Intelligent guide RNA (gRNA) with dual Toehold switch for conditional activation of the CRISPR / Cas9 system and targeted recognition of reporter gene DNA

Abstract

RNA detection plays an important role in various fields including medicine, agriculture, and biotechnology. Traditional RNA detection methods such as PCR and Northern blot have limitations such as time-consuming and non-specificity. Given the need for a rapid, simple, and specific method for RNA detection, an RNA sensor that uses the CRISPR-Cas9 gene editing system and strand displacement mechanism to detect specific RNAs has been proposed. This sensor can be used in vitro and in vivo for various applications including disease diagnosis, gene expression profiling, and genome editing. The platform is based on two main components, the CRISPR-Cas9 gene editing system and the strand displacement mechanism using two Toe-Hold sites. The reaction of this sensor with a trigger RNA strand activates the CRISPR-Cas9 system. This process leads to the cleavage of the target DNA, which can be observed in vitro on an agarose gel. Due to its specificity, it can be used for early disease detection, monitoring and diagnosis of viral infections. This platform offers an innovative method for RNA detection that, with continued development, will become a valuable tool for diagnosis, treatment and research in the future.

Description

Description of the invention Note: Items must be written only inside [ ], otherwise they will not be reviewed. Title of the invention (as stated in the declaration) Intelligent guide RNA (gRNA) with dual Toehold switch for conditional activation of the CRISPR / Cas9 system and targeted recognition of reporter gene DNA Technical background of the relevant invention This invention is related to the field of gene editing and synthetic biology. Technical problem and stating the objectives of the invention One of the most important issues that exist in all countries is the diagnostic problems in microbial and viral species, as well as the treatment of diseases of genetic origin. And in line with these two issues, many scientists have conducted extensive research. This project was started to solve two basic problems, one of which is the existence of a gap in diagnostic kits for detecting specific RNAs, and the other is to create an intelligent platform that recognizes the differences between different cells based on internal information (such as gene expression) and simultaneously destroys one of the cells or creates a targeted mutation in it. The complete solution to these problems requires other optimizations of this platform in the future, but the initial design of this platform and initial tests to utilize this diagnostic system have been successful and have the potential to solve the aforementioned problems by integrating the gene editing system called CRISPR-Cas9 with synthetic biology.This intelligent platform utilizes the rules found in nucleic acids, such as base pairing rules and strand displacement rules, to condition the activity of the CRISPR system and create a logic circuit that induces its cleavage activity in the presence of guide RNA. By using reporters such as luciferase in this design, this cleavage can be detected. A description of the state of the prior art and the history of developments related to the claimed invention. In the late 1990s and early 2000s, researchers such as Nadrian Seeman and Erik Winfree pioneered the field of DNA nanotechnology, demonstrating the programmable self-assembly of DNA molecules into complex nanostructures. These early studies laid the foundation for understanding DNA hybridization and strand displacement reactions as key mechanisms for controlling molecular interactions, making this process of great importance in the present product. One of the most important uses of this mechanism in diagnosis was used in 2014 by Green and Silver, who designed a molecular riboswitch to show that the presence of a synthetic RNA in the environment could be detected. By placing the ribosome binding site in a Stem-Loop structure, they prevented the ribosome from binding to its site and consequently producing a marker protein, and by introducing a synthetic RNA, they led to the opening of the structure and ultimately producing a signal. A little later, by replacing the RNA of viruses such as Zika instead of synthetic RNA, they demonstrated the ability to detect these viruses using this platform, so in one of their articles they presented a diagnostic kit that can detect the content of the virus RNA in a tube based on the riboswitch. The use of the translation system in this kit and the time-consuming nature and limitations in its diagnostic devices are the reasons why this kit is not widespread and commercialized. On the other hand, the present product, by utilizing the gene editing system, leads to a reduction in the time required for diagnosis and its further use for the treatment of patients in the future. The use of the CRISPR editing system in mutagenesis and genetic modification was first introduced by Doudna and Charpentier. In 2013, Mr. Zhang introduced the gRNA sequence by connecting two components of this system together. One of the applications of the CRISPR system for detecting the genetic material of viruses is the introduction of SHERLOCK, which is a method that tends to be replaced due to the multiple pathways and the use of several enzymatic steps for the amplification of the initial stimulus and the cost of this system. The integration of genome editing tools with other methods has led to the development of conditional control strategies for this system. As researchers began to explore different strategies for conditional control of CRISPR-Cas activity, including structural changes of gRNA, Cas protein activity, or complex formation. In 2019, Mr. Siu et al. introduced the concept of Toe-hold gated and succeeded in conditional cleavage in vitro by introducing a synthetic guide RNA. After that, other people introduced different examples of this platform using this concept. However, these studies had limitations in that they needed to provide synthetic RNA with a sequence similar to CrRNA to remove inhibition from the CrRNA region to cleave the target DNA or in general to activate the CRISPR system. They were limited in creating a platform by sensing any synthetic or natural RNA (with any specific sequence) with the specificity of targeting any gene, because to trigger the rules of strand displacement, the sequence of the sense RNA region was required to be similar to the gene targeting region (CrRNA).The current platform, introduced as Intelligent gRNA, attempts to overcome these limitations by providing two Toe-Hold positions. Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention One of the most important problems of recent research is the lack of a diagnostic platform for RNA regions, in which regard, activities have been carried out to develop diagnostic kits for specific RNA fragments, which are mostly based on the polymerase chain reaction or PCR. Although the use of these methods is widespread, the existence of false positives and the sometimes time-consuming nature of these methods have led researchers to replace them with other methods. One of these strategies is the integration of the gene editing system with synthetic biology, in such a way that the stimulus recognition feature of synthetic biology can be used by initiating the strand displacement process and the signal generation can be obtained from the function of the CRISPR system. Although the present product only refers to the diagnostic function and signal generation by the function of the CRISPR system, due to the use of the gene editing system, with other optimizations, this product has the potential to treat genetic diseases and even, as seen in Figure (1), distinguish healthy cells from cancerous cells and target cancer cells by cutting essential genes. The present product is known as an RNA sensor and miRNA-16 mimic RNA was used to evaluate its performance, which results in sensing this RNA, cutting the luciferase gene in the microtube or turning off the luciferase signal in Dh5 alpha bacteria. The processes used in this product are the CRISPR gene editing system and the Toe-Hold strand displacement process. The strand displacement mechanism is one of the most important mechanisms of molecular dynamics of nucleic acids, which alone has been able to accommodate a very wide part of structural biology and biosensors. This mechanism benefits from hydrogen bonding between DNA strands so that, according to the laws of energy, DNA strands are in their lowest energy state, which means that in this particular case they tend to reduce disorder by forming the most specific bonds. With this definition, it can be seen that if a DNA double strand is incompletely connected, it is conceivable that each strand is able to release the opposite strand, of course in the presence of a strand that brings more hydrogen bonds. In the aforementioned mechanism, the presence of a dangling strand or even unattached nucleotides is essential, which the attacking strands bind to perform strand displacement from these locations and gradually, by opening the remaining bonds with the previous strand and attaching their nucleotides to the target strand, they lead to their replacement in place of the complementary strand of the target. This dangling sequence or unattached nucleotides is called the Toe-Hold. Therefore, the entire mechanism along which strand displacement occurs is called the Toe-Hold Mediated Strand Displacement Figure (2). The second process is the CRISPR-Cas9 genome editing system, which has recently become one of the most important and influential tools in biology. The CRISPR system used in gene editing consists of two main components, namely nuclease protein and guide RNA or gRNA, so that the function of the nuclease component is to cut and the function of the gRNA is to identify the specific target and accompany the nuclease component for cutting, which has the ability to accompany the repair system and induce mutations in living systems. The gRNA component itself is divided into two parts, CrRNA, which is about 20 nucleotides and Scaffold-RNA, which is about 76 bases long, where the Cas9 nuclease enzyme binds to the gRNA from the Scaffold part, and the CrRNA part has the role of recognizing the specific DNA for cutting. The present study, by utilizing both processes, and employing two Toe-Hold sites, has created a smart sensor that can induce its cleavage activity only in the presence of a driving RNA. In this way, the CrRNA component of the gRNA is inhibited by a complementary strand and loses its target DNA recognition activity. To achieve this important design, the NUPACK bioinformatics site was used. In this platform, as shown in Figure (3), a number of bases are added to the 5' side of the gRNA, that is, upstream of the CrRNA. In this way, 5 nucleotides are used as a linker, then 22 nucleotides are used as the driving RNA binding site, of which 8 nucleotides of these 22 nucleotides are introduced as Toe-Hold (TH) and are located in the loop part of the structure. To reduce the pressure of the loop and prevent possible opening of the connections, 2 nucleotides are added to the loop part to make it flexible against possible pressures. Following this structure is the complementary strand, which blocks up to 15 nucleotides of CrRNA.The platform consists of 29 free bases, with the potential to initiate the strand exchange process only when the TH second is exposed, which leads to the release of the CrRNA from the binding and increases the potential to cleave the target DNA strand. In this platform, a mismatch is introduced upstream of the linker as the last nucleotide to facilitate the initiation of the strand exchange. As can be seen in Figure (8), the presence of the stimulatory RNA in the environment triggers the strand displacement reaction by binding to the first TH region, this binding leads to the opening of the bonds up to the protruding region (mismatch linker), which in turn leads to the exposure of the second TH region located in the complementary strand. And with the exposure of the second TH, the potential for CrRNA release from inhibition is created by triggering the second strand displacement process. This process is associated with the activation of the CRISPR system and causes the cleavage of the target DNA. Here, the miRNA-16 mimic sequence triggers this process and, when present in the environment, can cause the cleavage of the luciferase gene. It should be noted that the 20 nucleotide fragment of CrRNA was previously designed and tested to target the luciferase gene. The identification reaction to confirm this process has been performed at two levels: In-vitro in a test tube and In-cyto in the Dh5-alpha strain, but in general, the cleavage reaction at the In-vitro level is sufficient for the present product. The raw materials of this kit include purified Cas9 enzyme, enzyme buffer, IngRNA, Regular gRNA for controlling the cleavage test, driver RNA, luciferase gene as a marker, and also in this design, miRNA-16 is used as a driver RNA, on which the IngRNA sequence is designed. After designing the corresponding IngRNA, Regular gRNA and Mimic-miRNA-16, they can be ordered either as RNA or by adding a 20-base promoter called T7 promoter to the beginning of the 5' region of the template DNA fragments, IngRNA, Regular gRNA and Mimic-miRNA-16 sequences, which at 37 degrees and in the presence of T7 RNA polymerase according to the enzyme instructions, leads to the production of RNA from the DNA sequence. After that, treatment with DNase enzyme leads to the removal of remaining DNA in the medium. The phenol-chloroform method was used to purify the produced RNAs.Cas9 protein is also available in two ways, commercial purchase and cloning in a vector and extracting it from bacteria. In this product, Cas9 enzyme was expressed in a modified pET28a vector under IPTG induction at a concentration of 0.3 M and incubation time of 16 hours at 16 degrees in BL21 strain and purification was performed using a Ni-NTA column following the instructions. To perform the experiment, first we amplify the luciferase gene template using the LUF and LUR primers in the table, perform the PCR process, and purify the product. The reaction tubes and the concentrations of their ingredients are listed in Table (1). After combining the ingredients and placing the reaction tubes at 37 degrees for one hour, the products can be run on a 1.5% agarose gel to see the results. To validate this kit, a separate step was also performed inside the bacteria and the luciferase signal was measured in the presence of Trigger RNA. For this purpose, the expression of gRNA and IngRNA in the pTw vector was under the control of the pJ23119 promoter with Ori type A, and the expression of luciferase and mimic-miRNA was under the control of the arabinose-inducible promoter (araBAD promoter) and under the control of the IPTG-inducible promoter (Trc promoter) in the pCLw vector with Ori type C, respectively (Figure 5). Thus, by transferring both vectors into the Bl21 bacteria, two separate antibiotic screening steps were performed and the entry of each was confirmed by colony PCR. For the expression of various gRNAs, the pTw vector was used, which is a modified form of the pTargetF vector (addgene #62226). In this modified form, two Bbs1 enzyme cleavage sites were placed in front of the pJ23119 promoter by the Quick change PCR technique to make it easier and less expensive to construct forms expressing different gRNAs. Also, for the expression of the luciferase and Cas9 genes, as well as the expression of Trigger-RNA, the pCLw vector, which is a form of the pCas vector (addgene #62225), was used. In this modified form, the luciferase gene was replaced by the Lambda RED system genes in front of the inducible araBAD promoter (induced by adding arabinose to the medium) by the TEDA cloning method. Also, the miR-16 sequence was placed in front of the inducible promoter (Trc) by the same method (induced by adding IPTG to the medium). After introducing both pCLw and pTw plasmids in two different stages into BL21(DE3) bacteria and then screening for bacteria containing both plasmids, the bacteria were grown in LB liquid culture medium and the next day, the sub-culture process was performed with 500 microliters of medium containing bacteria added to 100 cc of medium containing spectinomycin and kanamycin antibiotics. After 3 hours, when their OD reached 0.5, luciferase induction was performed by adding arabinose at a final concentration of 0.1% and Trigger-RNA induction was performed by adding IPTG at a final concentration of 0.5 mM. After 6 hours, by separating the liquid medium from the bacteria and equalizing the concentration of bacteria using a light absorption device, lysis of the bacteria was performed using a sonicator. After two steps of separating cellular components by ultracentrifugation at 18,000 RPM, the supernatant was separated for measuring the luciferase signal and placed on ice. By adding 10 microliters of this solution to 10 microliters of the luciferin complex as a substrate, the luciferase signal was measured (Figure 6).The effectiveness of this system was investigated by comparing it with different expressions of gRNA or Trigger-RNA, the names and expressed sequences of which are given in Table (3). Confirmation of luciferase gene cleavage on agarose gel in the presence of Trigger-RNA Figure (4) was performed by extending the Intelligent RNA system into the living bacterial medium and reducing the luciferase signal as shown in Figure (6). Explanation of shapes, maps and diagrams Figure (1): Theoretical implications of the relevant platform for targeting differentiation between cancer and normal cells. The figure illustrates the potential applications of the IngRNA platform in specifically targeting cancer cells and having no effect on normal cells. Figure (2): Mechanism of strand displacement by targeting the Toe-Hold site. In this image, the binding of strands B and C leads to the creation of a Toe-Hold site for another strand called A, and when strand A attacks that site, due to the higher percentage of bases capable of binding to strand C compared to strand B, strand A replaces strand B. Figure (3): Schematic of platform activation in the presence of guide RNA. The sequences of both Toe-Hold and IngRNA components are also shown. Figure (4): Excision of the 1860 bp fragment by the platform in the presence of Trigger RNA. a) Schematic diagram of the cleavage of the luciferase DNA fragment into two fragments of 354 and 1506 bp. b) The result of in vitro cleavage on agarose gel in the presence and absence of Trigger RNA. The first well serves as a negative control and shows the Cas9 enzyme and Trigger-RNA. The second well serves as a positive control and shows the enzyme in the presence of Regular-gRNA. Well number 3 shows the contents of the test tube in the absence of Trigger-RNA on agarose gel. Well number 4 shows the contents of the test tube in the presence of Trigger-RNA. Figure (5): Map of plasmids used in this study. The pCLw vector is derived from the pCas9 vector, which has the pSC101 replication origin sequence, which is a low-copy C type, and also contains the sequences of genes expressing the Cas9 enzyme and resistance to the antibiotic kanamycin. Figure (6): Comparison of the effect of IPTG inducing Trigger-RNA expression on the IngRNA platform in targeting the luciferase gene. It shows a decrease in the luciferase signal to about 5000RLU at a concentration of 0.5Mm. Results were based on a p-value of less than 0.05 and three replicates in the experiments. Figure (7): The natural cleavage process of the gRNA_cas9 complex. Figure (8): The cleavage process by the IngRNA_cas9 complex, which occurs conditionally in the presence of guide RNA. Table (1): Reaction tubes for detecting miR-16 as Trigger-RNA. Table (2): Components and values ​​of the miR-16 detection reaction as Trigger-RNA. Table (3): Shows the names of plasmids with specific expression fragments and the sequences of these fragments. A clear and precise statement of the advantages of the claimed invention over prior inventions. Increased accuracy and specificity: Using two Toe-Hold positions instead of one position significantly increases the accuracy and specificity of the sensor. This is due to the need for two separate strands to be joined for the CRISPR system to activate, which greatly reduces the chance of accidental activation. Simplicity and ease of use: This product is presented as a diagnostic kit that is easy and quick to use. This kit does not require specialized equipment or complex technical knowledge. Usability in different environments: This product can be used in various environments, including the laboratory and inside the cell. This is due to the use of the CRISPR system, which exists naturally within cells. Potential therapeutic applications: In addition to diagnostic applications, this product also has potential for therapeutic applications. This is due to the CRISPR system's ability to edit the genome. Cost-effectiveness: This product is produced using simple methods and inexpensive raw materials. This makes this product accessible to a wide range of users. No false positives: Using the CRISPR system in particular completely eliminates the possibility of false positives. This is due to the mechanism of action of the CRISPR system, which only looks for specific DNA sequences. Description of at least one implementation method for implementing the invention This invention provides a smart RNA sensor that uses the CRISPR-Cas9 gene editing system and strand displacement mechanism to detect specific RNAs. The sensor can be used in the laboratory (In-vitro) and inside the cell (In-cyto) for various applications including disease diagnosis, gene expression monitoring, and genome editing. Materials and equipment: Purified Cas9 enzyme Enzyme buffer IngRNA (designed based on guide RNA sequence) Regular gRNA (cut control) Trigger RNA can be viral RNA or... Luciferase gene (as a marker) Steps to perform the test: Prepare the test tubes, positive control and negative control as introduced in Table (1) and prepare each of the tubes according to the components in Table (2) and then the reaction tubes are placed at 37 degrees for 30 to 60 minutes to perform the cutting process. Finally, the reaction products are loaded on a 1.5% gel and then the presence or absence of Trigger RNA is shown by the gel scanner. Explicit mention of the industrial application of the invention This platform has already demonstrated detection of miR-16 sequences by slicing the luciferase reporter gene sequence, indicating its potential in other applications, for example, with its optimizations, it can be used in Medical diagnosis: Early diagnosis of various diseases through identification of specific RNAs Study of gene expression and gene mutations Monitoring and diagnosing viral and bacterial infections Biotechnology: Genome editing and genetic engineering Production of new drugs and gene therapies Developing new methods for producing food and biomaterials Scientific research: Study of molecular mechanisms of diseases Investigating the function of genes and signaling pathways Developing new methods for biological research At present, further research is needed to determine the specific industrial applications of this invention and to develop it for large-scale use. Given the potentials mentioned, it can be expected that this invention will have significant industrial applications in various fields in the future. As mentioned, these are just a few examples of possible industrial applications of this invention. With continued research and development, it can be expected that new and innovative applications for this invention will be discovered in the future.

Claims

Claims What is claimed: (Items should be written only within [ ] [, otherwise they will not be considered.) Claim 1: An intelligent guide RNA (gRNA) for use in a CRISPR / Cas9-based gene editing system, comprising a DNA-targeting crRNA sequence, a scaffold sequence for binding to the Cas9 endonuclease, and two toehold switch sequences, both added to the 5ʹ end of the gRNA sequence; such that the gRNA remains in a inhibited and inactive state in the absence of a guide RNA and is activated only in the presence of a specific guide RNA through two toehold-mediated strand transfer steps, directing Cas9 to the target DNA. Claim 2: The intelligent guide RNA according to claim 1, wherein the guide RNA comprises a natural or synthetic RNA selected from miRNAs, viral RNAs, or pathogen-specific RNAs, and activation of the gRNA is dependent on the presence of this RNA. Claim 3: The smart guide RNA of claim 1, wherein the two toehold sequences act in a stepwise manner, such that binding of the guide RNA to the first toehold causes the second toehold to appear and initiate the second strand displacement process and release of the crRNA from the inhibition state. Claim 4: The smart guide RNA according to claim 1, wherein the target DNA is a luciferase reporter gene and activation of the gRNA results in cleavage of the luciferase gene DNA or silencing of the luciferase signal. Claim 5: The smart guide RNA according to claim 1, which is capable of being used in vitro or in cyto conditions. Claim 6: Claim 7: Claim 8: Claim 9: Claim 10: Claim 11: Claim 12: Claim 13: Claim 14: Claim 15: Claim 16: Claim 17: Claim 18: Claim 19:

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