CN111257297B

CN111257297B - RNA detection method based on Cas14a enzyme

Info

Publication number: CN111257297B
Application number: CN202010242856.5A
Authority: CN
Inventors: 杨治庆; 韦阳道; 万逸
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2023-06-16
Anticipated expiration: 2040-03-31
Also published as: CN111257297A

Abstract

The invention relates to a method for activating a Cas14a incidental cutting effect by RNA, in particular to the fields of analytical chemistry and rapid diagnosis of diseases. By utilizing the incidental cutting effect of the RNA activated Cas14a, the ssDNA fluorescent probe is cut, so that RNA detection is realized. The invention solves the problem that the cutting probe is RNA when the Cas system detects RNA, and enriches the application of Cas14a and the range of the detection field.

Description

RNA detection method based on Cas14a enzyme

Technical Field

The invention relates to an RNA detection method based on Cas14a enzyme, which is characterized by comprising the following steps: can be used in the field of biological analysis.

Background

CRISPR (Clustered regularly interspaced short palindromic repeats), known as regular clustered interval short palindromic repeats, is a system by which bacteria protect themselves against viruses [1]. The CRISPR-associated CRISPR-Cas system effector proteins currently discovered fall into two broad categories [2]. Wherein Cas9 cleaves target DNA with tracrRNA, guide crRNA, constituting an effector complex [3]; cas12a does not require tracr RNA, which alone uses crRNA as a guide to introduce staggered cleavage into the target double-stranded DNA, once the target is cleaved, the attendant cleavage activity of Cas12a is activated, allowing cleavage of the DNA single strand of the TTATT sequence [4]; cas13a is an RNA-guided RNase, and once the RNA target binds to the sgRNA, the "side cleavage" activity of Cas13a can be activated into a non-specific RNase [5]. Based on this ability to activate the accessory cleavage, CRISPR-Cas systems are widely used for detection of DNA, RNA and pathogenic microorganisms. The CRISPR-Cas14 enzyme is a novel CRISPR-Cas family protein discovered recently, has smaller molecular weight and target DNA cutting activity, can correspondingly provide an RNA activating Cas14a 'auxiliary cutting' activity to realize RNA detection, and has important significance in the field of biological analysis;

[1] Jackson, S. A.; McKenzie, R. E.; Fagerlund, R. D.; Kieper, S. N.; Fineran, P. C.; Brouns, S. J., CRISPR-Cas: Adapting to change. Science 2017, 356 (6333).

[2] Shmakov, S.; Smargon, A.; Scott, D.; Cox, D.; Pyzocha, N.; Yan, W.; Abudayyeh, O. O.; Gootenberg, J. S.; Makarova, K. S.; Wolf, Y. I.; Severinov, K.; Zhang, F.; Koonin, E. V., Diversity and evolution of class 2 CRISPR-Cas systems. Nat Rev Microbiol 2017, 15 (3), 169-182.

[3] Chen, J. S.; Doudna, J. A., The chemistry of Cas9 and its CRISPR colleagues. Nature Reviews Chemistry 2017, 1 (10).

[4] Max A. English; Luis R. Soenksen; Raphael V. Gayet; Helena de Puig; Nicolaas M. Angenent-Mari; Angelo S. Mao2; Peter Q. Nguyen; Collins, J. J., Programmable CRISPR-responsive smart materials. Science 2019.

[5] Abudayyeh, O. O.; Gootenberg, J. S.; Essletzbichler, P.; Han, S.; Joung, J.; Belanto, J. J.; Verdine, V.; Cox, D. B. T.; Kellner, M. J.; Regev, A.; Lander, E. S.; Voytas, D. F.; Ting, A. Y.; Zhang, F., RNA targeting with CRISPR–Cas13. Nature 2017, 550 (7675), 280-284。

disclosure of Invention

The invention aims at solving the problems of RNA detection of a CRISPR-Cas system, and provides an RNA detection method based on Cas14a enzyme, which adopts the following technical scheme:

adding RNA to be detected into a solution containing a single-stranded guide RNA (sgRNA) -Cas14a complex and a single-stranded DNA fluorescence quenching probe (F-ssDNA-Q), and realizing quantitative and qualitative analysis of the RNA by using the change intensity of a fluorescence signal; the preparation method of the sgRNA-Cas14a complex comprises the steps of mixing the sgRNA and the Cas14a enzyme, and performing enzyme synthesis in a range of 0-60 percent ^o C, mixed incubation is carried out for 0-300 minutes; the sgRNA concentration is 0-100mM; the Cas14a concentration is 0-100mM; the sequence of the sgRNA is UUC ACU GAU AAA GUG GAG AAC CGC UUC ACC AAA AGC UGU CCC UUA GGG GAU UAG AAC UUG AGU GAA GGU GGG CUG CUU GCA UCA GCC UAA UGU CGA GAA GUG CUU UCU UCG GAA AGU AAC CCU CGA AAC AAA UUC AUU UGA AAG AAU AAG GAA UGC AAC + interval sequence; the sequence of the single-stranded DNA fluorescence quenching probe (F-ssDNA-Q) is F- (T) _n -Q (n.gtoreq.5), e.g. n=5, sequence length F-TTTTT-Q; the spacer sequence is a sequence with a length of 5-40 bp which is complementary to the RNA to be detected.

Drawings

FIG. 1 is a bar graph of fluorescence intensity for different classes of microRNAs;

FIG. 2 is a bar graph of fluorescence intensity for different concentrations of miRNA-221;

FIG. 3 is a standard curve.

Detailed Description

The present invention is further illustrated by the following specific examples, which will help those of ordinary skill in the art to more fully understand the invention, but are not intended to limit the invention in any way;

example 1:

preparation of single stranded guide RNA (sgRNA) -Cas14a complex: 500 nM sgRNA, 500 nM Cas14a and 100nM FQ probe in 25 mM NaCl, 20 mM Tris-HCl, 1 mM DTT and 10 mM MgCl ₂ Mixing with 30 ^o Incubating for 30 minutes;

sgRNA sequence (red underlined is the spacer sequence): UUC ACU GAU AAA GUG GAG AAC CGC UUC ACC AAA AGC UGU CCC UUA GGG GAU UAG AAC UUG AGU GAA GGU GGG CUG CUU GCA UCA GCC UAA UGU CGA GAA GUG CUU UCU UCG GAA AGU AAC CCU CGA AAC AAA UUC AUU UGA AAG AAU AAG GAA UGC AACCAACAUCAGUCUGAUAACU

FQ probe sequence: fam-TTTTTTTTTTTT-BHQ1

Example 2:

RNA qualitative analysis: 100nM microRNA-221 and microRNA-145 are added into the incubated sgRNA-Cas14 and FQ solution in the example 1, fluorescence detection is carried out in a 96-well plate, qualitative detection of corresponding RNA is realized by fluorescence signal change, water is used for replacing RNA as a blank control group, and each group of experiments is repeated three times (see FIG. 1);

microRNA-221 sequence: UAGCUUAUCAGACUGAUGUUGA

microRNA-145 sequence: GUCCAGUUUUUCCAGGAAUCCU

As can be seen from FIG. 1, the fluorescence signals of the experimental group containing microRNA-221 are enhanced drastically, while the fluorescence signals of the microRNA-145 and the blank group are unchanged, which indicates that the method can be used for qualitative analysis of RNA;

example 3:

quantitative analysis of RNA: the microRNA-221 with different concentrations is put into the incubated sgRNA-Cas14 and FQ solution in the embodiment 1, fluorescence detection is carried out in a 96-well plate, and corresponding RNA qualitative analysis is realized through the fluorescence intensities corresponding to the microRNAs with different concentrations (see fig. 2 and 3);

as can be seen from FIG. 2, the fluorescence signal gradually increases with increasing concentration of microRNA-221, and as can be seen from FIG. 3, the logarithmic value of microRNA-221 concentration and the fluorescence signal value show a good linear relationship, R ² = 0.99721, which indicates that the method can be used for quantitative analysis of RNA.

Claims

1. An RNA detection method based on Cas14a enzyme is characterized in that: adding RNA to be detected into a solution containing the sgRNA-Cas14a complex and a single-stranded DNA fluorescence quenching probe F-ssDNA-Q, and realizing quantitative and qualitative analysis of the RNA by using the change intensity of a fluorescence signal;

the sequence of the sgRNA is TTCACTGATAAAGTGGAGAACCGCTTCACCAAAAGCTGTCCCTTAGGGGATTAGAACTTGAGTGAAGGTGGGCTGCTTGCATCAGCCTAATGTCGAGAAGTGCTTTCTTCGGAAAGTAACCCTCGAAACAAATTCATTTgaaaGAATGAAGGAATGCAAC + interval sequence; the spacer sequence is CAACAUCAGUCUGAUAACU;

uniformly mixing sgRNA, cas14a and FQ probes in NaCl, tris-HCl, DTT and MgCl2, and then incubating;

the sequence of the single-stranded DNA fluorescence quenching probe F-ssDNA-Q is F- (T) n-Q (n is more than or equal to 5);

the RNA detection method based on the Cas14a enzyme aims at microRNA-221 sequence: UAGCUUAUCAGACUGAUGUUGA, fluorescence signal is enhanced drastically, and can be used for qualitative analysis of RNA;

in addition, in RNA quantitative analysis, the logarithmic value of the concentration of microRNA-221 and the fluorescence signal value show a linear relationship, R ² = 0.99721, which indicates that the method can be used for quantitative analysis of RNA;

the preparation method of the sgRNA-Cas14a complex comprises the steps of mixing the sgRNA and the Cas14a enzyme, and performing enzyme synthesis in a range of 0-60 percent ^o C, mixed incubation is carried out for 0-300 minutes;

the sgRNA concentration is 0-100mM; the Cas14a concentration is 0-100mM;

the spacer sequence is a sequence containing a length of 5-40 bp complementary to the RNA to be detected.