WET LAB

Ideas and design

CRISPR (Clustered Regularly interspaced short palindromic Repeats), known as Clustered regularly interspaced short palindromic repeats, is an immune system that bacteria use to protect themselves from viruses. It's not just widely used in gene editing.

In recent years，Scientists from different countries have tried to develop new tools which are based on CRISPR-Cas system to detect nucleic acids.Fortunately,they successfully expanded the areas of CRISPR-Cas system and set up some effective platforms to detect[1].

Scientist describe a tandem nuclease assay using Cas13a and Csm6 to achieve both high sensitivity and fast signal generation without requiring a preceding target amplification step. This involved the design of potent, chemically stabilized activators for Csm6 as well as use of eight different CRISPR RNA (crRNA) sequences for Cas13a detection. they also show that this assay can be implemented in a portable device, consisting of a microfluidic chip and a compact detector, to detect viral RNA extracted from human samples. This indicates that the assay is robust and simple to adapt for use in point-of-care testing workflows. This also highlights the value of combining unrelated CRISPR–Cas effectors for sensitive, one-pot detection of RNA.[2].

Based on the above previous exploration, our project has selected Cas13a and Cas14a proteins, and we hope to build a more perfect and accurate detection platform. We will artificially design Peptide nucleic acids (PNA) for base complementary pairing with single base mutations, so as to improve the accuracy of target sequence detection and improve the specificity of the detection system, so as to realize the ability of single base recognition and detection. This scheme is an improvement of the existing nucleic acid detection method of CRISPR / Cas, system (Cas13a and Cas14a), and uses the signal amplification of csm6 protein for the detection of Cas13a and Cas14a proteins.

Validation of protein expression and activity

We successfully obtained the target protein using BL21 (DE3) expression, and successfully purified Cas13a, Cas14a and csm6 proteins using the protein purification instrument belonging to the State Key Laboratory of Marine Resources in the South China Sea. Later, we determined that the activity of the above three proteins was at high levels, and the restriction activity of both proteins was also tested, and the results also met our expected requirements for the protein.

Confirmation of “false positive” characterization

We validated the false-positive characterization of Cas13a1 proteins by designing the single-nucleotide polymorphism sequence of Target RNA to simulate the false-positive recognition representations generated in real situations. The recognition and cleavage of the artificially designed RNA by the Cas13a1 protein shows the trans-cleavage activity, and then the resulting fluorescence signal is reported by Relative Fluorescence unit (RFU). We can observe that each detected sequence exhibits a different fluorescence signal intensity, and that RM8, RM15 and RM19 are significantly larger than the target sequence. This indicates that false positives do exist and the specific effects on detection varies depending on the mutation sites.

Verification of clamp effect

Clamp is a system that we envisage to assist Cas13a, Cas14a proteins to avoid causing "false positive" characterization, and the principle is to artificially design complementary sequences of single-base mutant sequences, hoping to improve the accuracy of detection. We first designed the DNA chain as clamp, and found that the Relative fluorescence unit (RFU) of the experimental group adding DNA-clamp was significantly lower than that of the control group. Combined with the Delta-RFU analysis, Clamp did reduce the interference of the single base mutation sequence on the test results, but the effect did not meet our expectations. Ultimately, we determined that PNA has a better "yoke" effect than DNA[3]. To verify the quantitative effect, we set the quantitative PNA (100nM) and the gradient concentration of the target sequence and the mutation sequence binding, and found that the PNA interfered significantly more to the mutation sequence than the target sequence, and even after the mutation sequence concentration is less than 100nM, the RFU fell off a cliff. When PNA is relatively saturated with the mutant sequence. The magnitude of RFU change without PNA was not as pronounced as when PNA was added. At a certain concentration of PNA (100nM), the shielding effect of PNA on mutant sequence is relatively obvious at a lower concentration (<100nM), and the shielding effect of PNA gradually decreases as the concentration of mutant sequence increases; the interference rate of PNA to mutant sequence gradually decreases as the latter concentration increases until it approaches 0. The experiments also illustrate that our idea of artificially designing PNA to block the signal interference caused by false identification is feasible and enables accurate detection at the single-nucleotide level.

DRY LAB

Analysis and structure analysis of the physicochemical properties of proteins

Before deciding to perform the experiments with the cas13a protein and the csm6 proteins, we analyzed the physicochemical properties, the hydrophilic hydrophobicity, the stability of the protein structure, etc. For the hydrohydrophobicity part, we used the open-source program of Proscale to predict the hydrophobicity of cas13, csm6 from the amino acid composition. Because the amino acid sequence of the protein determines its hydrophilic / hydrophobicity, the hydrophobicity determines its solubility in water, the hydrophobicity is conducive to the protein folding to the inside to form a secondary structure, further form a domain, tertiary structure, and the strong hydrophobicity is more conducive to the protein forming a helix, increase stability. Later, we analyzed the physicochemical properties of the two proteins. whose basic profile is as follows, including the team model section.

Kinetic simulation of the PNA shielding reaction

The CRISPR / Cas system is a technique of targeting genes for modification by RNA-directed Cas proteins derived from bacteria-acquired immunity. The CRISPR / Cas group can be divided into three types, among which, the A isoform in the type III CRISPR-Cas system has an effector complex called Csm, which consists of multiple Cas proteins, together with the crRNA. The Csm complex not only cleaves the target RNA complementary to the crRNA, Moreover, the binding of the target RNA can activate the two novel enzymatic activities generated by the Csm complex, That is, the cleavage of the ssDNA during transcription and the activity of the synthetic cyclic oligo-adenylate cOA, The cOA acting as a second messenger can activate Csm6, Non-specific degradation of the RNA, Type III CRISPR can produce two cOA: cA6 and cA4, It can bind to the CARF domain on the cas protein, In turn, activating the HEPN domain for nonspecific cleavage, Cut off the predesigned fluorescence group with the quencher, Release of the fluorescent signal, Reference to the Team model section for detailed results.

Unwinding temperature prediction model based on near-neighbor method model and DNN neural network

In the experiment, we achieved the accuracy to single base specificity by blocking PNA for the mismatch chain, but different from using DNA as a chain, PNA, as a peptide nucleic acid, has the following advantages: When it is complementary to the nucleic acid chain, the unwinding temperature greatly increases the relative DNA / DNA binding; When a base mismatch occurs, a single base pair mismatch can reduce the Tm value by 8 to 20℃ (an average of 15℃), much higher than the reduced temperature during DNA / DNA binding. We propose to find a modeling method for the simulation predictions of the optimal unwinding temperature when screened with PNA.

Portable detection hardware

In our wet experiment, our team cut a single base and generated a fluorescent signal; to transform and apply our results, we imagined building a device that can identify the fluorescent signal. Different from the traditional microplate reader, which has a large size, large weight, high cost and an inconvenient need to connect to the computer, we have successfully developed a small and convenient microplate reader through 3D printing technology after completing the circuit design. This will help to make our project results much easier to use.

References

[1]Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018 Apr 27;360(6387):439-444. doi: 10.1126/science.aaq0179. Epub 2018 Feb 15. PMID: 29449508; PMCID: PMC5961727.

[2] Liu, T.Y., Knott, G.J., Smock, D.C.J. et al. Accelerated RNA detection using tandem CRISPR nucleases. Nat Chem Biol 17, 982–988 (2021).

[3] Egholm M, Buchardt O, Christensen L, et al. PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen-bonding rules[J]. Nature, 365(6446): 566-568（1993）.