基于CRISPR/Cas系统的生物传感器在动物疫病诊断中的应用

doi:10.11843/j.issn.0366-6964.2024.07.008

Abstract

Abstract:

Animal diseases not only seriously jeopardize the health of animals and hinder the healthy development of the breeding industry, but also pose a potential threat to human health. Therefore, it is vital to develop sensitive, specific and rapid diagnostic methods for disease prevention and control. The current gold standard for pathogens detection, PCR, has limitations such as long operational cycles, high instrumentation and technician requirements, cannot be applied to point-of-care testing. Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas) have the advantages of high sensitivity, high specificity and easy operation, which can provide a new method and opportunity for the diagnosis of animal diseases. CRISPR-Cas9 specificity in targeting nucleic acids and "collateral cleavage" activity of CRISPR-Cas12/Cas13 show significant promise in nucleic acid detection. In this review, the classification of CRISPR/Cas systems, the strategies adopted by three commonly used CRISPR/Cas systems applied to nucleic acid detection were summarized, as well as the latest research progress of their application in animal disease diagnosis, finally their advantages, challenges, and future development were analyzed, in the hope of providing a reference for the application of CRISPR/Cas biosensing platforms in animal diseases diagnosis.

Key words: CRISPR/Cas, biosensor, animal diseases, detection

CLC Number:

S854.43

Xiuqin CHEN, Su LIN, Shizhong ZHANG, Min ZHENG, Meiqing HUANG. Application of CRISPR/Cas-based Biosensors for Animal Diseases Diagnosis[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 2859-2876.

Figures/Tables 6

Table 1

Fig. 1

Table 2

Table 3

Application of CRISPR/Cas systems in the diagnosis of bacteria and fungi diseases"

靶标 Target	效应蛋白 Effector protein	扩增方法 Amplification	信号输出 Signal output	敏感性 Sensitivity	检测时间 Time	POCT	特点 Characteristic	参考文献 References
耐甲氧西林金黄色葡萄球菌 Methicillin-resistant Staphylococcus aureus	dCas9	无	F	10 CFU·mL^-1	＜30 min	是	采用dCas9/sgRNA复合物作为靶向材料，SYBR Green I作为荧光探针；无需使用细胞裂解物进行基因分离步骤	[68]
产单核细胞李氏杆菌 Listeria monocytogenes	LbCas12a	RPA	E	0.68×10^-18 mol·L^-1	＜2 h	否	-	[65]
副溶血性弧菌 Vibrio parahaemolyticus	LbCas12a	LAMP	F	30 copies·反应^-1	50 min	是	离心式芯片耦合CRISPR/Cas12a系统的集成核酸检测方法	[69]
副溶血性弧菌 Vibrio parahaemolyticus	LbCas12a	RPA	F	12.3 CFU·mL^-1	-	否	一个RPA扩增产物可以激活多CRISPR/Cas12a单元；克服了PAM限制的局限性	[70]
沙门菌 Salmonella	LbCas12a	无	C	1 CFU·mL^-1	＜3 h	是	利用无标记G-四链体作为报告系统；结果既可以用肉眼识别，也可以用智能手机辅助定量测量	[71]
鼠伤寒沙门菌 Salmonella Typhimurium	LbCas12a	LAMP	F	1 CFU·mL^-1	~2 h	否	首次将比率型荧光信号读数与CRISPR/Cas结合起来; 使用无标记DNA模板的银纳米簇将目标核酸信号转换为双色荧光	[72]
鼠伤寒沙氏菌 Salmonella Typhimurium	LbCas12a	末端脱氧核苷酸转移酶(Tdt)介导的信号放大	F	53 CFU·mL^-1	-	否	率先将尿嘧啶-DNA糖基化酶(UDG)消化技术与V型PCR(VPCR)相结合；首次将Tdt介导的信号放大技术用于预制备的多聚磁探针；构建的UDG-VPCR驱动的Cas12a-MRS(磁弛豫开关)具有四重特异性；解决了与核酸扩增相结合的Cas12a系统相关的气溶胶污染问题	[73]
7种食源性致病菌 Seven food-borne pathogens	LbCas12a	RPA	F	＜500 CFU·mL^-1	~1 h	是	开发了一种基于单管CRISPR/Cas12a-RPA的手指驱动微流体生物传感器；同时检测7种致病菌；解决了气溶胶污染问题	[74]
7种食源性致病菌 Seven food-borne pathogens	LbCas13a	RAA	液滴微流控	10 copies·μL^-1	~1 h	否	开发了一种基于单管CRISPR/Cas13a-RAA的液滴微流控平台，目标细菌鉴定通过液滴的颜色确定，通过泊松分布计算阳性液滴的百分比来计算目标细菌浓度。解决了气溶胶污染问题，且定量更准确	[75]
金黄色葡萄球菌 Staphylococcus aureus	LbCas12a	SDA	ECL	0.437×10^-18 mol·L^-1	-	否	卟啉Zr金属有机骨架作为共反应促进剂加速了电子转移并提高了ECL发光效率；设计有缺陷的T型连接结构用于SDA，消除了对目标DNA长度的考虑，具有更高的特异性和稳定性	[76]
炭疽杆菌 Bacillus anthracis	LbCas12a	RPA	F	2 copies·反应^-1	40 min	是	-	[77]
布鲁氏菌 Brucella spp.	LbCas12a	RPA	F/E	2 copies·反应^-1	-	否	-	[78]
幽门螺杆菌 Helicobacter pylori	LbCas12a	MIRA	F/LFA	1 copies·反应^-1 (F)；4 copies·反应^-1	45 min	是	-	[79]
犬小孢子菌和须癣毛癣菌 Microsporum canis, Trichophyton mentagrophytes	LbCas12a	RPA	F	100%	~30 min	是	-	[64]
念珠菌、曲霉菌和隐球菌 Candida, Aspergillus, Cryptococcus	LbCas12a	RT-RPA	纸基微流控	＜10 CFU·mL^-1	-	是	通过DNA智能水凝胶体系和葡萄糖氧化酶-辣根过氧化物酶级联反应进行信号的放大与转导，并最终利用纸基微流控芯片进行肉眼读取结果	[80]

Table 3

Table 4

Table 5

References 111

1	JONES K E , PATEL N G , LEVY M A , et al. Global trends in emerging infectious diseases[J]. Nature, 2008, 451 (7181): 990- 993. doi: 10.1038/nature06536
2	RODÓ X , SAN-JOSÉ A , KIRCHGATTER K , et al. Changing climate and the COVID-19 pandemic: more than just heads or tails[J]. Nat Med, 2021, 27 (4): 576- 579. doi: 10.1038/s41591-021-01303-y
3	ZHANG N R , WANG L L , DENG X Q , et al. Recent advances in the detection of respiratory virus infection in humans[J]. J Med Virol, 2020, 92 (4): 408- 417. doi: 10.1002/jmv.25674
4	WANG L Q , WANG X J , WU Y G , et al. Rapid and ultrasensitive electromechanical detection of ions, biomolecules and SARS-CoV-2 RNA in unamplified samples[J]. Nat Biomed Eng, 2022, 6 (3): 276- 285. doi: 10.1038/s41551-021-00833-7
5	ZHAO Y X , CHEN F , LI Q , et al. Isothermal amplification of nucleic acids[J]. Chem Rev, 2015, 115 (22): 12491- 12545. doi: 10.1021/acs.chemrev.5b00428
6	HU F , LIU Y F , ZHAO S H , et al. A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics[J]. Biosens Bioelectron, 2022, 202, 113994. doi: 10.1016/j.bios.2022.113994
7	ZANOLI L , SPOTO G . Isothermal amplification methods for the detection of nucleic acids in microfluidic devices[J]. Biosensors (Basel), 2013, 3 (1): 18- 43.
8	TIAN T , ZHOU X M . CRISPR-based biosensing strategies: technical development and application prospects[J]. Annu Rev Anal Chem, 2023, 16, 311- 332. doi: 10.1146/annurev-anchem-090822-014725
9	PANG B , XU J Y , LIU Y M , et al. Isothermal amplification and ambient visualization in a single tube for the detection of SARS-CoV-2 using loop-mediated amplification and CRISPR technology[J]. Anal Chem, 2020, 92 (24): 16204- 16212. doi: 10.1021/acs.analchem.0c04047
10	GOOTENBERG J S , ABUDAYYEH O O , LEE J W , et al. Nucleic acid detection with CRISPR-Cas13a/C2c2[J]. Science, 2017, 356 (6336): 438- 442. doi: 10.1126/science.aam9321
11	ARIZTI-SANZ J , FREIJE C A , STANTON A C , et al. Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2[J]. Nat Commun, 2020, 11 (1): 5921. doi: 10.1038/s41467-020-19097-x
12	FOZOUNI P , SON S , DÍAZ DE LEÓN DERBY M , et al. Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy[J]. Cell, 2021, 184 (2): 323- 333.e9. doi: 10.1016/j.cell.2020.12.001
13	LI Y R , LIU Y J , TANG X Q , et al. CRISPR/Cas-powered amplification-free detection of nucleic acids: current state of the art, challenges, and futuristic perspectives[J]. ACS Sens, 2023, 8 (12): 4420- 4441. doi: 10.1021/acssensors.3c01463
14	YANG H , ZHANG Y , TENG X C , et al. CRISPR-based nucleic acid diagnostics for pathogens[J]. Trends Analyt Chem, 2023, 160, 116980. doi: 10.1016/j.trac.2023.116980
15	HE Y W , HU Q Q , SAN S , et al. CRISPR-based biosensors for human health: a novel strategy to detect emerging infectious diseases[J]. Trends Analyt Chem, 2023, 168, 117342. doi: 10.1016/j.trac.2023.117342
16	李梦茹, 秦志强, 殷堃, 等. CRISPR/Cas系统在病原体核酸检测中的应用进展[J]. 中国血吸虫病防治杂志, 2023, 35 (1): 98-103, 110.
	LI M R , QIN Z Q , YIN K , et al. Application of CRISPR/Cas systems in the nucleic acid detection of pathogens: a review[J]. Chinese Journal of Schistosomiasis Control, 2023, 35 (1): 98-103, 110.
17	POURCEL C , SALVIGNOL G , VERGNAUD G . CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies[J]. Microbiology (Reading), 2005, 151 (3): 653- 663. doi: 10.1099/mic.0.27437-0
18	MAKAROVA K S , WOLF Y I , ALKHNBASHI O S , et al. An updated evolutionary classification of CRISPR-Cas systems[J]. Nat Rev Microbiol, 2015, 13 (11): 722- 736. doi: 10.1038/nrmicro3569
19	HSU P D , LANDER E S , ZHANG F . Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157 (6): 1262- 1278. doi: 10.1016/j.cell.2014.05.010
20	GILBERT L A , LARSON M H , MORSUT L , et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[J]. Cell, 2013, 154 (2): 442- 451. doi: 10.1016/j.cell.2013.06.044
21	CHEN J S , MA E B , HARRINGTON L B , et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity[J]. Science, 2018, 360 (6387): 436- 439. doi: 10.1126/science.aar6245
22	HUANG M Q , ZHOU X M , WANG H Y , et al. Clustered regularly interspaced short palindromic repeats/Cas9 triggered isothermal amplification for site-specific nucleic acid detection[J]. Anal Chem, 2018, 90 (3): 2193- 2200. doi: 10.1021/acs.analchem.7b04542
23	KI J , NA H K , YOON S W , et al. CRISPR/Cas-assisted colorimetric biosensor for point-of-use testing for African swine fever virus[J]. ACS Sens, 2022, 7 (12): 3940- 3946. doi: 10.1021/acssensors.2c02007
24	CAO Y M , LU X , LIN H S , et al. CoLAMP: CRISPR-based one-pot loop-mediated isothermal amplification enables at-home diagnosis of SARS-CoV-2 RNA with nearly eliminated contamination utilizing amplicons depletion strategy[J]. Biosens Bioelectron, 2023, 236, 115402. doi: 10.1016/j.bios.2023.115402
25	VAN DER VEER H J , VAN AALEN E A , MICHIELSEN C M S , et al. Glow-in-the-dark Infectious disease diagnostics using CRISPR-Cas9-based split luciferase complementation[J]. ACS Cent Sci, 2023, 9 (4): 657- 667. doi: 10.1021/acscentsci.2c01467
26	ZHANG Y H , QIAN L , WEI W J , et al. Paired design of dCas9 as a systematic platform for the detection of featured nucleic acid sequences in pathogenic strains[J]. ACS Synth Biol, 2017, 6 (2): 211- 216. doi: 10.1021/acssynbio.6b00215
27	ZHOU W H , HU L , YING L M , et al. A CRISPR-Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection[J]. Nat Commun, 2018, 9 (1): 5012. doi: 10.1038/s41467-018-07324-5
28	WANG M , ZHANG R , LI J M . CRISPR/cas systems redefine nucleic acid detection: principles and methods[J]. Biosens Bioelectron, 2020, 165, 112430. doi: 10.1016/j.bios.2020.112430
29	KNIGHT S C , XIE L Q , DENG W L , et al. Dynamics of CRISPR-Cas9 genome interrogation in living cells[J]. Science, 2015, 350 (6262): 823- 826. doi: 10.1126/science.aac6572
30	WANG X S , XIONG E H , TIAN T , et al. Clustered regularly interspaced short palindromic repeats/Cas9-mediated lateral flow nucleic acid assay[J]. ACS Nano, 2020, 14 (2): 2497- 2508. doi: 10.1021/acsnano.0c00022
31	CHO S W , KIM S , KIM Y , et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases[J]. Genome Res, 2014, 24 (1): 132- 141. doi: 10.1101/gr.162339.113
32	JINEK M , JIANG F G , TAYLOR D W , et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation[J]. Science, 2014, 343 (6176): 1247997. doi: 10.1126/science.1247997
33	WENG Z Y , YOU Z , YANG J , et al. CRISPR-Cas biochemistry and CRISPR-based molecular diagnostics[J]. Angew Chem Int Ed, 2023, 62 (17): e202214987. doi: 10.1002/anie.202214987
34	PARDEE K , GREEN A A , TAKAHASHI M K , et al. Rapid, low-cost detection of Zika virus using programmable biomolecular components[J]. Cell, 2016, 165 (5): 1255- 1266. doi: 10.1016/j.cell.2016.04.059
35	QIU X Y , ZHU L Y , ZHU C S , et al. Highly effective and low-cost microRNA detection with CRISPR-Cas9[J]. ACS Synth Biol, 2018, 7 (3): 807- 813. doi: 10.1021/acssynbio.7b00446
36	HAJIAN R , BALDERSTON S , TRAN T , et al. Detection of unamplified target genes via CRISPR-Cas9 immobilized on a graphene field-effect transistor[J]. Nat Biomed Eng, 2019, 3 (6): 427- 437. doi: 10.1038/s41551-019-0371-x
37	SWARTS D C , JINEK M . Mechanistic insights into the cis- and trans-acting DNase activities of Cas12a[J]. Mol Cell, 2019, 73 (3): 589- 600.e4. doi: 10.1016/j.molcel.2018.11.021
38	LIU L , LI X Y , WANG J Y , et al. Two distant catalytic sites are responsible for C2c2 RNase activities[J]. Cell, 2017, 168 (1-2): 121- 134.e12. doi: 10.1016/j.cell.2016.12.031
39	LI Y , LI S Y , WANG J , et al. CRISPR/Cas systems towards next-generation biosensing[J]. Trends Biotechnol, 2019, 37 (7): 730- 743. doi: 10.1016/j.tibtech.2018.12.005
40	LI S Y , CHENG Q X , WANG J M , et al. CRISPR-Cas12a-assisted nucleic acid detection[J]. Cell Discov, 2018, 4, 20.
41	ABUDAYYEH O O , GOOTENBERG J S , KONERMANN S , et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector[J]. Science, 2016, 353 (6299): aaf5573. doi: 10.1126/science.aaf5573
42	GOOTENBERG J S , ABUDAYYEH O O , KELLNER M J , et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[J]. Science, 2018, 360 (6387): 439- 444. doi: 10.1126/science.aaq0179
43	MYHRVOLD C , FREIJE C A , GOOTENBERG J S , et al. Field-deployable viral diagnostics using CRISPR-Cas13[J]. Science, 2018, 360 (6387): 444- 448. doi: 10.1126/science.aas8836
44	WANG N , ZHAO D M , WANG J L , et al. Architecture of African swine fever virus and implications for viral assembly[J]. Science, 2019, 366 (6465): 640- 644. doi: 10.1126/science.aaz1439
45	YANG B , SHI Z W , MA Y , et al. LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus[J]. Transbound Emerg Dis, 2022, 69 (4): e216-e223.
46	HE Q , YU D M , BAO M D , et al. High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system[J]. Biosens Bioelectron, 2020, 154, 112068. doi: 10.1016/j.bios.2020.112068
47	SHEN X T , XU W , OUYANG J , et al. Fluorescence resonance energy transfer-based nanomaterials for the sensing in biological systems[J]. Chin Chem Lett, 2022, 33 (10): 4505- 4516. doi: 10.1016/j.cclet.2021.12.061
48	XIE S Y , JI Z R , SUO T Y , et al. Advancing sensing technology with CRISPR: from the detection of nucleic acids to a broad range of analytes-A review[J]. Anal Chim Acta, 2021, 1185, 338848. doi: 10.1016/j.aca.2021.338848
49	杨芷翊, 王新凯, 史玉婷, 等. 基于RT-RAA的禽流感H5亚型核酸CRISPR-Cas13a检测方法的建立[J]. 畜牧兽医学报, 2023, 54 (9): 3803- 3811. doi: 10.11843/j.issn.0366-6964.2023.09.020
	YANG Z Y , WANG X K , SHI Y T , et al. Establishment of nucleic acid detection methods for avian influenza H5 subtype based on CRISPR-Cas13a and RT-RAA[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54 (9): 3803- 3811. doi: 10.11843/j.issn.0366-6964.2023.09.020
50	XU W , JIN T , DAI Y F , et al. Surpassing the detection limit and accuracy of the electrochemical DNA sensor through the application of CRISPR Cas systems[J]. Biosens Bioelectron, 2020, 155, 112100. doi: 10.1016/j.bios.2020.112100
51	WAN L , MA J F , YI J S , et al. CRISPR-empowered hybridization chain reaction amplification for an attomolar electrochemical sensor[J]. Chem Commun, 2022, 58 (63): 8826- 8829. doi: 10.1039/D2CC01155G
52	XU H P , PENG L J , WU J , et al. Clustered regularly interspaced short palindromic repeats-associated proteins13a combined with magnetic beads, chemiluminescence and reverse transcription-recombinase aided amplification for detection of avian influenza a (H7N9) virus[J]. Front Bioeng Biotechnol, 2023, 10, 1094028. doi: 10.3389/fbioe.2022.1094028
53	TANG Y D , QI L J , LIU Y C , et al. CLIPON: a CRISPR-enabled strategy that turns commercial pregnancy test strips into general point-of-need test devices[J]. Angew Chem Int Ed, 2022, 61 (12): e202115907. doi: 10.1002/anie.202115907
54	CHANG Y F , DENG Y , LI T Y , et al. Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR-Cas13a[J]. Transbound Emerg Dis, 2020, 67 (2): 564- 571. doi: 10.1111/tbed.13368
55	XU B R , GONG P , ZHANG Y , et al. A one-tube rapid visual CRISPR assay for the field detection of Japanese encephalitis virus[J]. Virus Res, 2022, 319, 198869. doi: 10.1016/j.virusres.2022.198869
56	WEI J , LI Y N , CAO Y L , et al. Rapid and visual detection of porcine parvovirus using an ERA-CRISPR/Cas12a system combined with lateral flow dipstick assay[J]. Front Cell Infect Microbiol, 2022, 12, 879887. doi: 10.3389/fcimb.2022.879887
57	LIAO K , PENG W Q , QIAN B X , et al. A highly adaptable platform powered by CRISPR-Cas12a to diagnose lumpy skin disease in cattle[J]. Anal Chim Acta, 2022, 1221, 340079. doi: 10.1016/j.aca.2022.340079
58	WEI J , WANG W J , YU Q , et al. MASTR Pouch: palm-size lab for point-of-care detection of Mpox using recombinase polymerase amplification and CRISPR technology[J]. Sens Actuators B Chem, 2023, 390, 133950. doi: 10.1016/j.snb.2023.133950
59	LIU S S , WANG C Y , WANG Z M , et al. Binding induced isothermal amplification reaction to activate CRISPR/Cas12a for amplified electrochemiluminescence detection of rabies viral RNA via DNA nanotweezer structure switching[J]. Biosens Bioelectron, 2022, 204, 114078. doi: 10.1016/j.bios.2022.114078
60	KHAN H , KHAN A , LIU Y F , et al. CRISPR-Cas13a mediated nanosystem for attomolar detection of canine parvovirus type 2[J]. Chin Chem Lett, 2019, 30 (12): 2201- 2204. doi: 10.1016/j.cclet.2019.10.032
61	YANG K K , ZHANG W Y , XU L , et al. Facile, ultrasensitive, and highly specific diagnosis of goose astrovirus via reverse transcription-enzymatic recombinase amplification coupled with a CRISPR-Cas12a system detection[J]. Poult Sci, 2022, 101 (12): 102208. doi: 10.1016/j.psj.2022.102208
62	YIN D D , YIN L , WANG J R , et al. Visual detection of duck tembusu virus with CRISPR/Cas13:a sensitive and specific point-of-care detection[J]. Front Cell Infect Microbiol, 2022, 12, 848365. doi: 10.3389/fcimb.2022.848365
63	HOFFMANN S , DEVLEESSCHAUWER B , ASPINALL W , et al. Attribution of global foodborne disease to specific foods: findings from a World Health Organization structured expert elicitation[J]. PLoS One, 2017, 12 (9): e0183641. doi: 10.1371/journal.pone.0183641
64	WANG H , WU Q , ZHOU M , et al. Development of a CRISPR/Cas9-integrated lateral flow strip for rapid and accurate detection of Salmonella[J]. Food Control, 2022, 142, 109203. doi: 10.1016/j.foodcont.2022.109203
65	LI F , YE Q H , CHEN M T , et al. An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for Listeria monocytogenes detection[J]. Biosens Bioelectron, 2021, 179, 113073. doi: 10.1016/j.bios.2021.113073
66	DONG J, WU X, HU Q, et al. An immobilization-free electrochemical biosensor based on CRISPR/Cas13a and FAM-RNA-MB for simultaneous detection of multiple pathogens[J]. Biosens Bioelectron, 2023, 241: 115673. [2024-05-21]. https://www.sciencedirect.com/science/article/pii/S0956566323006152?via%3Dihub.
67	WEI Y D , TAO Z Z , WAN L , et al. Aptamer-based Cas14a1 biosensor for amplification-free live pathogenic detection[J]. Biosens Bioelectron, 2022, 211, 114282. doi: 10.1016/j.bios.2022.114282
68	GUK K , KEEM J O , HWANG S G , et al. A facile, rapid and sensitive detection of MRSA using a CRISPR-mediated DNA FISH method, antibody-like dCas9/sgRNA complex[J]. Biosens Bioelectron, 2017, 95, 67- 71. doi: 10.1016/j.bios.2017.04.016
69	WU H , CHEN Y J , YANG Q Q , et al. A reversible valve-assisted chip coupling with integrated sample treatment and CRISPR/Cas12a for visual detection of Vibrio parahaemolyticus[J]. Biosens Bioelectron, 2021, 188, 113352. doi: 10.1016/j.bios.2021.113352
70	PENG Y B , XUE P P , WANG R J , et al. Engineering of an adaptive tandem CRISPR/Cas12a molecular amplifier permits robust analysis of Vibrio parahaemolyticus[J]. Talanta, 2024, 266, 125061. doi: 10.1016/j.talanta.2023.125061
71	YIN L J , DUAN N H , CHEN S , et al. Ultrasensitive pathogenic bacteria detection by a smartphone-read G-quadruplex-based CRISPR-Cas12a bioassay[J]. Sens Actuators B Chem, 2021, 347, 130586. doi: 10.1016/j.snb.2021.130586
72	MA L , WANG J J , LI Y R , et al. A ratiometric fluorescent biosensing platform for ultrasensitive detection of Salmonella typhimurium via CRISPR/Cas12a and silver nanoclusters[J]. J Hazard Mater, 2023, 443, 130234. doi: 10.1016/j.jhazmat.2022.130234
73	WEN J P , REN L Q , HE Q F , et al. Contamination-free V-shaped ultrafast reaction cascade transferase signal amplification driven CRISPR/Cas12a magnetic relaxation switching biosensor for bacteria detection[J]. Biosens Bioelectron, 2023, 219, 114790. doi: 10.1016/j.bios.2022.114790
74	XING G W , SHANG Y T , WANG X R , et al. Multiplexed detection of foodborne pathogens using one-pot CRISPR/Cas12a combined with recombinase aided amplification on a finger-actuated microfluidic biosensor[J]. Biosens Bioelectron, 2023, 220, 114885. doi: 10.1016/j.bios.2022.114885
75	SHANG Y T , XING G W , LIN J X , et al. Multiplex bacteria detection using one-pot CRISPR/Cas13a-based droplet microfluidics[J]. Biosens bioelectron, 2024, 243, 115771. doi: 10.1016/j.bios.2023.115771
76	LIU Y Q , WANG F Y , GE S , et al. Programmable T-junction structure-assisted CRISPR/Cas12a electrochemiluminescence biosensor for detection of Sa-16S rDNA[J]. ACS Appl Mater Interfaces, 2023, 15 (1): 617- 625. doi: 10.1021/acsami.2c18930
77	XU J H , BAI X R , ZHANG X L L , et al. Development and application of DETECTR-based rapid detection for pathogenic Bacillus anthracis[J]. Anal Chim Acta, 2023, 1247, 340891. doi: 10.1016/j.aca.2023.340891
78	XU J H , MA J F , LI Y W , et al. A general RPA-CRISPR/Cas12a sensing platform for Brucella spp. detection in blood and milk samples[J]. Sens Actuators B Chem, 2022, 364, 131864. doi: 10.1016/j.snb.2022.131864
79	WANG D , WANG D F , LIAO K , et al. Optical detection using CRISPR-Cas12a of Helicobacter pylori for veterinary applications[J]. Microchim Acta, 2023, 190 (11): 455. doi: 10.1007/s00604-023-06037-x
80	HUANG D , NI D S , FANG M J , et al. Microfluidic ruler-readout and CRISPR Cas12a-responded hydrogel-integrated paper-based analytical devices (μReaCH-PAD) for visible quantitative point-of-care testing of invasive fungi[J]. Anal Chem, 2021, 93 (50): 16965- 16973. doi: 10.1021/acs.analchem.1c04649
81	王梓乐, 高志强, 汪琳, 等. 我国动物寄生虫检测标准现状分析与建议[J]. 寄生虫与医学昆虫学报, 2020, 27 (2): 121- 128. doi: 10.3969/j.issn.1005-0507.2020.02.010
	WANG Z L , GAO Z Q , WANG L , et al. Current standards for inspection of food for animal parasites in China: analyses and suggestions[J]. Acta Parasitology et Medica Entomologica Sinica, 2020, 27 (2): 121- 128. doi: 10.3969/j.issn.1005-0507.2020.02.010
82	AKHOUNDI M , KUHLS K , CANNET A , et al. A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies[J]. PLoS Negl Trop Dis, 2016, 10 (3): e0004349. doi: 10.1371/journal.pntd.0004349
83	GAO H , FENG M M , LI F , et al. G-Quadruplex DNAzyme-substrated CRISPR/Cas12 assay for label-free detection of single-celled parasitic infection[J]. ACS Sens, 2022, 7 (10): 2968- 2977. doi: 10.1021/acssensors.2c01104
84	BENGTSON M , BHARADWAJ M , FRANCH O , et al. CRISPR-dCas9 based DNA detection scheme for diagnostics in resource-limited settings[J]. Nanoscale, 2022, 14 (5): 1885- 1895. doi: 10.1039/D1NR06557B
85	CHALMERS R M , KATZER F . Looking for Cryptosporidium: the application of advances in detection and diagnosis[J]. Trends Parasitol, 2013, 29 (5): 237- 251. doi: 10.1016/j.pt.2013.03.001
86	LI Y , DENG F , HALL T , et al. CRISPR/Cas12a-powered immunosensor suitable for ultra-sensitive whole Cryptosporidium oocyst detection from water samples using a plate reader[J]. Water Res, 2021, 203, 117553. doi: 10.1016/j.watres.2021.117553
87	MACGREGOR S R , MCMANUS D P , SIVAKUMARAN H , et al. Development of CRISPR/Cas13a-based assays for the diagnosis of Schistosomiasis[J]. eBioMedicine, 2023, 94, 104730. doi: 10.1016/j.ebiom.2023.104730
88	CHENG P P , WU Y T , GUO S S , et al. RPA assay coupled with CRISPR/Cas12a system for the detection of seven Eimeria species in chicken fecal samples[J]. Vet Parasitol, 2022, 311, 109810. doi: 10.1016/j.vetpar.2022.109810
89	SIMA N , DUJEANCOURT-HENRY A , PERLAZA B L , et al. SHERLOCK4HAT: a CRISPR-based tool kit for diagnosis of human African trypanosomiasis[J]. eBioMedicine, 2022, 85, 104308. doi: 10.1016/j.ebiom.2022.104308
90	WANG X F , CHENG M , YANG S Q , et al. CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood[J]. Parasit Vectors, 2023, 16 (1): 256. doi: 10.1186/s13071-023-05868-0
91	LEI R , LI L M , WU P S , et al. RPA/CRISPR/Cas12a-based on-site and rapid nucleic acid detection of Toxoplasma gondii in the environment[J]. ACS Synth Biol, 2022, 11 (5): 1772- 1781. doi: 10.1021/acssynbio.1c00620
92	YU F C , ZHANG K H , WANG Y L , et al. CRISPR/Cas12a-based on-site diagnostics of Cryptosporidium parvum Ⅱd-subtype-family from human and cattle fecal samples[J]. Parasit Vectors, 2021, 14 (1): 208. doi: 10.1186/s13071-021-04709-2
93	HUANG T J , LI L , LI J H , et al. Rapid, sensitive, and visual detection of Clonorchis sinensis with an RPA-CRISPR/Cas12a-based dual readout portable platform[J]. Int J Biol Macromol, 2023, 249, 125967. doi: 10.1016/j.ijbiomac.2023.125967
94	SHEN Y , HU K , YUAN M Z , et al. Progress and bioapplication of CRISPR-based one-step, quantitative and multiplexed infectious disease diagnostics[J]. J Appl Microbiol, 2023, 134 (3): lxad035. doi: 10.1093/jambio/lxad035
95	YANG J , BARUA N , RAHMAN M N , et al. Rapid SARS-CoV-2 variants enzymatic detection (SAVED) by CRISPR-Cas12a[J]. Microbiol Spectr, 2022, 10 (6): e0326022. doi: 10.1128/spectrum.03260-22
96	JOUNG J , LADHA A , SAITO M , et al. Detection of SARS-CoV-2 with SHERLOCK one-pot testing[J]. N Engl J Med, 2020, 383 (15): 1492- 1494. doi: 10.1056/NEJMc2026172
97	CHEN Y J , SHI Y , CHEN Y , et al. Contamination-free visual detection of SARS-CoV-2 with CRISPR/Cas12a: a promising method in the point-of-care detection[J]. Biosens Bioelectron, 2020, 169, 112642. doi: 10.1016/j.bios.2020.112642
98	WANG R , QIAN C Y , PANG Y N , et al. opvCRISPR: one-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection[J]. Biosens Bioelectron, 2021, 172, 112766. doi: 10.1016/j.bios.2020.112766
99	LU S H , TONG X H , HAN Y , et al. Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a[J]. Nat Biomed Eng, 2022, 6 (3): 286- 297. doi: 10.1038/s41551-022-00861-x
100	HU M L , LIU R H , QIU Z Q , et al. Light-start CRISPR-Cas12a reaction with caged crRNA enables rapid and sensitive nucleic acid detection[J]. Angew Chem Int Ed, 2023, 62 (23): e202300663. doi: 10.1002/anie.202300663
101	GUK K , YI S , KIM H , et al. Hybrid CRISPR/Cas protein for one-pot detection of DNA and RNA[J]. Biosens Bioelectron, 2023, 219, 114819. doi: 10.1016/j.bios.2022.114819
102	DENG F , LI Y , LI B T , et al. Increasing trans-cleavage catalytic efficiency of Cas12a and Cas13a with chemical enhancers: application to amplified nucleic acid detection[J]. Sens Actuators B Chem, 2022, 373, 132767. doi: 10.1016/j.snb.2022.132767
103	YUE H H , SHU B W , TIAN T , et al. Droplet Cas12a assay enables DNA quantification from unamplified samples at the single-molecule level[J]. Nano Lett, 2021, 21 (11): 4643- 4653. doi: 10.1021/acs.nanolett.1c00715
104	CHEN X Q , LIU X L , YU Y , et al. FRET with MoS₂ nanosheets integrated CRISPR/Cas12a sensors for robust and visual food-borne parasites detection[J]. Sens Actuators B Chem, 2023, 395, 134493. doi: 10.1016/j.snb.2023.134493
105	CHEUNG K M , ABENDROTH J M , NAKATSUKA N , et al. Detecting DNA and RNA and differentiating single-nucleotide variations via field-effect transistors[J]. Nano Lett, 2020, 20 (8): 5982- 5990. doi: 10.1021/acs.nanolett.0c01971
106	CHOI J H , SHIN M , YANG L T , et al. Clustered regularly interspaced short palindromic repeats-mediated amplification-free detection of viral DNAs using surface-enhanced Raman spectroscopy-active nanoarray[J]. ACS Nano, 2021, 15 (8): 13475- 13485. doi: 10.1021/acsnano.1c03975
107	LI Z Y , UNO N , DING X , et al. Bioinspired CRISPR-mediated cascade reaction biosensor for molecular detection of HIV using a glucose meter[J]. ACS Nano, 2023, 17 (4): 3966- 3975. doi: 10.1021/acsnano.2c12754
108	LIU Y L , WANG Y W , MA L R , et al. A CRISPR/Cas12a-based photothermal platform for the portable detection of citrus-associated Alternaria genes using a thermometer[J]. Int J Biol Macromol, 2022, 222, 2661- 2669. doi: 10.1016/j.ijbiomac.2022.10.048
109	ZHANG K , FAN Z Q , YAO B , et al. Exploring the trans-cleavage activity of CRISPR-Cas12a for the development of a Mxene based electrochemiluminescence biosensor for the detection of Siglec-5[J]. Biosens Bioelectron, 2021, 178, 113019. doi: 10.1016/j.bios.2021.113019
110	LI X P , CHEN X J , MAO M X , et al. Accelerated CRISPR/Cas12a-based small molecule detection using bivalent aptamer[J]. Biosens Bioelectron, 2022, 217, 114725. doi: 10.1016/j.bios.2022.114725
111	LI J J , YANG S S , ZUO C , et al. Applying CRISPR-Cas12a as a signal amplifier to construct biosensors for non-DNA targets in ultralow concentrations[J]. ACS Sens, 2020, 5 (4): 970- 977. doi: 10.1021/acssensors.9b02305

亚型Subtypes	Ⅱ型Type Ⅱ	Ⅴ型Type Ⅴ		Ⅵ型Type Ⅵ
效应蛋白 Effector protein	Cas9	Cas12a	Cas14a/b	Cas13a/b
核酸酶结构域 Nuclease domains	RuvC、HNH	RuvC	RuvC	HEPN^a
向导RNA Guide RNA	tracrRNA^b、crRNA^c	crRNA	tracrRNA、crRNA	crRNA
间隔区长度/nt Spacer length	18~24	18~25	20~40	22~30
PAM/PFS	3′ GC-rich PAM^d	5′ AT-rich PAM	-	3′ PFS^e: non-G
结合的靶标 Target binding	dsDNA	DNA (ds/ss^f)	DNA (ds/ss)	ssRNA
反式切割底物 Trans-cleavage substrates	-	ssDNA	ssDNA	ssRNA

检测方法 Detection method	敏感性 Sensitivity	特异性 Specificity	检测速度 Speed	POCT	可操作性 Operability	检测病原体的广度 Breadth of pathogens	成本 Cost
PCR	高	较高	h	否	中等	广	中等
等温扩增^a Isothermal amplification	中等	中等	min~h	是	容易	广	较高
基因测序 Gene sequencing	中等	高	数小时至数天	否	最难	无限	高
CRISPR/Cas	可以很高	高	min~h	是	较容易	广	可以很低

[1]	CHONG Qian, ZHAO Xuehui, CAO Qing, XUE Huiwen, GOU Huitian. Research Progress of Applied Research on Listeria monocytogenes Phage [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1819-1826.
[2]	QIU Meiyu, ZHANG Xuemei, ZHANG Ning, LIU Mingjun. Approach and Application of Prime Editing System [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1345-1355.
[3]	ZHANG Shaohua, WANG Shuai, ZOU Yang, LIU Zhongli, CAI Xuepeng. Advances in Detection Approaches for Ovine Haemonchosis [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1499-1510.
[4]	WANG Jiali, YANG Fan, SHAO Wenhua, HUANG Mengyao, CAO Weijun, PU Xiuying, ZHANG Wei, ZHENG Haixue. Construction of Tollip Knockout Pig Kidney Cell Line [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1810-1818.
[5]	ZHANG Chenjian, LI Yinxia, DING Qiang, LIU Weijia, WANG Huili, HE Nan, WU Jiashun, CAO Shaoxian. Efficient Preparation of CRISPR/Cas9-mediated Goat SOCS2 Gene Edited Embryos [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 129-141.
[6]	YANG Heng, LI Zhanhong, SONG Zi'ang, GAO Lin, LI Zhuoran, LIAO Defang, XIAO Lei, LI Huachun. Development and Application of Serotyping Real-time Quantitative RT-PCR Methods for Palyam Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 395-400.
[7]	JIANG Lingling, NIU Xiaoxia, LIU Qiang, ZHANG Gang, WANG Pu, LI Yong. Analysis of Infection Status of Beef Cattle Diarrhea-related Virus in Ningxia [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3863-3871.
[8]	LIU Hua, YIN Dongdong, SHAO Ying, SONG Xiangjun, WANG Zhenyu, PAN Xiaocheng, TU Jian, HE Changsheng, ZHU Liangqiang, QI Kezong. Detection of Porcine Epidemic Diarrhea Virus by Recombinase Aided Amplification Combined with CRISPR/Cas13a [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3991-3997.
[9]	HU Xiangyun, CAO Yanhong, LÜ Lingyan, LIU Zheng, HUANG Facai, WU Zhuyue, XIAO Zhengzhong. Nanobodies and Their Research Status in Veterinary Field [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3164-3172.
[10]	ZHANG Ping, ZHUANG Linlin, ZHANG Di, DONG Yongyi, SHENG Zhongwei, WANG Chengming, XU Bu, DOU Xinhong, GONG Jiansen. Research Progress on Molecular Detection Methods of Salmonella [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3217-3229.
[11]	CHEN Hongjian, CAO Yan, FAN Jie, GAN Rongxuan, SONG Wenbo, YU Shengwei, YANG Ting, ZHAO Yanxia, WEI Chunyan, XIE Rui, HUA Lin, PENG Zhong, WU Bin. Prevalence and Phylogenetic Analysis of Pseudorabies Virus within Pig Slaughterhouses in Hubei Province of China during 2020-2022 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2972-2981.
[12]	LI Zhaoyan, GAO Jiang, GUO Shihui, ZHAO Ruqian, MA Wenqiang. Advances in Detection Methods and Control Solutions for Feline Allergens [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2272-2279.
[13]	CHEN Yang, MENG Linchun, GUO Mengjiao, ZHANG Chengcheng, BO Zongyi, CHU Dianfeng, CAO Yongzhong, WU Yantao, ZHANG Xiaorong. Establishment and Preliminary Application of Indirect ELISA Method and HI Test for Detection of Mycoplasma Gallisepticum Antibody [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2062-2072.
[14]	Lü Qiao, ZHAO Zhongyi, YIN Dewei, LIU Yumeng, WANG Wei, ZHENG Min, HU Shiyue, ZHAO Chenchen, ZHANG Xinyu, LEI Xiaoxiao, LU Jingyi, SUN Wenchao, LAN Tian. Establishment and Preliminary Application of a Real-time RT-RAA for Detection of Transmissible Gastroenteritis Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2208-2214.
[15]	ABI-kehamo, TANG Cheng, YANG Chen, YANG Falong. Research Advances in Kobuvirus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 900-913.

Application of CRISPR/Cas-based Biosensors for Animal Diseases Diagnosis

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 6

References 111

Related Articles 15

Recommended Articles

Metrics

Comments