Overview

Our ultimate goal is to build a single-base, amplification-free, portable set of detection platforms. In the engineering success page, we first need to implement the most important and experimentally verified part, the single-base accuracy part. We learned from our review and experiments that Cas13a and Cas14a can tolerate 1-2 base mutations in target sequence recognition, specifically, if the target RNA or DNA has single nucleotide polymorphism in the detection system, it will lead to unclear signal source (from target sequence or mutated sequence), and subsequently will produce a "false positive "false positive" characterization.

To address this problem, we thought of combining single base mutation sequence with complementary paired CLAMP to shield the mutation sequence and enable Cas protein to recognize the target sequence more accurately. At the same time, to ensure the stability of the CLAMP-mutation sequence, we will use peptide nucleic acid (PNA) as the backbone of CLAMP, which will reduce the intensity of the "false positive" signal and make it easier for us to identify the source of the signal.

To achieve amplification-free detection, we will use the trans cleavage activity of Cas13a and Cas14a to cleave and degrade the labeled nucleic acid by coupling with TtCsm6 and Csm6 activator (A4-U6 oligonucleotide) to generate fluorescent signals (through the linkage between the proteins to transmit and amplify the signal in the process), coupled with the base complementary pairing PNA "CLAMP" to shield the single base mutation sequence, this detection system has a specificity, sensitivity and high efficiency that no single protein detection means has.

Engineering Cycle

CYCLE1：

Expression of cas and csm6 proteins.

parts:BBa_K4223018、BBa K4223008

1.1 DESIGN：

His tag-MBP-Cas14a9 (BBa_K4223018) makes an important part of our project and we use it for efficient and high quality Cas14a protein expression and purification extraction. MBP helps to increase the yield of soluble protein in E. coli, and TEV is used as an enzymatic site to purify MBP-Cas14a recombinant protein after His tag binding to a Ni column, which retains the pure Cas14a protein and passed through the heparin column.

1.2 BUILD

His tag-MBP-Cas14a1 purification Day1 1.Preparation of LB medium 500ml*4 2.Shake the bacteria to recover. Take LB medium 3ml*2 (TEV,MBP) + bacteria night 30μl + antibiotic (Amp) ampicillin 3μl Constant temperature shaker 37℃ 180rpm 12-16h Day2 1.Sterilization of culture medium 2. Expansion, induction. Take sterilized LB medium*4 (TEV*2,MBP*2) +500μl of antibiotics (Amp) +2ml of the night before the bacteria Shake at 37℃ 180rpm for 6-8h Shaken TEV and MBP TEV+IPTG 50μl*2 37℃ 180rpm 12h MBP+IPTG 100μl*2 18℃ 180rpm 12h Day3 1. Bacteria-breaking lysis. Take TEV, MBP high-speed centrifugation 10000 rpm 5min, discard supernatant add lysis solution 5ml / tube (operation in the ice box, the action should be fast), blow well, three tubes in one tube Use ultrasonic crushing bacteria 25% power on 4s off 10s working time 50-60min 2. Preparation. Purified water equilibrium solution eluent (wash once) 20% ethanol sequential extraction on the machine, wash the machine (full water wash once) on the HIS nickel column (1ml/min), wash the column, the tube into the corresponding reagent bottle syringe filter membrane sample 3. Purification of proteins. TEV and MBP max speed 5ml/min A1 pure water A2 equilibrium solution B eluent A3 20% ethanol When the machine is turned off, water wash once, unload the column (1ml/min) ethanol wash once

4. Measure protein concentration (kit) Measure protein concentration (kit), 96-well plate, 100ul BCA working solution per well, 1ul Cu reagent 10ul protein, two protein two-well, measure absorbance, about 0.4 is normal value

5. Mix digestion with protein concentration 1:1 equal volume digestion (TEV slightly more) two tubes mixed, 4 ° c overnight Day4. 1. Protein purification Ultra-filter tube, with filtered water, centrifuge 3800r 30min twice Equilibrium solution 30min twice Top sample, centrifugation, 30min 3800r until protein is consumed, replenish equilibrium solution 3 times Loading the machine Wash the machine as before, wash the column to wash the equilibrium solution once more, heparin column 1ml/min (pay special attention to sample evacuation)

Csm6 purification Day1 1.Preparation of LB medium 500ml*2 Sterilization 2.Shake the bacteria to recover. Take LB medium 3ml*2 +bacterial night 30μl+carboxymycin 3μl Constant temperature shaker 37℃ 180rpm 12-16h Day2 1. Expansion, induction. Take sterilized LB medium + 500μl of caramycin + 3ml of ante-night bacterium night Shaking bed 37℃ 180rpm 6-8h Shake the good bacteria solution Csm6+IPTG 100μl 18℃ 180rpm 12h Day3 1. Bacteria breaking lysis. Take the bacterium solution high-speed centrifugation 10000rps 5min, discard the supernatant add lysate, blowing uniformly, three tubes in one tube Use ultrasonic crushing bacteria 25% power on 4s off 10s working time 50-60min 2. Preparation. Pure water equilibrium solution eluent (wash once) 20% ethanol sequential extraction on the machine, cleaning machine (full water wash once) on the HIS nickel column (1ml/min), wash the column, the tube into the corresponding reagent bottle syringe filter membrane sample 2. Collect the sample Run gel verification SDS-page protein gel electrophoresis 1.Gel preparation 2.Sample processing 3.Add maker and sample 4.Add running buffer to electrophoresis according to the procedure 5.Staining of gel plate Decolorization 6.Observation

1.3 TEST

To further examine the protein activity of Cas14a and Csm6,

we set up the system without the addition of target DNA as the control group and the system with the addition of target DNA as the experimental group. The fluorescence assay was performed on 4 groups of samples at 37°C using an enzymatic standard (excitation wavelength 492 nm, emission wavelength 520 nm). Three parallel samples of each group were measured and the variance and mean were calculated and plotted with Origin 9.0. As shown in the figure, the validation of the cleavage activity of cas14a1 and csm6 proteins was finally completed.

1.4 LEARN

Here, we successfully expressed Cas14a and csm6 proteins, which were verified to be ready for use after activity. However, we did not get the expected results when expressing Cas13a protein, and the expressed Cas13a concentration was very low, which was not improved after several attempts to change the conditions of different reaction systems, so we considered asking other teams for help or purchasing Cas13a protein in future experiments.

We will then perform a false positive test in the next step

to complete the proof of concept of the experimental idea done in the wet experiment.

CYCLE2：

False-positive tests

2.1 DESIGN：

In the previous cycle, we successfully expressed the desired Cas and Csm6 proteins and presented the problem with a corresponding solution. We will then confirm the "false positive" characterization in the next cycle to determine the interference of the mutant sequence with the clear signal source when the target sequence coexists with the mutant sequence in the reaction system.

We designed two 22-base-long target ssDNA sequences and target ssRNAs, respectively, and both designed 12 sequences with inversions at odd sites, respectively, as mutant sequences in our laboratory:

2.2 BUILD

We took the target and mutant sequences and added them to the reaction system of Cas13a and Cas14a, which was carried out as follows.

Cas14a Reaction Buffer (20 mM tris-hcl, 20 mM NaCl, pH 9.0) 15 uL: Cas14a （final 500nM） &sgRNA (final 500nM) in Reaction buffer ; 6 uL: Mg ion ( final 10mM) & FQ (final 400nM in reaction buffer); 9 uL: T or Mx & Clamps in reaction buffer. (2x) buffer: 20 mM tris-hcl, KCl 100 mM, pH=8.2. 20 uL /cell： 10uL buffer(2x); 0.8uL 500nM Cas13a; 0.1uL 5uM crRNA; 0.67uL 150mM Mg2+(ca. 5mM Mg2+ final.) 37 ℃ Incubation 10-15min, add probe 0.6uL（10uM） FQ ; （12.17 uL in total ca. 12 uL) Cas13a Continuous system (2x) buffer: 20 mM HEPEs, pH=7.5. 20 uL /cell： 10uL buffer(2x); 0.8uL 500nM Cas13a;0.1uL 5uM crRNA; 0.67uL 150mM Mg2+(ca. 5mM Mg2+ final.) 1uL 200mM KCL; 37 ℃ Incubation 10-15min, add probe 0.6uL（10uM） FQ , 0.5uL Csm6; （total 13.67 uL ) Ac & Target or clamps or mutants (in DEPC water) total 6.33 uL。 Target or clamps or mutants (in DEPC water) total 7.83 uL。

2.3 TEST

The recognition and cleavage of the target sequences by Cas14a and Cas13a proteins, thus exhibiting trans cleavage activity, and consequently the fluorescence signal report, we show as Relative fluorescence unit (RFU). We can observe that each detected sequence exhibits a different fluorescence signal intensity, and M7, M20, RM8, RM15 and RM19 are significantly larger than the target sequence. This indicates that false positive characterization does exist and the specificity of the detection is affected differently depending on the mutation site.

2.4 LEARN:

In this engineering cycle, we validated the false positive characterization of the assay using Cas13a and Cas14a proteins from the previous engineering cycle, and after we concluded that false positives do exist and interfere with the source of the signal, we started to design a solution to the problem - using PNA-CLAMP to mask the mismatched sequences

Circle3：

Blocking of mismatched sequences using PNA

3.1 DESIGN

Here we plan to provide a solution to the false positive problem that occurred in the last engineering cycle. We will use synthetic target sequences with mutant sequences in the wet lab to simulate the processing of samples in a real environment.

Here we plan to provide a solution to the false positive problem that occurred in the last engineering cycle. We will use synthetic target sequences with mutant sequences in the wet lab to simulate the processing of samples in a real environment.

3.2 BUILD

During this BUILD period, we tested multiple sets of different base mismatches and the shielding effect of PNA and DNA on the sequences at different sites and different lengths. However, since PNA is expensive, it is costly and time-consuming to design DNA/PNA for each of the above mentioned mutant sequences for shielding, so we selected the more representative mutant sequences for our experiments.

3.3 TEST

Taking Cas14a1 as an example, based on the experimental results in Cycle 2 (Confirmation of "false positive" characterization), sequences with mutation sites at 5', -5, 7, 20-3' bases were selected from a series of sequences with single-base mutations. -5, 7, 20-3' bases, which showed high RFU in the false positive mock assay, were selected as typical sequences to demonstrate the role of the shackle system (clamp). Initially, we used the designed complementary DNA as clamp as a pre-experiment to verify the preliminary role of Clamp. As illustrated by the experimental results presented in the figure below: the Relative fluorescence unit (RFU) of the experimental group with the addition of DNA-clamp was significantly lower than that of the control group, and combined with the Delta-RFU analysis, Clamp did reduce the interference of mutant sequences on the assay results.

But just DNA as clamp is not enough for the goal of our project. So we designed PNA as clamp and validated it in the same way. We also compared the control group with DNA-clamp and PNA-clamp to make the data more reliable. The above two experiments also demonstrate that our idea of designing "Clamp" to avoid the defect of misidentification of target sequences by Cas13a and Cas14a due to similar mutated sequences can be realized.

Although we have verified that PNA as clamp has better effect than DNA as clamp, however, we are not yet sure to what extent the shielding effect of using PNA as clamp on single base mutation false sequences can be achieved, and it is not clear what effect different concentrations of PNA have on the effect of target sequence detection. So, we set a certain amount of PNA (100nM) and gradient concentrations of target and mutant sequences in combination, and found that the interference of PNA on mutant sequences was significantly greater than that of target sequences, and even after the concentration of mutant sequences was less than 100nM, its RFU dropped in a precipitous manner. When PNA was relatively saturated in the mutant sequence, it was able to play an almost complete shielding role. In the absence of PNA addition, the magnitude of RFU change of both was not as obvious as when PNA was added.

In the system of Cas13a, as shown in the figure below, the shielding effect of PNA-CLAMP on complementary sequences gradually increased as the concentration of PNA-CLAMP increased (0-100 nM) for a certain amount of the shielded sequence, indicating that the shielding effect increased with the amount of PNA-CLAMP within a certain concentration range.

In the data in the figure below, we show that when PNA-CLAMP coexists with target and mutant sequences, only the signal of mutant sequences is significantly shielded, while target sequences are largely unaffected. It indicates that the application of PNA-CLAMP in single-base detection platform is feasible and has predictable good results.

3.4 LEARN

Instrument

Currently, in vitro nucleic acid assays based on CRISPR technology are in the initial development stage. Existing PCR nucleic acid assays require special instrumentation, laboratory testing sites and specialized technical staff, and have strict zoning requirements for laboratories. In this environment, we decided to develop a portable enzyme marker based on our project to validate and promote our project. For more details, please see the Hardware page (please tie the wiki hardware link to Hardware at the Expo)

Outlook

In the era of the epidemic, we are quite sure that our project has strong applications. However, even if our protocol is feasible, there are still many issues and challenges to really develop it to maturity. How to further improve the sensitivity and specificity of the nucleic acid assay, how to combine and simplify the reagent components, how to make the CRISPR-based nucleic acid assay home-based/private, how to further reduce the cost, how to combine the CRISPR nucleic acid assay technology with the instrumentation to automate the rapid detection, these are all things we are committed to achieve.

We will continue to innovate and breakthrough ourselves, and not end with IGEM, and finally build a perfect testing platform to make our own contribution to improve the society!