CN117143968A - Non-modified CRISPR/Cas12a multiple performance adjusting method - Google Patents

Abstract

The invention relates to flexible regulation of multiple performances of a CRISPR/Cas12a system, in particular to regulation of CRISPR/Cas12a activity and specificity, and a method for detecting and quantifying nucleic acid by a one-pot method based on CRISPR/Cas12a and Primer Exchange Reaction (PER). The invention utilizes an External RNA Accessory (ERA) kit to bind to a partial spacer of crRNA to form different toehold-containing double-stranded structures, thereby converting simple pairing binding of activator to crRNA into a conditional reaction determined by strand displacement. Attaching ERA of different structure, length and mismatch can change the kinetics and thermodynamics of ERA-crRNA complex reaction with activator, thereby precisely controlling the activation degree of Cas nuclease, and further controlling a series of properties of activity, speed, specificity, programmability, scalability, sensitivity, etc.

Description

Non-modified CRISPR/Cas12a multiple performance adjusting method

Technical Field

The invention relates to flexible regulation of multiple performances of a CRISPR/Cas12a system, in particular to regulation of CRISPR/Cas12a activity and specificity, and a method for detecting and quantifying nucleic acid by a one-pot method based on CRISPR/Cas12a and Primer Exchange Reaction (PER).

Background

The revolutionary technique of clustering regularly spaced short palindromic repeats (CRISPR) has deeply changed the development patterns of the molecular biology field including gene editing, intracellular imaging, transcriptional regulation, gene therapy, molecular diagnostics, construction of molecular biochemical loops, and the like. However, as the application range is increasingly expanded, the inherent performance of CRISPR/Cas alone is difficult to meet the diversified demands of increasingly sub-divided application fields, and more research is gradually focused on how to change the performance of CRISPR/Cas to better adapt to different demands. CRSIPR RNA (crRNA) responsible for recognition and Cas nuclease responsible for cleavage are core components of CRISPR/Cas, and there have been many efforts to achieve enhancement or attenuation of CIRSPR/Cas performance by modifying both components, but as research goes deep and demand grows, emerging short plates are blocking their next stage of development. First, it is difficult to develop new Cas nucleases or make editing changes by protein engineering, which is difficult to match with the rapid development of the segment application field. Modification of crrnas is relatively easy, but crrnas are designed with sequence-environmental dependencies. More importantly, the very high cleavage efficiency of CRISPR/Cas in a short time also makes the tunable range very narrow, most approaches can only control the "on/off" shift of CRISPR/Cas activity.

The popularity of global epidemic has deeply changed the paradigm of the nucleic acid detection field, during which the CRISPR/Cas12a detection strategy after isothermal nucleic acid amplification (INA) shows tremendous potential for molecular diagnostic applications. The SHERLOCK, DETECTR, HOLMESv, CRISDA and other systems based on this strategy have been used for high-sensitivity rapid detection of viruses, bacteria, DNA methylation, SNP typing and the like. However, separate nucleic acid pre-amplification procedures and multiple manual manipulation steps complicate the testing procedure and transfer of amplified products may also cause cross-contamination issues. However, if the CRISPR/Cas12a system is directly integrated with the INA, cas12a with strong back-cleavage capability will cleave not only the Cas-reporter, but also the primer and template. This natural incompatibility results in low detection efficiency of the "one-pot" method, which remains a challenge in clinical applications. Zhou Xiaoming et al temporarily inactivate Cas12a by introducing a PC-linker modified Protective oligonucleotide chain(s), re-using the light activated CRISPR program when the INA program in the same tube is over. This spatially continuous but temporally isolated strategy is more convenient, controllable, and may be a better solution, but still requires many chemical modifications and external stimuli.

Toehold mediated strand displacement reaction (TMSD) is an important basis for DNA dynamic nanotechnology. In TMSD, the invader strand binds to the toehold on the double-stranded substrate, initiates subsequent branching migration, and eventually replaces the protecting strand on the substrate, reaching the thermodynamically most stable state. TMSD follows precise watson-crick base pairing, not only with strict specificity but also with a very broad range of adjustability. A wide variety of modulation schemes have been developed to alter the rate, yield, specificity, etc. of TMSD reactions and these methods are generic and quite tolerant of systems of different sequence composition. In previous studies, we found that treatment of the activator into a double strand containing toehold was more specific, suggesting that there may be potential for further binding of TMSD to CRISPR/Cas.

Here we designed an External RNA Accessory (ERA) kit for Cas12a that would not activate Cas12a, but would bind to part of the spacer of the crRNA, forming a different toehold-containing double-stranded structure, thereby converting the simple pairing binding of the activator to the crRNA into a conditional response determined by TMSD. Attaching ERA of different structure, length and mismatch can change the kinetics and thermodynamics of ERA-crRNA complex reaction with activator, thereby precisely controlling the activation degree of Cas nuclease, and further controlling a series of properties of activity, speed, specificity, programmability, scalability, sensitivity, etc. In this work, we have studied the fine tuning method of CRISPR/Cas performance and explored the compatibility and heterogeneity of DNA dynamic nanotechnology with CRISPR/Cas systems. Interestingly, we also realized spatially continuous but time-isolated CRISPR activation control through ERA, thereby overcoming the problem of mutual rejection of CRISPR/Cas signal output module and amplification module in isothermal one-pot detection, improving sensitivity and signal-to-noise ratio of isothermal one-pot detection, and demonstrating great potential for fine control of CRISPR performance.

Disclosure of Invention

The invention aims to develop a non-modified CRISPR/Cas12a multiple performance regulating method, which is characterized by comprising the following steps of:

era-binding CRISPR/Cas12a

ERA is first mixed with crRNA and TE/Mg ²⁺ The solution was made up to a volume of 18 μl, annealed from 95 ℃ to 4 ℃ in a PCR instrument, then Cas12a was added and incubated for 1h at 25 ℃ to obtain ERA-crRBA-Cas12a complex.

2. Fluorescence detection and gel electrophoresis analysis flow

In testing the activation efficiency of CRISPR/Cas12aAdding a single-stranded activator with complete match or single base mismatch and rCutSmartbuffer, cas-reporter, DTT and ERA-crRBA-Cas12a complex incubated in step (1) into an EP tube, and using 12.5mmol/LTE/Mg ²⁺ The solution was made up to a volume of 20 μl; the fluorescence change (temperature 25 ℃) was then monitored in a Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (QIAGEN, germany); in the electrophoresis analysis, 15mL of 8% non-denaturing polyacrylamide gel electrophoresis is firstly prepared: 7.9mL of double distilled water, 4mL of 30% acrylamide, 3mL of TBE (5X), 0.11mL of 10% ammonium persulfate and 0.01mL of TEMED are added, and after being quickly and reversely mixed, the prepared liquid gum is poured into a gum bed and stored at 37 ℃ until the gel is solidified; fixing the gel on an electrophoresis tank, and adding 1 XTBE buffer; annealing crRNA and ERA at a ratio of 1:1 to obtain double-chain crRNA/ERA, adding corresponding single-chain activator, incubating at 37 ℃ for 30min, and mixing 10 mu L of reaction product with 2 mu L of loading buffer (6×); simultaneously, 10 mu L of 500N crRNA, a perfect matching activator (PM) and ERA are taken and mixed with 2 mu L of loading buffer (6X); taking 5 μl of the mixed sample and adding to the sample well; electrophoresis with Beijing Liuyi DYY-6C electrophoresis apparatus under 130V and 90mA conditions for 1.5 hr; finally, after 20min staining with 4S GelRed nucleic acid dye (1×), the gel was observed using a gel imaging system (Bio-Rad, USA).

ERA-assisted "one-pot" detection of CRISPR/Cas12a and PER

PER reaction system: 10nmol/L target, 1 XThermopol buffer, 10mmol/L MgSO ₄ Buffer solution, 10nmol/L of protective agent/hairpin template double-stranded probe, 200nmol/L of primer, 100 mu mol/L of dNTPs and 0.2Ubst large-fragment polymerase; 10×,2 μ L rCutSmart buffer, 5 μmol/L,1 μLCas-reporter (5 μmol/L,1 μL), 100mmol/L,0.2 μLDTT (100 mmol/L,0.2 μL) and 2 μmol/L,0.8 μL ERA-crRBA-Cas12a complex (2 μmol/L,0.8 μL) were added; 12.5mmol/L TE/Mg ²⁺ The solution was made up to a volume of 20 μl; the fluorescence change (temperature 37 ℃) was then monitored in a Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (QIAGEN, germany); and finally, determining the existence condition of the target and the signal to noise ratio through analysis.

The concentration and amount of ERA in the step (1) were 20. Mu. Mol/L and 2. Mu. L.

The concentration and amount of crRNA in step (1) was 20. Mu. Mol/L and 4. Mu.L.

TE/Mg in the step (1) ²⁺ The concentration of (C) was 12.5mmol/L.

The amount of Cas12a used in step (1) is 20. Mu. Mol/L, 2. Mu.L.

The amount of the single-stranded activator used in the step (2) is 100nmol/L and 8. Mu.L.

The amount of rCutSmartbuffer in step (2) was 10X, 2. Mu.L.

The amount of Cas-reporter in step (2) is 5. Mu. Mol/L, 1. Mu.L.

The amount of DTT used in the step (2) was 100mmol/L and 0.2. Mu.L.

The concentration of ERA-crRBA-Cas12a complex used in step (2) is 2. Mu. Mol/L, 0.2. Mu.L.

According to the invention, external RNA accessory kits (ERA) with different directions and different lengths are respectively designed according to the crRNA sequence, and the activity of the Cas12a is regulated and controlled through the length and the direction of the ERA. ERA binds to the spacer region of crRNA to form RNA double-stranded substrates with different lengths of toehold, and the exposed single-stranded region on crRNA-spacer serves as the toehold domain for target strand invasion (FIGS. 1-3). The PI domain of the Toehold near Cas12a protein is 5'Toehold and the PI domain near Nuc domain is 3' Toehold. As shown in fig. 1, the reverse cut rate of Cas12a is continuously slowed down with shortening of the toehold, indicating that the activity of Cas12a can be regulated by the length and direction of the toehold. The elimination of the mismatch by strand displacement can reduce the enthalpy, and the gibbs free energy change is negative, and the thermodynamic drive can also accelerate the kinetics of branch migration due to the coupling of kinetics and thermodynamics near toehold, namely, the branch migration after toehold combination. We continued to verify that the elimination of mismatches by ERA can provide a thermodynamic driver for ERA-based Cas12a activation, as shown in fig. 4-5, with the elimination of mismatches by ERA the rate of back-cut activation of Cas12a can be further enhanced.

CRISPR/Cas12a is often activated by mismatch activators to produce false positive signals, so that the improvement of the identification specificity of Cas12a is of great significance for further promoting the clinical application of CRISPR/Cas12 a. We designed four different location mismatched activators (MM-1, MM-2, MM-3, MM-4), introducing ERA in different orientations and lengths, and reducing the binding affinity of crRNA and activator by toehold-mediated strand displacement (TMSD) increased the sensitivity to mismatch (FIGS. 6-7). The closer the mismatch is to the toehold, the more pronounced the inhibition of ERA-controlled Cas12a activation. Toehold exchange strand displacement allows the number of hydrogen bond formations and breaks before and after the reaction to be substantially unchanged by introducing reverse Toehold, so that the net enthalpy change (ΔH) is small, which can be fine-tuned to positive, negative, or even zero by controlling the length and direction of ERA (forward (f) and reverse Toehold (r)), eliminating the high dependence of discrimination on mismatch positions (MM-1, MM-2, MM-3, MM-4) (FIGS. 8-9).

In the traditional one-pot method, CRISPR/Cas signal output and INA amplification are directly integrated, amplification and Cas activation are continuous in space and time, the activator yield is low enough to activate part of Cas12a to destroy the primer and the template indiscriminately at the initial stage of the amplification process, negative feedback is formed to the amplification process, and finally only lower signal output is realized.

Toehold can be used as a controller for delayed activation of Cas12a controlled by ERA, so that indiscriminate trans-cleavage of Cas12a is misplaced with INA program, time is strived for INA amplification of activation chain, and final activation efficiency is improved. Taking PER as an example (fig. 10), when ERA is not used, the reverse cleavage activity of Cas12a causes damage to the PER system, and the signal of a one-step method is obviously reduced; when ERA is used, trans-cleavage of Cas12a is delayed, and signal output by the one-step method is significantly improved with amplified dislocation of PER (fig. 11 to 12).

Drawings

FIG. 1 is a schematic diagram of the principle of ERA modulating Cas12a activity by length and direction

FIG. 2 is a representation of the chain-transfer feasibility of polyacrylamide gel electrophoresis

FIG. 3 is a real-time fluorescent amplification curve of ERA through length and direction modulation of Cas12a activity

FIG. 4 is a schematic representation of the principle of ERA modulating Cas12a activity by mismatch elimination

FIG. 5 is a dotted line graph of mismatch elimination on ERA modulating Cas12a activity

FIG. 6 is a schematic diagram of the principle of Cas12 a-specific modulation by TMSD

FIG. 7 is a discrimination factor-time heat graph of increasing Cas12a single base specificity by TMSD

FIG. 8 is a schematic diagram of the principle of Cas12 a-specific modulation by toehold exchange

FIG. 9 is a discrimination factor-time heat graph of increasing Cas12a single base specificity by toehold exchange

FIG. 10 is a schematic diagram of the principle of PER

FIG. 11 is a schematic illustration of ERA-assisted "one-pot" detection principle

FIG. 12 is a signal-to-noise ratio improvement for ERA supported "one-pot" detection

Detailed Description

A method for regulating multiple performances of unmodified CRISPR/Cas12a is developed, which is characterized by comprising the following steps:

era-binding CRISPR/Cas12a

ERA was first mixed with crRNA, the volume was made up to 18 μl with TE/mg2+ solution, annealed from 95 ℃ to 4 ℃ in PCR instrument, then Cas12a was added, and incubated for 1h at 25 ℃ to obtain ERA-crRBA-Cas12a complex.

2. Fluorescence detection and gel electrophoresis analysis flow

When testing the activation efficiency of CRISPR/Cas12a, adding a single-stranded activator with perfect match or single base mismatch to the EP tube with rCutSmartbuffer, cas-reporter, DTT and the ERA-crRBA-Cas12a complex incubated in step (1), the volume was made up to 20 μl with 12.5mmol/LTE/mg2+ solution; the fluorescence change (temperature 25 ℃) was then monitored in a Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (QIAGEN, germany); in the electrophoresis analysis, 15mL of 8% non-denaturing polyacrylamide gel electrophoresis is firstly prepared: 7.9mL of double distilled water, 4mL of 30% acrylamide, 3mL of TBE (5X), 0.11mL of 10% ammonium persulfate and 0.01mL of TEMED are added, and after being quickly and reversely mixed, the prepared liquid gum is poured into a gum bed and stored at 37 ℃ until the gel is solidified; fixing the gel on an electrophoresis tank, and adding 1 XTBE buffer; annealing crRNA and ERA at a ratio of 1:1 to obtain double-chain crRNA/ERA, adding corresponding single-chain activator, incubating at 37 ℃ for 30min, and mixing 10 mu L of reaction product with 2 mu L of loading buffer (6×); simultaneously, 10 mu L of 500N crRNA, a perfect matching activator (PM) and ERA are taken and mixed with 2 mu L of loading buffer (6X); taking 5 μl of the mixed sample and adding to the sample well; electrophoresis with Beijing Liuyi DYY-6C electrophoresis apparatus under 130V and 90mA conditions for 1.5 hr; finally, after 20min staining with 4S GelRed nucleic acid dye (1×), the gel was observed using a gel imaging system (Bio-Rad, USA).

ERA-assisted "one-pot" detection of CRISPR/Cas12a and PER

PER reaction system: 10nmol/L target, 1×Thermopol buffer, 10mmol/LMgSO4 buffer, 10nmol/L protector/hairpin template double-stranded probe, 200nmol/L primer, 100. Mu. Mol/L dNTPs and 0.2Ubst large fragment polymerase; 10×,2 μLrCutSmartbuffer, 5 μmol/L,1 μLCas-report (5 μmol/L,1 μL), 100mmol/L,0.2 μLDTT (100 mmol/L,0.2 μL) and 2 μmol/L,0.8 μLERA-crRBA-Cas12a complex (2 μmol/L,0.8 μL) were added; the volume was replenished to 20 μl with 12.5mmol/LTE/mg2+ solution; the fluorescence change (temperature 37 ℃) was then monitored in a Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (QIAGEN, germany); and finally, determining the existence condition of the target and the signal to noise ratio through analysis.

The concentration and amount of ERA in the step (1) were 20. Mu. Mol/L and 2. Mu. L.

The concentration and amount of crRNA in step (1) was 20. Mu. Mol/L and 4. Mu.L.

TE/Mg in the step (1) ²⁺ The concentration of (C) was 12.5mmol/L.

The amount of Cas12a used in step (1) is 20. Mu. Mol/L, 2. Mu.L.

The amount of the single-stranded activator used in the step (2) is 100nmol/L and 8. Mu.L.

The amount of rCutSmartbuffer in step (2) was 10X, 2. Mu.L.

The amount of Cas-reporter in step (2) is 5. Mu. Mol/L, 1. Mu.L.

The amount of DTT used in the step (2) was 100mmol/L and 0.2. Mu.L.

The concentration of ERA-crRBA-Cas12a complex used in step (2) is 2. Mu. Mol/L, 0.2. Mu.L.

Claims (10)

1. A method for modifying multiple performance of CRISPR/Cas12a without modification, comprising the steps of:

(1) ERA binding CRISPR/Cas12a

ERA is first mixed with crRNA and TE/Mg ²⁺ The volume of the solution was made up to 18 μl, annealed from 95 ℃ to 4 ℃ in a PCR instrument, then Cas12a was added and incubated for 1h at 25 ℃ to obtain ERA-crRBA-Cas12a complex;

(2) Fluorescence detection and gel electrophoresis analysis flow

When testing the activation efficiency of CRISPR/Cas12a, adding a perfectly matched or single base mismatched single-stranded activator with rCutSmartbuffer, cas-reporter, DTT and ERA-crRBA-Cas12a complex incubated in step (1) to an EP tube, using 12.5mmol/LTE/Mg ²⁺ The solution was made up to a volume of 20 μl; the fluorescence change (temperature 25 ℃) was then monitored in a Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (QIAGEN, germany); in the electrophoresis analysis, 15mL of 8% non-denaturing polyacrylamide gel electrophoresis is firstly prepared: 7.9mL of double distilled water, 4mL of 30% acrylamide, 3mL of TBE (5X), 0.11mL of 10% ammonium persulfate and 0.01mL of TEMED are added, and after being quickly and reversely mixed, the prepared liquid gum is poured into a gum bed and stored at 37 ℃ until the gel is solidified; fixing the gel on an electrophoresis tank, and adding 1 XTBE buffer; annealing crRNA and ERA at a ratio of 1:1 to obtain double-chain crRNA/ERA, adding corresponding single-chain activator, incubating at 37 ℃ for 30min, and mixing 10 mu L of reaction product with 2 mu L of loading buffer (6×); simultaneously, 10 mu L of 500N crRNA, a perfect matching activator (PM) and ERA are taken and mixed with 2 mu L of loading buffer (6X); taking 5 μl of the mixed sample and adding to the sample well; electrophoresis with Beijing Liuyi DYY-6C electrophoresis apparatus under 130V and 90mA conditions for 1.5 hr; finally, after 20min staining with 4S GelRed nucleic acid dye (1×), the gel was observed using a gel imaging system (Bio-Rad, usa);

(3) ERA-assisted "one-pot" detection of CRISPR/Cas12a and PER

PER reaction system: 10nmol/L target, 1 XThermopol buffer, 10mmol/L MgSO ₄ Buffer solution, 10nmol/L protective agent/hairA template double-stranded probe, 200nmol/L primer, 100 mu mol/L dNTPs and 0.2Ubst large fragment polymerase; 10×,2 μ L rCutSmart buffer, 5 μmol/L,1 μLCas-reporter (5 μmol/L,1 μL), 100mmol/L,0.2 μLDTT (100 mmol/L,0.2 μL) and 2 μmol/L,0.8 μL ERA-crRBA-Cas12a complex (2 μmol/L,0.8 μL) were added; 12.5mmol/L TE/Mg ²⁺ The solution was made up to a volume of 20 μl; the fluorescence change (temperature 37 ℃) was then monitored in a Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (QIAGEN, germany); and finally, determining the existence condition of the target and the signal to noise ratio through analysis.

2. The DNA dynamic nanotechnology based external RNA accessory kit of claim 1, steplessly modulating multiple performance strategies of CRISPR/Cas12 a.

3. The precise control strategy of CRISPR/Cas12a without modification of claim 1, wherein the concentration and amount of crRNA in step (1) of the preparation method is 20 μmol/L,4 μl.

4. The precise control strategy of CRISPR/Cas12a without modification according to claim 1, which is prepared by the process of TE/Mg in step (1) ²⁺ The concentration of (C) was 12.5mmol/L.

5. The precise control strategy of CRISPR/Cas12a without modification of claim 1, wherein Cas12a is used in the preparation method step (1) in an amount of 20 μmol/L,2 μl.

6. The precise control strategy of CRISPR/Cas12a without modification of claim 1, wherein the amount of single-stranded activator used in step (2) of the preparation method is 100nmol/L,8 μl.

7. The CRISPR/Cas12a precise control strategy without modification according to claim 1, wherein the rCutSmartbuffer is used in step (2) of the preparation method in an amount of 10×,2 μl.

8. The precise control strategy of CRISPR/Cas12a without modification of claim 1, wherein the Cas-reporter is used in step (2) of the preparation method in an amount of 5 μmol/L,1 μl.

9. The precise control strategy of CRISPR/Cas12a without modification according to claim 1, wherein the amount of DTT used in step (2) of the preparation method is 100mmol/L,0.2 μl.

10. The precise CRISPR/Cas12a control strategy without modification according to claim 1, which is prepared by the method step (2) in which ERA-crRBA-Cas12a complex is used in a concentration of 2 μmol/L,0.2 μl.