Temperature-controlled amplification combined with CRISPR applications

● The target nucleic acid was enriched by constant temperature (MIRA) amplification.

● The amplified product binds to Cas12 crRNA, activating the accessory cleavage function of Cas12 (trans cleavage), and cleaves the ssDNA report probe.

● The fluorescence probe is cut to release fluorescence signals, which can be detected by a fluorescence meter or observed by the naked eye under blue light.

● The colloidal gold probe is cut to release corresponding groups, and the results can be observed through a test strip.

Principle of MIRA and Cas12a detection system:

The target gene is enriched by constant temperature amplification, Cas12-crRNA complex recognizes the target gene, activates the cutting activity of Cas protein, which cuts the reporter probe and releases the fluorescent model, and the result reading can be fluorescence curve, naked eye observation under blue light lamp; the colloidal gold probe is cut, it can be determined by the result of the coloring method of the test strip.

● The amplification product of MIRA basic amplification is a double stranded DNA sequence containing the T7 promoter.

● By converting T7 transcriptase into ssRNA, the Cas13a crRNA complex recognizes the target ssRNA, activates transcleavage activity, and cleaves the report probe.

● The fluorescence probe is cut to release fluorescence signals, which can be detected by a fluorescence meter or observed by the naked eye under blue light.

● The colloidal gold probe is cut to release corresponding groups, and the results can be observed through a test strip.

Principle of MIRA and Cas12a detection system:

The target gene is enriched by constant temperature amplification, Cas12-crRNA complex recognizes the target gene, activates the cutting activity of Cas protein, which cuts the reporter probe and releases the fluorescent model, and the result reading can be fluorescent curve, blue.

MIRA combined with Cas13a detection principle:

Enrich the target gene product by constant temperature amplification, the difference with Cas12 front-end amplification is that the constant temperature amplification product of Cas13a front-end has T7 promoter sequence, which is transcribed into single-stranded RNA by T7 transcriptase, and this single-stranded RNA is recognized by Cas13-crRNA complex, and the cleavage activity of Cas13 is activated, which cleaves the reporter probe. The result presentation is fluorescent, naked-eye interpretation, and colloidal gold like Cas12.

Naked eye observation under the light lamp; colloidal gold probe is cut, it can be determined by the test strip color development method results.

● Primer screening is the key to establishing the MIRA+CRISPR system.

● The sensitivity of the CRISPR detection system is determined by the basic amplification sensitivity of MIRA.

The sensitivity of different primer combinations varies, and the optimal combination is F1+R1

Whether it is Cas12 or Cas13 detection can not be separated from the front-end nucleic acid amplification, so the front-end MIRA-based nucleic acid amplification is crucial, and at the same time, the sensitivity of the front-end nucleic acid amplification determines the sensitivity of the back-end Cas protein detection.

How to improve the sensitivity of front-end MIRA amplification?

First of all, the most basic is the primer screening, different primer combinations amplification efficiency, sensitivity is different, must be screened to the optimal primer combination before entering the Cas system detection. There are fluorescent type, naked eye reading, colloidal gold type and so on.

Naked eye observation under the light lamp; colloidal gold probe is cut, it can be determined by the test strip color development method results.

MIRA basic amplification: Incubate at 42 ℃ for 20 minutes.

Obtaining amplification products

Fluorescence collection: 37 ℃, 30s/cycle, 20min.

System preparation for MIRA+cas12a

MIRA amplification system, including: buffer buffer, upstream and downstream primers, template, start buffer, water supplemented to 50ul system

Cas12a cutting system, including: buffer Buffer, Cas12a protein, thermostatic amplification product, crRNA, etc.

Note: MIRA thermostatic amplification product is directly added to the cas protein cutting system without any treatment.

MIRA+cas13a system preparation

MIRA basic amplification system, including: buffer buffer, upstream and downstream primers, templates, starter buffer, and water supplemented to 50ul system.

Cas13a system including: cas13a protein, thermostable amplification product, crRNA, reporter probe, RNAase inhibitor, buffer required for transcription, T7 transcriptase, auxiliaries needed for A/C/G/U transcription, and so on.

Again, the MIRA amplification product can be added directly to the Cas13a cutting system without any processing.

Comparison of the two cutting systems:

Cas12a requires a PAM region with TTTV, cas13a does not;

The cas12a protein is activated for cleavage activity by double-stranded DNA, Cas13a is activated for cleavage activity by single-stranded RNA;

cas12a cleaves single-stranded DNA and therefore the reporter probe is designed for single-stranded DNA;

Similarly cas13a cleaves single-stranded RNA and the reporter probe is designed for single-stranded RNA;

Front-end MIRA amplification, the primer design difference is that Cas13a needs to be added to the T7 promoter sequence in the upper primer; the final test strips used in the test strips are CRISPR-specific test strips, rather than ordinary nucleic acid detection test strips.

MIRA combined with CRISPR detection application scenarios are diversified and can be applied to many aspects such as community hospitals, community monitoring stations, customs and quarantine, home self-testing, etc., as it is not constrained by temperature conditions, equipment limitations and environmental conditions.

Impact factor: 29.234

Content Introduction:

Using Cas12a's suboptimal PAM to quickly and sensitively detect SARS-COV-2 RNA.

The MIRA+Cas12 one tube method, named SPAMC, has the advantages of reducing cover opening, preventing pollution, high sensitivity, and short detection time.

Published by Mr. Yin Hao's group at Wuhan University, a platform for MIRA combined with cas12 one-tube assay was established.

Suboptimal PAM

Cas12a cis cutting activity ↓

Stable temperature amplification of substrates in a short period of time ↑

Sensitivity and reaction speed ↑

In this article, a series of validations were done to improve the sensitivity and detection time of the one-tube method using suboptimal PAM regions.

Speculation 1: In the one-tube assay reaction, CRISPR detection, i.e., Cas protein cleavage and thermostatic amplification, may have a competitive relationship, and the strong binding of standard PAM to cas protein biases this competitive relationship in favor of cas protein cleavage, resulting in delayed or stopped amplification due to the lack of thermostatic amplification substrate, which ultimately leads to delayed signal and decreased sensitivity.

How to reduce Cas protein binding activity?

Switching to suboptimal PAM leads to the following inference?

Suboptimal PAM → cas12a cis-cleavage activity is reduced → more cas protein is activated by the increase of thermostatic amplification substrate in a short period of time → sensitivity and reaction speed are increased.

The standard PAM is TTTV (V represents A/C/G), and the suboptimal PAM area is VTTV.

Two-step method

One tube method

For example, the Orf1ab gene showed better VTTV results in the suboptimal PAM region compared to TTTV in the one tube reaction.

Suboptimal PAM: GTTG, CTTA

Standard PAM: TTTG, TTTA

In order to verify the above speculation, the authors explored the suboptimal PAM; first of all, what would be the result if the first nucleotide of the standard PAM is changed to other?

The authors took the new crown Orf gene as an example, and carried out two-step and one-step data validation on the suboptimal PAM with the first nucleotide changed, and found that the standard PAM was superior to the suboptimal PAM in the two-step method, and the suboptimal PAM was detected better than the standard PAM in the one-step method, which indicated that the change of the first nucleotide of the standard PAM was meaningful for the one-tube method.

So is the 2nd/3rd/4th nucleotide change of standard PAM superior?

Is there any regularity in PAM base changes?

Exploring the second nucleotide of PAM, the conventional PAM region TTTA, and the second best PAM are TATA, TGTA, and TCTA

Test results: The second nucleotide is T/C (pyrimidine), and the one tube reaction is faster and the fluorescence intensity is higher; On the contrary, G/A (purine) results in weaker detection.

The second nucleotide of the standard PAM was also changed and verified, and the T was changed to A/G/C, and it was found that the one-tube method reacted better when the second base was C. The second nucleotide of the standard PAM was also changed and verified.

A series of verification was also done on the change of the 3rd/4th nucleotide of standard PAM, and it was found that there is also a certain pattern, and the specific results can be seen in Mr. Yin Hao's article.

All of the above validations are based on the data derived from the new crown of the ORF gene, E gene, N gene as the detection of genes, only for the RNA type nucleic acid test can illustrate this law?

The authors performed a DNA type nucleic acid test here selected HPV18 to verify this pattern and found it to be applicable. See the article for details of the results.

After a large number of tests, it was finally concluded that more than 80% of the suboptimal PAMs would have enhanced fluorescence signal intensity and shorter detection time in the one-tube reaction. The optimal suboptimal PAM is VTTV, and the second optimal is TCTV.

It is verified that the suboptimal PAM can enhance the fluorescence model and shorten the detection time, so it must have some effect on the sensitivity!

SARS-CoV-2: Orf1ab gene as an example

Suboptimal PAM: Sensitivity 0.234fM

Standard PAM: Sensitivity 2.34fM

Suboptimal PAM: Sensitivity 0.3255fM

Standard PAM: Sensitivity 32.55fM

The authors next performed some sensitivity tests and found that sub-optimal PAMs can also be 10-100 times more sensitive compared to standard PAMs.

HCMV virus sensitivity detection

The authors also did a sensitivity test with an actual sample of human cytomegalovirus and also compared the sensitivity with QPCR, which was 10 times better than Qpcr.

Fluorescence curve detection of high sensitivity whether the same naked eye can be observed, the authors carried out the application of the expansion, found that the reaction 15-20 minutes naked eye reading sensitivity of 24cp / reaction, and the sensitivity of Qpcr is consistent.

Colloidal gold strips were also tested, and the sensitivity was slightly worse compared to fluorescence.

All the experiments done by the authors in the front-end were performed with MIRA kit, and they also considered whether this method is applicable to other thermostatic amplification kits, so they made a comparison between AmpFuture kit and TwistDx kit:.

There was almost no difference in the sensitivity of the front-end basic amplification test, but the electrophoretic data of AmpFuture kit had clearer and brighter bands;

We also performed the thermostatic amplification + Cas12a one-tube assay, and the result was that our kit was better than the TwistDx kit in terms of sensitivity and fluorescence detection intensity.

The final conclusion is that MIRA reagent is more compatible with Cas12a buffer environment.

The established sPAMC detection method was compared with other detection methods, the advantage lies in; the reaction of a tube method, do not need other conditions to limit the higher sensitivity, up to 1cp/ul, the reaction time is shorter 15-20min.

The whole article speaks down the author is to do a lot of experimental verification, and do the results are better, summarized in the following points: 1, high sensitivity, short detection time, the actual project tested human cytomegalovirus, new coronavirus, sensitivity and QPCR comparable to the detection time in 10-15min.

2, the authors also focused on verifying the better compatibility of our reagents with cas proteins.

Impact factor: 20.693

Content Introduction: MIRA combined with Cas13a has established a colloidal gold strip assay (LFA) for the detection of SARS-CoV-2.

The second article is "MIRA combined with cas13a dual colloidal gold test strip assay", which will hopefully shed some light on our subsequent research.

Experimental procedure: nucleic acid extraction → MIRA base amplification enrichment substrate → T7 transcriptase transcribes double-stranded DNA into single-stranded RNA, cas13a-crRNA complex recognizes and activates the cleavage activity → cleavage reporter probe (fluorescence assay/test strip assay)

Optimization of the detection system, in addition to the MIRA front-end thermostatic amplification primer screening that has been emphasized before, there are some optimizations of the CRISPR system, first of all, you need to screen the crRNA, and from the results, you can see that different crRNA sequences will also have a certain impact on the detection. Here, the new crown N gene, N-8, was chosen as the optimal crRNA.

Optimization of conditions for CRISPR fluorescence detection methods

- Probe selection: probe1

- Final concentration of MgCl 2: 20uM

- T7 RNA polymerase: 1.5U/uL

- RNase inhibitor: 0.8U/uL

- Taurine: 10uM

- MIRA primer concentration: 5uM

The authors dual colloidal gold test strip detection principle is where we are more concerned, and also may provide some new ideas with us:

The T line detects the N gene, and the C2 line serves as a quality control line for detecting the internal reference.

Here the N gene is amplified by MIRA and then recognized and cleaved by cas13a, and detected by T line with high sensitivity;

The internal reference gene is amplified by MIRA, and the amplified product carries digoxin and TAMRA, which is detected by C2 line;

The significance of introducing the internal reference is to ensure the validity of the sample and prevent false negatives.

So what is the result of this design detection?

Dual test strip results showed sensitivity at 0.25cp/UL with no cross-reactivity and good specificity.

A fluorescent type test was also done and the sensitivity was also at 0.25cp/UL with no cross-reactivity and good specificity.

The significance of the final article is that the sensitivity is high, it can be used for early screening determination, ensure the validity of the sample, and at the same time, this immunochromatographic method does not need to be used with the instrumentation, which reduces the cost, and can be carried out in a simple environment.

After more than ten years of technical precipitation, Amp-future bio-team has developed Multienzyme Isothermal Rapid Amplification (MIRA), which represents the future trend of bio-assay technology. We have also developed "Ultra Rapid Nucleic Acid Release Technology", "Dual-Channel Constant Temperature Fluorescence Detector", and "All-in-One Fully-Automated Closed Detection System" with MIRA as the core platform technology, "Self-testing nucleic acid detection device" and a series of ancillary products, and plans to launch "microfluidic chip technology", "small automated detection system" and other aspects of the new products, and strive to We are planning to launch new products such as "microfluidic chip technology" and "small automated detection system", etc. We will strive to create a total solution in the field of rapid molecular detection, and fully realize the features of MIRA technology, such as high sensitivity, high specificity, speed and simplicity, low risk of contamination, and on-site operation.