CN111257297A

CN111257297A - RNA detection method based on Cas14a enzyme

Info

Publication number: CN111257297A
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: 2020-06-09
Anticipated expiration: 2040-03-31
Also published as: CN111257297B

Abstract

The invention discloses a method for activating Cas14a subsidiary cleavage effect by RNA, and particularly relates to the fields of analytical chemistry and rapid disease diagnosis. The RNA detection is realized by utilizing the accessory cleavage effect of the RNA activated Cas14a to cleave the ssDNA fluorescent probe. The invention solves the problem that the cutting probe is RNA when the Cas system detects RNA, and enriches the range of the application and detection field of Cas14 a.

Description

RNA detection method based on Cas14a enzyme

Technical Field

The invention relates to a 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) is a system used by bacteria to protect themselves against viruses [1 ]. The CRISPR-associated CRISPR-Cas system effector proteins currently found fall into two broad categories [2 ]. Wherein Cas9, tracrRNA and guide crRNA form an effector complex to cut the target DNA [3 ]; cas12a does not require tracr RNA, it uses crRNA alone as a guide to introduce staggered cleavage into the target double-stranded DNA, the attendant cleavage activity of Cas12a can be activated once the target is cleaved, and DNA single strand of the TTATT sequence can be cleaved [4 ]; cas13a is an RNA-guided RNase that can be activated as a non-specific RNase by the "nicking" activity of Cas13a once the RNA target binds to the sgRNA [5 ]. Based on this property of activating collateral cleavage, the CRISPR-Cas system is widely used for the detection of DNA, RNA and pathogenic microorganisms. The CRISPR-Cas14 enzyme is a novel CRISPR-Cas family protein discovered recently, has small molecular weight and has the activity of targeted DNA cleavage, and the invention can correspondingly provide an RNA-activated Cas14a 'additional cleavage' activity to realize the detection of RNA, and has important significance to 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-Cassystems. Nat Rev Microbiol 2017, 15 (3), 169-182.

[3]Chen, J. S.; Doudna, J. A., The chemistry of Cas9 and its CRISPRcolleagues. 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 withCRISPR–Cas13. Nature 2017, 550 (7675), 280-284。

disclosure of Invention

The invention aims to solve the problem of RNA detection by a CRISPR-Cas system, and provides a method for detecting RNA based on Cas14a enzyme, which adopts the following technical scheme for realizing the aim:

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 is to mix sgRNA and Cas14a enzyme at 0-60%^oC, mixing and incubating for 0-300 minutes; the concentration of the sgRNA is 0-100 mM; the Cas14a concentration is 0-100 mM; the sequence of the sgRNA is UUC ACU GAUAAA GUG GAG AAC CGC UUC ACC AAA AGC UGU CCC UUA GGG GAU UAG AAC UUG AGU GAAGGU GGG CUG CUU GCA UCA GCC UAA UGU CGA GAA GUG CUU UCU UCG GAA AGU AAC CCUCGA AAC AAA UUC AUU UGA AAG AAU AAG GAA UGC AAC + spacer sequence; the sequence of the single-chain DNA fluorescence quenching probe (F-ssDNA-Q) is F- (T)_n-Q (n.gtoreq.5), such as n =5, the sequence length being F-TTTTT-Q; the spacer sequence is a sequence with a length complementary with the RNA to be detected by 5-40 bp.

Drawings

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

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

FIG. 3 is a standard curve.

Detailed Description

The present invention is further illustrated by the following specific examples, which are intended to provide a more complete understanding of the invention by one of ordinary skill in the art, but are not intended to be limiting in any way;

example 1:

preparation of single stranded guide rna (sgrna) -Cas14a complex: 500 nM sgRNA, 500 nM Cas14a, and 100nM FQ probes in 25 mM NaCl, 20 mM Tris-HCl, 1 mM DTT, and 10 mM MgCl₂Mixing with 30 deg.C^oC, incubation for 30 minutes;

sgRNA sequence (spacer in underlined red): UUC ACU GAU AAA GUG GAG AAC CGC UUC ACCAAA AGC UGU CCC UUA GGG GAU UAG AAC UUG AGU GAA GGU GGG CUG CUU GCA UCA GCCUAA UGU CGA GAA GUG CUU UCU UCG GAA AGU AAC CCU CGA AAC AAA UUC AUU UGA AAGAAU AAG GAA UGC AACCAACAUCAGUCUGAUAACU

FQ probe sequence: Fam-TTTTTTTTTTTT-BHQ1

Example 2:

RNA qualitative analysis: adding 100nM microRNA-221 and microRNA-145 into the sgRNA-Cas14 and FQ solution incubated in example 1, performing fluorescence detection in a 96-well plate, realizing qualitative detection of corresponding RNA by using fluorescence signal change, using water to replace RNA as a blank control group, and repeating each group of experiments three times (see figure 1);

microRNA-221 sequence: UAGCUUAUCAGACCUGAUUGUAGA

The sequence of microRNA-145: GUCCAGUUUCCCCAGGAAUCCCU

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

example 3:

RNA quantitative analysis: putting microRNAs-221 with different concentrations into sgRNA-Cas14 and FQ solutions incubated in example 1, performing fluorescence detection in a 96-well plate, and realizing qualitative analysis of corresponding RNAs through the fluorescence intensities corresponding to the microRNAs with different concentrations (see figures 2 and 3);

as can be seen from FIG. 2, the fluorescence signal is gradually enhanced with the increase of the concentration of the microRNA-221, and as can be seen from FIG. 3, the logarithm of the concentration of the microRNA-221 and the fluorescence signal show a good linear relationship, R²=0.99721, indicating that the method can be used for quantitative analysis of RNA.

Claims

1. A method for detecting RNA based on Cas14a enzyme, which is characterized in that: 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.

2. The method of claim 1 for detecting RNA based on Cas14a enzyme, wherein the method comprises the following steps: the preparation method of the sgRNA-Cas14a complex is to mix sgRNA and Cas14a enzyme at 0-60%^oC, mixing and incubating for 0-300 minutes.

3. The method of claim 1 for detecting RNA based on Cas14a enzyme, wherein the method comprises the following steps: the concentration of the sgRNA is 0-100 mM; the Cas14a concentration was 0-100 mM.

4. The method for activating CRISPR-Cas14a enzyme collateral cutting effect by RNA according to claim 1, characterized in that: the sequence of the sgRNA is TTCACTGATAAAGTGGAGAACCGCTTCACCAAAAGCTGTCCCTTAGGGGATTAGAACTTGAGTGAAGGTGGGCTGCTTGCATCAGCCTAATGTCGAGAAGTGCTTTCTTCGGAAAGTAACCCTCGAAACAAATTCATTTgaaaGAATGAAGGAATGCAAC + spacer sequence.

5. The method for activating CRISPR-Cas14a enzyme collateral cutting effect by RNA according to claim 1, characterized in that: 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), if n =5, the sequence length is F-TTTTT-Q.

6. The sgRNA sequence according to claim 5, characterized in that: the spacer sequence is a sequence with a length complementary to the RNA to be detected and 5-40 bp.