR arrayPAMtracrRNAtracrRNACas9Cas1Cas2Csn2ABCRISPR/Cas systems have been divided largely into two classes,six types and 48 subtypes(Figure 4).33 Class 1 systems include Types I,III,and IV,which use multiprotein complexes to destroy nucleic acids,while class 2 systems include Types II,V,and VI,which

23、 use single proteins.34Below is a brief overview of natural Cas nucleases and engineered variants that have been adopted for use as therapeutics.Class 1 CRISPR/Cas systems Class 1 systems require multiple Cas proteins to come together in a complex to mediate interference against foreign genetic elem

24、ents,limiting gene editing applications of class 1 systems.Class 1 systems are further divided into three CRISPR/Cas types based on the presence of a specific signature protein:Type I contains Cas3,Type III contains Cas10,and Type IV contains Csf1,a Cas8-like protein.35Class 2 CRISPR/Cas systems Unl

25、ike the Class 1 CRISPR/Cas system,the effector module of the Class 2 CRISPR/Cas system is only a single protein with multiple domains and functions.36 Class 2 CRISPR/Cas systems are further divided into three types:Type II,Type V,and Type VI.CRISPR classificationType IIType IIIType VsgRNAHNHTarget s

26、equenceREC lobeCas9Linker loopsgRNA(crRNA+tracrRNA)RuvCdsDNAPAM5335NUC lobe35Cas12asgRNATarget sequenceREC lobeLinker loopcrRNARuvCdsDNAPAM533355NUC lobessDNACollateral activity3ssDNACas12f/Cas14Target sequenceREC lobeLinker loopcrRNANUC lobessDNACollateral activitysgRNA553Type VIRNA3Cas13Target seq

27、uenceREC lobeLinker loopcrRNAPFSHEPNNUC lobessDNACollateral activitysgRNA553Class IIClass ICsf ComplexType IVcrRNAPAM533355Nuclease?CasDinGdsDNACas3Type IcrRNALinker loopPAMHDsgRNA533355dsDNAssDNACollateral activityTarget sequenceCascade ComplexCas10-Csm/Cmr ComplexcrRNACoding strandTemplate strandc

28、rRNACas10RNAPTranscriptCsm3553DNA55Figure 4.Schematic depiction of various CRISPR/Cas system types and their cleavage characteristics.The CRISPR/Cas system is classified into two classes based on the effector molecules involved.Class I systems are characterized by multi-unit effectors,while Class II

29、 systems involve a single effector.Each class is further divided into three types,distinguished by their catalytic domain and target nucleotide specificity.The illustration was created using www.BioR.CRISPR INSIGHT REPORT|7Therapeutic possibilities with CRISPRThe concept of gene therapy was introduc

30、ed by Friedmann and Roblin in 1972.37 Zinc finger nucleases(ZFN)and transcription activator-like effector nucleases(TALENs)were developed as mainstream tools to evaluate the possibility of targeting or editing genes to treat and cure diseases.However,both methods have unfavorable characteristics,mak

31、ing the development of effective gene therapy challenging.3841Since then,CRISPR/Cas has been defined and explored as a potential therapeutic approach for disorders previously believed to be incurable or difficult to treat.These include certain types of cancer,infectious diseases,and various genetic

32、disorders,among others.CRISPR/Cas technology has various key applications in the field of therapeutics,the most prominent of which is the ability to correct or replace mutated or disease-causing gene(s).42 As of today,numerous CRISPR-based therapeutics are in the pre-clinical stage of development,an

33、d many are undergoing clinical trials to validate their safety and efficacy for diverse disease conditions,with one CRISPR therapeutic being U.S.FDA approved.CRISPR therapeutics publication landscape at a glanceA quantitative analysis of data from the CAS Content Collection(19952024)revealed 39,000

34、academic journal articles and over 14,000 patents on the application of CRISPR to therapeutics.Publications sharply rose in 2008 and have steadily increased ever since,with an average growth rate of 54%in the last decade.This total rise in publications is primarily led by academic journal articles;h

35、owever,patents have shown a larger average yearly growth rate of 72%in the last decade,compared to 50%for journals,demonstrating an increase in commercial interest.Key commercial assignees of patents in the field of CRISPR therapeutics include Regeneron Pharmaceuticals in the U.S.,CRISPR Therapeutic

36、s in Switzerland,and Shandong Shunfeng Biotechnology in China,while non-commercial activity is led by the Chinese Academy of Sciences,a group of 124 individual research institutions(Figure 5).43CRISPR INSIGHT REPORT|7Figure 5.Leading commercial(A)and non-commercial(B)patent assignees in the field of

37、 CRISPR between 20042023.BANumber of patent publicationsChinese Academy of SciencesThe Broad InstituteUniversity of CaliforniaHarvard CollegeChinese Academy of Agricultural SciencesHuazhong Agricultural UniversityStanford UniversityZhejiang UniversityJiangnan UniversityChina Agricultural UniversityM

38、assachusetts Institute of TechnologyAcademy of Military Medical Science Peoples Liberation Army of ChinaZhejiang UniversityYangzhou UniversityThe General Hospital Corporation020040015035010030050250450ChinaUnited StatesNumber of patent publicationsRegeneron Pharmaceuticals Inc.CRISPR Therapeutics AG

39、Shandong Shnufeng Biotechnology Co.,Ltd.Editas Medicine Inc.Emendo Bio Inc.Beam Therapeutics Inc.Shenzhen Biocan Technologies Co.,Ltd.Mammoth Biosciences Inc.Flagship Pioneering Inc.Arbor Biotechnologies Inc.Intellia Therapeutics Inc.Childrens Medical Center CorporationChina Tobacco Yunnan Industria

40、l Co.,Ltd.Metagenomi Technologies LLCNanjing Superyears Gene Technology Co.,Ltd0408030702060105090ChinaSwitzerlandUnited StatesHot topics in the CRISPR publication landscape:therapeutics targeting diseasesAmong the various diseases or disorders currently being investigated,the majority of documents

41、in the CRISPR therapeutics dataset focus on cancer and infectious diseases(Figure 6).The publication trend also shows a significant increase in the number of CRISPR articles focused on these two conditions.Other conditions investigated include blood disorders,genetic disorders,nervous system disorde

42、rs,cardiovascular diseases,respiratory diseases,immune diseases,and metabolic disorders.35%24%22%14%11%9%8%25%10%9%7%4%3%3%3%1%1%4%4%2%Infectious diseasesBlood disordersGenetic disordersNervous system disordersCardiovascular diseasesRespiratory diseasesImmune diseasesMetabolic disordersDigestive sys

43、tem disordersCancerJournal(outer donut),Patent(inner pie),ACRISPR INSIGHT REPORT|9CancerImmuneBloodMetabolicInfectiousRespiratoryGeneticCardiovascularNervous systemDigestive systemFigure 6.(A)Distribution and(B)time trends for CRISPR documents co-occurring with various disease conditions.Data includ

44、es journal and patent publications from the CAS Content Collection.B1,4001,2001,000800600200400Number of journal publicationsPublication year020112013201520172019202120234504003503002502001501005002011201520192013201720212023500400300200100Number of patent publicationsPublication year020112013201520

45、172019202120232502001001505002011201520192013201720212023Figure 7A.Publication frequencies of potential gene targets occurring in the CRISPR dataset retrieved from the CAS Content Collection.Data includes patent and journal publications for the period 19952024 and is based on CAS indexing.Note that

46、data for 2024 is incomplete due to the time of data extraction and encompasses data for January to June.Figure 7B.Time trends of some of the most highly occurring potential gene targets in the CRISPR dataset retrieved from the CAS Content Collection.Data includes patent and journal publications for

47、the period 2014-2023 and is based on CAS indexing.Regarding target genes,TP53,c-myc,the hemoglobin beta subunit(HBB)and c-Ki-Ras are most commonly associated with CRISPR publications.In particular,TP53 has seen a rapid increase over the last few years(Figure 7).GenesYear50040030020010009075604530150

48、Number of publicationsNumber of publicationsTP53c-mycHBBc-Ki-RasBRCA1DMDBcl-2ERBB1ERBB2CDKN2ACCL2CFTRAPCTGFB1TRPAX6ICAM1N-rasHPRTHLA-ACXCL1RB1NF1HLA-BIL-6COL1A1 SOD1CXCL10LDLRRAG1APOEHLA-CIL1BFasMDM22014201520172016201920182021202020232022TP53c-mycBRCA1ERBB1c-Ki-RasHBBCCL2ERBB2Bcl-2DMDCDKN2ACRISPR I

49、NSIGHT REPORT|11Figure 8.Co-occurrence of cancer subtypes with genes in the CRISPR dataset retrieved from the CAS Content Collection.Data includes patent and journal publications for the period 19952024.CancerFundamentally,cancer originates from activating and inactivating mutations or from the dysr

50、egulation of the epigenome.At the cellular level,altered metabolism,cell structure,and migration facilitate the expansion of cancer cells.Eventually,cancer cells circumvent the immune defense mechanism of the host and co-exist with normal cells.Understanding the complex genomic,cellular,and tissue l

51、evel changes is crucial for the developing more effective treatment options and improving outcomes.CRISPR has had a significant impact on our understanding of cancer biology and is continuously driving new discoveries in the field.44Multiple gene candidates are being studied in cancer in the context

52、 of CRISPR.Figure 8 below shows the co-occurrence of cancer types and genes found in the CRISPR dataset retrieved from the CAS Content Collection,and Table 1 shows the variety of applications of CRISPR to cancer therapy.Table 1.Approaches to using CRISPR/Cas technology in cancer.Therapeutic Correcti

53、ng driver mutations in oncogenes or tumor suppressor genes4554 Modifying or silencing epigenetic markers5559 Supporting immunotherapy6064 Targeting mutations that determine drug response or susceptibility6571 Inactivating carcinogenic viral infections7276Disease pathology Creating tumor models and o

54、rganoids195197 Creating high-throughput genetic screens198200Breast cancerAcute myeloid leukemiaLiver cancerLung cancerRectal cancerMelanomaGlioblastomaKidney cancerProstate cancerTP53:120BRCA1:32ERCC1:18c-Ki-Ras:50c-myc:36ERCC2:16APC:26ERBB2:15ERBB1:33XRCC1:13ERCC3:12N-ras:12Bcl-2:12NF1:10c-Ki-Ras2

55、:9CDKN2A:8ERCC6:8RB1:6MGMT:5HBB:4VHL:4ICAM1:4LPL:4CCL2:4JUN:4CXCL1:4fos:4Cancer subtypeGeneInfectious diseases Infectious diseases represent the second largest subset of CRISPR-related documents in the CAS Content Collection.Of the journal and patent publications mentioning human diseases,infectious

56、 diseases account for 25%and 22%,respectively.Over the past few years,there has been a significant increase in the number of documents on infectious diseases and CRISPR/Cas technology.The majority of these documents focus on anti-viral studies.CRISPR/Cas technology has emerged as a promising alterna

57、tive to develop therapeutics against various pathogens via the following methods:7779 Targeting the pathogen genes required for replication,or entering or infecting the host cells Altering the host genes required by the pathogen to cause infection Modifying the genes responsible for drug resistance

58、or susceptibilityBlood disorders CRISPR/Cas technology possesses an interesting possibility of providing a cure for patients with inherited monogenic blood diseases like sickle cell anemia and-thalassemia.As previously highlighted,Casgevy was the first U.S.FDA-approved CRISPR therapeutic and is an a

59、utologous gene therapy that targets the BCL11A gene,preventing red blood cells from adopting the sickle shape.80 Other gene-targeting therapies for the treatment of sickle cell anemia and-thalassemia are currently in clinical trials.81 Genetic disorders Among the many promising possibilities of usin

60、g CRISPR-based therapeutics,their translational use in monogenic human genetic diseases has the potential to provide long-term therapy with a single treatment.Genetic disorders can be treated with the help of CRISPR by editing the defective(disease-causing)gene or by editing the enhancer or regulato

61、r of the defective gene.Numerous studies have shown promising results by using these two approaches.Genetic disorders for which CRISPR-based therapeutics have been investigated include Duchenne muscular dystrophy(Dystrophin DMD gene),Huntingtons disease(Huntingtin HTT gene),and glaucoma(MYOC).8284Ne

62、rvous system disorders The publication rate of CRISPR-related documents on Alzheimers and Parkinsons diseases has increased in recent years.CRISPR/Cas9 technology has gained popularity in the field of neurodegenerative diseases due to its short experimental duration and easy molecular engineering re

63、quirements.It is currently being extensively utilized for building disease models,identifying pathogenic genes through screening,and for targeted therapy.8591 CRISPR in practiceClinical developmentOver the last decade,CRISPR has advanced significantly in clinical research,with numerous trials launch

64、ed to explore its potential in therapeutics,and the first CRISPR-based medicine was approved in just 11 years.1416Figure 9A shows the steady increase in the number of CRISPR-based therapeutics in development over the past decade.There are currently 142 CRISPR therapeutics in different stages of deve

65、lopment.Of these,10%are in Phase I,11%in Phase II,and 1%in Phase III clinical trials.The majority(77%)are in the pre-clinical stage(Figure 9B).CRISPR INSIGHT REPORT|13CRISPR pre-clinical research and clinical trials are targeting a wide range of diseases,from rare genetic disorders and blood disease

66、s to various forms of cancer and infectious diseases such as human immunodeficiency virus(HIV),tuberculosis,and COVID-19(Figure 10).Figure 9.(A)Year-wise distribution of CRISPR-based therapeutics in pre-clinical and clinical trials.(B)Current status of CRISPR-based therapeutics in various stages of

67、development.Data taken from Pharmaproject Citeline Clinical Intelligence.Note that data for 2024 is incomplete due to time of data extraction and encompasses data for January to June.Figure 10.Distribution of CRISPR-based therapeutics under development among different disease groups.The stacked bar

68、shows the split of therapeutics among various stages of development for each disease group.The percentage distribution among disease groups has been mentioned in parentheses.Data from Pharmaproject Citeline Clinical Intelligence.Note that data for 2024 is incomplete due to time of data extraction an

69、d encompasses data for January to June.ABNumber of CRISPR-based therapeutics in development16014012010080604020020152016201720182019Year2020 2021 2022 2023 2024Pre-clinical110Phase l14Phase II15Phase III 2Registered 1Disease groupNumber of CRISPR-based therapeuticsAnticancerBlood and clottingSensory

70、MusculoskeletalAlimentary/metabolicNeurologicalCardiovascularAnti-infectiveImmunologicalRespiratoryDermatological010203040Pre-clinicalPhase IPhase IIPhase III Registered (12%)(9%)(7%)(12%)(9%)(4%)(10%)(8%)(3%)(1%)(25%)The distribution of CRISPR-based therapeutics in pre-clinical development and clin

71、ical trials is shown in Figures 11A and 11B,respectively.Most pre-clinical research and clinical trials are focused on using CRISPR-related therapies as anticancer treatments,with solid cancers being the primary target.Figure 11A.Distribution of CRISPR-based therapeutics in the pre-clinical stage of

72、 development across disease groups,individual diseases,and their biological targets.Data retrieved from Pharmaproject Citeline Clinical Intelligence in June 2024.NOS,not specified;NA,not available.Disease groupDisease nameTarget nameNOS:55NA:16Dystrophin:3DDR1:1PTK7:1SLC39A6:1DNA pol:1CD70:1NFE2L2:1

73、GPC3:1CD83:1PRPF31:1OTOF:1TGFBI:1VEGF-A:1DUX4L1:1LAMA1:1HAO1:1GAA:1C9orf72-SMCR8:1STMN2:1SOD1:1UBE3A:1TRR:1PMP22:1SCN9A:1Factor VIII:1Factor IX:1ALAS:1PCSK9:1AGT:1CFTR:1SERPINA1:1PRE-CLINICALAnticancer:22Sensory:14Solid cancer:12NOS cancer:12HM:2NSCLC:1NCC:1BCL:1BC:1ALM:1ODs:1RP:1CRD:1CD:1AMD:1DMD:7

74、CMD:1DM:2HD:3UC:1PH:1IBD:3AS:1CMT:1HA:2HeFH:2DCM:1HTN:1HSV:1NOS BI:1CF:1RTI:1NOS ROs:1CIPN:1SCD:1HCM:1FH:1HIV:1HBV:1JCV:1AAT:1hATTR:1Neutropenia:1Thalassaemia:1AD:1RIGI:1T1D:2ALS:5HB:3EB:1ATTRv amyloidosis:2Pompes disease:1Porphyria:1NOS HLD:2CDs,MDD,GAD:1Staph bacteria:1FSHD:2MD:1Retinoschisis:1NOS

75、 MD:1Hearing loss:1Alimentary/metabolic:12Neurological:11Blood and clotting:9Cardiovascular:8Anti-infective:7Unspecified:6Respiratory:4Dermatological:1Musculoskeletal:12CRISPR INSIGHT REPORT|15Figure 11B.Distribution of CRISPR-based therapeutics in the clinical stages(Phase I,II and III)of developme

76、nt across disease groups,individual diseases,and their biological targets.Data retrieved from Pharmaproject Citeline Clinical Intelligence in June 2024.NOS,not specified;NA,not available.Disease groupClinical trial phaseDisease nameTarget namePhase I:14VEGF-A:2TNFRSF17:2IL3RA:1PIk4:1USP1:2CD19:3PD-1

77、:1SOCS1:1ANGPTL3:1HBB:1NA:8CD70:1Lp(a):1NOS:3HBG1:1KLKB1 gene:1TTR:1Phase II:15Phase III:2Sensory:2Retinopathy:1BC,OC,PC,BPC cancer:1Solid cancer:2SCC,NSCLC:1HeFH/HoFH:1GI,NSCLC:1Thalassaemia:3AMD:1AML:1NHL:2SCD:4FH:1UTI:1HIV/AIDS:1T1D:1HAE:1hATTR:1BCL/TCL:1CVD NOS:1ALL:1BCL:1MM:2Anticancer:13Cardio

78、vascular:3E.coli prophylaxis:1Blood and clotting:7Anti-infective:3Alimentary/Metabolic:1Immunological:1Neurological:1Disease names:AML:Acute myeloid leukemia,NHL:non-Hodgkins lymphoma,MM:multiple myeloma,NOS:unspecified,SCD:sickle cell disease,FH:familial hypercholesterolemia,HeFH/HoFH:heterozygous/

79、homozygous familial hypercholesterolaemia,CVD:cardiovascular disease,AMD:wet age-related macular degeneration,BC:breast cancer,OC:ovarian cancer,PC:pancreatic cancer,BPH:benign prostatic hyperplasia,GI:gastrointestinal cancer,NSCLC:non-small cell lung cancer,SCC:squamous cell carcinoma,ALL:acute lym

80、phocytic leukemia,BCL:B-cell lymphoma,TCL:T-cell lymphomas,HAE:hereditary angioedema,HIV/AIDS:human immunodeficiency virus/acquired immunodeficiency syndrome,T1D:type 1 diabetes,UTI:urinary tract Infection,hATTR:hereditary transthyretin amyloidosis,HD:hereditary disease of metabolism,UC:ulcerative c

81、olitis,HD:Hepatic dysfunction,PH:primary hyperoxaluria,IBD:Inflammatory bowel disease,RIGI:Radiation-induced gastric injury,HM:Hematological Malignancies,HCC:hepatocellular carcinoma,NOS BI:unspecified bacterial Infection,HBV:hepatitis-B virus,HSV:herpes simplex virus,JCV:John Cunningham virus,RTI:r

82、espiratory tract Infection,Staph:staphylococcal,HA:haemophilia A,HB:haemophilia B,DCM:dilated cardiomyopathy,HCM:hypertrophic cardiomyopathy,HLD:Hyperlipidaemia,HTN:hypertension,EB:epidermolysis bullosa,NOS AD:unspecified autoimmune disease,CMD:congenital muscular dystrophy,DMD:Duchennes muscular Dy

83、strophy,FSHD:facioscapulohumeral muscular dystrophy,NOS MD:unspecified muscular dystrophy,MD:myotonic muscular dystrophy,AD:Alzheimers disease,ALS:amyotrophic lateral sclerosis,AS:angelman syndrome,CDs:Cognitive disorders,MDD:major depressive disorder,GAD:generalized anxiety disorder,CMT:CharcotMari

84、etooth disease,CIPN:chemotherapy-induced peripheral neuropathy,CF:cystic fibrosis,AAT:alpha-1 antitrypsin deficiency,NOS RDs:unspecified respiratory diseases,ODs:ocular disorders,CRD:cone-rod dystrophy,MD:macular dystrophy,RP:retinitis pigmentosa,CD:corneal dystrophy.Target names:IL3RA:Interleukin 3

85、 receptor alpha,CD19:CD19 molecule,TNFRSF17:TNF receptor superfamily member 17,Plk4:polo-like kinase 4,USP1:ubiquitination-specific proteases,NA:not applicable,NOS:unspecified,ANGPTL3:Angiopoietin-like protein 3,VEGF-A:vascular endothelial growth factor A,Lp(a):lipoprotein(a),SOCS1:suppressor of cyt

86、okine signaling 1,CD19:CD19 molecule,PD-1:programmed cell death 1,CD70:CD70 molecule,KLKB1:kallikrein B1,HBB:hemoglobin subunit beta,HBG1:hemoglobin subunit gamma 1,TTR:transthyretin,HAO1:hydroxyacid oxidase 1,GAA:glucosidase alpha,GPC3:glypican 3,NFE2L2:nuclear factor,erythroid 2 like 2,DDR1:discoi

87、din domain receptor tyrosine kinase 1,PTK7:protein tyrosine kinase 7(inactive),SLC39A6:solute carrier family 39 member 6,DNA pol:DNA polymerase theta,Factor VIII:coagulation factor VIII,Factor IX:coagulation factor IX,ALAS:5-aminolevulinate synthase 1,PCSK9:proprotein convertase subtilisin/kexin typ

88、e 9,AGT:angiotensinogen,LAMA1:laminin subunit alpha 1,DUX4L1:double homeobox 4 like 1(pseudogene),C9orf72-SMCR8:C9orf72-SMCR8 complex subunit,STMN2:stathmin 2,SOD1:superoxide dismutase 1,UBE3A:ubiquitin protein ligase E3A,PMP22:peripheral myelin protein 22,SCN9A:sodium voltage-gated channel alpha su

89、bunit 9,CFTR:CF transmembrane conductance regulator,SERPINA1:serpin family A member 1,PRPF31:pre-mRNA processing factor 31,OTOF:otoferlin,TGFBI:transforming growth factor beta induced.Delivery systems:The portal for therapeutic useDespite the potential of CRISPR,its therapeutic application faces som

90、e challenges,particularly in the area of cellular delivery systems.Effective and safe delivery of CRISPR components such as the Cas9 nuclease and gRNA to target cells and tissues is paramount for achieving the desired therapeutic outcomes while minimizing off-target effects and immune responses.9294

91、The choice of delivery method can significantly influence the efficiency,specificity,and safety of CRISPR-mediated gene editing.Carriers currently used for delivery fall into three general groups:viral vectors,non-viral vectors,and physical delivery methods(Figure 12).9599 Viral vectors dominate the

92、 publication landscape,with adeno-associated vectors(AAVs)being the most commonly represented.Electroporation and microinjection are the most widely used physical delivery methods.ADelivery systemsCRISPR/Cas9 systemsGene editingViral vectorsNon-viral vectorsPhysicaldeliveryAdeno-associatedvirusMicro

93、injectionLipid nanoparticleAdenovirusElectroporationPolymer nanoparticleLentivirusPlasmid DNASonoproationHydrodynamicinjectionCas9 RNPGold nanoparticleCell penetration peptideCas9 mRNA+sgRNACRISPR INSIGHT REPORT|17Table 2.Attributes of molecular diagnostic methods NGS,PCR,and CRISPR/Cas.138148cDNA,c

94、omplimentary deoxyribonucleic acid;CRISPR/Cas,clustered regularly interspaced short palindromic repeats/CRISPR associated proteins;DNA,deoxyribonucleic acid;PCR,polymerase chain reaction;RNA,ribonucleic acid.The utility of CRISPR/Cas technology is not limited to disease modification and has rapidly

95、evolved into a powerful tool for disease diagnosis.138140 Its ability to detect specific genetic sequences is invaluable for identifying infectious diseases,genetic disorders,and even cancers.Quantitative polymerase chain reaction(qPCR),isothermal amplification,and next-generation sequencing(NGS)are

96、 currently used in routine clinical diagnostics,141144 but all have pitfalls.144146 Therefore,the CRISPR/Cas system can open a new window of possibilities for genetic diagnostics that integrates the ease of use and cost efficiency of isothermal amplification with the diagnostic accuracy of qPCR.More

97、over,the CRISPR/Cas system can fulfill the ASSURED criteria(affordable,sensitive,specific,user-friendly,rapid,equipment-free,delivered)set by the World Health Organization147 for infectious disease diagnostics.Table 2 compares the three molecular detection methods.CRISPR diagnosticsMethodTime requir

98、ed Response proceduresAdvantagesDisadvantagesNGS20 hoursLibrary preparation,NGS sequencing,bioinformatics analysisComprehensive analysis of all nucleic acids,rapid and preliminary identification of new pathogensExpensive equipment,complex operation,and not all genomes are availableqPCR1.5 hoursRever

99、se transcription,RNA-cDNA hybridization denaturation,PCR amplification Gold standard,currently the most common detection methodRequires complex laboratory infrastructure and specialized technical personnelCRISPR/Cas0.6 hoursDNA amplification,Cas reactionLow cost,high sensitivity,no need for complex

100、instruments and equipment,fast and convenient for field testingNot widely used in clinical trials,pending clinical validationFigure 12.(A)Schematic representation of CRISPR/Cas9 delivery systems.(B)Distribution of documents related to CRISPR delivery systems.Data includes journal and patent publicat

101、ions from the CAS Content Collection from 19952024.100137Physical delivery 36%Viral vectors 42%Non-viral vectors 22%Adeno-associated 22%Hydrodynamic injection 2%Sonoporation 2%Electroporation 17%Microinjection 15%CPP 2%Gold NP 5%Polymer NP 4%Adenovirus 6%Lentivirus 14%Lipid NP 11%BData from the CAS

102、Content Collection shows more than 6,500 journal articles and nearly 3,000 patent publications on the application of CRISPR technology in disease diagnosis from 2004 to 2023 which is 17%and 21%of total journal articles and patent publications,respectively,related to CRISPR therapeutics(Figure 13).Th

103、e publication trend of CRISPR in disease diagnosis has shown a significant increase in recent years,reflecting its growing importance as a diagnostic tool in molecular biology and medical research.Since the discovery of CRISPRs potential in 2012,there has been an exponential rise in journal and pate

104、nt publications,especially after 2015.A notable increase in publications(44%)between 2020 and 2022 is observable and coincides with the COVID-19 pandemic.Patent publications on CRISPR-based disease diagnostics have also surged in recent years,paralleling the technologys rapid adoption in both resear

105、ch and clinical applications.Figure 13.Journal and patent publications trends on CRISPR-based disease diagnostics from the CAS Content Collection from 2004 to 2023.1,6001,4001,2001,0008006004002000YearNumber of publications20042005200620072008200920102011201220132014201520162017201820192020202120222

106、023JournalsPatentsJournals6,668Patents2,933CRISPR INSIGHT REPORT|19Figure 14.Time trends of publications related to artificial intelligence(AI)in the CRISPR dataset.Data includes journal and patent publications from the CAS Content Collection for the period 20102024.Note that data for 2024 is incomp

107、lete due to the time of data extraction and encompasses data for January to June.With the rise of artificial intelligence(AI),interest in its application to CRISPR/Cas technology has increased,as reflected by the growth of publications over the last decade.The incorporation of AI into CRISPR-mediate

108、d gene editing promises to improve both the precision and efficiency of the process.AI also possesses the ability to decipher complex genetic information,facilitate disease diagnosis,and inform on prognosis,going hand-in-hand with CRISPR capabilities.149AI may be used to design novel Cas proteins,ai

109、d in high-throughput screening of sgRNAs,and predict on-and off-target efficacy of particular CRISPR/Cas models.150170 CRISPR and artificial intelligenceTo read more about the application of AI to CRISPR/Cas technology,refer to the following review articles:Dixit,S,et al.Advancing genome editing wit

110、h artificial intelligence:opportunities,challenges,and future directions.Front Bioeng Biotechnol 2023,11,1335901.DOI:10.3389/fbioe.2023.1335901.Lee,M.Deep learning in CRISPR-Cas systems:a review of recent studies.Front Bioeng Biotechnol 2023,11,1226182.DOI:10.3389/fbioe.2023.1226182.6005004003002001

111、000YearNumber of publications201020112012201320142015201620172018201920202021202220232024JournalsPatentsIn the past decade,capital investment in CRISPR/Cas technology has seen a remarkable increase,with a sharp increase starting in 2018 and persisting until 2021,with investments exceeding a staggeri

112、ng$11 billion USD in 2021(Figure 15A).An overwhelming majority of these investments involved companies originating in the U.S.(96%).Other key players in terms of geographical distribution,though of much smaller magnitude,included Switzerland,China,and Japan(Figure 15B).Biotechnology,pharmaceuticals,

113、and drug discovery together account for almost 90%of the capital invested(Figure 15C).Other industries with smaller contributions include agricultural chemicals,diagnostic equipment,and laboratory services,among others(Figure 15C).The largest influx of capital in the biotechnology industry occurred

114、with two major spikes in 2019 and 2021.A record number of deals were made in 2021,with some of the largest involving Century Therapeutics and Mammoth Biosciences($150 million),and Caribou Biosciences and AgBiome($100 million).The most consistent influx in capital appears to be in drug discovery unti

115、l 2021,with a plunge in 2022 and an uptick after 2022 (Figure 15D).Commercial interest in CRISPR technologyABCCapital invested(USD)Number of dealsYear14B12B10B8B6B4B2B080706050403020100201320142015201620172019202220232024201820202021China(1%)Switzerland(2%)Japan(0.4%)Others(1%)U.S.(96%)Laboratory se

116、rvices(0.4%)Diagnostic equipment(1%)Drug delivery(0.1%)Biotechnology(52%)Decision/risk analysis(1%)Agricultural chemicals(1%)Discovery tools(healthcare)(2%)Specialty chemicals(7%)Drug discovery(9%)Pharmaceuticals(27%)Primary industry codesCRISPR INSIGHT REPORT|21Figure 15.Commercial interest in CRIS

117、PR/Cas technology.(A)Capital invested in and number of deals in CRISPR.(B)Geographical distribution of capital invested in CRISPR.(C)Breakdown of capital invested as per primary industry codes and(D)their time trends over the last decade(20132024).Data from PitchBook Data,Inc.Data has not been revie

118、wed by PitchBook analysts.DBiotechnologyDrug discoveryAgricultural chemicalsDrug delivery12B10B8B6B2B4BCapital invested(USD)Year0201320142015201620172018201920202021202220232024900M750M600M450M300M150M0201320172020 20212014 2015 20162018 2019202220242023Diagnostic equipmentDespite the widespread acc

119、eptance of CRISPR/Cas technology in gene editing,owing to its versatility and ease of use,several challenges remain.These include the ethical implications of genetic tampering,171175 potential for off-target effects,176183 packaging and delivery,184186 DNA damage and toxicity,187189 and regulatory h

120、urdles.EthicsGenetic manipulation of eukaryotic organisms raises several ethical dilemmas.Jennifer Doudna,one of the pioneers behind CRISPR/Cas technology,expressed her concern at the 2016 American Association for the Advancement of Science Annual Meeting,stating that one of her biggest fears is“wak

121、ing up one morning and reading about the first CRISPR baby,”which would undoubtedly create public backlash and regulatory uproar.171 In 2018,her fears were realized when Chinese researcher He Jiankui claimed that he used CRISPR to alter the DNA of seven embryos of couples,where the males were HIV ca

122、rriers,to immunize the babies against HIV.This resulted in the birth of two twin girls,the first CRISPR babies.172,173In 1979,Beauchamp and Childress proposed the four principles of biomedical ethics:beneficence,nonmaleficence,respect for autonomy,and justice.174 In summary,the proposed“treatment”sh

123、ould result in a positive outcome/benefit(beneficence),avoid or minimize harm as much as possible(nonmaleficence),require informed consent from patients(autonomy),and ensure equitable access to treatment(justice).When looking at applications and study of CRISPR/Cas genome editing,researchers should

124、take these principles into consideration.175Other ethical concerns are legal regulations,the use of the technology at home by communities without medical supervision(biohackers),175 and the use of CRISPR/Cas for non-therapeutic purposes like enhancements and eugenics.Off-target effectsIn the natural

125、 setting,CRISPR/Cas systems have evolved to tolerate mismatches between the gRNA and the target DNA to a certain extent.However,this property is not applied to genome engineering applications in the laboratory,as it may result in the alteration of off-target sites.Numerous studies have reported off-

126、target activity at sites,ranging from a single base mismatch,to sites containing multiple consecutive mismatches or even nucleotide insertions or deletions.176179 High-throughput profiling studies exploring off-target effects have shown that their frequency is consistently lower in vivo as compared

127、with isolated genomic DNA.182183Packaging and delivery In vivo delivery of CRISPR/Cas9 into mammalian cells is generally accomplished using viral vectors.AAVs remain the preferred choice due to their low immunogenicity and high transduction efficiency.However,AAVs have limited packaging capacity,mak

128、ing it difficult to package all of the components required for successful application.184,185Another limiting factor for most gene editing components is their safe,efficient,and targeted delivery to the specific organ or tissue.If CRISPR/Cas9 components are delivered via a systemic approach,they may

129、 get destroyed and cleared by the body before reaching their target site.186DNA-damage toxicity and immunotoxicityCRISPR-based gene editing involves cutting DNA,which can inadvertently trigger cell death and growth inhibition rather than the intended genetic edit.187 Multiple simultaneous off-target

130、 edits can ultimately result in genomic rearrangements such as inversions,deletions,and chromosomal translocations and trigger DNA damage and stress response pathways.188190Immunogenic toxicity is another known limitation of any gene editing technology,including CRISPR/Cas.Pre-existing antibodies ag

131、ainst Cas9 and reactive T cells have been identified in humans,and Cas9 immunity has been associated with compromised therapeutic outcomes in various disease models.191194Regulatory hurdlesThe regulation of CRISPR-based gene editing varies between countries,and guidelines are still under development

132、 in some areas.In some countries,one regulatory agency oversees gene therapy,while other agencies regulate genetically modified organisms,creating a complex regulatory landscape for CRISPR-based therapeutics.Additionally,the long-term effects and safety of CRISPR-based therapeutics are not yet fully

133、 understood,which may contribute to lengthy and complex approval processes for these novel therapies.CRISPR:What challenges remain?CRISPR INSIGHT REPORT|23Concluding remarksSince the first use of CRISPR-based gene editing,the field has evolved at an exceptional pace,resulting in a plethora of public

134、ations exploring applications in disease management and diagnostics,as well as the identification of genes underlying various disorders.A considerable number of CRISPR-related publications appear to be connected to cancer and infectious diseases,while other diseases such as blood,genetic and nervous

135、 system disorders are also explored in the context of CRISPR/Cas technology.The use of CRISPR/Cas systems in disease diagnostics has also seen a surge,most notably after 2019.All of the research and development in the field has translated to a considerable increase in commercial interest in CRISPR-b

136、ased diagnostics and therapeutics over the last few years.Currently,more than 140 CRISPR-based therapeutics are in various stages of clinical trials,with approximately a quarter of them targeting a range of cancer subtypes.Despite the great strides and widespread acceptance of CRISPR/Cas technology

137、in gene editing,a few challenges remain in applying it for therapeutic purposes.The ongoing refinement of existing CRISPR components continues to improve the efficiency and specificity of CRISPR-based therapeutics.Expanding the targeting capabilities and optimizing delivery systems continue to aid i

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