RNA detection and transcription-dependent editing with reprogrammed tracrrnas

Abstract

To provide a method for detecting a specific RNA.SOLUTION: The present invention relates to methods for detecting at least one sensed RNA in a cell, tissue, and / or sample using at least one non-naturally occurring tracrRNA specifically hybridizing with the sensed RNA and at least one tracrRNA-dependent CRISPR nuclease enzyme binding to at least one target nucleic acid, as well as respective systems and diagnostic and therapeutic uses thereof.SELECTED DRAWING: Figure 1-1

Description

Technical Field

[0001] The present invention relates to a method for detecting at least one target RNA in a cell, tissue, and / or sample using at least one non-naturally occurring tracrRNA that specifically hybridizes with the target RNA, and at least one tracrRNA-dependent CRISPR nuclease enzyme that binds to at least one target nucleic acid, as well as respective systems, and their diagnostic and therapeutic uses.

Background Art

[0002] Almost all archaea and about half of bacteria have short palindromic repeat (CRISPR)-CRISPR associated genes (Cas) adaptive immune systems that form clusters and have regular intervals, by which prokaryotes are protected from viruses and other foreign invaders with nucleic acid genomes. The CRISPR-Cas system is functionally classified into class 1 and class 2 according to the composition of the effector complex. Class 2 consists of single effector nucleases, and the practice of genome editing has been realized by the utilization of class 2 CRISPR-Cas systems including type II, type V, and type VI CRISPR-Cas systems. Type II and type V can be used for DNA or RNA targeting, whereas type VI is used for RNA targeting (see, for example, Non-Patent Document 1). Type II and type V Cas effector nucleases usually depend on protospacer adjacent motif (PAM) as the first step in target DNA recognition, and the effector nuclease directly binds to the PAM sequence via protein-DNA interaction and then unwinds the downstream DNA sequence. Next, the effector protein collates the degree of base pairing between one strand of the DNA target and the guide part of the CRISPR RNA (crRNA). If the complementarity between the two is sufficient, target cleavage is driven. The PAM sequence is known to be quite different not only between systems but also between other similar nucleases, and it has been shown that Cas proteins can be manipulated to change PAM recognition (Non-Patent Document 2). In addition to DNA targeting, some type II and type V single effector nucleases such as Cas9 of C. jejuni, Cas9 of N. meningitidis, Cas9 of S. aureus, and Cas12f1 derived from uncultured archaea have also been shown to target ssDNA and / or RNA (Non-Patent Documents 3, 4, 5). In these cases, PAM was not required.Some nucleases, such as Cas9 (SpyCas9) of S. pyogenes (S. pyogenes), were unable to immediately target ssDNA or RNA. However, by supplying oligonucleotides to generate a double-stranded PAM region, SpyCas9 was able to bind to and cleave a single-stranded target (Non-Patent Document 6).

[0003] Mature crRNAs are important elements in CRISPR-Cas defense against invading genomes. These short RNAs contain unique guide sequences that direct Cas protein(s), if multiple, to homologous invading nucleic acids and destroy them. CRISPR RNAs (crRNAs) are naturally encoded in CRISPR arrays that contain conserved alternating repeats and spacers. This array is often transcribed into a long pre-crRNA, which is then processed into individual crRNAs composed of parts of repeat-spacer units. Different types of CRISPR-Cas systems have evolved distinct crRNA maturation mechanisms. A number of subtypes (II-A, II-B, II-C, V-B, V-C, etc.) have evolved their own crRNA biosynthesis pathways, where trans-activating CRISPR RNA (tracrRNA encoded within the CRISPR locus) base pairs with each repeat sequence of the pre-crRNA to form double-stranded RNA duplexes (Non-Patent Document 7). The crRNA-tracrRNA duplex is cleaved by the housekeeping endoribonuclease RNase III, and a system-specific single effector nuclease binds. This nuclease then uses the crRNA within the RNA duplex to direct DNA targeting, and in some cases RNA targeting. To date, a number of tracrRNA-dependent CRISPR nucleases have been reported, including Cas9, Cas12b1 / C2c1, Cas12b2, Cas12e / CasX, Cas12f1 / Cas14a, Cas12g, and Cas12k. The Cas12k nuclease is unique in that it functions in conjunction with a series of transposon genes to perform RNA-guided insertion of DNA templates (Non-Patent Document 8). In each of these cases, the tracrRNA:crRNA duplex also induces sequence-specific dsDNA cleavage by Cas9 and other tracrRNA-dependent CRISPR nucleases when engineered as a single RNA chimera called a single guide RNA (sgRNA) (see Non-Patent Document 9 and Non-Patent Document 10).

[0004] The CRISPR-Cas9 system of Francisella novicida exhibits PAM-dependent interactions between Cas9 and its endogenous DNA targets, which rely on noncanonical small CRISPR-associated RNAs (scaRNAs) and tracrRNAs. Intrinsically, scaRNAs are thought to direct Cas9 to genomic DNA targets with partial complementarity, resulting in transcriptional repression. ScaRNAs can be reprogrammed to repress other genes and can also induce DNA cleavage by extending complementarity through engineering against exogenous targets (Non-Patent Document 11). Considering that scaRNAs are encoded within the CRISPR-Cas system like crRNAs, the only previously known source of RNA capable of directing a CRISPR nuclease to its intended target will be derived solely within the CRISPR-Cas system.

[0005] In a recent publication by the inventors (Non-Patent Document 12), the RNAs co-immunoprecipitated with Campylobacter jejuni Cas9 nuclease (CjeCas9) were sequenced, revealing that a subset of RNAs complementary to the guide portion of crRNAs are encoded within the CRISPR locus of this bacterium. The inventors performed similar co-immunoprecipitation experiments in a separate strain of C. jejuni. The resulting unpublished dataset included a set of RNAs that did not show complementarity to crRNAs, but instead shared complementarity with the anti-repeat portion of tracrRNA (Figure 1). The composition and size of these RNAs were similar to those of crRNAs. From these insights, it was suggested that mature crRNAs (which direct Cas9 to their targets) could be derived from messenger RNAs and other RNAs (e.g., ribosomal RNAs, transfer RNAs, small RNAs, antisense RNAs, small nucleolar RNAs, microRNAs, piwiRNAs, long non-coding RNAs, spliced introns, circular RNAs) in addition to those encoded by the CRISPR array. In the context of the present invention, these RNAs are designated and / or referred to as non-canonical crRNAs (ncrRNAs).

[0006] Base pairing between the CRISPR repeat and the tracrRNA anti-repeat forms an RNA duplex that is bound by the tracrRNA-dependent CRISPR nuclease. The exact secondary structure of the duplex varies, and may show complete complementarity or may contain characteristic bulges. Regardless of the exact secondary structure, this secondary structure could be maintained as part of the sgRNA for Cas9 from Streptococcus pyogenes, and the sequence of this region of the sgRNA could be modified (Non-Patent Document 13). Therefore, the inventors hypothesized that by changing the sequence of the anti-repeat portion of tracrRNA to be complementary to other RNAs, this portion of each RNA could become a mature crRNA (Figure 2).

[0007] The idea of changing the anti-repeat region of tracrRNA, especially converting any RNA into ncrRNA, has not yet been investigated. The region downstream of the anti-repeat region has been modified in terms of sgRNA and has been shown to accommodate several changes without disrupting the function of sgRNA (Non-Patent Document 13). Furthermore, Scott, T et al. (Non-Patent Document 14) showed that changing the region outside the anti-repeat region could improve the overall editing activity exhibited by the Cas9 RNP, but the mechanism was unclear. However, these regions are involved in recognition by Cas9 and not in pairing with crRNA or any other RNA.

[0008] Patent Document 1 relates to compositions and methods utilizing nucleic acid sequences having a genetically modified trans-activating crRNA (tracrRNA) sequence in which at least one uracil nucleotide of the tracrRNA sequence is replaced with a nucleotide other than uracil, and / or nucleic acid sequences having a guide RNA (gRNA) sequence in which one or more cytosine nucleotides and / or one or more uracil nucleotides of the gRNA sequence are modified nucleotides. Compositions further comprising an RNA-guided DNA endonuclease enzyme, such as Cas9 or Cpfl or a class II CRISPR endonuclease or variants thereof, such as inactivated Cas9 (dCas9) or mutant Cas9 nickase (D10A) are disclosed.

[0009] Patent Document 2 relates to compositions comprising a synthetic two-part aptamer-containing guide RNA, and methods of using the synthetic two-part aptamer-containing guide RNA in a CRISPR / Cas activator system. Methods of targeted transcriptional activation, targeted transcriptional repression, targeted epigenomic modification, targeted genomic modification, or targeted genomic locus visualization in eukaryotic cells are recited in the claims.

[0010] Patent Document 3 discloses chemically modified crRNA and tracrRNA, crRNA and tracrRNA having a portion conjugated to the 5' and / or 3', and crRNA and tracrRNA having a modification in the repeat region of crRNA or the anti-repeat region of tracrRNA. Also provided are methods of using crRNA and tracrRNA for genome editing using CRISPR nuclease, and kits for carrying out the above methods. The crRNA and tracrRNA are chemically modified while maintaining the effectiveness of SpyCas9-based genome editing in cultured human cells.

[0011] Tautvydas Karvelis et al. (in Non-Patent Document 15) disclose that the Cas9-crRNA complex of the CRISPR3-Cas system of Streptococcus thermophilus DGCC7710 functions as an RNA-guided endonuclease with crRNA-directed target sequence recognition and protein-mediated DNA cleavage. An additional RNA molecule, tracrRNA (trans-activating CRISPR RNA), co-purifies with the Cas9 protein isolated from a heterologous E. coli strain having the CRISPR3-Cas system of S. thermophilus DGCC7710. tracrRNA is required for Cas9-mediated DNA interference both in vitro and in vivo, and Cas9 specifically promotes duplex formation between the precursor crRNA (pre-crRNA) transcript and tracrRNA in vitro. Furthermore, the housekeeping RNase III contributes to the major pre-crRNA-tracrRNA duplex cleavage for mature crRNA biosynthesis. However, RNase III is not required in the processing of short pre-crRNAs transcribed from minimal CRISPR arrays containing a single spacer. The ternary Cas9-crRNA-tracrRNA complex assembled in vitro cleaves DNA. This reference further identifies the molecular basis for crRNA-based reprogramming of Cas9 to specifically cleave any target DNA sequence for high-precision genome surgery. The disclosed processes of crRNA maturation and effector complex assembly are said to contribute to the further development of a Cas9-reprogrammable system for genome editing applications.

[0012] Nevertheless, all of Patent Document 1, Patent Document 2, Patent Document 3, and Non-Patent Document 15 modify the tracrRNA as part of the standard crRNA:tracrRNA duplex used for DNA targeting. These references attempt to improve the existing duplex representing the truncated crRNA and tracrRNA, but the present invention uses a non-naturally occurring tracrRNA modified to hybridize to a non-crRNA naturally occurring RNA.

[0013] The presence of RNA binds to an active tracrRNA-dependent CRISPR nuclease directed to a specific target by the ncrRNA, as any RNA can be used to generate the ncrRNA. Thus, reprogramming of tracrRNA has the potential to detect specific RNA transcripts through sequence-specific targeting activity. This relationship is different from the other two CRISPR technologies used for nucleic acid detection based on Cas12a and Cas13. Specifically, Chen et al. (Non-Patent Document 16) disclose that RNA-induced DNA binding causes non-specific single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. By combining Cas12a ssDNase activation with isothermal amplification, a method called "DNA endonuclease-targeted CRISPR trans reporter" (DETECTR) was created. Separately, Abudayyeh et al. (Non-Patent Document 17) demonstrated the RNA-induced RNase activity of the putative type VI CRISPR effector LshC2c2 (current Cas13a). Gootenberg et al. (Non-Patent Document 18) further disclose that by using this "collateral effect" to establish CRISPR-based diagnosis (CRISPR-Dx), rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity can be provided. This Cas13a-based molecular detection platform is called Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), and it has been used to detect specific strains of Zika virus and dengue virus, identify genotypes of pathogenic bacteria and human DNA, and identify mutations in cell-free tumor DNA. Although both technologies can output the presence of the detected nucleic acid, they both rely on non-specific nuclease activity as the output. In contrast, the present invention relies on sequence-specific nuclease activity determined by the RNA to be detected.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0015]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Summary of the Invention

Problems to be Solved by the Invention

[0016] The object of the present invention is to apply the above progress in CRISPR in the field of molecular diagnosis, particularly for detecting a specific RNA. Other objects and advantages will become apparent by further examining this specification with reference to the attached examples.

Means for Solving the Problem

[0017] In its first aspect, the object of the present invention is a method for detecting at least one target RNA in a cell, tissue, and / or sample, comprising: a) contacting the sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes to a first portion of the target RNA, wherein a second portion of the target RNA specifically hybridizes to at least one target nucleic acid, and the at least one non-naturally occurring tracrRNA hybridizes or is capable of hybridizing to the target RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of a dsRNA cleaving enzyme; b) detecting the binding of the at least one tracrRNA-dependent CRISPR nuclease enzyme to the at least one target nucleic acid; comprising: c) wherein the binding detects the at least one target RNA in the cell, tissue, and / or sample, is solved by providing a method.

[0018] In its second aspect, the object of the present invention is a method for detecting at least one target RNA in a cell and / or sample, comprising: a) contacting the sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes to a first portion of the target RNA; Here, the second portion of the detected RNA specifically hybridizes with at least one target nucleic acid, and the at least one non-naturally occurring tracrRNA hybridizes with or can hybridize with the detected RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme that optionally further includes the presence of a dsRNA cleaving enzyme. b) detecting cleavage of the at least one target nucleic acid in the sample by the nuclease enzyme; comprising c) where detection of the cleavage of the at least one target nucleic acid solves the problem by providing a method for detecting the at least one detected RNA in the cell, tissue, and / or sample.

[0019] In its third aspect, the problem of the present invention is a method for detecting at least one detected RNA in a cell, tissue, and / or sample, a) contacting the sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with the first portion of the detected RNA; Here, the second portion of the detected RNA specifically hybridizes with at least one target nucleic acid, and the at least one non-naturally occurring tracrRNA hybridizes with or can hybridize with the detected RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme that optionally further includes the presence of a dsRNA cleaving enzyme. b) nicking or cleaving the at least one target nucleic acid in the sample with the nuclease; c) detectably editing the at least one target nucleic acid, including, for example, non-homologous end joining (NHEJ) repair, microhomology-mediated end joining (MMEJ), homology-directed repair (HDR), prime editing, base editing, or RNA editing; d) For example, at least one method selected from the detection of the presence and / or length of indel or gene editing, the detection of the activation / suppression of a gene or other genetic element encoded by the at least one target nucleic acid, and / or the detection of the prime editing or base editing, for appropriately detecting the editing of the at least one target nucleic acid; comprising; e) Here, it is solved by providing a method in which the detection of the editing of at least one target nucleic acid detects the at least one detectable RNA in the cell, tissue, and / or sample.

[0020] In a preferred embodiment of the present invention, the above methods can be combined. For example, at least one detectable RNA in the cell, tissue, and / or sample can be detected based on the binding, cleavage, and / or editing of the at least one nucleic acid DNA.

[0021] In its fourth aspect, the problem of the present invention is a method for discovering a medical condition in a mammal related to the presence of at least one detectable RNA, the expression of the detectable RNA, and / or a mutation (s) in the detectable RNA, comprising implementing a method according to any of the above aspects, and discovering the medical condition based on the presence of the at least one detectable RNA detected, the expression of the detectable RNA, and / or a mutation (s) in the detectable RNA. This is solved by providing a method.

[0022] In its fifth aspect, the problem of the present invention is a detection system for a detectable RNA, comprising: a) a non-naturally occurring tracrRNA designed to bind to at least one portion of the detectable RNA further comprising a portion that specifically hybridizes to a first portion of the target nucleic acid; and b) at least one tracrRNA-dependent CRISPR nuclease enzyme. This is solved by providing a detection system.

[0023] In that sixth aspect, the problem of the present invention is solved by providing an unnatural-produced tracrRNA nucleic acid molecule comprising a portion containing an anti-repeat region sequence designed to specifically hybridize to a preselected test RNA sequence through formation of a complex that mimics a natural crRNA:tracrRNA duplex. The unnatural-produced tracrRNA nucleic acid molecule can be a part of a complex comprising the unnatural-produced tracrRNA nucleic acid molecule according to the present invention, at least one tracrRNA-dependent CRISPR nuclease enzyme, and at least one test RNA, and can further bind to a target DNA nucleic acid molecule comprising a sequence based on the at least one test RNA sequence or a sequence designed based thereon.

[0024] In that seventh aspect, the problem of the present invention is solved by providing the use of an unnatural-produced tracrRNA nucleic acid molecule or a test RNA detection system according to the above aspects for carrying out the method according to any of the above aspects, in particular for detecting a test RNA, a viral test RNA, a test RNA transcribed from a disease marker, or for generating an expression profile regarding one or more test RNAs.

[0025] In that first aspect, the problem of the present invention is a method for detecting at least one test RNA in a cell, tissue, and / or sample, a) contacting the sample with at least one unnatural-produced tracrRNA comprising a portion that specifically hybridizes to a first portion of the test RNA, wherein a second portion of the test RNA specifically hybridizes to at least one target nucleic acid, and the at least one unnatural-produced tracrRNA hybridizes or can hybridize to the test RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising an RNA cleavage enzyme such as RNase III, b) detecting the binding of the at least one tracrRNA-dependent CRISPR nuclease enzyme to the at least one target nucleic acid; comprising c) providing a method wherein the binding results in detection of the at least one RNA to be detected in the cell, tissue, and / or sample.

[0026] In this first aspect of the invention, detection of the at least one RNA to be detected in the cell, tissue, and / or sample is dependent on detection of the binding of the at least one tracrRNA-dependent CRISPR nuclease enzyme to the at least one target nucleic acid. Cas enzymes bind to target nucleic acids independent of their ability to cleave the target nucleic acid, and this freedom is used to detect the at least one RNA to be detected based on the binding activity.

[0027] The binding of the at least one tracrRNA-dependent CRISPR nuclease enzyme to the at least one target nucleic acid can be detected by any suitable detection method known to those skilled in the art, examples of which can include chromatin immunoprecipitation (ChIP) methods using antibodies against the nuclease and PCR primers for the target DNA sequence. Antibodies are used to selectively precipitate protein-DNA complexes from other genomic DNA fragments and protein-DNA complexes. PCR primers enable specific amplification and detection of the target nucleic acid sequence. The amount of the target nucleic acid sequence can be quantified by quantitative PCR (qPCR) techniques. The ChIP assay can be modified to an array-based format (ChIP-on-chip) or direct sequencing of DNA captured by the immunoprecipitated protein (ChIP-seq).

[0028] Other methods include DNA electrophoretic mobility shift assay (EMSA) or pull-down assay, which are used to selectively extract protein-DNA complexes from samples. Typically, pull-down assays use DNA probes labeled with high-affinity tags such as biotin, by which the probes can be recovered or immobilized. After the biotinylated DNA probe is complexed with proteins from cell lysates in a reaction similar to that used in EMSA, it can be used to purify the complex using agarose or magnetic beads. Next, the protein is eluted from the DNA and detected by Western blot or identified by mass spectrometry. Alternatively, the protein can be labeled with an affinity tag, or the DNA-protein complex can be isolated using an antibody against the target protein (similar to the supershift assay). In this case, the unknown DNA sequence to which the protein binds is detected by Southern blotting, PCR analysis, or sequence analysis. A microplate capture assay containing immobilized DNA probes, which is a hybrid of the DNA pull-down assay and enzyme-linked immunosorbent assay (ELISA), can be used to capture specific protein-DNA interactions and confirm the identity and relative amounts of the proteins using target-specific antibodies. Reporter assays can also be used to provide real-time in vivo readouts of translational activity for target promoters that can be blocked or induced by nuclease binding. The reporter gene is a fusion of the DNA sequence of the target promoter and the DNA sequence of the reporter gene. The DNA sequence of the promoter is customized by the researcher, and the DNA sequence of the reporter gene encodes a protein with detectable properties such as firefly luciferase, Renilla luciferase, alkaline phosphatase, or green fluorescent protein. These genes produce enzymes only when the target promoter is activated. Subsequently, the enzyme emits fluorescence or catalyzes a substrate to produce light, color change, or other reactions that can be detected by a spectrometer.Signals from reporter genes are used as indirect determinants of the transcription or translation of endogenous proteins driven from the same promoter.

[0029] In embodiments of this aspect of the invention, preferably, at least one tracrRNA-dependent CRISPR nuclease enzyme has its cleavage activity at least substantially inactivated. For example, both the RuvC and HNH nuclease domains can be inactivated by point mutations (e.g., D10A and H840A in Cas9 (SpCas9) of S. pyogenes), resulting in a Cas9 (dCas9) molecule without nuclease activity that cannot cleave target nucleic acids. Nevertheless, the dCas9 molecule retains the ability to bind to target DNA based on the sgRNA targeting sequence. In the case of a single-stranded target, disruption of the HNH domain is sufficient to prevent cleavage but not sufficient to prevent binding (Non-Patent Document 12). Even when the target sequence contains mismatches, particularly in the PAM distal region of the target, cleavage by the nuclease is avoided (Cas9 gRNA engineering for genome editing, activation and repression. Nat Methods. 2015;12(11):1051-1054). Similar to Cas9, the RuvC domain of Cas12 nuclease can be mutated to convert the nuclease into a programmable DNA-binding protein (Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems. Mol Cell. 2016;62(1):137-147), and introduction of mismatches can prevent cleavage but retain target binding (Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nat Methods. 2019;16(9):887-893).

[0030] The method according to the present invention detects at least one target RNA in a cell, tissue, and / or sample. In the context of the present invention, the target RNA is any target RNA detected using the method according to the present invention. Usually, preferably, the target RNA is a single-stranded RNA molecule, such as messenger RNA, ribosomal RNA, transfer RNA, small RNA, antisense RNA, small nucleolar RNA, microRNA, piwiRNA, long non-coding RNA, spliced intron, and circular RNA. The RNA may be of natural origin or artificially produced. The single-stranded target RNA may be derived from human cells, animal cells, plant cells, cancer cells, infected cells, or diseased cells, and / or may be derived from viruses, parasites, helminths, fungi, protozoa, bacteria, or pathogenic bacteria. The target RNA includes a first portion that specifically hybridizes with a first portion of a non-naturally produced tracrRNA produced and used in the method of the present invention, and a second portion that specifically hybridizes with at least one nucleic acid molecule.

[0031] The target RNA may include two or more first portions and second portions, and the non-naturally produced tracrRNA is designed accordingly. In the context of the present invention, the target nucleic acid can be any suitable target nucleic acid, but preferably includes target DNA, or target RNA or target ssDNA. This is because some Cas9 and Cas12 nucleases can target these nucleic acids.

[0032] The detected RNA may further include a 5' end and / or a 3' end that extends from the above-mentioned first part and extends outside the complex formed between the detected RNA, the non-naturally occurring tracrRNA, and at least one tracrRNA-dependent CRISPR nuclease enzyme (see Figure 2), and may include a part recognized by a dsRNA-cleaving enzyme and / or a 5' end cleaved or digested by other enzymes (see Figure 2). The 3' end is preferred because it can bring about an extension to the hybridizing part in the complex, thereby further stabilizing the molecule(s).

[0033] The detected RNA includes at least one second part that specifically hybridizes with at least one part of at least one target nucleic acid. In the context of the first aspect of the method according to the present invention, when the at least one tracrRNA-dependent CRISPR nuclease enzyme in the form of the complex (see Figure 2) between the detected RNA, the non-naturally occurring tracrRNA, and the at least one tracrRNA-dependent CRISPR nuclease enzyme binds to the at least one target nucleic acid, the at least one detected RNA in the cell, tissue, and / or sample is detected. As described above, the detection of the complex can be direct (e.g., by an antibody) or indirect using an appropriate assay described. The detection of the complex may include quantitative detection, that is, the amount of the detected RNA in the cell, tissue, and / or sample is determined using the amount of the detected complex.

[0034] The detected RNA can be labeled at either the 3' end and / or the 5' end and / or by including a marker within the RNA molecule / strand. Each method is known to those skilled in the art. The detected RNA may be a combination of RNA and parts of DNA and / or PNA.

[0035] Certain portions of the nucleic acid molecules used in the method according to the invention have been found to hybridize specifically with complementary portions in other molecules and / or are designed to do so. As is known to those skilled in the art, hybridization conditions and washing conditions are important for this purpose. High stringency hybridization can be carried out when the sequences are 100% complementary. Nevertheless, according to the invention, the hybridizing and / or specifically hybridizing portions are at least 80% complementary, preferably more than 90%, more preferably more than 95% and most preferably 100% complementary. The stringency of hybridization is determined by the hybridization temperature and the salt concentration in the hybridization buffer, with high temperature and low salt being more stringent. A commonly used washing solution is SSC (Saline Sodium Citrate, a mixture of Na citrate and NaCl). Hybridization can be carried out in solution, and more commonly, at least one component may be on a solid support, such as nitrocellulose paper. In a protocol often used, blocking reagents such as casein from non-fat dry milk or bovine serum albumin are often used in combination with denatured fragmented salmon sperm DNA (or other very complex heterologous DNA) and a surfactant such as SDS. Often, very high concentrations of SDS are used as a blocking agent. The temperature may be between 42°C and 65°C or higher, and the buffer may be 3×SSC, 25 mM HEPES (pH 7.0), 0.25% SDS (final).

[0036] The RNA cleavage enzyme cleaves the hybridized region formed between the first portion of the RNA to be detected and the anti-repeat portion of the non-native tracrRNA, or processes the other single-stranded end of the ncrRNA. This enzyme is preferably RNase III derived from E. coli, but may also be RNase III derived from other bacteria or archaea, as well as other dsRNA cleavage enzymes such as Drosha, Dicer, DCL1, Rntp1, or Pac1p. RNA cleavage enzymes such as RNase A can also cleave ssRNA.

[0037] Next, another aspect of the present invention relates to a method of transcriptional recording in vitro or in vivo, particularly in a multiplexed configuration (see, for example, FIGS. 13 and 19-22). In in vivo transcriptional recording, using the methods of the present invention described herein, the (respective) RNA to be detected present is converted into a readable signal or modification. In each example, the RNA to be detected is converted to ncrRNA via a non-native tracrRNA, which then directs the tracrRNA-dependent CRISPR nuclease to a target "reporter" DNA. This "reporter" DNA is edited, resulting in a detectable marker, modification, or "edit" that is maintained ("preserved") within the DNA. Examples are indels, markers introduced via HDR, base editing, prime editing, or RNA editing. As one readout, the expression of a reporter gene can be regulated. A fusion between dCas9 and the transcriptional activation domain VP16 is shown in FIG. 13B, but many other forms of expression regulation are possible (e.g., fusion of other regulatory domains to nucleases, use of pairs of nucleases with or without catalytic activity and partially mutated guide sequences, use of scaffold sgRNAs that recruit regulatory domains). Consistent with the robustness of the designs for the other methods disclosed herein, surprisingly, the transcriptional recording method was also found to be quite robust with respect to high G / C content and non-canonical sequences (see, for example, FIGS. 20-22, and also the following examples).

[0038] Previous examples of transcriptional recording detect environmental signals or non-specifically record the transcriptional profile of cells. Environmental recording relies on an inducible promoter that expresses a CRISPR acquisition protein or a guide RNA that drives editing. In contrast, transcriptional recording with a non-native tracrRNA can record one or multiple specific transcripts within a cell. This recording does not require an inducible promoter, and the non-native tracrRNA determines which transcripts are recorded. Multiple non-native tracrRNAs can also be combined with their corresponding DNA targets to perform multiplexed recording.

[0039] Perli SD (in Perli SD. Continuous genetic recording with self-targeting CRISPR-Cas in human cells. Science. 2016 Sep 9;353(6304):aag0511. doi: 10.1126 / science.aag0511. Epub 2016 Aug 18. PMID: 27540006) discloses a self-contained analog memory device that longitudinally records molecular stimuli as DNA mutations in human cells. This device consists of a self-targeting guide RNA (stgRNA), which enables local and continuous DNA mutagenesis in response to stgRNA expression by repeatedly directing the Cas9 nuclease activity of Streptococcus pyogenes towards the DNA encoding the stgRNA. Programmable and multiplexed memory storage in human cells induced by exogenous inducers or inflammation is demonstrated both in vitro and in vivo. This tool, the Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE), provides a clear strategy for investigating cell biology in vivo and enables continuous progression of targeted DNA sequences.

[0040] Farzadfard F, et al. (in Single-Nucleotide-Resolution Computing and Memory in Living Cells. Mol Cell. 2019 Aug 22;75(4):769-780.e4. doi: 10.1016 / j.molcel.2019.07.011. PMID: 31442423; PMCID: PMC7001763) disclose DOMINO, a robust and expandable platform for encoding logic and memory in bacterial and eukaryotic cells. DOMINO uses a single-nucleotide-resolution read-write head efficient for DNA manipulation to convert the DNA of living cells into an addressable, readable, and writable medium for computation and memory. DOMINO operators enable analog and digital molecular recording for signaling dynamics and long-term monitoring of cellular events. Furthermore, since multiple operators can be hierarchically organized and interconnected to encode order-independent sequential and temporal logic, the combination, order, and timing of molecular events within cells can be recorded and controlled. They believe that DOMINO provides a foundation for building robust and sophisticated computational and memory genetic circuits for a number of biotechnological and biomedical applications.

[0041] Tang, W., & Liu, D. R. (In Rewritable multi-event analog recording in bacterial and mammalian cells. Science. 2018 Apr 13;360(6385):eaap8992. doi: 10.1126 / science.aap8992. Epub 2018 Feb 15. PMID: 29449507; PMCID: PMC5898985) disclosed that Cas9 nuclease and base editors can be used to record the amplitude, duration, and order of stimuli as stable changes in the content of both genomic DNA and extrachromosomal DNA (see Perspectives by Ho and Bennett). Recording of multiple stimuli, including exposure to antibiotics, nutrients, viruses, and light, as well as Wnt signaling, has been achieved in living bacterial and human cells. The recorded memory can be erased and re-recorded over multiple cycles.

[0042] Finally, Schmidt, F. et al. (In Transcriptional recording by CRISPR spacer acquisition from RNA. Nature 562, 380 - 385 (2018). https: / / doi.org / 10.1038 / s41586-018-0569-1) enabled DNA-based memory of transcriptional information by using CRISPR spacer acquisition to capture intracellular RNA and convert it to DNA. In Escherichia coli, defined stimuli such as RNA viruses or any sequence, and complex stimuli such as oxidative stress, have been shown to result in quantifiable transcriptional recordings that are stored within the cell population. They showed that transcriptional recording enabled the classification and description of complex cellular behaviors and the identification of the precise genes that govern various cellular responses. In the future, it may be possible to reconstruct the transcriptional history that explains complex cellular behaviors or pathological states using RNA recording via CRISPR spacer acquisition followed by deep sequencing (Record-seq).

[0043] A method of transcriptional recording, particularly in a multiplexed configuration, for in vitro or in vivo transcriptional recording of at least one target DNA in a cell, tissue, and / or sample. a) Contacting the sample, cell, or tissue with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with a first portion of the RNA to be detected. Here, a second portion of the RNA to be detected specifically hybridizes with the at least one target DNA, and the at least one non-naturally occurring tracrRNA hybridizes or can hybridize with the RNA to be detected in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of a dsRNA cleavage enzyme such as RNase III. b) Nicking or cleaving the at least one target nucleic acid in the sample with the nuclease. c) Detectably editing the at least one target DNA, including, for example, non-homologous end joining (NHEJ) repair, microhomology-mediated end joining (MMEJ), homology-directed repair (HDR), detectable markers, detectable modifications, base editing, prime editing, or RNA editing. d) Optionally, appropriately detecting the editing of the at least one target DNA, including at least one method selected from, for example, detection of the presence and / or length of indels or gene editing, detection of activation / suppression of a gene or other genetic element encoded by the at least one target nucleic acid, and / or detection of the prime editing or base editing. Including e) Here, a method is preferred in which detection of the editing of the at least one target DNA records the transcription of the at least one RNA to be detected in the cell, tissue, and / or sample. Preferred embodiments and components are similar to other methods described herein.

[0044] Preferred uses of the transcription recording according to the present invention relate to the analysis of the microbiota and / or microbial sentinels (i.e., commensal bacteria that function as reporters for non-invasive measurements, see, for example, Stewart JR, et al. The coastal environment and human health: microbial indicators, pathogens, sentinels and reservoirs. Environ Health. 2008 Nov 7;7 Suppl 2(Suppl 2):S3. doi: 10.1186 / 1476-069X-7-S2-S3. PMID: 19025674; PMCID: PMC2586716), and the tracking of the spread of viruses or bacteria in vivo, such as the whole organism, or based on samples taken, for example, from the environment (e.g., the spread of viruses or resistant bacteria detected in wastewater samples, etc.).

[0045] During studies in the context of the present invention using the 84-21 strain of Campylobacter jejuni (Cje), the inventors evaluated the RNA co-immunoprecipitated with CjeCas9. One set shared complementarity to crRNA2 in that strain, while the other set shared complementarity to the anti-repeat portion of the trans-acting CRISPR RNA (tracrRNA) responsible for crRNA maturation. This binding was similar to the way tracrRNA processes crRNA from the repeat to form mature crRNA. From these insights, the first indication was given that mature crRNAs (which direct Cas9 to their targets) can be derived from mRNAs and other RNAs other than those encoded by the CRISPR array, although crRNAs were thought to be derived only within the CRISPR array and the CRISPR-Cas system.

[0046] Surprisingly, in the context of the present invention, it has been found that the tracrRNA-dependent CRISPR nuclease enzyme system can be designed in a specific manner to provide a highly versatile and specific diagnostic tool. An essential element of this tool is the design of a non-naturally produced tracrRNA. Specific modifications of tracrRNA, and thus "reprogramming", enable the production of ncRNAs specialized for a purpose. Interestingly, the tracrRNA-dependent CRISPR nuclease enzyme system has been found to still be functional despite the expectation that reprogramming of tracrRNA might disrupt the recognition and activity of Cas9.

[0047] In the context of the present invention, the RNAs identified using a motif complementary to the tracrRNA anti-repeat had an approximate size of the processed crRNA (36 nucleotides to 37 nucleotides). In one specific example (derived from the fliF gene), the ncRNA was consistent with the structure of the crRNA (guide of approximately 24 nucleotides + processed repeat of approximately 12 nucleotides). A method according to the present invention is preferred, wherein the portion of the at least one non-naturally produced tracrRNA that specifically hybridizes to the first portion of the RNA to be detected hybridizes for 10 nucleotides or more, preferably 11 nucleotides or more, more preferably approximately 12 nucleotides or more. A range between 9 nucleotides and 15 nucleotides is a preferred range, 10 nucleotides to 14 nucleotides is more preferred, and 12 nucleotides is most preferred. As described above, an extension to the hybridizing portion in the complex (5' extension of tracrRNA, 5' extension to ncRNA) is possible and preferred, providing the advantage of more stable formation to the complex.

[0048] The non-naturally occurring tracrRNA used in the present invention includes a sequence that specifically hybridizes with the first portion of the target RNA and forms a stem-like secondary structure that basically mimics the original repeat:anti-repeat stem formed between the crRNA and the tracrRNA. The method according to the present invention is preferred, wherein the portion of the at least one non-naturally occurring tracrRNA that specifically hybridizes with the first portion of the target RNA hybridizes with 10 nucleotides or more, preferably 11 nucleotides or more, more preferably about 12 nucleotides or more.

[0049] The method according to the present invention is preferred, wherein the first portion of the target RNA contains a PAM (see FIG. 14). As an example, in the case of the Cas12 nuclease, the PAM is present in the repeat portion upstream of the guide portion of the obtained ncrRNA, while in the case of Cas9, the PAM is present in the repeat portion downstream of the guide portion of the ncrRNA. By selecting a target having an appropriate PAM, the generated crRNA can target its own DNA site, that is, the ncrRNA can target the DNA from which the ncrRNA originated. Thereby, editing is possible only when the gene is transcribed. Using this strategy, for example, editing can be significantly limited only in specific tissues according to the gene expression profile.

[0050] In a preferred embodiment of the method according to the present invention, for example, the nuclease can be modified to recognize a broader range of PAM sites by replacing a major region in the PAM-interacting (PI) domain of Cas9 with the corresponding region in a related series of Cas9 orthologs (see, for example, Ma et al., Engineer chimeric Cas9 to expand PAM recognition based on evolutionary information. Nat Commun. 2019 Feb 4;10(1):560. doi: 10.1038 / s41467-019-08395-8). This expands the possibility of genomic targets within the cell.

[0051] As described above, the non-naturally occurring tracrRNA used in the present invention includes a sequence that specifically hybridizes with the first portion of the test RNA to form a stem-like secondary structure that basically mimics the original repeat:anti-repeat stem. In a preferred embodiment of the method according to the present invention, this stem-like secondary structure can be extended (i.e., 3' to the mRNA and 5' to the non-naturally occurring tracrRNA) to create a dsRNA, and thus an RNase III recognition site or domain. For example, when a detectable group is attached to the stem extension that can be cleaved by RNase activity, this structure can be used in a particular assay. The inventors evaluated mismatches in the repeat-anti-repeat stem recognized and cleaved by RNase III. The ncrRNA mutant was expressed together with tracrRNA. Mutations were introduced into the ncrRNA while preserving the tracrRNA. Overall, large-scale mutations can be tolerated. Without wishing to be bound by theory, this suggests that RNase III may not be essential for Cas9 activity.

[0052] The inventors further analyzed how much the ncrRNA can deviate from the conventional crRNA, and found that the "robustness" of the system related to the stem-like secondary structure that basically mimics the original repeat:anti-repeat stem in the natural double strand was shown (see above). Particular attention was paid to the base stem bound by Cas9. Bulges were created by mutating the repeats, thereby preserving the tracrRNA. The results indicate that bulges can be tolerated to a considerable extent, especially when the bulge is smaller, when it is far from the base of the stem, or when it is on the repeat side. Very similar results were seen when adapting to some mismatches within the stem. See Figures 5-9 for supporting data.

[0053] The inventors further analyzed how much the ncrRNA can deviate from the conventional crRNA, and found that the "robustness" of the system related to the stem-like secondary structure that basically mimics the original repeat:anti-repeat stem in the natural double strand was shown (see above). Next, particular attention was paid to the base stem bound by Cas12. Bulges were created by mutating the repeats, thereby preserving the tracrRNA. The results indicate that bulges can be tolerated to a considerable extent, especially when the bulge is smaller, when it is far from the base of the stem, or when it is on the repeat side. Very similar results were seen when adapting to some mismatches within the stem. See Figures 23-25 for supporting data.

[0054] Therefore, as shown in the two examples of Cas9 and Cas12, the ncrRNA can deviate from the conventional crRNA, which indicates the "robustness" of the system related to the stem-like secondary structure that basically mimics the original repeat:anti-repeat stem in the natural double strand (see above).

[0055] Furthermore, the effect of reprogramming tracrRNA to convert various RNAs into crRNA was tested. Using sgRNA and CjeCas9 in TXTL, the changes to the repeat:anti-repeat duplex were evaluated and it was found that all changes could be accommodated with little effect on target cleavage (see Figure 4). In particular, the duplex between the repeat and the anti-repeat can accommodate various sequences with little effect on the targeting activity by CjeCas9 (Figure 4). The mutations were found to have only a limited effect on the time to target cleavage. Also, the duplex between the repeat and the anti-repeat can accommodate several bulges on either side as part of the DNA targeting by CjeCas9 (Figure 5), and the duplex between the repeat and the anti-repeat can similarly accommodate mismatches as part of the DNA targeting by CjeCas9 (Figure 6). Most of the duplexes maintained the targeting activity despite the presence of bulges. The region of the repeat:anti-repeat duplex involved in RNase III processing can also accommodate mismatches and bulges as part of the DNA targeting by CjeCas9 (Figure 7). Figure 8 shows that extensions to the 5' or 3' end of the crRNA have only a limited effect on the targeting activity by CjeCas9. Similar rules apply to Cas12 and other enzymes as disclosed herein (see Figures 22-25).

[0056] Furthermore, the engineered tracrRNA that converts the detected RNA into the active crRNA has been shown to function well with CjeCas9 (Figure 9), SpyCas9 (Figure 10), and Sth1Cas9 (Figure 11). According to the present invention, the engineered tracrRNA can be designed to induce the formation of the active ncrRNA used by CjeCas9 from the detected RNA (Figure 9). After design, the results of TXTL show DNA targeting induced by the detected RNA and the engineered tracrRNA. Each engineered tracrRNA is designed to hybridize to a specific region of the RNA being tested. Overall, all five engineered tracrRNAs tested produced strong cleavage activity. In another aspect, the engineered tracrRNA can be designed to induce the formation of the active ncrRNA used by SpyCas9 from the detected RNA (Figure 10). Again, all five engineered tracrRNAs tested produced strong cleavage activity. Thus, other Cas9s different from CjeCas9 (SpyCas9, Sth1Cas9) can be used with the reprogrammed tracrRNA to convert the detected RNA into the active crRNA. Again, as disclosed herein, similar rules apply to Cas12 and other enzymes.

[0057] A second portion of the detected RNA specifically hybridizes with at least one target nucleic acid that defines the nucleic acid target of the Cas protein, resulting in a crRNA-like ncrRNA. At least one engineered tracrRNA hybridizes or can hybridize with the detected RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme.

[0058] Non-naturally occurring tracrRNAs can be produced according to standard methods, such as by synthesis, produced by in vitro transcription, and / or cloned into a plasmid. The tracrRNAs may be derived from naturally occurring sequences, which are then modified to engineer the sequences within the molecule as desired. The tracrRNAs may further comprise labels or modified nucleotides, such as additional modifications like inosine, etc.

[0059] In a second aspect thereof, the problem of the present invention is a method for detecting at least one target RNA in a cell and / or a sample, comprising: a) contacting the sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes to a first portion of the target RNA, wherein a second portion of the target RNA specifically hybridizes to at least one target nucleic acid, and the at least one non-naturally occurring tracrRNA hybridizes or is capable of hybridizing to the target RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of an ssRNA and / or dsRNA cleavage enzyme such as an RNase III enzyme; b) detecting cleavage of the at least one target nucleic acid in the sample by the nuclease enzyme; comprising: c) wherein detection of the cleavage of the at least one target nucleic acid results in detection of the at least one target RNA in the cell, tissue, and / or sample.

[0060] The basic principle of this second aspect regarding the components used is as described above. In an aspect of the present invention, detection of the at least one target RNA in the cell, tissue, and / or sample depends on detection of cleavage of the at least one target nucleic acid in the sample by the nuclease enzyme, and detection of the cleavage of the at least one target nucleic acid results in detection of the at least one target RNA in the cell, tissue, and / or sample.

[0061] Detection of binding products and / or cleavage products based on the target nucleic acid can be direct (e.g., detection of size changes in cleaved nucleic acids) or indirect using appropriate fluorophores with or without a quencher. Cleavage of the nucleic acid releases a detectable fluorophore, changes are detected by passing the cleaved nucleic acid through a nanopore sequencer, the fluorescently labeled nuclease can be localized, and / or cleavage affects a detectable electric current. Detection of the cleaved product may sometimes involve quantitative detection, i.e., the amount of the detected fragment(s) is used to determine the amount of the RNA to be detected in the cell, tissue, and / or sample.

[0062] Detection of binding can be indirect based on regulation of reporter gene expression. A nuclease without at least one catalytic activity can directly suppress transcription by binding to the promoter region or coding region of the reporter gene. Alternatively, the nuclease can recruit regulatory domains responsible for local regulation of gene expression, such as domains that upregulate expression (e.g., VP16, VPR) or domains that downregulate expression (e.g., KRAB). These domains can be operably linked to a nuclease without at least one catalytic activity. These domains can be recruited through interaction with a binding domain fused to a nuclease without at least one catalytic activity and / or a tracrRNA.

[0063] In its third aspect, the object of the present invention is a method for detecting at least one RNA to be detected in a cell, tissue, and / or sample, a) contacting the sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes to a first portion of the RNA to be detected; Here, the second portion of the detected RNA specifically hybridizes with at least one target nucleic acid, and the at least one non-naturally occurring tracrRNA hybridizes with or can hybridize with the detected RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, which optionally further includes the presence of an ssRNA and / or dsRNA cleavage enzyme such as an RNase III enzyme. b) nicking or cleaving the at least one target nucleic acid in the cell, tissue, and / or sample with the nuclease; c) detectably editing the at least one target nucleic acid, including, for example, non-homologous end joining (NHEJ) repair, microhomology-mediated end joining (MMEJ), homology-directed repair (HDR), prime editing, base editing, or RNA editing; d) appropriately detecting the editing of the at least one target nucleic acid, including at least one method selected from, for example, detection of the presence and / or length of indels or gene editing, detection of activation / suppression of a gene or other genetic element encoded by the at least one target nucleic acid, and / or detection of the prime editing or base editing; comprising; e) solved by providing a method wherein detection of at least one target nucleic acid detects the at least one detected RNA in the cell, tissue, and / or sample.

[0064] The basic principle of this third aspect regarding the components used is as described above. In this aspect of the present invention, the detection of the at least one target RNA in the cell, tissue, and / or sample depends on detecting the cleavage of the at least one target nucleic acid in the sample by the nuclease enzyme, editing the at least one cleaved target nucleic acid to be detectable, and further detecting the editing of the at least one target nucleic acid as appropriate. Here, by detecting the editing of the at least one target nucleic acid, the at least one target RNA in the cell, tissue, and / or sample is detected. Therefore, this aspect depends on the detection of changes to a target nucleic acid that is controlled or caused ( "driven") by the target RNA.

[0065] Editing the at least one cleaved target nucleic acid to be detectable may preferably include non-homologous end joining (NHEJ) repair, microhomology-mediated end joining (MMEJ), homology-directed repair (HDR), and / or base editing. For example, cells or single-cell zygotes typically use the error-prone non-homologous end joining (NHEJ) repair pathway to repair DNA cleaved by Cas9, which randomly inserts or deletes DNA bases to repair double-strand breaks. The most common indels by NHEJ repair are in the range of 1 nucleotide to 15 nucleotides, preferably 1 nucleotide to 9 nucleotides. There are a number of repair pathways known to those skilled in the art that can be employed depending on cell type and conditions.

[0066] The detection of the editing of the at least one target nucleic acid may include, for example, at least one method selected from the detection of the presence and / or length of indels or gene editing, the activation / suppression of genes or other genetic elements encoded by the at least one target nucleic acid, and / or the detection of base editing, for example, by sequencing, restriction digestion, and / or detection of length changes of the target nucleic acid. As described above, the detection of cleavage products based on the target nucleic acid can be direct (e.g., detection of size changes of the cleaved nucleic acid) or indirect using an appropriate phosphor. Cleavage of the nucleic acid releases a detectable phosphor, and changes are detected by passing the cleaved nucleic acid through a nanopore sequencer, the fluorescently labeled nuclease can be localized, and / or the cleavage affects a detectable electric current. Detection of cleavage products may include quantitative detection, i.e., the amount of the detected fragment(s) is used to determine the amount of the RNA to be detected in the cell, tissue, and / or sample.

[0067] The length of the indel to be detected is between about 1 nucleotide and 15 nucleotides, more preferably between about 1 nucleotide and 9 nucleotides, and optionally the indel can be detected by sequencing, restriction digestion, and / or detection of length changes of the target nucleic acid, and by qPCR. The method according to the invention is preferred. In a preferred embodiment, this makes it possible to improve the efficiency of gene editing by so-called iterative self-targeting. The presence of an indel almost always allows re-targeting of the sequence, leading to re-attack. Since some indels do not interfere with the re-attack, this approach may then lead to larger-sized indels and / or higher homologous recombination frequencies. In practice, this approach advantageously allows follow-up of lineage tracing.

[0068] Detectable base editing corresponds to distinct editing means. Generally, base editors fall into two classes: cytosine base editors (CBEs) and adenine base editors (ABEs). Cytosine base editors are created by fusing Cas9 nickase or catalytically inactive "dead" Cas9 (dCas9) to a cytidine deaminase such as APOBEC. Base editors are targeted to specific loci by this system and can convert cytidine to uridine within a small editing window near the PAM site. Subsequently, uridine is converted to thymidine by base excision repair, resulting in a change from C to T (or G to A on the opposite strand). Adenine base editors are engineered to convert adenosine to inosine, which is treated by the cell similarly to guanosine, thus resulting in a change from A to G (or T to C). Adenine DNA deaminase does not exist in nature but was created by directed evolution of TadA from Escherichia coli, which is a tRNA adenine deaminase. Similar to cytosine base editors, an evolved TadA domain is fused to the Cas9 protein to create adenine base editors.

[0069] Both types of base editors are available with a number of Cas9 variants that include high-fidelity Cas9 (see below). Further progress has been made by optimizing the expression of the fusion, modifying the linker region between the Cas variant and the deaminase to adjust the editing window, or adding fusion products such as a DNA glycosylase inhibitor (UGI) or the Gam protein from bacteriophage Mu (Mu-GAM) to increase product purity. Many base editors are designed to function within a very narrow window proximal to the PAM sequence, but some useful base editing systems are well-suited for directed evolution applications because they result in a wide range of single nucleotide variants (somatic hypermutation) within a broader editing window. Examples of these base editing systems include targeted AID-mediated mutagenesis (TAM) and CRISPR-X, where Cas9 is fused to activation-induced cytidine deaminase (AID).

[0070] In a preferred embodiment of the method according to the present invention, the above method is carried out in vivo, for example, in cells, tissues, or bacteria, fungi, plants, or animals, or in a sample in vitro. The sample may be a solid sample or a liquid sample and may be selected from a sample containing cells and a cell-free in vitro sample. The cells are preferably plant cells or animal cells, such as mammalian cells, preferably human cells.

[0071] The sample can be a biological sample, preferably a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph fluid, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, serous effusion, pus, or a biological sample obtained from a swab of the skin surface or mucosal surface. In one aspect, the cells, tissues, and / or samples may be crude samples and / or one or more nucleic acid molecules may not be purified or amplified from the sample prior to application of the method. In another aspect, the cells, tissues, and / or samples may be purified or partially purified (concentrated) samples and / or one or more nucleic acid molecules may be purified or amplified from the sample prior to application of the method. In another aspect, the cells may be part of an environmental sample such as air, natural water bodies (e.g., rivers, lakes, oceans), wastewater, or soil.

[0072] The method according to the present invention may be partially or fully automated and may be carried out, for example, fully or partially by a robot. The method according to the present invention may require the use of a computer and respective databases for executing and / or analyzing the obtained results.

[0073] In one aspect, several or even more target nucleic acids are analyzed in one or more samples, tissues, and / or cells, preferably a number of samples, tissues, and / or cells. In one aspect, several or even more detectable RNAs are detected in one or more samples, tissues, and / or cells, preferably several or even more samples, tissues, and / or cells.

[0074] In a preferred embodiment of the method according to the present invention, in one or more samples, tissues, and / or cells, preferably in several or even more samples, tissues, and / or cells, two or more non-naturally occurring tracrRNAs that specifically hybridize to various portions of the RNA to be detected are selected, designed, produced (manufactured, see above), and used.

[0075] This system can target, for example, anywhere from two to seven loci by cloning multiple tracrRNAs into a single plasmid. These multiplex tracrRNA vectors can be used in the context of the present invention in combination with any of the above-described CRISPR nucleases as appropriate.

[0076] In a preferred embodiment of the method according to the present invention, the at least one RNA to be detected is single-stranded or initially double-stranded. In the context of the present invention, the RNA to be detected is any target RNA detected using the method according to the present invention. Usually, preferably, the RNA to be detected is a single-stranded RNA molecule such as mRNA or non-coding RNA. This RNA may be of natural origin or artificially produced. The single-stranded RNA to be detected may be derived from human cells, animal cells, plant cells, immune cells, cancer cells, infected cells, or diseased cells, and / or may be derived from viruses, parasites, helminths, fungi, protozoa, bacteria, or pathogenic bacteria.

[0077] In a preferred embodiment of the method according to the present invention, the at least one RNA to be detected is derived from a virus selected from the group consisting of Zika virus, human immunodeficiency virus (HIV), hepatitis B virus, hepatitis C virus, herpes virus, coronavirus, influenza, herpes simplex virus type I, herpes simplex virus type II, papillomavirus, rabies virus, cytomegalovirus, human serum parvo-like virus, respiratory syncytial virus, varicella-zoster virus, measles virus, adenovirus, human T cell leukemia virus, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, bluetongue virus, Sendai virus, feline leukemia virus, reovirus, poliovirus, simian virus 40, murine mammary tumor virus, dengue virus, rubella virus, West Nile virus, and yellow fever virus.

[0078] In a preferred embodiment of the method according to the present invention, the at least one RNA to be detected is derived from a pathogenic bacterium selected from Mycobacterium tuberculosis, Streptococcus agalactiae, Staphylococcus aureus resistant to methicillin, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, and Brucella abortus.

[0079] In a preferred embodiment of the method according to the present invention, the at least one RNA to be detected is derived from a gene whose transcription and / or expression is modified in response to external factors such as, for example, metabolic factors or signals, hormones, pathogens, toxins, drugs, aging, and / or biotic or abiotic stress.

[0080] In a preferred embodiment of the method according to the invention, the RNA to be detected is selected to be specific to an environment, species, strain, disease, cell, and / or tissue. In this aspect, the method of the invention serves to identify and / or classify cells or organisms based on the selected RNA to be detected. At least one RNA to be detected is preferably selected from viral infections, such as coronavirus infections, infections by pathogens, metabolic diseases, cancer, neurodegenerative diseases, aging, drugs, and biological or abiotic stress-related pathologies.

[0081] In a preferred embodiment of the method according to the invention, prior to step a), at least one RNA to be detected can be added to the cells, tissues, and / or samples, and / or the method further comprises at least one step selected from in vitro transcription from DNA to RNA, reverse transcription from RNA to DNA, and optimally subsequent in vitro transcription from the DNA to RNA. This can be done to provide appropriate or desired signal amplification. Usually, the RNA to be detected in the cells, tissues, or samples is present in the range of about 500 fM to about 1 μM, for example about 500 fM to about 1 nM, preferably in the range of about 1 pM to about 1 nM. Optimally, the method can detect a single molecule per cell, tissue, and / or sample.

[0082] In another preferred embodiment of the method according to the present invention, for example, as listed in Non-Patent Document 7 (the listing of which forms part of this specification by reference), any tracrRNA-dependent CRISPR nuclease enzyme can be used as long as the tracrRNA is required for its binding function. Preferably, the nuclease is selected from type II Cas9 and type V Cas12 nuclease enzymes, and the recently identified CasX (now type V-E) (Liu et al. CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature, 2019 Feb; 566(7743): 218-223). Examples are type II Cas9 nucleases selected from the group consisting of II-A, II-B, and II-C, and types of Cas12 nucleases selected from the group consisting of type V-B, V-C, V-D, V-E, V-F, V-G, and V-K. Usually, an appropriate plasmid system is used to introduce and express the Cas nuclease. Type V-A, identified as a system in type V first known in 2013 in the human pathogenic bacterium Francisella tularensis, has the characteristic effector protein Cas12a (previously called Cpf1). Type V-B and V-C were identified by Shmakov et al. in 2015, type V-F was identified by Harrington et al. in 2018, and types V-G, V-H, and V-I were identified by Yan et al. in 2019.By querying uncultured bacteria in groundwater and sediment samples via genome-resolved metagenomics, V-D and V-E types were identified in 2017 by Burstein and colleagues (Tong B, et al. The Versatile Type V CRISPR Effectors and Their Application Prospects. Front Cell Dev Biol. 2021 Feb 4;8:622103. doi: 10.3389 / fcell.2020.622103. PMID: 33614630; PMCID: PMC7889808).

[0083] Deltcheva et al. (in Deltcheva et al., CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011; 471(7340):602-607. doi:10.1038 / nature09886) reported identifying a trans-encoded small RNA, tracrRNA, with 24-nucleotide complementarity to the repeat region of the crRNA precursor transcript by differential RNA sequencing of the human pathogen Streptococcus pyogenes. They showed that tracrRNA induces crRNA maturation through the activities of the widely conserved endogenous endoribonuclease RNase III and the CRISPR-associated Cas9 (Csn1) protein, and all of these components were found to be essential for protecting S. pyogenes from phage-derived DNA. tracrRNA pairs with the repeat region, providing a substrate for RNase III. All examples of type V-B / C / E / F / G / K nucleases using sgRNA are effectively dependent on tracrRNA. Next, the cleaved RNA duplex is bound by Cas9, and the crRNA portion induces DNA targeting. tracrRNA has also been identified in subtype V-B systems, where the cleavage enzyme has not yet been characterized (Liu L, Chen P, Wang M, Li X, Wang J, Yin M, Wang Y. 2017C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism. Mol. Cell 65, 310-322).

[0084] In yet another preferred embodiment of the method according to the invention, at least one tracrRNA-dependent CRISPR nuclease enzyme is modified, e.g., mutated, and / or is a recombinant fusion protein. A given RNA targeting sequence will have additional sites of partial homology throughout the genome or, e.g., as part of an in vitro diagnosis, there may be a number of target nucleic acids that resemble each other. These sites are called off-targets and need to be considered when designing the ncrRNA. In addition to optimizing the RNA design, the specificity of CRISPR can also be enhanced by modifications to Cas9. When high specificity is important, a dual nickase approach that results in a double nick-induced double-strand break can be used, and this can also be combined with HDR-mediated gene editing for specific gene editing. Another approach is the use of mutant Cas nucleases called high-fidelity enzymes. For example, the phage-assisted continuous evolution (PACE) method results in xCas9 3.7, which has seven mutations in the REC2, REC3, and PAM interaction domains, enabling not only an expansion of PAM recognition but also an increase in specificity and a decrease in off-target activity. Thus, the tracrRNA-dependent CRISPR nuclease enzyme can be mutated in the PAM interaction domain to change PAM recognition.As another example, by screening E. coli cells transformed with a pooled library of SpCas9 variants, mutant variants such as so-called Sniper-Cas with less off-target activity than wild-type Cas9 can be identified (Directed evolution of CRISPR-Cas9 to increase its specificity. Nat Commun. 2018 Aug 6;9(1):3048, Lee, J., Jung, M. h., Jeong, E., Lee, J. K. Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution. J. Vis. Exp. (144), e59202, doi:10.3791 / 59202 (2019)).

[0085] For example, Cas9 can be fused with a transcriptional repressor or activator, and targeting these dCas9 fusion proteins to the promoter region causes robust transcriptional repression (CRISPR interference, i.e., CRISPRi) or activation (CRISPRa) of downstream target genes. The simplest dCas9-based activator and repressor consist of dCas9 directly fused to a single transcriptional activator (e.g., VP64) or repressor (e.g., KRAB). Furthermore, for stronger activation of target genes in mammalian cells, co-expression of epitope-tagged dCas9 and an antibody-activator effector protein (e.g., the SunTag™ system), dCas9 sequentially fused to several different activation domains (e.g., dCas9-VPR), or co-expression of dCas9-VP64 with a modified scaffold sgRNA and an additional RNA-binding helper activator (e.g., the SAM activator) and other activation strategies have been developed. Importantly, unlike genome modification induced by Cas9 or Cas9 nickase, gene activation or repression mediated by dCas9 is reversible because it does not permanently modify genomic DNA. dCas9 has also been used for genome-wide screening to activate or repress gene expression in mouse and human cells. Synthetic CRISPR-Cas gene activators have been developed for bacteria by recruiting activator proteins using a scaffold RNA containing a gRNA and an RNA hairpin. CRISPRi may be adapted to diverse bacterial species using a modular system called Mobile-CRISPRi. In this system, CRISPRi is introduced into bacteria using conjugation and stably integrated into the chromosome.

[0086] In another aspect of the method according to the present invention, the first portion of the detected RNA comprises a protospacer adjacent motif (PAM). As an example, in the case of Cas12 nuclease, the PAM is present within the repeat portion upstream of the guide portion of the obtained ncrRNA, while in the case of Cas9, the PAM is present in the repeat portion downstream of the guide portion of the ncrRNA. By selecting a target having an appropriate PAM, the generated crRNA can target its own site. This enables editing only when the gene is being transcribed. This strategy significantly limits editing, for example, for a specific tissue, depending on the expression profile of the gene to be targeted.

[0087] As described above, in a preferred embodiment of the method according to the present invention, the detected RNA and the non-naturally produced tracrRNA result in a recognition site or domain for a dsRNA, and thus a dsRNA-cleaving enzyme, such as an RNase III enzyme. An ssRNA-cleaving enzyme such as RNase A can also be applied to facilitate further processing or remove RNA that has not been converted into ncrRNA. Preferably, the substrate for the dsRNA-cleaving enzyme in the detected RNA has a length of 10 nucleotides or more, for example, up to about 50 nucleotides or more, preferably 11 nucleotides or more, more preferably about 12 nucleotides or more. A range between 9 and 15 nucleotides is a preferred range, 10 to 14 nucleotides is more preferred, and 12 nucleotides is most preferred.

[0088] In another aspect, the hybridizing portion of the nucleic acid molecule used in the method of the present invention is complementary up to at least 80%, preferably more than 90%, more preferably more than 95%, and most preferably 100% to each other. As discussed above, surprisingly, it has been found that ncrRNA can deviate from conventional crRNA, indicating the "robustness" of the system with respect to a stem-like secondary structure that basically mimics the original repeat:anti-repeat stem, with particular attention paid to the base stem bound by Cas9 and Cas12 (see FIGS. 20 to 25). The results indicate that bulges can be tolerated to a significant extent, especially when the bulge is smaller, farther from the base of the stem, or on the repeat side. Very similar results were seen when adapting some mismatches within the stem. Thus, the nucleotide sequence of the at least one portion of the tracrRNA that specifically hybridizes to the first portion of the detected RNA is produced and / or modified to be complementary up to at least 80%, preferably more than 90%, more preferably more than 95%, and most preferably 100% to the first portion with respect to the detected RNA.

[0089] In another aspect of the method according to the present invention, the method at least partially includes quantitative analysis. Thus, a step preferably includes detecting the amount of at least one modified target nucleic acid in the sample, tissue, and / or cell, such as a cleaved and / or edited product and / or the at least one detected RNA, and the amount of the target nucleic acid bound thereto. Preferably, the amount per sample, tissue, and / or cell is determined in comparison to a control. Quantitative assays are known to those skilled in the art and may include absorbance (e.g., UV spectrophotometry) and / or fluorescence assays, and real-time PCR. The assay can quantify the amount(s) and / or ratio(s) of the nucleic acid(s) (which may be plural) to be quantified (at least one modified target nucleic acid and / or the at least one detected RNA) as a single value (e.g., as a result of the assay used or at the "end" thereof), or can monitor the change over time in the nucleic acid, i.e., preferably further includes detecting the change in the amount of the modification of the at least one target nucleic acid (e.g., cleaved and / or edited product) and / or the at least one detected RNA in the sample, tissue, and / or cell, particularly in comparison to a control.

[0090] In yet another aspect of the method according to the present invention, a number of labels and / or markers are used. The markers can be used for both nucleic acid molecules and protein components (e.g., nucleases and / or fusions) that form part of the assay. The labels and markers can not only be included in the components of the assay (particularly nucleic acids and / or proteins), but can also constitute moieties attached either covalently or non-covalently.

[0091] Next, another aspect of the present invention is a method for discovering a medical condition in a cell, tissue, or organism, such as a mammal, preferably a human, wherein the medical condition is related to the presence of at least one detected RNA, the expression of the detected RNA, and / or a mutation (s) in the detected RNA. This method involves performing the method according to the present invention as described above and discovering the medical condition based on the presence of at least one detected RNA detected, the expression of the detected RNA, and / or a mutation (s) in the detected RNA.

[0092] Medical conditions that can be discovered using the present invention are those related to at least one detected RNA molecule. As described above, the detected RNA can itself constitute the origin of a medical condition or disease, for example, in the case of an infection of the cell, tissue, and / or sample being tested, such as a viral infection such as a coronavirus infection, a bacterial infection, and / or a fungal infection. Other medical conditions may be indirectly related to at least one detected RNA molecule, for example, in the case of RNA that is abnormally transcribed (present or discovered), expressed, processed (e.g., spliced), and / or mutated. The detected RNA molecule may be present in an increased or decreased amount compared to a healthy control (e.g., a control based on a group of healthy samples or diseased samples).

[0093] In a preferred embodiment of the method according to the invention, the at least one RNA to be detected is single-stranded or initially double-stranded. The single-stranded RNA to be detected may be derived from / associated with human cells, animal cells, plant cells, immune cells, cancer cells, infected cells, or diseased cells, and / or may be derived from a virus (see above), parasite, worm, fungus, protozoan, bacterium, or pathogenic bacterium (see above). In a preferred embodiment of the method according to the invention, the at least one RNA to be detected is such that its transcription and / or expression is modified in response to external factors such as, for example, metabolic factors or signals, hormones, pathogens, toxins, drugs, aging, and / or biological or abiotic stress, and is thus derived from / associated with genes whose transcription and / or expression is modified in response to external factors. Thus, the at least one RNA to be detected is preferably associated with a pathological condition selected from viral infections, such as coronavirus infection, infections by pathogens, metabolic diseases, cancer, neurodegenerative diseases, aging, drugs, and biological or abiotic stress. Usually, the presence of the RNA to be detected, or an increase or decrease in its amount, serves as an indicator of the presence of the above-mentioned disease or pathological condition. Another aspect of the method relates to monitoring the amount or presence of the RNA to be detected during the treatment of the above-mentioned individual, patient, or organism, in particular the individual, patient, or organism from which the cell, tissue, and / or sample was obtained.

[0094] In yet another aspect of the invention, the method of the invention comprises detecting said one or more RNAs to be detected as part of a set of RNAs that constitute a gene signature (i.e., a set of RNAs to be detected that are specific to an organism, cell, and / or tissue). Thus, the method detects one or more gene signatures, where each gene signature identifies an organism, cell, and / or tissue such as a plant, animal, or microbial species, one or more phenotypes of a plant, animal, or microbe, or both. Subsequently, the method of the invention can be used to identify an expression profile for a set of RNAs that constitute a gene signature, one or more RNAs to be detected, where said signature is specific to an organism, cell, and / or tissue such as a plant, animal, or microbial species, one or more phenotypes of a plant, animal, or microbe, or both (see also above for transcriptional records).

[0095] Examples of preferred in vitro diagnostic formats for the method of the invention are microarrays, particularly microarrays using double-stranded DNA that monitors many DNAs at once, and lateral flow assays. Lateral flow assays are known to those skilled in the art and operate on the same principle as enzyme-linked immunosorbent assays (ELISAs). Essentially, in these tests, a liquid sample flows along the surface of a pad containing reactive molecules that indicate a visual positive or negative result.

[0096] As described above, in a preferred embodiment of the method according to the present invention, prior to step a), at least one RNA to be detected can be added to the cells, tissues, and / or samples, and / or the method further comprises at least one step selected from in vitro transcription from DNA to RNA, reverse transcription from RNA to DNA, and optimally, subsequent in vitro transcription from the DNA to RNA. By doing this, appropriate or desired signal amplification can be achieved. Usually, the RNA to be detected in the cells, tissues, or samples is present in the range of about 500 fM to about 1 μM, for example, in the range of about 500 fM to about 1 nM, and preferably, in the range of about 1 pM to about 1 nM. Optimally, this method can detect a single molecule per cell, tissue, and / or sample.

[0097] Next, yet another aspect of the present invention is a method of treating a disease or medical condition in a cell, tissue, or organism, such as a mammal, preferably a human, wherein the condition is related to the presence of at least one detected RNA, the expression of the detected RNA, and / or a mutation (s) in the detected RNA. This method provides an appropriate treatment, particularly a specific drug treatment, to the cell, tissue, or organism, and includes performing the method according to the present invention as described above and modifying the treatment of the disease or medical condition based on the presence of the at least one detected RNA, the expression of the detected RNA, and / or a mutation (s) in the detected RNA that has been detected. Medical conditions that can be discovered using the present invention are those related to at least one detected RNA molecule. As described above, the detected RNA can itself constitute the origin of a condition or disease, for example, in the case of an infectious disease, such as a viral infection such as a coronavirus infection, a bacterial infection, and / or a fungal infection, in a cell, tissue, and / or sample being tested. Other conditions may be indirectly related to at least one detected RNA molecule, for example, in the case of RNA that is abnormally transcribed (present or found), expressed, processed, and / or mutated. The detected RNA molecule can be present in an increased or decreased amount compared to a healthy control (e.g., a control based on a group of healthy samples or diseased samples).

[0098] In a preferred embodiment of the method according to the present invention, the at least one RNA to be detected is single-stranded or initially double-stranded. The single-stranded RNA to be detected may be derived from / associated with human cells, animal cells, plant cells, immune cells, cancer cells, infected cells, or diseased cells, and / or may be derived from a virus (see above), parasite, worm, fungus, protozoan, bacterium, or pathogenic bacterium (see above). In a preferred embodiment of the method according to the present invention, the at least one RNA to be detected is such that its transcription and / or expression is modified in response to external factors such as metabolic factors or signals, hormones, pathogens, toxins, drugs, aging, and / or biological or abiotic stress, etc. Thus, the at least one RNA to be detected is preferably associated with a pathological condition selected from viral infections, such as coronavirus infections, infections by pathogens, metabolic diseases, cancer, neurodegenerative diseases, aging, drugs, and biological or abiotic stress. Usually, the presence of the RNA to be detected, or an increase or decrease in its amount, serves as an indicator of the presence of the above diseases or pathological conditions. Another aspect of the method relates to monitoring the amount or presence of the RNA to be detected during the treatment of the above individual, patient, or organism, particularly the original individual, patient, or organism from which the cell, tissue, and / or sample was obtained.

[0099] In the case of drug treatment, the reprogrammed tracrRNA will result in a quantitative readout of a specific target RNA in a patient sample that directly informs the course of treatment. For example, the reprogrammed tracrRNA can be used to detect viral sequences in a blood sample or sputum to identify a specific virus that infects a patient, such as a coronavirus infection. The identified virus will directly inform the course of treatment. For example, if rhinovirus is identified, a more conservative treatment plan including fluid and rest will result, while if coronavirus is identified, intensive monitoring and containment will be required, whereas if Ebola is identified, immediate medical intervention will be required. Similarly, the reprogrammed tracrRNA can be used to detect specific RNAs in a cancer biopsy to identify important markers of cancer progression and drug sensitivity. Subsequently, this information will enable the physician to know how to proceed with the course of treatment. Finally, the reprogrammed tracrRNA can be used with samples containing infectious bacteria or fungal pathogens (e.g., Clostridioides difficile in a fecal sample, Pseudomonas aeruginosa in a sputum sample) to identify specific markers of antibiotic resistance and determine the best mode of antibiotics to administer to the patient.

[0100] The above method for treating a disease or medical condition in a cell, tissue, or organism, such as a mammal, preferably a human, wherein the condition is related to the presence of at least one detectable RNA, particularly viral RNA or DNA, the expression of the detectable RNA, and / or a mutation (s) in the detectable RNA. In one embodiment of the method, a reprogrammed (e.g., specific for a viral target nucleic acid) tracrRNA according to the invention is used to detect viral sequences in a blood sample or sputum, and to identify a specific virus such as the coronavirus that infects the patient. Next, the attending physician treats the detected viral infection with an antiviral chemotherapeutic agent and / or a biologic. During the course of treatment, the reprogrammed tracrRNA provides a quantitative readout of a specific target RNA in a patient sample that directly informs about the course and effectiveness / success of the treatment. Next, the attending physician adjusts the treatment accordingly, i.e., gives more antiviral chemotherapeutic agent and / or biologic as needed. This treatment schedule can be repeated as needed.

[0101] Also, when a virus such as rhinovirus is identified, a more conservative treatment plan including fluids and rest may result, but when coronavirus is identified, intensive monitoring and containment are required, while when Ebola is identified, immediate medical intervention is required.

[0102] Similarly, as described above, a reprogrammed (e.g., specific for a cancer target nucleic acid) tracrRNA can be used to detect specific RNAs in a cancer biopsy and identify important markers of cancer progression and drug sensitivity. Subsequently, this information will enable the physician to know how to proceed with the course of treatment.

[0103] Finally, again as above, the reprogrammed (e.g., specific to a bacterial or fungal target nucleic acid) tracrRNA can be used with a sample containing an infectious bacterial or fungal pathogen (e.g., Clostridioides difficile in a fecal sample, Pseudomonas aeruginosa in a sputum sample) to identify specific markers of antibiotic resistance and determine the best mode of antibiotic to administer to a patient.

[0104] In another preferred embodiment, the object of the present invention is solved by providing a non-naturally produced tracrRNA nucleic acid molecule comprising an anti-repeat region sequence designed to specifically hybridize to a preselected RNA sequence to be detected through the formation of a complex that mimics the natural crRNA:tracrRNA duplex. The non-naturally produced tracrRNA nucleic acid molecule can be part of a complex comprising the non-naturally produced tracrRNA nucleic acid molecule according to the present invention, at least one tracrRNA-dependent CRISPR nuclease enzyme, and at least one RNA to be detected, and can further bind to a target DNA nucleic acid molecule comprising a sequence based on the at least one RNA sequence to be detected, or a sequence designed based thereon.

[0105] Accordingly, the non-naturally produced tracrRNA nucleic acid molecule can be part of a complex comprising the non-naturally produced tracrRNA nucleic acid molecule according to the present invention, at least one tracrRNA-dependent CRISPR nuclease enzyme, and at least one RNA to be detected, and can further bind to a target DNA nucleic acid molecule comprising a sequence based on the at least one RNA sequence to be detected, or a sequence designed based thereon.

[0106] In the context of the present invention, a non-naturally occurring tracrRNA nucleic acid molecule comprises an anti-repeat region sequence that is designed to specifically hybridize to a pre-selected target RNA sequence through formation of a complex that mimics the native crRNA:tracrRNA duplex. This is meant to imply that the non-naturally occurring tracrRNA nucleic acid molecule and the pre-selected target RNA sequence molecule form a duplex that is structurally similar to the consensus structure of the crRNA:tracrRNA duplex disclosed in the literature. For example, the crRNA:tracrRNA duplex can form a lower stem, bulge, and upper stem module in the case of Cas9, a lower stem and upper stem module in the case of Cas12b, and a stem and triple helix in the case of Cas12e. The crRNA contains a spacer module and the tracrRNA contains a nexus and terminal hairpin (see, for example, Briner AE, et al. Guide RNA functional modules direct Cas9 activity and orthogonality. Mol Cell. 2014 Oct 23;56(2):333-339. doi: 10.1016 / j.molcel.2014.09.019. Epub 2014 Oct 16. PMID: 25373540, or Crawley, A.B., Henriksen, E.D., Stout, E. et al. Characterizing the activity of abundant, diverse and active CRISPR-Cas systems in lactobacilli. Sci Rep 8, 11544 (2018). https: / / doi.org / 10.1038 / s41598-018-29746-3).

[0107] In another preferred embodiment, the object of the present invention is a detection system for a target RNA, comprising: a) a non-naturally occurring tracrRNA designed to bind to at least one portion of the target RNA further comprising a portion that specifically hybridizes to a first portion of the target nucleic acid; and b) at least one tracrRNA-dependent CRISPR nuclease enzyme. A detection system for detecting several target RNAs in parallel, comprising a set of several non-naturally occurring tracrRNAs for the several target RNAs, is preferred. Another detection system comprises several non-naturally occurring tracrRNAs that hybridize at several positions on one target RNA.

[0108] This aspect of the present invention provides a component for implementing the method according to the present invention as a detection system, for example, as part of a diagnostic kit, such as the non-naturally occurring tracrRNA nucleic acid molecule according to the present invention. Preferably, the detection system is provided in one or more containers and includes appropriate enzymes, buffers, and additives, as well as instructions for use. These components may be at least partially immobilized on a substrate, where the substrate can be exposed to the cells, tissues, and / or samples. The detection system can be applied to a plurality of separate positions on a substrate made of a flexible material, such as a chip. The substrate made of a flexible material can be a paper substrate, a cloth substrate, or a flexible polymer-based substrate.

[0109] Examples of preferred in vitro diagnostic formats for the method of the present invention are microarrays, especially microarrays using double-stranded DNA that monitors many DNAs at once, and lateral flow assays. Lateral flow assays are known to those skilled in the art and operate on the same principle as enzyme-linked immunosorbent assays (ELISAs). Essentially, in these tests, a liquid sample flows along the surface of a pad containing reactive molecules that indicate a visual positive or negative result.

[0110] In another preferred embodiment, the object of the present invention is to implement the method according to any one of the above embodiments, in particular for detecting a detected RNA, a viral detected RNA, a detected RNA transcribed from a disease marker, and / or generating an expression profile for one or more of the above-described detected RNAs. This is solved by providing the use of a non-naturally occurring tracrRNA nucleic acid molecule or a detected RNA detection system according to the above embodiments.

[0111] The examples described herein include diagnosing medical conditions and informing the course of treatment, identifying SNPs associated with diseases such as healthy outcomes or acute sepsis, determining the identity, pathogenicity factors, resistance markers, SNPs, virus detection of pathogens, i.e., identity, and / or virus variants (see, e.g., SARS CoV-2 disclosed herein), diagnosing cancer, determining mutations and / or SNPs in cancer samples such as biopsies, identifying contaminated microorganisms in drinking water, identifying contaminated viruses or microorganisms in fermented products or cell cultures, identifying mutants in plants or insects, or identifying the major microbial members in a mixed community (e.g., in the intestine, soil, water), such as microbiota and / or microbial sentinels (i.e., commensal bacteria that function as reporters for non-invasive measurements), in particular analysis of identity, relative abundance, resistance markers, metabolic genes, genes specific to phylum / genus / species / strain, etc., and tracking the spread of viruses or bacteria in vivo, e.g., throughout an organism or based on samples taken from the environment, e.g., the spread of viruses or resistant bacteria detected in wastewater samples, etc. It can be used for a wide range of applications.

[0112] In the context of the present invention, unless otherwise explicitly stated, the term "about" shall mean a value within ±10% of a given value.

[0113] Starting from the characterization of the native CRISPR-Cas9 system in the bacterial pathogen C. jejuni, the inventors have found that cellular transcripts can be converted into aberrant crRNAs through hybridization with tracrRNA. This discovery adds ncrRNA to the list of RNA guides found in nature, including crRNAs, small CRISPR-Cas associated RNAs (scaRNAs) that pair similarly to crRNA, tracrRNA anti-repeats, and "natural" sgRNAs formed by transcription upstream of tracrRNA. Importantly, all of these previous examples are encoded within the CRISPR-Cas locus.

[0114] The inventors further demonstrated that an engineered tracrRNA can link the presence of a target RNA to sequence-specific DNA targeting by Cas9. The target RNA was otherwise not related to Cas9 or the CRISPR-Cas system. This enables engineered tracrRNAs for in vivo applications using Cas9, such as multiplexed transcription recording or transcription-dependent editing. The most direct application includes in vitro multiplexed RNA detection by LEOPARD as disclosed herein. In a preferred embodiment, LEOPARD provides an expandable multiplexing in a single reaction and the first diagnostic platform that includes Cas9 while adding to existing CRISPR diagnostic platforms based on Cas12a or Cas13.

[0115] Due to the inventors' reliance on gel electrophoresis, a proof-of-principle for multiplexed detection was achieved. However, more advanced implementations such as existing microarray-based technologies are also possible, or next-generation sequencing chips can be extended to associate DNA sequences with specific spatial positions and monitor millions of targets in parallel. Since the number of DNA molecules within a given cluster is limited, any approach will increase assay sensitivity, where the detection of the RNA to be detected is well below the 40 nM used in an exemplary gel-based assay. DNA targets linked to phosphors with different spectra and cleavable quenchers can also be used as sequence-specific and expandable readouts.

[0116] By incorporating isothermal pre-amplification similar to existing CRISPR diagnostics, means to enhance sensitivity will also be provided. With further development, LEOPARD will become a powerful diagnostic tool not only for virus detection but also for other applications that require multiplexed readouts such as screening for cancer mutations, identifying pathogens and antibiotic resistance markers, or determining gene expression profiles for drug sensitivity, as also discussed herein.

[0117] In this specification, non-native tracrRNAs are exemplified in conjunction with Cas9 and Cas12 nuclease, which also depend on tracrRNAs (see also Figures 9-11 and Figures 22-25). By extending tracrRNA reprogramming to these and other nucleases known to those skilled in the art and described herein, the available repertoire of nucleases is expanded and their unique attributes such as signal amplification or programmable translocation are incorporated.

[0118] While systematically perturbing the canonical crRNA:tracrRNA duplex, the inventors found that many deviations outside the 5' end of the repeat, especially, are tolerated by Cas9 and Cas12 and yet lead to targeting of the designed DNA target. However, despite this promiscuity, targeting is still determined by the requirements for the upstream guide sequence and the adjacent PAM. Thus, both anti-repeat hybridization and guide-dependent DNA targeting limit the potential for off-targeting. Correspondingly, the inventors did not observe any detectable off-targeting when evaluating numerous viral RNAs using LEOPARD or when introducing total yeast RNA (FIGS. 18B, 18D). Design rules for unnatural tracrRNAs that explain potential off-targeting and on-target activity serve to advance the conversion of any RNA into a sequence-specific guide for CRISPR technology, similar to existing sgRNA design algorithms.

[0119] As described herein, the present invention relates particularly to the following items.

[0120] Item 1. A non-naturally produced tracrRNA nucleic acid molecule comprising a portion comprising an anti-repeat region sequence designed to specifically hybridize to a preselected RNA sequence to be detected through formation of a complex that mimics a native crRNA:tracrRNA duplex.

[0121] Item 2. A complex comprising the non-naturally produced tracrRNA nucleic acid molecule according to Item 1, at least one tracrRNA-dependent CRISPR nuclease enzyme, and at least one RNA to be detected.

[0122] Item 3. The non-naturally produced tracrRNA nucleic acid molecule according to Item 2, further bound to a target DNA nucleic acid molecule comprising a sequence based on at least one RNA sequence to be detected or a sequence designed based on the RNA sequence to be detected.

[0123] Item 4. A method for detecting at least one target RNA in a cell, tissue, and / or sample, comprising: a) contacting the sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes to a first portion of the target RNA; wherein a second portion of the target RNA specifically hybridizes to at least one target nucleic acid, particularly DNA, RNA, or ssDNA, and the at least one non-naturally occurring tracrRNA hybridizes or is capable of hybridizing to the target RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of a dsRNA-cleaving enzyme such as RNase III; b) detecting the binding of the at least one tracrRNA-dependent CRISPR nuclease enzyme to the at least one nucleic acid DNA; and c) wherein the binding detects the at least one target RNA in the cell, tissue, and / or sample.

[0124] Item 5. The method according to Item 4, wherein the nuclease has its cleavage activity and / or nicking activity inactivated.

[0125] Item 6. A method for detecting at least one target RNA in a cell and / or sample, comprising: a) contacting the sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes to a first portion of the target RNA; wherein a second portion of the target RNA specifically hybridizes to at least one target nucleic acid, and the at least one non-naturally occurring tracrRNA hybridizes or is capable of hybridizing to the target RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of a dsRNA-cleaving enzyme such as RNase III; b) detecting cleavage of the at least one target nucleic acid in the sample by the nuclease enzyme; comprising c) a method, wherein detection of the cleavage of the at least one target nucleic acid detects the at least one detectable RNA in the cell, tissue, and / or sample.

[0126] Item 7. The detection of the at least one target nucleic acid that is bound and / or cleaved includes detecting a signal of a suitable label such as a dye or a phosphor, or a change in conductivity, and / or detecting the at least one cleaved target nucleic acid fragment itself, according to any one of Items 4 to 6.

[0127] Item 8. A method for detecting at least one detectable RNA in a cell, tissue, and / or sample, a) contacting the sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with a first portion of the detectable RNA, wherein a second portion of the detectable RNA specifically hybridizes with at least one target nucleic acid, and the at least one non-naturally occurring tracrRNA hybridizes or is capable of hybridizing with the detectable RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme that optionally further comprises the presence of a dsRNA cleavage enzyme such as RNase III; b) nicking or cleaving the at least one target nucleic acid in the sample with the nuclease; c) detectably editing the at least one target nucleic acid, including, for example, non-homologous end joining (NHEJ) repair, microhomology-mediated end joining (MMEJ), homology-directed repair (HDR), base editing, or prime editing; d) For example, at least one method selected from the detection of the presence and / or length of indel or gene editing, the detection of the activation / suppression of a gene or other genetic element encoded by the at least one target nucleic acid, and / or the detection of the prime editing or base editing, appropriately detecting the editing of the at least one target nucleic acid; comprising e) A method, wherein the detection of the editing of the at least one target nucleic acid detects the at least one detectable RNA in the cell, tissue, and / or sample.

[0128] Item 9. The method according to any one of Items 4 to 8, wherein the method is performed in vivo or in vitro.

[0129] Item 10. The method according to any one of Items 4 to 9, wherein the sample is selected from a sample containing cells and a cell-free in vitro sample, and wherein the cells are preferably plant cells or animal cells, such as mammalian cells, preferably human cells.

[0130] Item 11. The method according to any one of Items 4 to 10, wherein several or a large number of detectable RNAs are detected in one or more samples, tissues, and / or cells.

[0131] Item 12. The method according to any one of Items 4 to 11, wherein the at least one detectable RNA is single-stranded.

[0132] Item 13. The method according to any one of Items 4 to 12, wherein the at least one detectable RNA is specific to an environment, species, strain, disease, cell, and / or tissue, or is related to a pathological condition selected from viral infections, such as coronavirus infection, infections by pathogens, metabolic diseases, cancer, neurodegenerative diseases, aging, drugs, and biological or abiotic stresses.

[0133] Item 14. The method according to any one of Items 4 to 13, further comprising adding the at least one detectable RNA to the cells, tissues, and / or samples before step a), and / or transcribing DNA into the detectable RNA before step a).

[0134] Item 15. The at least one tracrRNA-dependent CRISPR nuclease enzyme is selected from type II Cas9 and type V Cas12 nuclease enzymes, for example, type II Cas9 nuclease selected from the group consisting of II-A, II-B, and II-C, and type Cas12 nuclease of the type selected from the group consisting of V-B, V-C, V-D, V-E, V-F, V-G, and V-K. The method according to any one of Items 4 to 14.

[0135] Item 16. The method according to any one of Items 4 to 15, wherein the at least one tracrRNA-dependent CRISPR nuclease enzyme is a recombinant fusion protein.

[0136] Item 17. The portion of the at least one non-naturally occurring tracrRNA that specifically hybridizes to the first portion of the detectable RNA hybridizes with 10 nucleotides or more, preferably 11 nucleotides or more, more preferably about 12 nucleotides or more. The method according to any one of Items 4 to 16.

[0137] Item 18. The first portion of the detectable RNA comprises a protospacer adjacent motif (PAM). The method according to any one of Items 4 to 17.

[0138] Item 19. The method according to any one of Items 4 to 18, wherein the non-naturally occurring tracrRNA further comprises at least one portion that hybridizes with the detectable RNA to provide a substrate for a dsRNA cleavage enzyme.

[0139] Item 20. The method according to item 19, wherein the substrate of the dsRNA-cleaving enzyme in the detected RNA has a length of 10 nucleotides or more, preferably 11 nucleotides or more, more preferably about 12 nucleotides or more.

[0140] Item 21. The method according to any one of items 4 to 20, wherein the hybridizing portion is complementary up to at least 80%, preferably more than 90%, more preferably more than 95%, and most preferably 100% complementary.

[0141] Item 22. The nucleotide sequence of the at least one portion of the tracrRNA that specifically hybridizes to the first portion of the detected RNA is produced and / or modified to be complementary up to at least 80%, preferably more than 90%, more preferably more than 95%, and most preferably 100% complementary to the first portion of the detected RNA. The method according to any one of items 4 to 21.

[0142] Item 23. The method according to any one of items 4 to 22, wherein two or more non-naturally occurring tracrRNAs are produced and used that specifically hybridize to different portions of the detected RNA.

[0143] Item 24. The method according to any one of items 4 to 23, further comprising detecting the amount of the at least one target nucleic acid and / or the at least one detected mRNA in the sample, tissue, and / or cell, preferably the amount per sample, tissue, and / or cell.

[0144] Item 25. The method according to item 24, further comprising detecting a change in the amount of the at least one target nucleic acid and / or the at least one detected mRNA in the sample, tissue, and / or cell compared to a control.

[0145] Item 26. The method according to any one of Items 8 to 25, wherein the length of the detected indel is between 1 nucleotide and 14 nucleotides, more preferably between 5 nucleotides and 9 nucleotides, and optionally the indel can be detected by sequencing and / or qPCR.

[0146] Item 27. The method according to any one of Items 4 to 26, wherein a plurality of labels and / or markers are used.

[0147] Item 28. A method for discovering a medical condition in a mammal related to the presence of at least one detected RNA, the expression of the detected RNA, and / or a mutation (s) in the detected RNA, comprising performing the method according to any one of Items 4 to 27, and discovering the medical condition based on the presence of the at least one detected RNA, the expression of the detected RNA, and / or a mutation (s) in the detected RNA.

[0148] Item 29. The method according to Item 25, wherein the at least one detected RNA is specific to an environment, species, strain, disease, cell, and / or tissue, or is related to a condition selected from viral infections such as coronavirus infection, infectious diseases caused by pathogens, metabolic diseases, cancer, neurodegenerative diseases, aging, drugs, and biological or abiotic stress.

[0149] Item 27. The method according to Item 25 or 26, further comprising adding the at least one detected RNA to the cell, tissue, and / or sample before step a), and / or transcribing DNA into the detected RNA before step a).

[0150] Item 28. A detected RNA detection system comprising: a) a non-naturally occurring tracrRNA designed to bind to at least one portion of the detected RNA further comprising a portion that specifically hybridizes to a first portion of a target nucleic acid; and b) at least one tracrRNA-dependent CRISPR nuclease enzyme.

[0151] Item 29.c) The detected RNA detection system according to Item 28, further comprising at least one target nucleic acid molecule containing a label for detecting cleavage of the target nucleic acid.

[0152] Item 30.d) The detected RNA detection system according to Item 28 or 29, further comprising at least one dsRNA cleavage enzyme such as RNase III enzyme.

[0153] Item 31. The detected RNA detection system according to any one of Items 28 to 30, wherein at least one of the above components a) to d) is encoded on a nucleic acid vector system.

[0154] Item 32. Use of the detected RNA detection system according to any one of Items 28 to 31 for carrying out the method according to any one of Items 1 to 26, particularly for detecting a detected RNA, a viral detected RNA, a detected RNA transcribed from a disease marker, or for generating an expression profile regarding one or more detected RNAs.

[0155] Here, the present invention will be further described in the following examples with reference to the accompanying drawings, but it is not intended to be limited thereto. For the present invention, all references cited herein are incorporated by reference in their entirety and form a part of this specification. This disclosure includes a sequence listing containing SEQ ID NOs: 1 to 128 as a detailed description part, which is also incorporated by reference in its entirety and forms a part of this specification.

[0156] In the figure, the non-naturally occurring tracrRNA according to the present invention may be referred to as "rptr".

Brief Description of the Drawings

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Mode for Carrying Out the Invention

Examples

[0158] Construction and Growth of C. jejuni Mutant Strains C. jejuni mutant strains (deletion, chromosomally 3xFLAG-tagged) were constructed using double-crossover homologous recombination (High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacter jejuni isolates. PLoS Genet. 2013;9(5):e1003495). Briefly, PCR products with 500-bp homologous ends or genomic DNA with the desired mutation were introduced into C. jejuni using electroporation or natural transformation, respectively, as previously described (High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacter jejuni isolates. PLoS Genet. 2013;9(5):e1003495). C. jejuni strains were grown routinely at 37°C under microaerobic (10% CO 2 , 5% O 2 ) conditions on Mueller-Hinton agar plates supplemented with both at 10 μg / ml vancomycin or in Brucella broth (BB) with shaking.

[0159] RIP-seq of Cas9-3xFLAG in C. jejuni Co-immunoprecipitation (Co-IP) combined with RNA-seq (RIP-seq) to identify direct RNA-binding partners of Cas9-3xFLAG in C. jejuni was performed essentially as previously described (Non-Patent Document 12).

[0160] Briefly, at mid-exponential phase (OD 600C. jejuni CG8421 cas9-3xFLAG and WT (untagged, control) strains grown to an optical density at 600 nm (OD600) of 0.6 were lysed, and co-immunoprecipitation (Co-IP) of chromosomally tagged Cas9-3xFLAG was performed using an anti-FLAG antibody and protein A-sepharose beads. Cells were harvested by centrifugation, and the pellet was resuspended in 1 mL of buffer A (20 mM Tris-HCl (pH 8.0), 150 mM KCl, 1 mM MgCl 2 -1 -1 -1 -1Lysis of the remaining cells was achieved by a second lysis for 5 minutes. Centrifugation was repeated, and this second supernatant was combined with the first supernatant. The combined supernatant was centrifuged again at 15200×g for 30 minutes at 4°C to clarify it, and the resulting supernatant (lysate fraction) was transferred to a new tube. The lysate was incubated with 35 μL of anti-FLAG antibody (monoclonal anti-FLAG M2, Sigma, number F 1804, RRID:AB_262044) on a rocker at 4°C for 30 minutes. Next, 75 μL of protein A-Sepharose (Sigma, number P6649) pre-washed with buffer A was added, and the mixture was shaken at 4°C for an additional 30 minutes. After centrifugation, the supernatant was removed, and the pelleted beads were washed 5 times with 0.5 mL of buffer A. Finally, 500 μL of buffer A was added to the beads, and RNA and protein were separated using phenol-chloroform-isoamyl alcohol. Approximately 1000 ng of RNA was recovered from the aqueous phase from each CoIP. 100 μL of 1× protein loading buffer was added to the final protein sample (CoIP sample) pelleted with the beads. To verify the success of CoIP, protein samples obtained during various stages of coIP (cultures, lysates, supernatants, washes, and CoIP) were analyzed using Western blot.

[0161] Preparation of cDNA Library and Deep Sequencing The remaining gDNA was removed using DNase I treatment on the coIP RNA obtained above. The cDNA library for Illumina sequencing was constructed by vertis Biotechnologie AG (http: / / www.vertis-biotech.com) in Germany in a strand-specific manner as previously described (High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacter jejuni isolates. PLoS Genet. 2013;9(5):e1003495). Briefly, the RNA samples were polyadenylated using poly(A) polymerase. Next, after removing the 5'-triphosphates using tobacco acid pyrophosphatase (TAP), RNA adapters were ligated to the resulting 5'-monophosphates. First-strand cDNA was synthesized using M-MLV reverse transcriptase with an oligo(dT) adapter primer. The cDNA concentration was increased to 10 ng / μL - 20 ng / μL in a PCR-based amplification step using high-fidelity DNA polymerase. For all libraries, DNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and subsequently analyzed by capillary electrophoresis.

[0162] Library-specific barcodes for multiplex sequencing were included as part of the 3'-sequencing adapter. The cDNA inserts were flanked by the TruSeq_sense_primer (SEQ ID NO: 1) and the TruSeq_antisense_NNNNNN_primer (NNNNNN = 6-nucleotide barcode for multiplexing) (SEQ ID NO: 2). The samples were run in single-read mode on an Illumina HiSeq instrument for approximately 100 cycles.

[0163] Analysis of RIP-seq sequencing data To ensure high array quality, Illumina reads in FASTQ were trimmed with a cut-off phred score of 20 by the program fastq_quality_trimmer from version 0.0.13 of the FASTX Toolkit. After trimming, version 0.4.3 of the pipeline READemption was applied for the following analysis steps: the poly(A) tail sequence was clipped from the 3'-end of the reads, size filtering was applied to exclude read sequences shorter than 12 nucleotides, and the remaining collection of reads was mapped to the self-curated C. jejuni CG8421 genome with a 95% accuracy cut-off using version 0.2.0 of segemehl.

[0164] Coverage plots corresponding to the number of mapped reads per nucleotide were generated using READemption. Reads mapped to multiple positions contributed only slightly to the coverage values. Each read with a minimum overlap of 10 nucleotides was counted with a value based on the number of positions to which the read was mapped. When a read overlapped with more than two annotations, the value was divided by the number of regions and counted individually for each region (e.g., 1 / 3 for a read mapped to three positions).

[0165] To visualize in the Integrated Genome Browser (IGB), the coverage files were normalized by the number of reads that could be mapped from each library. To restore the original data range, each graph was then multiplied by the minimum number of reads mapped across the two libraries.

[0166] Peak detection and combined motif analysis To automatically define the RNA regions or peaks bound to Cas9 from the Cas9-3xFLAG coIP dataset, we applied an in-house tool, "sliding_window_peak_calling_script", based on a custom-developed sliding window approach to detect the binding regions of CsrA in C. jejuni (The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni. Nat Commun. 2016;7:11667). The script is deposited on Zenodo at https: / / doi.org / 10.5281 / zenodo.49292. This script is written in Python 3 and requires the installation of the Python 3 packages numpy and scipy to execute.

[0167] Briefly, the "sliding_window_peak_calling_script" software uses the rRNA-normalized wiggle files of the Cas9-3xFLAG and control coIP libraries as input to determine sites showing continuous enrichment of the Cas9-3xFLAG-tagged library compared to the control. Identification of enriched regions is based on four parameters: the minimum fold change (FC) required for enrichment, a coefficient multiplied by the 90th percentile of the wiggle graph reflecting the minimum required expression (MRE) in the tagged library, the window size (WS) in nucleotide units calculated in a sliding window approach, and the nucleotide step size (SS) defining the step by which the window moves along the genomic axis. All consecutive windows meeting the enrichment requirement are grouped into a single peak region. Peak detection is performed separately for the forward and reverse strands of each replicon. For the Cas9-3xFLAG coIP dataset, the following parameters were used: FC = 5, MRE = 3, WS = 25, and SS = 5. Next, MEME was used to predict the consensus motif based on the peak sequences. Next, two highly enriched motifs were compared to the sequences of crRNA and tracrRNA to infer their potential binding modes.

[0168] Northern blot analysis For northern blot analysis, 100 μg of total RNA samples treated with DNaseI were loaded into each lane. After separation on a 6% polyacrylamide (PAA) gel containing 7 M urea, the RNA was transferred to a Hybond-XL membrane (GE-Healthcare) by electroblotting. After blotting, the RNA was UV cross-linked to the membrane and hybridized with γ32P-ATP end-labeled DNA oligonucleotides.

[0169] Plasmid construction All primer gBlocks used in this study were ordered from Integrated DNA Technologies (IDT, Coralville, USA). The NEBuilder (TM) HiFi DNA Assembly Cloning Kit was used for plasmid construction by Gibson assembly. The relevant sequence numbers (sequence numbers 1 to 96) can be found in the attached sequence listing, which is hereby incorporated by reference in its entirety and forms part of this specification. The Q5 site-directed mutagenesis kit was used to perform small insertions and nucleotide substitutions. Unless otherwise specified, all nucleases used were expressed in plasmids having a Cm selectable marker and an origin of replication (ORI) of p15A. All plasmids of sgRNA or tracrRNA-crRNA or tracrRNA-ncrRNA were expressed in plasmids having an Amp selectable marker and an ORI of ColE1, and all targeting plasmids were expressed in plasmids having a Kan selectable marker and an ORI of pSC101.

[0170] For the construction of plasmids of Cas9 and Cas12, the SpyCas9 and codon-optimized CjeCas9 genes of E. coli were PCR amplified from plasmids pCB843 and pCB588 and then inserted into the PCR-linearized vector pCBS583 by Gibson assembly, respectively.

[0171] To construct plasmids encoding sgRNAs with 5' overhangs of various lengths, gBlocks encoding sgRNAs targeting fliF or 1093 with various 5' overhangs and their variants were inserted into the PCR-linearized vector pCSM180 by Gibson assembly, respectively. In the case of crRNAs with 5' overhangs in the dual RNA guide (tracrRNA / crRNA) system, a tracrRNA expression cassette was obtained by PCR amplification using the template of CJfr0018 of the gBlock and the primer pair of CJpr0304 / CJpr0305 (SEQ ID NO: 3 to SEQ ID NO: 4), and homologous arms were introduced using the vector and the crRNA cassette. Cassettes expressing fliF-targeting crRNA and its 5' overhang variants were obtained by PCR amplification using the templates of CJfr0022, CJfr0070, and CJfr0071 and the primer pair of CJpr0110 / CJpr0291 (SEQ ID NO: 5 to SEQ ID NO: 9), and homologous arms were introduced using the vector and the tracrRNA cassette. The tracrRNA expression cassette and the cassette expressing crRNA with a 5' overhang were inserted into the PCR-linearized vector pCSM180 by Gibson assembly, respectively.

[0172] To construct a plasmid encoding a crRNA with a 3' overhang in the dual RNA system, a similar method was implemented. The only difference was that a cassette expressing fliF-targeting crRNA and its 3' overhang variants was obtained by PCR amplification using the templates of CJfr0080, CJfr0081, and CJfr0176 (SEQ ID NO: 10 to SEQ ID NO: 12).

[0173] To construct the reprogrammed ncrRNA plasmids for CjeCas9, SpyCas9, and Sth1Cas9, peak 113 containing mRNA (spanning genes CJ8421_04970, CJ8421_04975, and CJ8421_04980) was used as a template for the tracrRNA modified to be targeted. This peak was one of the RNAs that were confirmed to be highly enriched as part of the CjeCas9 co-immunoprecipitation. The modified tracrRNA and the gBlocks encoding the mRNA contained in peak 113 were PCR amplified from plasmid pCJ174 and inserted into the PCR-linearized vector pCSM180 by Gibson assembly.

[0174] To construct the self-targeting ncrRNA for CjeCas9, the mRNA contained in peak 113 and the reporter gene GFP were placed on one plasmid, and the modified tracrRNA was placed on the other plasmid. The PAM sequence was introduced at the base of the tracrRNA-ncrRNA repeat-anti-repeat stem using Q5 site-directed mutagenesis.

[0175] For all targeted plasmid constructs, Q5 site-directed mutagenesis using the template of pCSM168 was used to insert the target sequence upstream of the promoter OR2-OR1 or between the promoter OR2-OR1 and the CDS of GFP.

[0176] In vitro DNA cleavage assay using TXTL The TXTL system was used to characterize ncrRNAs and to characterize how tracrRNAs can be reprogrammed / modified. This system functions by introducing linear or plasmid DNA encoding each component into E. coli. The E. coli lysate then transcribes and translates these components based on E. coli signals (e.g., promoter, RBS). The biosynthesis and activity of crRNA (or ncrRNA) can be monitored over time by using a GFP reporter containing a target adjacent to the PAM of the crRNA. This assay was performed as previously described (Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System. Mol Cell. 2018;69(1):146-157.e3). All DNA fragments or plasmids used in the assay should not contain nucleases (DNase, RNase) and inhibitors of the TXTL machinery. Reporter plasmids encoding Cas9, sgRNA / dual-RNA, and deGFP were added to the myTXTL Sigma 70 Master Mix (Arbor Biosciences) at final concentrations of 1.5 nM, 0.5 nM, and 1 nM, respectively. When expressing linear fragments, GamS (Arbor Biosciences) was supplemented at a final concentration of 5 μM, and the fliF-targeted plasmid (pCJ002) with a target sequence inserted between the promoter and the coding sequence of deGFP was used. When expressing genes driven by the T7 promoter, the plasmid encoding T7 RNA polymerase was supplemented at a final concentration of 0.5 nM. Fluorescence was measured in a BioTek Synergy Neo2 plate reader using a 96-well V-bottom plate (Corning Costar 3357) equipped with a 485 nm excitation filter and a 528 nm emission filter. Time-course measurements were performed at 29 °C for 16 h with 3-min intervals between readings.

[0177] Reprogramming of tracrRNA The mRNA contained in peak 113 was used as a template for reprogramming against the target of tracrRNA. Regions containing AT-rich sequences preferentially recognized by Rnase III of E. coli were selected as targets for the reprogrammed tracrRNA (In vivo cleavage rules and target repertoire of RNase III in Escherichia coli. Nucleic Acids Res. 2018;46(19):10380-10394). These regions contained AT-rich motifs and were 25 base pairs in length for CjeCas9 and 38 base pairs in length for SpyCas9 and Sth1Cas9. In the case of CjeCas9, the anti-repeat portion of the original tracrRNA was replaced with a sequence complementary to each selected target. In the case of SpyCas9 and Sth1Cas9, four or three extra nucleotides were introduced between the 6th and 7th nucleotides or between the 9th and 10th nucleotides (counting from the bottom up in the duplex) in the anti-repeat portion of tracrRNA to form the bulges necessary to maintain the activity of SpyCas9 and Sth1Cas9 (Non-Patent Document 13). Correspondingly, the sequences of 20 to 24 base pairs upstream of these base pairing regions were treated as spacers.

[0178] Plasmid removal assay in E. coli The plasmid removal assay was performed as previously described (Non-Patent Document 12). A 40 nM sgRNA, ncrRNA plasmid was electroporated into an E. coli strain containing Cas9 and a targeted plasmid or a non-targeted plasmid. After recovery in SOC medium for 1 hour, 3 μl of 10-fold serial dilutions of these transformants were spotted onto LB plates containing Cm+Amp+Kan antibiotics and incubated overnight at 37°C. The fold reduction in transformation efficiency was calculated by dividing the non-targeted CFU by the targeted CFU using colonies from countable spots.

[0179] In vitro RNA transcription by T7 RNA polymerase RNA was transcribed in vitro at 37°C for 4 hours using the HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs) according to the product manual. The transcribed RNA was purified using RNA Clean & Concentrator-25 (Zymo Research), and residual DNA was removed by on-column DNase I treatment according to the product manual.

[0180] In vitro RNA transcription and detection using LEOPARD The RNA to be detected and the unnatural tracrRNA were encoded in a synthetic gBlock starting with the T7 promoter and transcribed using the HiScribe T7 in vitro transcription kit (New England Biolabs, catalog number E2040S). The transcribed RNA was purified using RNA Clean & Concentrator-25 (Zymo Research), and residual DNA was removed by on-column DNase I treatment. The relevant DNA target was PCR amplified from the targeted plasmid using Cy5-labeled forward primers paired with various reverse primers.

[0181] The detected RNAs and their associated non-natural tracrRNAs were annealed by heating at 95 °C for 2 minutes using a thermocycler and then gradually cooling to 25 °C over 45 minutes at a 1:1 molar ratio in 1× NEB3.1 buffer (New England Biolabs, catalog number B7203S). Next, SpyCas9 (New England Biolabs, catalog number M0386M) was added and the mixture was incubated at room temperature for 25 minutes to promote RNP assembly. Next, the DNA target was added and the reaction was incubated at 37 °C for 1 hour to promote DNA cleavage.

[0182] To investigate the effect of addition of RNase on the ncrRNA, ShortCut™ RNase III (New England Biolabs, catalog number M0245L) was added at a final concentration of 33.3 U / ml and / or RNase A (Thermo Fisher Scientific, catalog number EN0531) was added at a final concentration of 166.7 μg / ml before or during the DNA cleavage step. Next, Proteinase K (New England Biolabs, catalog number P8107S) was added at a final concentration of 25.8 U / ml and the reaction was incubated at room temperature for 15 minutes to digest Cas9. For the reaction with unlabeled DNA, the final concentration of the DNA target was 10 nM, while the final concentrations of the detected RNA, non-natural tracrRNA, and SpyCas9 were all 200 nM. The unlabeled DNA products were separated on a 2% agarose gel and visualized with an Intas GelStick imager. For the reaction with Cy5-labeled DNA, the final concentration of the DNA target was 2 nM, while the final concentration of each detected RNA and non-natural tracrRNA was 40 nM. The final concentration of SpyCas9 was 40 nM for individual RNA detection and 360 nM for multiplexed RNA detection. For multiplexed RNA detection, all nine non-natural tracrRNAs and DNA targets were present in a single reaction tube to detect viral RNA individually or in sets of three or five.

[0183] The products obtained in the case of individual or multiplexed RNA detection were loaded together with Novex™ TBE HiDensity buffer without tracking dye and separated on a 4%–20% Novex™ TBE gel (Invitrogen, catalog number EC6225BOX).

[0184] Simultaneous detection of RNA from various respiratory viruses in one test, and discrimination of SARS-CoV-2 and its dominant D614G variant with single-base resolution using LEOPARD In vitro transcription and detection of RNA using LEOPARD were performed as shown above. In the case of multiplexed RNA detection, all the unnatural tracrRNAs and DNA targets were present in a single reaction tube to detect viral RNA individually or in sets of three or five. The products obtained in the case of individual or multiplexed RNA detection were loaded together with Novex™ TBE HiDensity buffer without tracking dye and separated on a 4%–20% Novex™ TBE gel (Invitrogen, catalog number EC6225BOX). For the specific detection of the D614G mutation in the SARS-CoV-2 spike protein and for individual products in experiments using total yeast RNA, the products were loaded onto a 2% agarose gel. Images for gels with Cy5 labeling were captured on an Amersham Typhoon™ Biomolecular Imager (GE Healthcare). Figure 18E shows the specific detection of the D614G point mutation within the spike protein of SARS-CoV-2. Each detected T7 transcribed RNA contained the ncrRNA sequence of interest with a 50-nucleotide extension at either end.

[0185] The D614G mutation in the spike protein of SARS-CoV-2 serves as a proof of function example because this mutation is due to a single nucleotide change (A23403G) and is associated with increased infectivity and its worldwide spread. By placing this nucleotide change within the target seed region, the inventors were able to detect WT or D614G RNA using one non-natural tracrRNA in combination with either the WT or D614G target (Figure 18E). The inventors found that when each target was tested individually, the matching DNA target was preferentially cleaved. However, when two targets were combined in a single reaction, distinguishable cleavage was obtained only for the matching target, presumably due to preferential binding and cleavage of the complete target by Cas9. Thus, LEOPARD can provide multiplexed RNA detection with single-base resolution in a single reaction.

[0186] Identification of the anti-repeat regions for various tracrRNAs As part of the design of non-natural tracrRNAs, the sequence of the anti-repeat region of the tracrRNA is varied to be complementary to the region of the RNA to be detected. Identification of these regions is relatively straightforward and can be achieved, for example, by using common RNA folding algorithms such as NUPACK or mfold to evaluate the base pairing ability of the tracrRNA with one of the repeats within the CRISPR array. Alternatively, this region can be determined experimentally by evaluating the RNA structure through common probing approaches (e.g., using DMS, SHAPE) after hybridizing the purified repeat and the purified tracrRNA. The anti-repeat region is the region that is partially or fully base paired with the repeat region and should start near the 5' end of the tracrRNA sequence. See, for example, Figures 9-11.

[0187] Use of non-natural tracrRNAs for RNA detection in vitro RNA collections are extracted from samples using common approaches for purifying RNA, such as the GenEasy RNA extraction kit sold by Sigma-Aldrich or the RNeasy RNA extraction kit sold by Qiagen. Samples can be derived from humans or animals (e.g., blood, sputum, biopsy), plants (e.g., excised roots, excised leaves), soil, water, or air.

[0188] The set of RNAs detected within each sample is selected based on the specific requirements of the user. The accompanying set of non-native tracrRNAs is designed by varying the sequence of the anti-repeat portion of the tracrRNA to be complementary to a portion of the RNA. Figures 9-11 show the regions of the tracrRNA to be varied when using CjeCas9, SpyCas9, or Sth1Cas9, but a similar approach can be extended to other tracrRNA-dependent CRISPR nucleases. When using E. coli RNase III, A / T-rich sequences are generally preferred, but other variants of RNase III or other dsRNA-cleaving enzymes (e.g., Rntp1, Pac1p, DCL1, Dicer, Drosha) can also be used based on their sequence preferences. To better facilitate RNA:RNA hybridization and enzymatic cleavage, the complementary regions can also be extended or reduced. Multiple non-native tracrRNAs can also be designed to recognize various regions of the RNA to be detected, thereby generating multiple ncrRNAs from a single transcript. The upstream portion of the region in the RNA to be detected that pairs with the non-native RNA becomes the guide sequence for DNA targeting. The length of this region varies depending on the nuclease (e.g., 20 nucleotides to 24 nucleotides across CjeCas9, SpyCas9, and Sth1Cas9 as shown in Figures 9-11). These RNAs are made by in vitro transcription from a DNA template or by custom synthesis by standard commercial suppliers (e.g., IDT).

[0189] Next, a dsDNA target is designed for each ncrRNA. The target contains the guide sequence of the ncrRNA adjacent to an appropriate PAM recognized by the CRISPR nuclease (e.g., NNNNACAC for CjeCas9, NGG for SpyCas9, NNAGAA for Sth1Cas9). For visual depictions specifically regarding CjeCas9, SpyCas9, and Sth1Cas9, refer to FIGS. 9-11. The dsDNA target is prepared by annealing custom-synthesized DNA oligonucleotides (e.g., from IDT) or custom-synthesized dsDNA (e.g., gBlocks from IDT). As part of the synthesis process, various chemical moieties can be added to use the DNA target as a part for in vitro RNA detection such as a fluorophore and a quencher, or as a tag for click chemistry to add other moieties or covalently link the DNA to a surface.

[0190] This reaction also requires a purified tracrRNA-dependent nuclease (e.g., SpyCas9), and optionally a dsRNA-cleaving enzyme (e.g., RNase III). The nuclease may be non-catalytic or catalytically active depending on whether the detection step requires binding or cleavage of the target nucleic acid.

[0191] As part of a representative detection assay, RNA purified from a sample is added to a binding buffer that promotes RNA hybridization (e.g., 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl 2、and mixed with a set of non-natural tracrRNAs in 0.05% Tween-20 (pH 7.8), incubated at 37°C for 10 minutes to form duplexes between the RNAs. Next, tracrRNA-dependent CRISPR nuclease and dsRNA-cleaving enzyme are added, and the mixture is incubated at the same temperature for an additional 20 minutes. During this step, the dsRNA-cleaving enzyme cleaves RNA duplexes such as the RNA duplex formed between the RNA to be detected and the non-natural tracrRNA, and a ribonucleoprotein complex forms between the tracrRNA-dependent nuclease and the cleaved RNA duplex. Next, by exposing the resulting mixture to a DNA target, the ribonucleoprotein complex can target each DNA target. The DNA targets can be added as soluble molecules or immobilized on a solid surface (e.g., magnetic beads, on a silicon chip). Next, a washing step is added (e.g., using binding buffer) to remove RNA, dsRNA-cleaving enzyme, unbound ribonucleoprotein complex, and any cleaved and non-immobilized portions of the DNA target.

[0192] Finally, the DNA targets are evaluated to measure the extent to which they are recognized by the ribonucleoprotein complex (see FIG. 12 for various representative examples). Various formats can be used here. For example, the DNA target may include a fluorophore fused to one end and a quencher fused to the other end or nearby on another surface (e.g., when the DNA target is immobilized on a silicon chip). Next, upon cleavage of the target, a portion of the DNA fused to the quencher is released, thereby generating a fluorescent signal. Changes in the size of the DNA can also be measured by separating the DNA by size without requiring a change in the fluorescent signal (e.g., capillary electrophoresis, gel electrophoresis). As another example, the fluorophore and quencher can be attached to either strand on the same side. Nicking of the non-target strand by a mutated form of the tracrRNA-dependent CRISPR nuclease (e.g., Cas9n) releases the strand fused to the quencher, thereby allowing the quencher to diffuse away and generate a fluorescent signal. As a final example, a fluorophore or dye (e.g., FITC, Texas Red) can be fused to the tracrRNA-dependent CRISPR nuclease or tracrRNA, where the nuclease does not cleave its DNA target (e.g., by mutation to the endonuclease domain or coding for a mismatch distal to the PAM within the target). By localizing the target to a specific location (e.g., on a silicon chip), the fluorescence / colorimetric intensity at that location reflects the bound ribonucleoprotein complex for that target. This localization can be detected spatially using various approaches such as using a camera or a photodiode array.

[0193] In all of these examples, the measured output is linked to a specific DNA target and thus functions as an indirect readout of the presence of the detected RNA. A quantitative readout can be provided to identify the concentration of each detected RNA in the sample based on fluorescence intensity. For example, when there is a cluster of molecules encoding the same DNA target sequence, the fluorescence intensity will correspond to the number of DNA molecules recognized by the ribonucleoprotein complex encoding the appropriate ncrRNA and thus the number of detected RNA molecules that generated the ncrRNA.

[0194] In addition to using dsDNA targets, ssDNA and RNA targets can also be used as part of the diagnostic platform. A number of Cas9 and Cas12 nucleases (e.g., CjeCas9, NmeCas9, SauCas9) have been shown to recognize ssDNA and RNA, where recognition requires base pairing to the guide region of the guide RNA but does not require a PAM. Since a fluorophore is attached to one end of the target and a quencher is attached to the other end, a similar detection configuration can be used where cleavage of the target releases the quencher, or a nuclease conjugated with a fluorophore can localize fluorescence upon target binding.

[0195] Some Cas9 nucleases (e.g., SpyCas9) cannot recognize ssDNA or RNA unless a double-stranded PAM sequence is present adjacent to the target. This could be incorporated into the target design by using an ssDNA target with a fixed sequence and flanking sequences for the PAM. Another ssDNA oligo can then be hybridized to the fixed sequence to provide flanking sequences with a double-stranded PAM and a single-stranded target.

[0196] The examples described in this specification can be used for a wide range of applications such as diagnosing medical conditions and informing the course of treatment, identifying SNPs associated with healthy outcomes or diseases, identifying contaminating microorganisms in drinking water, identifying contaminating viruses or microorganisms in fermented products or cell cultures, identifying mutants of plants or insects, or identifying the major microbial members in a mixed community (e.g., gut, soil, water).

[0197] Use of an unnatural tracrRNA for in vivo RNA recording For this application, an unnatural tracrRNA is used to record the presence of one or more target RNAs in a cell in the form of DNA editing or a change in the expression of a measurable reporter gene.

[0198] The tracrRNA-dependent nuclease and the unnatural tracrRNA are encoded in a linear DNA, plasmid, or viral construct for delivery and cellular expression. The tracrRNA can be expressed separately as individual expression constructs or in a single construct having intervening RNA cleavage sequences (e.g., tRNA, ribozyme).

[0199] The DNA corresponding to the target of the generated ncrRNA may be encoded in the same vector or a separate vector, where its composition will depend on how the nuclease is used to record the detected RNA. For representative examples, see Figures 13 and 19-22. In one example, DNA editing (e.g., indel formation, base editing) functions as a readout and only needs to encode a single DNA target for each non-native tracrRNA. In the case of homology-dependent repair or homologous recombination, the recombinase template also exists in the form of an oligonucleotide, linear dsDNA, or circular DNA in cells containing the desired edit flanked by homology arms. Alternatively, the nuclease can drive the expression of a reporter gene such as a fluorescent RNA aptamer (e.g., RNA spinach), a fluorescent protein (e.g., GFP), or an enzyme that produces a visual output (e.g., LacZ coupled to X-gal). Next, the nuclease regulates the expression of the reporter (e.g., blocking transcription by targeting the promoter or coding region, recruitment of RNA polymerase by a fusion or mobilized activation domain such as SoxS in bacteria or VP16 in eukaryotic cells).

[0200] This construct can express a dsRNA-cleaving enzyme (e.g., RNase III from E. coli) for ncrRNA biosynthesis. However, virtually all cells express dsRNA-cleaving enzymes that can be used instead. There may also be cases where no dsRNA-cleaving enzyme is needed at all.

[0201] For detection and recording, the DNA construct is introduced into cells by standard means (e.g., electroporation, chemical transformation, conjugation, transfection, viral delivery), and the DNA can exist transiently only (e.g., by transient transfection) or can be stably maintained (e.g., under selection or integrated into chromosomal DNA). Next, the components are expressed from the construct and the presence of the RNA to be detected is actively detected. When RNA is present, this is converted into ncrRNA, which induces the tracrRNA-dependent nuclease to the target DNA reporter. In the case of a reporter that depends on expression, the reporter activity can be measured using standard techniques (e.g., fluorescence by flow cytometry or a fluorescence microtiter plate reader). In the case of a reporter that depends on DNA editing, the target can be sought via standard approaches that directly or indirectly output the DNA sequence (e.g., Sanger sequencing, next-generation sequencing).

[0202] As an example, a non-native tracrRNA was combined with a base editor derived from SpyCas9 (e.g., BE3) to detect the expression of a multidrug resistance gene (e.g., encoding NMD-1) or a stress response gene (e.g., encoding RyhB, an sRNA induced by iron starvation) in E. coli. The non-native tracrRNA was designed to hybridize to the portion of the RNA to be detected as shown in FIG. 10. The accompanying guide sequence of the ncrRNA was used in the design of the target DNA and was selected to contain multiple internal Cs that could be edited by BE3. This target DNA was then encoded in a plasmid (e.g., pBAD18) together with the expression constructs for BE3 and the non-native tracrRNA. This plasmid was then transformed into an E. coli strain and cultured with or without another plasmid encoding NMD-1 or in the presence of an iron chelator. The plasmid was then isolated, the target DNA was amplified, and Sanger sequencing analysis was performed. Even when the plasmid encoding NMD-1 was removed or additional iron was added to the growth medium so that the RNA to be detected was no longer expressed, at the target site, the expected conversion from multiple Cs to multiple Ts was encoded in the strain with the expressed RNA to be detected. Also, no crosstalk was seen between the two non-native tracrRNAs and the target site, and detection was multiplexed by encoding the non-native tracrRNA and the target and exposing the cells to both the NMD-1 plasmid and the iron chelator. This example can be easily extended to other bacteria in which base editing has been shown to function, as well as to eukaryotic cells such as yeast, animal cells, human cells, or plant cells.

[0203] In parallel with the above example, a non-natural tracrRNA can be combined with a CRISPR transposon based on Cas12k to perform transcription-dependent recording. Cas12k, transposon components (TnsB, TnsC, TnsQ), an insertion template flanked by transposon repeats (e.g., encoding a reporter gene or an antibiotic resistance marker), and a non-natural tracrRNA can be encoded within an expression plasmid together with plasmid DNA encoding the corresponding target site. Using this construct, the presence of the RNA to be detected will drive the insertion of the template proximal to the target sequence. Next, the insertion can be detected by PCR or sequencing.

[0204] Use of non-natural tracrRNA for transcription-dependent targeting Another use of non-natural tracrRNA is that it requires editing of an intracellular DNA target only when the gene is actively transcribed. As part of this use, the non-natural tracrRNA is designed to hybridize to a region of the RNA to be detected that encodes a PAM sequence recognized by a tracrRNA-dependent CRISPR nuclease. The PAM is specifically located at the 5' or 3' end of the hybridizing portion of the RNA to be detected for type II or type V CRISPR nucleases, respectively. By encoding the PAM at this position, the resulting ncrRNA can direct the tracrRNA-dependent CRISPR nuclease to the DNA locus encoding the RNA to be detected. Since the RNA to be detected is an essential component in this process, the DNA locus will be targeted only when the RNA to be detected is present and thus when the DNA locus is transcribed. Figure 14 illustrates this concept, while Figure 15 shows a proof-of-concept using TXTL.

[0205] To achieve transcription-dependent targeting in cells, the tracrRNA-dependent nuclease and the non-natural tracrRNA are encoded in a plasmid, or a viral construct, for delivery and cellular expression. The tracrRNA can be expressed separately as an individual expression construct, or in a single construct having an intervening RNA cleavage sequence (e.g., tRNA, ribozyme). The nuclease can drive indel formation or homologous recombination in the presence of a DNA repair template, or the nuclease can generate other edits when modified to function as a base editor or a prime editor. Alternatively, a nuclease without catalytic activity can regulate the expression of the gene of interest by blocking transcription or by mobilizing KRAB to drive transcriptional repression.

[0206] This construct can express a dsRNA-cleaving enzyme (e.g., RNase III from E. coli) for ncrRNA biosynthesis. However, virtually all cells express dsRNA-cleaving enzymes that can be used instead.

[0207] For example, transcriptional-dependent editing could be implemented in the context of gene drive in mosquitoes only when the target locus is actively transcribed. The non-natural tracrRNA is designed to generate ncrRNA from the relevant target locus (e.g., the doublesex gene), and the hybridizing region in the target RNA encodes a PAM (e.g., NGG at the 5' end of this region when using SpyCas9). The tracrRNA-dependent CRISPR nuclease, the non-natural tracrRNA, and any gene cargo are encoded in an expression construct that is inserted into the target locus via standard genetic manipulation techniques for mosquitoes. When the resulting mosquitoes mate and produce offspring, the gene drive is expected to be transmitted only to cells in which the locus is actively transcribed. The ultimate effect is limited to the tissue types to which the gene drive is transmitted, and an additional layer of control is introduced depending on the expression profile of the locus.

[0208] Multiple non-native tracrRNAs can be designed against the doublesex gene, encoded within the gene drive locus, to improve the frequency of transmission and reduce the frequency of drive failure due to the formation of disruptive indels at the target site.

[0209] The tracrRNA can be reprogrammed to induce Cas9 activity by the target RNA. The conversion of cellular RNAs to ncRNAs was based on sequences with extensive complementarity to the tracrRNA anti-repeat, similar to the natural biosynthesis of crRNAs. If the anti-repeat sequence of the tracrRNA can be altered to hybridize to other RNAs while maintaining a structure suitable for Cas9 recognition, the resulting non-native tracrRNA can drive the conversion of any expressed cellular RNA to an ncRNA, thereby leading Cas9 to the matching DNA target (Figure 17A).

[0210] Originally, CjeCas9 recognizes a perfect RNA duplex formed between the crRNA repeat and the tracrRNA anti-repeat. Based on mutational analysis of the crRNA repeat, the inventors had already observed that the crRNA repeat:tracrRNA anti-repeat duplex could accommodate several mutations. Thus, the inventors mutated both sides of the duplex in the sgRNA of fliF while preserving the secondary structure and then evaluated GFP silencing in TXTL (s26 - s31, Figure 17B). GFP silencing was maintained even when the entire sequence of the duplex was exchanged (s31, Figure 17B). Next, the inventors reprogrammed the tracrRNA anti-repeat to form a complete 25-base pair duplex with three putative ncrRNAs (derived from fliF, CJ8421_04280, CJ8421_04970) that showed the most modest GFP silencing in TXTL. In all three cases, GFP repression was significantly enhanced by the non-native tracrRNA compared to the WT tracrRNA, although the repression was not as strong as that by the canonical crRNA:tracrRNA pair (p = 1×10 -6 ~0.0011) (Figure 17C). Finally, the inventors reprogrammed the tracrRNA anti-repeat to base pair with a completely new region of the mRNA (Figure 17D). Starting from the mRNA encoding the CDS of CJ8421_04970, the inventors designed five different non-native CjeCas9 tracrRNAs that hybridize to various positions (n1 - n5) in the mRNA (Figure 17D). Four of these non-native tracrRNAs resulted in a significant decrease in GFP levels compared to the non-targeting crRNA control (p = 6×10 -7 ~0.002). Importantly, GFP expression was restored by mutation of the predicted seed region, scrambling of the tracrRNA anti-repeat, or replacement of CjeCas9 with FnCas12a (Figure 17D). Thus, non-native tracrRNAs can be used to induce DNA targeting by CjeCas9 to the target RNA.

[0211] Considering the functionality of the unnatural tracrRNA of CjeCas9, the inventors asked whether the tracrRNAs for other Cas9 homologs could be similarly reprogrammed. The inventors selected the well-characterized Cas9 of Streptococcus pyogenes (SpyCas9) and the CRISPR1 Cas9 of Streptococcus thermophilus (Sth1Cas9) as examples. In both cases, the inventors devised design rules for the unnatural tracrRNA based on the known secondary structure of the crRNA:tracrRNA duplex and the preference of RNase III for cleaving double-stranded RNAs with AT-rich sequences. All 10 unnatural tracrRNAs tested significantly reduced GFP levels compared to the non-targeting crRNA control (p = 1×10 -7 ~1×10 -4 ). As before, GFP expression was restored by disrupting the seed sequence in the ncrRNA guide, scrambling the tracrRNA anti-repeat, or exchanging any of the Cas9s for FnCas12a. In many cases, the degree of GFP silencing approached that of the targeting crRNA control (Figure 17D). The inventors also evaluated the targeting plasmid removal by the unnatural tracrRNA in E. coli for all three Cas9 orthologs and found that each could induce efficient plasmid removal for at least one of the tested unnatural tracrRNAs (Figure S8B). Overall, the tracrRNAs for various Cas9 orthologs can be converted to unnatural tracrRNAs to induce DNA targeting based on the presence of selected cellular RNAs.

[0212] The reprogrammed tracrRNA enables sequence-specific multiplexed RNA detection by Cas9. By linking DNA targeting to the target RNA, the unnatural tracrRNA provides unique opportunities for RNA detection and various paradigms for CRISPR diagnostics. Current CRISPR diagnostics rely on Cas12a or Cas13 to search for double-stranded DNA or RNA targets in a sample, where target recognition induces non-specific cleavage of a fluorescent reporter single-stranded DNA or RNA. The non-specific readout effectively limits one test to one target sequence. In contrast, the unnatural tracrRNA converts the RNA to be detected into an ncrRNA, which induces Cas9 to bind to or cleave the matching DNA. The Cas9 binding or cleavage of the DNA sequence will indicate whether the associated RNA is present in the sample. Since the sequence of each DNA target is unique, multiple target sequences could be monitored in parallel in one test. The inventors named the resulting diagnostic platform LEOPARD (Leveraging Engineering tracrRNAs and On-target DNAs for PArallel RNA Detection) (Figure 18A).

[0213] To initiate the evaluation of LEOPARD, the inventors performed a purified in vitro reaction using T7-transcribed RNA, commercially available Cas9 protein, and a linear DNA target. The inventors started with RNA corresponding to one of the synthetic ncrRNA loci within CJ8421_04970 (Figure 17D) and separated the cleaved DNA target from the uncleaved DNA target by size using gel electrophoresis. It was found that DNA target cleavage occurred even when the ncrRNA sequence was extended at either end upon introduction of an annealing step in which the non-native tracrRNA hybridizes to the T7-transcribed ncrRNA. Moreover, in the annealing step, not only was it unnecessary to add RNase III or RNase A, but it was even confirmed that it partially inhibited apparent DNA cleavage. Efficient cleavage could occur even with 100-fold excess total yeast RNA, but was limited to the presence of ncrRNA (Figure 4B). Thus, LEOPARD reports the presence of a specific target RNA based on DNA target cleavage.

[0214] To fully realize the multiplexing potential of LEOPARD, it is necessary to monitor multiple DNA targets simultaneously. Therefore, the inventors devised a readout scheme based on separating distinct cleavage products from pooled DNA targets by gel electrophoresis (Figure 18C). Each target is labeled with a phosphor at one end and yields only two visualizable products, cleaved and uncleaved. Next, applying this scheme, nine approximately 150-nucleotide RNA fragments related to respiratory viruses, including two from SARS-CoV-2 coronavirus (the pathogen of COVID-19), six from other coronaviruses, and one from influenza H1N1, were specifically detected (Figure 18B, Figure 18D). The inventors found that each DNA target was cleaved by Cas9 only in the presence of the corresponding RNA. Furthermore, since this scheme enabled the specific detection of three or five RNA fragments in the same reaction, the ability of LEOPARD for multiplexed detection was verified. Finally, the question was asked whether LEOPARD could detect even a single nucleotide difference when viral RNA sequences were selected to minimize homology.

Explanation of Symbols

[0215] Drawing Translation Figure 1 Campylobacter jejuni 84-21 Campylobacter jejuni 84-21 Pull down CjCas9 Pull-down of CjCas9 Sequence RNAs enriched from pull down RNA sequences enriched from pull-down motif Motif cellular RNA Cellular RNA RNase III cleavage site RNase III cleavage site processed tracrRNA Processed tracrRNA Figure 2 Sensed RNA Sensed RNA Non-natural tracrRNA with reprogrammed anti-repeat processed non-natural tracrRNA Figure 3 non-targeting targeting reaction time Targeting site Figure 4 Top Upper center lower center Bottom Reaction time (h) Figure 5 terminator 1 nt Bulge in anti-repeat strand 2 nt Bulge in anti repeat strand Reaction time (h) 1 nt Bulge in repeat strand 2 nt Bulge in repeat strand Figure 6 terminator 1 bp mismatch Reaction time (h) 2 bp mismatch 2-base pair mismatch G:U wobble base pair G:U wobble base pair Figure 7 mutated crRNA mutated crRNA 1 mismatch 1 mismatch 300 nt overhang 300-nucleotide overhang 2 mismatches and 2 GU wobble base pairing 2 mismatches and 2 GU wobble base pairings Multiple mismatches Multiple mismatches 2 mismatches & 2 GU base pairing 2 mismatches and 2 GU base pairings Reaction time (h) Reaction time (h) tracrRNA-strand bulge in cutting site tracrRNA-strand bulge at the cutting site crRNA-strand bulge in cutting site crRNA-strand bulge at the cutting site spacer-proximal tracrRNA-strand bulge in recognition site spacer-proximal tracrRNA-strand bulge in the recognition site spacer-proximal crRNA-strand bulge in recognition site spacer-proximal crRNA-strand bulge in the recognition site tracrRNA-strand bulge tracrRNA-strand bulge crRNA-strand bulge crRNA-strand bulge Figure 8 Overhang Overhang Terminator Terminator Reaction time (h) Reaction time (h) +1,000 nts +1000 nucleotides +150 nts +150 nucleotides +50 nts +50 nucleotides +300 nts +300 nucleotides Figure 9 sensed RNA Detected RNA Non-natural tracrRNA with reprogrammed anti-repeat Non-natural tracrRNA with reprogrammed anti-repeat DNA target DNA target target Target non-target strand Non-target strand target strand Target strand mRNA associated with peak 113 mRNA associated with peak 113 loci targeted by non-natural tracrRNA Loci targeted by non-natural tracrRNA Locus 1 Locus 1 Locus 2 Locus 2 Locus 3 Locus 3 Locus 4 Locus 4 Locus 5 Locus 5 Reaction time (h) Reaction time (h) Figure 10 sensed RNA Detected RNA Non-natural tracrRNA with reprogrammed anti-repeat Non-natural tracrRNA with reprogrammed anti-repeat DNA target DNA target target Target non-target strand Non-target strand target strand Target strand mRNA associated with peak 113 loci targeted by non-natural tracrRNA Locus 1 Locus 2 Locus 3 Locus 4 Locus 5 Reaction time (h) Figure 11 sensed RNA Non-natural tracrRNA with reprogrammed anti-repeat DNA target target non-target strand target strand mRNA associated with peak 113 loci targeted by non-natural tracrRNA Locus 1 Locus 2 Locus 3 Locus 4 Locus 5 Reaction time (h) Figure 12 target DNA processed non-natural tracrRNA Figure 13 Non-natural tracrRNA with reprogrammed anti-repeat Non-natural tracrRNA with reprogrammed anti-repeat Sensed RNA Sensed RNA Reporter DNA Reporter DNA Reporter editing (e.g. through indel formation) Reporter editing (e.g. through indel formation) Reporter expression Reporter expression Figure 14 Non-natural tracrRNA with reprogrammed anti-repeat Non-natural tracrRNA with reprogrammed anti-repeat Target DNA Target DNA Transcription-dependent editing (e.g. through indel formation) Transcription-dependent editing (e.g. through indel formation) Figure 15 Cas9 cleavage Cas9 cleavage RecBCD degradation RecBCD degradation non-targeting non-targeting targeting targeting reaction time reaction time mRNA associated with peak 113 mRNA associated with peak 113 loci targeted by non-natural tracrRNA loci targeted by non-natural tracrRNA Locus 2 Locus 2 Locus 4 Locus 4 locus 2 without PAM locus 2 without PAM Locus 2 with PAM: Locus 2 containing PAM Reaction time (h): Reaction time (hours) Locus 4 without PAM: Locus 4 not containing PAM Locus 4 with PAM: Locus 4 containing PAM Figure 16 Non-natural tracrRNA with reprogrammed anti-repeat: Non-natural tracrRNA with reprogrammed anti-repeat Sensed RNA: Detected RNA Target plasmid: Target plasmid Loss of antibiotic resistance: Loss of antibiotic resistance E. coli: E. coli Transformation efficiency: Transformation efficiency Non-targeting: Non-targeting Targeting: Targeting Figure 17 RNA-of-interest: RNA of interest Reprogrammed tracrRNA (Rptr): Reprogrammed tracrRNA (Rptr) tracrRNA anti-repeat: tracrRNA anti-repeat cellular RNA: Cellular RNA DNA target: DNA target natural ncrRNA: Natural ncrRNA CJ8421_04970 mRNA (1,189 nts): CJ8421_04970 mRNA (1,189 nucleotides) predicted ncrRNAs derived from Rptrs: Predicted ncrRNAs derived from Rptr Figure 18 LEOPARD - Leveraging Engineered tracrRNAs and On-target DNAs for PArallel RNA Detection LEOPARD - Using Engineered tracrRNAs and On-target DNAs for Parallel RNA Detection Pool of RNAs from sample Pool of RNAs from the sample Cas9, Rptrs, DNA targets Cas9, Rptrs, DNA targets Detection by monitoring each DNA target Detection by monitoring each DNA target DNA target DNA target n4 ncrRNA (50-nt overhangs) n4 ncrRNA (50-nucleotide overhangs) yeast total RNA Yeast total RNA Mass ratio (yeast RNA:n4 ncrRNA + Rptr) Mass ratio (yeast RNA:n4 ncrRNA + Rptr) DNA targets DNA targets Resolved DNA products Resolved DNA products Uncleaved products Uncleaved products RNA source RNA source SARS-CoV-2 (locus1) SARS-CoV-2 (locus1) SARS-CoV-2 (locus2) SARS-CoV-2 (locus2) Influenza H1N1 Influenza H1N1 Uncleaved DNA Uncleaved DNA Cleaved DNA Cleaved DNA RNA genome RNA genome D614G (S protein) D614G (S protein) Sensed RNA Sensed RNA Figure 19 Sth1nCas9 base editor Sth1nCas9 base editor target target target plasmid target plasmid Peak 113 mRNA locus2 Peak 113 mRNA locus 2 Percentage of base editing(%) Percentage of base editing (%) Figure 20 Preferred preceding base T Preferred preceding base T percentage of base editing(%) percentage of base editing (%) guide:target guide:target Non-preferred preceding base A Non-preferred preceding base A Guide Guide Target Target Figure 21 Percentage of base editing(%) Percentage of base editing (%) Region hybridizing with the Rptr Region hybridizing with the Rptr Figure 22 percentage of base editing(%) percentage of base editing (%) without xlyose without xylose with xlyose with xylose Figure 23 Spacer Spacer Triplex region Triplex region Antirepeat Antirepeat Repeat Repeat Joint loop Joint loop Figure 24 Spacer Spacer Duplex region Double-stranded region Lower stem Lower stem Bulge Bulge Repeat-antirepeat duplex Repeat - anti-repeat double-stranded region Upper stem Upper stem Bulge removed Removal of bulge Figure 25 Triplex region Triple-stranded region cellular RNA Cellular RNA DNA target DNA target CJ8421_04970 mRNA (1,189 nts) CJ8421_04970 mRNA (1189 nucleotides) predicted ncrRNAs derived from Rptrs Predicted ncrRNAs derived from Rptrs Duplex region Double-stranded region

Claims

1. A non-naturally occurring tracrRNA nucleic acid molecule comprising a portion that contains an anti-repeat region sequence that is designed to specifically hybridize to a preselected detectable RNA sequence through the formation of a complex that mimics the naturally occurring crRNA:tracrRNA duplex.

2. A complex comprising the non-naturally occurring tracrRNA nucleic acid molecule of claim 1, at least one tracrRNA-dependent CRISPR nuclease enzyme, and at least one detectable RNA.

3. The non-naturally occurring tracrRNA nucleic acid molecule of claim 2, further bound to a target DNA nucleic acid molecule comprising a sequence based on at least one detectable RNA sequence or a sequence designed based on the detectable RNA sequence.

4. 1. A method for detecting at least one detectable RNA in a cell, tissue, and / or sample, comprising: a) contacting the sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes to a first portion of the detection RNA; wherein the second portion of the detectable RNA specifically hybridizes to at least one target nucleic acid, and the at least one non-naturally occurring tracrRNA hybridizes or is capable of hybridizing to the detectable RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of at least one RNA cleaving enzyme, such as RNase III; b) detecting binding of the at least one tracrRNA-dependent CRISPR nuclease enzyme to the at least one target nucleic acid; Including, c) wherein binding detects the at least one detectable RNA in the cell, tissue, and / or sample; and Preferably, said nuclease has its cleavage and / or nicking activity inactivated.

5. 1. A method for detecting at least one detectable RNA in a cell and / or sample, comprising: a) contacting the sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes to a first portion of the detection RNA; wherein the second portion of the detectable RNA specifically hybridizes to at least one target nucleic acid, and the at least one non-naturally occurring tracrRNA hybridizes or is capable of hybridizing to the detectable RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of at least one RNA cleaving enzyme, such as RNase III; b) detecting cleavage of the at least one target nucleic acid in the sample by the nuclease enzyme; Including, c) A method, wherein said at least one detectable RNA is detected in said cell, tissue and / or sample by detection of said cleavage of at least one target nucleic acid.

6. The method of claim 4 or 5, wherein detection of the bound and / or cleaved at least one target nucleic acid comprises detecting a change in the signal of a suitable label, such as a dye, fluorophore, or electrical conductivity, and / or detecting the cleaved at least one target nucleic acid fragment itself.

7. 1. A method for detecting at least one detectable RNA in a cell, tissue, and / or sample, comprising: a) contacting the sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes to a first portion of the detection RNA; wherein the second portion of the detectable RNA specifically hybridizes to at least one target nucleic acid, and the at least one non-naturally occurring tracrRNA hybridizes or is capable of hybridizing to the detectable RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of at least one RNA cleaving enzyme, such as RNase III; b) nicking or cleaving the at least one target nucleic acid in the sample with the nuclease; c) detectably editing the at least one target nucleic acid, including, for example, non-homologous end joining (NHEJ) repair, microhomology-mediated end joining (MMEJ), homology-directed repair (HDR), base editing, prime editing, or RNA editing; d) suitably detecting said edits of said at least one target nucleic acid, for example comprising at least one method selected from the presence and / or length of an indel or gene edit, the activation / repression of a gene or other genetic element encoded by said at least one target nucleic acid, and / or the detection of said prime edit or base edit; Including, e) A method, wherein said at least one detectable RNA is detected in said cell, tissue and / or sample by detecting said editing of said at least one target nucleic acid.

8. 1. A method for recording transcription of at least one target DNA in a cell, tissue, and / or sample, comprising: a) contacting the sample, cell, or tissue with at least one non-naturally occurring tracrRNA that comprises a portion that specifically hybridizes to a first portion of a detection RNA; wherein the second portion of the detectable RNA specifically hybridizes to the at least one target DNA, and the at least one non-naturally occurring tracrRNA hybridizes or is capable of hybridizing to the detectable RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of at least one RNA cleaving enzyme, such as RNase III; b) nicking or cleaving the at least one target nucleic acid in the sample with the nuclease; c) detectably editing the at least one target DNA, including non-homologous end joining (NHEJ) repair, microhomology-mediated end joining (MMEJ), homology-directed repair (HDR), a detectable marker, a detectable modification, base editing, prime editing, or RNA editing; d) optionally detecting said edits of said at least one target DNA, including at least one method selected from the presence and / or length of indels or gene edits, detection of activation / repression of genes or other genetic elements encoded by said at least one target nucleic acid, and / or detection of said prime edits or base edits; Including, e) A method wherein detection of said editing of said at least one target DNA results in recording of transcription of said at least one detectable RNA in said cell, tissue and / or sample.

9. The method according to any one of claims 4 to 8, wherein several or a large number of detectable RNAs are detected in one or more samples, tissues and / or cells.

10. 10. The method of any one of claims 4 to 9, wherein the at least one detectable RNA is environment, species, strain, disease, cell and / or tissue specific or associated with a pathology selected from a viral infection, a coronavirus infection, an infection by a pathogen, a metabolic disease, cancer, a neurodegenerative disease, aging, a drug, and a biotic or abiotic stress.

11. The method according to any one of claims 4 to 10, further comprising the step of adding the at least one detectable RNA to the cell, tissue and / or sample prior to step a) and / or transcribing DNA into detectable RNA prior to step a).

12. 12. The non-naturally occurring tracrRNA nucleic acid molecule or method of any one of claims 1 to 11, wherein the at least one tracrRNA-dependent CRISPR nuclease enzyme is selected from Cas9 type II and Cas12 type V nuclease enzymes, Cas9 nuclease type II selected from the group consisting of II-A, II-B, and II-C, and Cas12 nuclease type VB, VC, VD, VE, VF, VG, and VK.

13. The non-naturally occurring tracrRNA nucleic acid molecule or method according to any one of claims 1 to 12, wherein the portion of the at least one non-naturally occurring tracrRNA that specifically hybridizes with the first portion of the detectable RNA hybridizes with 10 or more, 11 or more, or 12 or more nucleotides, and preferably the first portion of the detectable RNA comprises a protospacer adjacent motif (PAM).

14. The non-naturally occurring tracrRNA nucleic acid molecule or method of any one of claims 1 to 13, wherein the nucleotide sequence of the at least one portion of the tracrRNA that specifically hybridizes to a first portion of the detected RNA is produced and / or modified to be at least 80% complementary, more than 90% complementary, more than 95% complementary, or 100% complementary to the first portion of the detected RNA.

15. The non-naturally occurring tracrRNA nucleic acid molecule or method according to any one of claims 1 to 14, wherein two or more non-naturally occurring tracrRNAs are generated and used, each of which specifically hybridizes with a different portion of the detection RNA.

16. The method according to any one of claims 4 to 15, further comprising detecting the amount of said at least one target nucleic acid and / or said at least one detectable mRNA in said samples, tissues and / or cells, preferably the amount per sample, tissue and / or cell, and preferably further comprising detecting a change in the amount of said at least one target nucleic acid and / or said at least one detectable mRNA in said samples, tissues and / or cells compared to a control.

17. 17. A method for discovering a medical condition in a mammal associated with the presence of at least one detectable RNA, the expression of said detectable RNA and / or the mutation(s) in said detectable RNA, comprising carrying out a method according to any one of claims 4 to 16 and discovering said medical condition based on the presence of said at least one detectable RNA, the expression of said detectable RNA and / or the mutation(s) in said detectable RNA being detected, wherein preferably said at least one detectable RNA is environment, species, strain, disease, cell and / or tissue specific or associated with a condition selected from viral infection, coronavirus infection, infection by a pathogen, metabolic disease, cancer, neurodegenerative disease, ageing, drugs and biotic or abiotic stress, optionally further comprising the step of adding said at least one detectable RNA to said cell, tissue and / or sample prior to step a) and / or transcribing DNA into detectable RNA prior to step a).

18. A detectable RNA detection system comprising: a) a non-naturally occurring tracrRNA designed to bind to at least one portion of the detectable RNA, the portion further comprising a portion that specifically hybridizes to a first portion of a target DNA; b) at least one tracrRNA-dependent CRISPR nuclease enzyme, preferably further comprising c) at least one target nucleic acid molecule comprising a label for detecting cleavage of the target nucleic acid; and optionally, d) at least one RNA cleavage enzyme, such as an RNase III enzyme.

19. Use of a non-naturally occurring tracrRNA nucleic acid molecule according to any one of claims 1 to 3 or a detectable RNA detection system according to claim 18 for carrying out a method according to any one of claims 4 to 17, in particular for detecting a detectable RNA, a viral detectable RNA, a detectable RNA transcribed from a disease marker, or for generating an expression profile for one or more detectable RNAs.

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