Mechanism Study and Application of CRISPR/Cas12a-based Biosensing Platform

doi:10.13560/j.cnki.biotech.bull.1985.2024-0118

Abstract

Abstract:

CRISPR/Cas12a system can accurately identify specific single-stranded DNA or double-stranded DNA containing PAM sequences with programmable guide RNA. Then, the non-specific single strand DNA can be cleaved rapidly at the same time as the target substrate, which shows great prospects in biosensing. In recent years, the CRISPR/Cas12a system has been widely used for assisted detection of biomarkers, and its biomarker sensing platform has been applied in the construction of biomolecular sensors such as visualized cell pathways and in vivo diagnostics. Based on the principles of CRISPR/Cas12a biosensing platform construction, this paper summarizes the variants of CRISPR/Cas12a system under different application scenarios, then highlights and summarizes the applications of in vitro and in vivo sensing platforms based on this system, and further discusses the characteristics of different sensing platforms. Finally, the paper summarizes and prospects the advantages and limitations of current applications, aiming to provide a certain reference for the research and application in this field.

Key words: CRISPR/Cas12a, biosensing in vivo, biosensing in vitro, accurately identifying

CHEN Mo-yan, ZHU Cheng. Mechanism Study and Application of CRISPR/Cas12a-based Biosensing Platform[J]. Biotechnology Bulletin, 2024, 40(7): 90-98.

Figures/Tables 3

Fig. 1 Principles of CRISPR/Cas12a The Cas12a-crRNA complex cleaved target dsDNA（cis）and non-complementary ssDNA（trans）after recognizing PAM sequences. Target ssDNA that specifically recognizes crRNA is also able to activate the cis-cleavage and trans-cleavage activity of Cas12a, while the activity is not restricted by the PAM sequence

Fig. 2 A Biosensing platform based on Cas12a variant A: Schematic of bacterial interference assay used to identify variants with altered PAM specificity. B: A diagram illustrating the double-stranded-break-induced gain-of-expression assay used in subsequent figures. A construct with an in-frame（+1）crRNA target sequence（orange）preceding an out-of-frame（+3）luciferase gene（light green）is stably integrated into the genomes of HEK293T cells. Upon cleavage by a Cas12a/crRNA complex, non-homologous end-joining（NHEJ）repair places approximately one-third of the downstream luciferase genes in frame（dark green）, enabling their expressions. The activities of 11 luciferase reflects the efficiency of Cas12a-mediated cleavage. C: A list of target sequences used in subsequent panels with their preceding PAMs（blue）. D: Histograms illustrating the number of GUIDE-seq detected off-target sites for AsCas12a variants on sites with canonical TTTV PAMs or non-canonical PAMs. E: Schematic of LAMP. Six distinct regions（F1, F2, F3, B1, B2, and B3）in the target locus are recognized by four core primers, which have a black arrow at their 3' ends to indicate extension by the DNA polymerase. FIP is indicated by a dark green rectangle joined to a slanted light blue rectangle. BIP is indicated by a dark brown rectangle joined to a slanted light purple rectangle. In addition, the F3 displacement primer is indicated by a red rectangle, while the B3 displacement primer is indicated by an orange rectangle. The letter “c” appended to each region name indicates the reverse complementary sequence. After LAMP optimization process, writter incorporated swarm primers, whose sequences are equivalent to F1c and B1c. Moreover, to demonstrate how a mismatch at the 5' end of FIP can affect the LAMP reaction, add a yellow asterisk to track the progression of the mismatch. F: The red arrow indicates the test bands, while the green arrow indicates the control bands. Ratios of test band intensity to control band intensity are given under each dipstick

Table 1 CRISPR/Cas12a-based biosensing strategy

检测样品 Detection sample	LOD	组合反应方法 Combined reaction method	信号输出方式 Signal output manner	时间 Time
DNA病毒/RNA病毒 DNA viruses/RNA viruses^[17]	10^-18 mol/L	PCR	荧光读数 Fluorescent readout	1 h
人乳头瘤病毒 Human papillomavirus^[16]	10^-18 mol/L	RPA	荧光读数 Fluorescent readout	1 h
葡萄红色斑点病毒 Grapevine red-blotch virus^[27]	10^-17 mol/L	PCR	视觉比色读数 Visual colorimetric readout（aunp）	45 min
沙门氏菌Salmonella^[35]	1 CFU/mL	RPA	视觉色度读数 Visual colorimetric readout（TMB）	3 h
人乳头瘤病毒 Human papillomavirus^[34]	5×10^-11 mol/L	/	电化学读数 Electrochemical readout	1 h
大肠杆菌O157:H7 Escherichia coli O157:H7^[33]	19 CFU/mL	Aptamer	电化学读数 Electrochemical readout	1 h
非洲猪瘟病毒 African swine fever virus^[36]	20 copies	RAA	横向流动条读数 Lateral flow strip readout	1 h
大鼠肉瘤病毒癌基因Kirsten rat sarcoma viral oncogene^[37]	2.6×10^-11 mol/L	Poly-invertase-DNA immobilized magnetic Beads	便携式血糖仪读数 Portable glucose meter readout	2 h
心肌钙蛋白 Cardiac troponins I^[37]	7.5 ng/mL	Poly-invertase-DNA immobilized magnetic Beads	便携式血糖仪读数 Portable glucose meter readout	2 h
SARS冠状病毒2型 SARS-CoV-2^[38]	1.6×10^-18 mol/L	RPA	妊娠试纸条读数 Pregnancy test strip readout	30 min
尿酸 Uric acid^[32]	10^-8 mol/L	Allosteric transcription Factors	荧光读数 Fluorescent readout	25 min
对羟基苯甲酸 P-hydroxybenzoic acid^[32]	1.8×10^-9 mol/L	Allosteric transcription Factors	荧光读数 Fluorescent readout	25 min
胞外体 Exosome^[39]	10³ particles/μL	CD63 aptamer	荧光读数 Fluorescent readout	2 h
ATP^[32]	4.75×10^-6 mol/L	Aptamer	便携式荧光计读数 Portable fluorimeter readout	15 min
Na^+[32]	10^-10 mol/L	Aptamer	便携式荧光计读数 Portable fluorimeter readout	15 min

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