Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (7): 90-98.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0118
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Received:
2024-01-03
Online:
2024-07-26
Published:
2024-07-30
Contact:
ZHU Cheng
E-mail:2020226044@tju.edu.cn;cheng_zhu@tju.edu.cn
CHEN Mo-yan, ZHU Cheng. Mechanism Study and Application of CRISPR/Cas12a-based Biosensing Platform[J]. Biotechnology Bulletin, 2024, 40(7): 90-98.
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
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[ | 10-18 mol/L | PCR | 荧光读数 Fluorescent readout | 1 h |
人乳头瘤病毒 Human papillomavirus[ | 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[ | 10-17 mol/L | PCR | 视觉比色读数 Visual colorimetric readout(aunp) | 45 min |
沙门氏菌Salmonella[ | 1 CFU/mL | RPA | 视觉色度读数 Visual colorimetric readout(TMB) | 3 h |
人乳头瘤病毒 Human papillomavirus[ | 5×10-11 mol/L | / | 电化学读数 Electrochemical readout | 1 h |
大肠杆菌O157:H7 Escherichia coli O157:H7[ | 19 CFU/mL | Aptamer | ||
非洲猪瘟病毒 African swine fever virus[ | 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[ | 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[ | 7.5 ng/mL | |||
SARS冠状病毒2型 SARS-CoV-2[ | 1.6×10-18 mol/L | RPA | 妊娠试纸条读数 Pregnancy test strip readout | 30 min |
尿酸 Uric acid[ | 10-8 mol/L | Allosteric transcription Factors | 荧光读数 Fluorescent readout | 25 min |
对羟基苯甲酸 P-hydroxybenzoic acid[ | 1.8×10-9 mol/L | |||
胞外体 Exosome[ | 103 particles/μL | CD63 aptamer | 荧光读数 Fluorescent readout | 2 h |
ATP[ | 4.75×10-6 mol/L | Aptamer | 便携式荧光计读数 Portable fluorimeter readout | 15 min |
Na+[ | 10-10 mol/L |
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[ | 10-18 mol/L | PCR | 荧光读数 Fluorescent readout | 1 h |
人乳头瘤病毒 Human papillomavirus[ | 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[ | 10-17 mol/L | PCR | 视觉比色读数 Visual colorimetric readout(aunp) | 45 min |
沙门氏菌Salmonella[ | 1 CFU/mL | RPA | 视觉色度读数 Visual colorimetric readout(TMB) | 3 h |
人乳头瘤病毒 Human papillomavirus[ | 5×10-11 mol/L | / | 电化学读数 Electrochemical readout | 1 h |
大肠杆菌O157:H7 Escherichia coli O157:H7[ | 19 CFU/mL | Aptamer | ||
非洲猪瘟病毒 African swine fever virus[ | 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[ | 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[ | 7.5 ng/mL | |||
SARS冠状病毒2型 SARS-CoV-2[ | 1.6×10-18 mol/L | RPA | 妊娠试纸条读数 Pregnancy test strip readout | 30 min |
尿酸 Uric acid[ | 10-8 mol/L | Allosteric transcription Factors | 荧光读数 Fluorescent readout | 25 min |
对羟基苯甲酸 P-hydroxybenzoic acid[ | 1.8×10-9 mol/L | |||
胞外体 Exosome[ | 103 particles/μL | CD63 aptamer | 荧光读数 Fluorescent readout | 2 h |
ATP[ | 4.75×10-6 mol/L | Aptamer | 便携式荧光计读数 Portable fluorimeter readout | 15 min |
Na+[ | 10-10 mol/L |
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