Patent Application
20230175078
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 said 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.
Almost all archaea and about half of bacteria possess clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated genes (Cas) adaptive immune systems, which protect prokaryotes against viruses and other foreign invaders with nucleic-acid genomes. The CRISPR-Cas system is functionally divided into classes 1 and 2 according to the composition of the effector complexes. Class 2 consists of a single-effector nuclease, and routine practice of genome editing has been achieved by the exploitation of Class 2 CRISPR-Cas systems, which include the type II, V, and VI CRISPR-Cas systems. Types II and V can be used for targeting DNA or RNA, while type VI is employed for targeting RNA (see, for example, Koonin EV and Makarova KS Origins and evolution of CRISPR-Cas systems Philos Trans R Soc Lond B Biol Sci. 2019 May 13;374(1772):20180087). Types II and V Cas effector nucleases commonly rely on protospacer-adjacent motifs (PAMs) as the first step in target DNA recognition, and the effector nucleases directly bind the PAM sequence through protein-DNA interactions and subsequently unzip the downstream DNA sequence. The effector proteins then interrogate the extent of base pairing between one strand of the DNA target and the guide portion of the CRISPR RNA (crRNA). Sufficient complementarity between the two drives target cleavage. PAM sequences are known to vary considerably not only between systems but also between otherwise similar nucleases, and it was shown that Cas proteins can be engineered to alter PAM recognition (Leenay and Beisel Deciphering, Communicating, and Engineering the CRISPR PAM. J Mol Biol. 2017 Jan 20;429(2): 177-191). In addition to targeting DNA, some Type II and V single-effector nucleases such as the C. jejuni Cas9, the N. meningitidis Cas9, the S. aureus Cas9, and the Cas12f1 from an uncultured archaeon have also been shown to target ssDNA and/or RNA (RNA-dependent RNA targeting by CRISPR-Cas9. Elife. 2018;7:e32724; DNase H Activity of Neisseria meningitidis Cas9. Mol Cell. 2015;60(2):242-255; Programmed DNA destruction by miniature CRISPR-Cas14 enzymes). In these cases, no PAM was required. Some nucleases such as the S.pyogenes Cas9 (SpyCas9) were not able to immediately target ssDNA or RNA, although providing an oligonucleotide to generate a double-stranded PAM region allowed SpyCas9 to bind the single-stranded target and cleave it (Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature. 2014;516(7530):263-266).
Mature crRNAs are key elements in CRISPR-Cas defense against genome invaders. These short RNAs contain unique guide sequences that guide the Cas protein(s) to the cognate invading nucleic acids for their destruction. The CRISPR RNAs (crRNAs) are naturally encoded in CRISPR arrays comprising alternating conserved repeats and spacers. The array is often transcribed into a long pre-crRNA, and then this RNA is processed into the individual crRNAs composed of a portion of a repeat-spacer unit. 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 a unique crRNA biogenesis pathway, in which a trans-activating CRISPR RNA (tracrRNA, encoded within the CRISPR-Cas locus) base pairs with each repeat sequence of the pre-crRNA to form a double-stranded RNA duplex (Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol.2020; 18(2):67-83). The crRNA-tracrRNA duplex is cleaved by the housekeeping endoribonuclease RNase III and is bound by the single-effector nuclease specific to the system. The nuclease then uses the crRNA within the RNA duplex to guide DNA and, in some cases, RNA targeting. To-date, multiple tracrRNA-dependent CRISPR nucleases have been reported, including Cas9, Cas12b1/C2c1, Cas12b2, Cas12e/CasX, Cas12f1/Cas14a, Cas12g, and Cas12k. The Cas12k nucleases are unique because they work in concert with a set of transposon genes for RNA-directed insertion of a DNA template (RNA-guided DNA insertion with CRISPR-associated transposases. Science. 2019;365(6448):48-53). In each of these cases, the tracrRNA:crRNA duplex, when engineered as a single RNA chimera called a single-guide RNA (sgRNA), also directs sequence-specific dsDNA cleavage by Cas9 and other tracrRNA-dependent CRISPR nucleases (see Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012); Engineering of CRISPR-Cas12b for human genome editing. Nat Commun. 2019;10(1):212).
The CRISPR-Cas9 system of Francisella novicida exhibits a PAM-dependent interaction of Cas9 with its endogenous DNA targets that is dependent on a non-canonical small CRISPR-associated RNA (scaRNA) and tracrRNA. Naturally, the scaRNA appears to direct Cas9 to genomic DNA targets with partial complementarity, resulting in transcriptional repression. scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, can also direct DNA cleavage (Catalytically Active Cas9 Mediates Transcriptional Interference to Facilitate Bacterial Virulence Mol Cell. 2019 Aug 8;75(3):498-510.e5). Given that the scaRNA is encoded within the CRISPR-Cas system similar to crRNAs, the only previously known source of RNAs that could guide the CRISPR nucleases to its intended targets would only come from within the CRISPR-Cas system.
In a recent publication of the inventors (CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacterjejuni Cas9. Mol Cell. 2018;69(5):893-905.e7), they sequenced RNAs that co-immunoprecipitated with the Cas9 nuclease of Campylobacterjejuni (CjeCas9), which had revealed a subset of RNAs that exhibited complementarity to the guide portion of the crRNAs encoded within the CRISPR locus of this bacterium. The inventors performed a similar co-immunoprecipitation experiment in a separate strain of C.jejuni. The resulting unpublished dataset included a set of RNAs that did not exhibit complementarity to the crRNA and instead shared complementarity to the anti-repeat portion of the tracrRNA (
Base pairing between the CRISPR repeat and the tracrRNA anti-repeat form the RNA duplex bound by tracrRNA-dependent CRISPR nucleases. The exact secondary structure of duplex varies, with some duplexes exhibiting perfect complementarity while others contain characteristic bulges. Regardless of the exact secondary structure, maintaining this secondary structure as part of sgRNAs for the Cas9 from Streptococcuspyogenes allowed for modification of the sequence of this region of the sgRNAs (Guide RNA functional modules direct Cas9 activity and orthogonality. Mol Cell. 2014;56(2):333-339). The inventors therefore hypothesized that the sequence of the anti-repeat portion of the tracrRNA could be changed to be complementary to other RNAs, allowing this portion of each RNA to become a mature crRNA (
The concept of altering the anti-repeat region of the tracrRNA remains to be been explored, particularly for converting any RNA into an ncrRNA. The region downstream of the anti-repeat region has been modified in the context of sgRNAs and shown to be accommodate some changes without disrupting the function of the sgRNA (Guide RNA functional modules direct Cas9 activity and orthogonality. Mol Cell. 2014;56(2):333-339). Furthermore, Scott, T. et al (Improved Cas9 activity by specific modifications of the tracrRNA. Sci Rep. 2019;9(1):16104) showed that a region outside of the anti-repeat region could be changed to improve the overall editing activity exhibited by a Cas9 RNP, although the mechanism was unclear. However, these regions are involved in recognition by Cas9 and are not involved in pairing with a crRNA or any other RNA.
WO 2019/126037 relates to compositions and methods that utilize a nucleic acid sequence that has a genetically modified trans-activating crRNA (tracrRNA) sequence, where at least one uracil nucleotide of the tracrRNA sequence is replaced with a nucleotide other than uracil, and/or a nucleic acid sequence that has a guide RNA (gRNA) sequence wherein one or more cytosine nucleotides and/or one or more uracil nucleotides of said gRNA sequence are modified nucleotides. Disclosed is a composition further comprising an RNA-guided DNA endonuclease enzyme, such as Cas9 or Cpfl or a Class II CRISPR endonuclease or a variant thereof, such as deactivated Cas9 (dCas9) or mutated Cas9 nickase (D10A).
US 2019/032053 relates to compositions comprising synthetic two-part aptamer-containing guide RNAs and methods of using said synthetic two-part aptamer-containing guide RNAs with CRISPR/Cas activator systems. Methods for targeted transcription activation, targeted transcription repression, targeted epigenome modification, targeted genome modification, or targeted genomic locus visualization in a eukaryotic cell are claimed.
WO 2019/183000 discloses chemically modified crRNAs and tracrRNAs, crRNAs and tracrRNAs with 5’ and/or 3’ conjugated moieties, and crRNAs and tracrRNAs with modifications in the repeat region of the crRNA or the anti-repeat region of the tracrRNA. Methods of using the crRNAs and tracrRNAs for genome editing with a CRISPR nuclease and kits for performing the same are also provided. crRNA and tracrRNA were chemically modified while retaining the efficacy of SpyCas9-based genome editing in cultured human cells.
Tautvydas Karvelis et al. (in: crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophiles. RNA BIOLOGY, vol. 10, no. 5, 27 Mar. 2013, pages 841-851, DOI: 10.4161/ma24203), disclose that the Cas9-crRNA complex of the Streptococcus thermophilus DGCC7710 CRISPR3-Cas system 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 the heterologous E. coli strain carrying the S. thermophilus DGCC7710 CRISPR3-Cas system. 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 primary pre-crRNA-tracrRNA duplex cleavage for mature crRNA biogenesis. RNase III, however, is not required in the processing of a short pre-crRNA transcribed from a minimal CRISPR array containing a single spacer. An in vitro-assembled ternary Cas9-crRNA-tracrRNA complex cleaves DNA. The reference further specifies the molecular basis for crRNA-based re-programming of Cas9 to specifically cleave any target DNA sequence for precise genome surgery. The processes for crRNA maturation and effector complex assembly as disclosed are said to contribute to the further development of the Cas9 re-programmable system for genome editing applications.
Nevertheless, WO 2019/126037, US 2019/032053, WO 2019/183000, and Tautvydas Karvelis et al. all modify tracrRNAs as part of a standard crRNA:tracrRNA duplex used for DNA targeting. The references try to improve the existing duplex reflecting a truncated crRNA and tracrRNA, whereas the present invention uses modified non-naturally occurring tracrRNA to hybridize to naturally occurring RNAs that are not crRNAs.
The ability to generate an ncrRNA from any RNA links the presence of an RNA to an active tracrRNA-dependent CRISPR nuclease guided to specific targets by the ncrRNA. Therefore, reprogramming tracrRNAs holds the potential to sense specific RNA transcripts through sequence-specific targeting activity. This link is distinct from the two other CRISPR technologies used for nucleic acid detection that are based on Cas12a and Cas13. Specifically, Chen et al. (CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018;360(6387):436-439) disclose that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. Combining Cas12a ssDNase activation with isothermal amplification created a method termed “DNA endonuclease-targeted CRISPR trans reporter” (DETECTR). Separately, Abudayyeh et al. (C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016;353(6299):aaf5573) demonstrated the RNA-guided RNase activity of the putative type VI CRISPR-effector LshC2c2 (now Cas13a). Gootenberg et al. (Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017 Apr 28;356(6336):438-442) further disclose that this “collateral effect” could be used to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. This Cas13abased molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), has been used to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. While both technologies can output the presence of a detected nucleic acid, they both rely on non-specific nuclease activity as the output. In contrast, the present invention relies on a sequence-specific nuclease activity dictated by the sensed RNA.
It is an object of the present invention to apply the above advancements in CRISPR in the field of molecular diagnostics, in particular for detecting certain RNAs. Other objects and advantages will become apparent upon further studying the present specification with reference to the accompanying examples.
In a first aspect thereof, the object of the present invention is solved by providing a method for detecting at least one sensed RNA in a cell, tissue, and/or sample, said method comprising: a) contacting said sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with a first portion of said sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with at least one target nucleic acid; wherein said at least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed 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 binding of said at least one tracrRNA-dependent CRISPR nuclease enzyme to said at least one target nucleic acid; c) wherein said binding detects said at least one sensed RNA in said cell, tissue and/or sample.
In a second aspect thereof, the object of the present invention is solved by providing a method_for detecting at least one sensed RNA in a cell and/or sample, said method comprising: a) contacting said sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with a first portion of said sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with at least one target nucleic acid; wherein said at least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed 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 cleavage of said at least one target nucleic acid in said sample by said nuclease enzyme; c) wherein detecting said cleavage of at least one target nucleic acid detects said at least one sensed RNA in said cell, tissue and/or sample.
In a third aspect thereof, the object of the present invention is solved by providing a method for detecting at least one sensed RNA in a cell, tissue and/or sample, said method comprising: a) contacting said sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with a first portion of said sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with at least one target nucleic acid; wherein said least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of a dsRNA-cleaving enzyme; and b) nicking or cleaving of said at least one target nucleic acid in said sample by said nuclease, c) detectably editing said at least one target nucleic acid comprising, for example, non-homologous end joining (NHEJ) repair, microhomology-mediated end joining (MMEJ), homologous-directed repair (HDR), prime editing, base editing, or RNA editing, and d) suitably detecting said editing of said at least one target nucleic acid comprising, for example, at least one method selected from detecting the presence and/or length of indels or gene edits, the activation/repression of a gene or other genetic element as encoded by said at least one target nucleic acid, and/or the detection of said prime or base editing; e) wherein detecting at least one edited target nucleic acid detects said at least one sensed RNA in said cell, tissue and/or sample.
In a preferred embodiment of the present invention, the above methods can be combined, e.g. the at least one sensed RNA in said cell, tissue and/or sample can be detected based on binding, cleavage and/or editing of said at least one nucleic acid DNA.
In a fourth aspect thereof, the object of the present invention is solved by providing a method for detecting a medical condition in a mammal, wherein said condition is related to the presence of, expression of and/or mutation(s) in at least one sensed RNA, comprising performing the method according to any of the aspects as above, and detecting said medical condition based on the presence of, expression of and/or mutation(s) in said at least one sensed RNA as detected.
In a fifth aspect thereof, the object of the present invention is solved by providing a detection system for a sensed RNA comprising a) a non-naturally occurring tracrRNA designed to bind to at least one portion of said sensed RNA, said sensed RNA further comprising a portion that specifically hybridizes with a first portion of a target nucleic acid, and b) at least one tracrRNA-dependent CRISPR nuclease enzyme.
In a sixth aspect thereof, the object of the present invention is solved by providing a non-naturally occurring tracrRNA nucleic acid molecule, comprising an anti-repeat region sequence that is designed to specifically hybridize to a preselected sensed RNA sequence through forming a complex that mimics the natural crRNA:tracrRNA duplex. The non-naturally occurring tracrRNA nucleic acid molecule may be part of a complex comprising the non-naturally occurring tracrRNA nucleic acid molecule according to the present invention, at least one tracrRNA-dependent CRISPR nuclease enzyme, and at least one sensed RNA, and may be further bound to a target DNA nucleic acid molecule comprising a sequence based or designed based on the at least one sensed RNA sequence.
In a seventh aspect thereof, the object of the present invention is solved by providing the use of the non-naturally occurring tracrRNA nucleic acid molecule or sensed RNA detection system according to the aspect as above for performing a method according to any of the aspects as above, in particular for detecting a sensed RNA, a viral sensed RNA, a sensed RNA transcribed from a disease marker, or for generating an expression profile for one or more sensed RNAs.
In the first aspect thereof, the object of the present invention is solved by providing a method for detecting at least one sensed RNA in a cell, tissue, and/or sample, said method comprising: a) contacting said sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with a first portion of said sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with at least one target nucleic acid; wherein said at least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of an RNA-cleaving enzyme, such as RNase III; b) detecting binding of said at least one tracrRNA-dependent CRISPR nuclease enzyme to said at least one target nucleic acid; c) wherein said binding detects said at least one sensed RNA in said cell, tissue and/or sample.
In this first aspect of the present invention, the detection of said at least one sensed RNA in said cell, tissue and/or sample relies on the detection of binding of said at least one tracrRNA-dependent CRISPR nuclease enzyme to said at least one target nucleic acid. The Cas enzymes bind target nucleic acid independently of their ability to cleave target nucleic acid, and this flexibility is used in order to detect said at least one sensed RNA based on said binding activity.
Binding of said at least one tracrRNA-dependent CRISPR nuclease enzyme to said at least one target nucleic acid can be detected by any suitable detection method as known to the person of skill, and may include chromatin immunoprecipitation (ChIP) methods using an antibody against the nuclease and PCR primers for the DNA sequence of interest. The antibody is used to selectively precipitate the protein-DNA complex from the other genomic DNA fragments and protein-DNA complexes. The PCR primers allow specific amplification and detection of the target nucleic acid sequence. Quantitative PCR (qPCR) technique allows the amount of target nucleic acid sequence to be quantified. The ChIP assay is amenable to array-based formats (ChIP-on-chip) or direct sequencing of the DNA captured by the immunoprecipitated protein (ChIP-seq).
Other methods comprise DNA electrophoretic mobility shift assays (EMSA), or pull-down assays that are used to selectively extract a protein-DNA complex from a sample. Typically, the pull-down assay uses a DNA probe labeled with a high affinity tag, such as biotin, which allows the probe to be recovered or immobilized. A biotinylated DNA probe can be complexed with a protein from a cell lysate in a reaction similar to that used in the EMSA and then used to purify the complex using agarose or magnetic beads. The proteins are then eluted from the DNA and detected by western blot or identified by mass spectrometry. Alternatively, the protein may be labeled with an affinity tag, or the DNA-protein complex may be isolated using an antibody against the protein of interest (similar to a supershift assay). In this case, the unknown DNA sequence bound by the protein is detected by Southern blotting, PCR analysis, or sequencing analysis. A hybrid of the DNA pull-down assay and enzyme-linked immunosorbent assay (ELISA), the microplate capture assay, can be used that involves immobilized DNA probes to capture specific protein-DNA interactions and confirm protein identities and relative amounts with target-specific antibodies. Also, reporter assays can be used that provide a real-time in vivo readout of translational activity for a promoter of interest that may be blocked or induced by binding of the nuclease. Reporter genes are fusions of a target promoter DNA sequence and a reporter gene DNA sequence. The promoter DNA sequence is customized by the researcher and the reporter gene DNA sequence codes for a protein with detectable properties such as firefly luciferase, Renilla luciferase, alkaline phosphatase, or green fluorescent protein. These genes produce enzymes only when the promoter of interest is activated. The enzyme, in turn, fluoresces or catalyzes a substrate to produce either light, a color change, or other reaction that can be detected by spectroscopic instrumentation. The signal from the reporter gene is used as an indirect determinant for the transcription or translation of endogenous proteins driven from the same promoter.
In an embodiment of this aspect of the present invention, preferably the at least one tracrRNA-dependent CRISPR nuclease enzyme is inactivated, at least substantially, in its cleaving activity. For example, both the RuvC and HNH nuclease domains can be rendered inactive by point mutations (e.g. D10A and H840A in S.pyogenes Cas9 (SpCas9)), resulting in a nuclease dead Cas9 (dCas9) molecule that cannot cleave target nucleic acid. Nevertheless, the dCas9 molecule retains the ability to bind to target DNA based on the sgRNA targeting sequence. In the case of single-stranded targets, disruption of the HNH domain would be sufficient to prevent cleavage but not binding (CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9. Mol Cell. 2018;69(5):893-905.e7). Cleavage by the nuclease is also avoided if the target sequence contains mismatches, particularly in the PAM-distal region of the target (Cas9 gRNA engineering for genome editing, activation and repression. Nat Methods. 2015;12(11):1051-1054). Similar to Cas9, the RuvC domain for Cas12 nucleases 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 introducing mismatches can prevent cleavage but preserve target binding (Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nat Methods. 2019;16(9):887-893).
The method according to the present invention detects at least one sensed RNA in a cell, tissue, and/or sample. In the context of the present invention, a sensed RNA is any RNA of interest to be detected using the methods according to the present invention. Usually and preferably, the sensed RNA is a single-stranded RNA molecule, such as a messenger RNA, ribosomal RNAs, transfer RNAs, small RNAs, antisense RNAs, small nucleolar RNAs, microRNAs, piwiRNAs, long non-coding RNAs, spliced introns, and circular RNAs. The RNA can be of natural origin or artificially produced. The single stranded sensed RNA can be from a human cell, an animal cell, a plant cell, a cancerous cell, an infected cell, or a diseased cell, and/or can be derived from a virus, a parasite, a helminth, a fungus, a protozoan, a bacterium, or a pathogenic bacterium. The sensed RNA comprises a first portion that specifically hybridizes with a first portion of a non-naturally occurring tracrRNA as generated and used in the methods of the present invention, and a second portion that specifically hybridizes with at least one nucleic acid molecule.
The sensed RNA may comprise two or more of the first and second portions, and the non-naturally occurring tracrRNAs are designed accordingly. In the context of the present invention, a target nucleic acid can be any suitable target nucleic acid, but preferably comprises target DNA, or target RNA or target ssDNA, as some Cas9 and Cas12 nucleases can target these nucleic acids.
The sensed RNA may additionally comprise 5′ and/or 3′ ends extending from said first portion and extending outside of the complex as formed between the sensed RNA, the non-naturally occurring tracrRNA and the least one tracrRNA-dependent CRISPR nuclease enzyme (see
The sensed RNA comprises at least one second portion thereof that specifically hybridizes with at least one portion of at least one target nucleic acid. In the context of the first aspect of the method according to the present invention, this binding of said at least one tracrRNA-dependent CRISPR nuclease enzyme in form of the complex between the sensed RNA, the non-naturally occurring tracrRNA and the at least one tracrRNA-dependent CRISPR nuclease enzyme (see
The sensed RNA may be labelled, either at the 3′- and/or 5′-end, and/or by including a marker inside the RNA molecule/strand. Respective methods are known to the person of skill. The sensed RNA may also be a combination of RNA with portions of DNA and/or PNA.
Certain portions of the nucleic acid molecules as used in the methods according to the present invention are found and/or designed to specifically hybridize with complementary portions in other molecules. As known to the person of skill, for this, the hybridization and washing conditions are critical. If the sequences are 100% complementary, then a high stringency hybridization may be carried out. Nevertheless, according to the invention, the portions that hybridize and/or specifically hybridize are complementary to at least 80%, preferably complementary to more than 90%, more preferably to more than 95%, and most preferably are 100% complementary. The stringency of hybridization is determined by the hybridization temperature and the salt concentration in the hybridization buffer, and high temperature and low salt is more stringent. A commonly used washing solution is SSC (Saline Sodium Citrate, a mixture of NaCitrate and NaCl). Hybridization may be carried out in solution or – more commonly – at least one component may be on a solid-phase support, e.g., nitrocellulose paper. Frequently used protocols employ a blocking reagent, such as casein from nonfat dried milk or bovine serum albumin, often in combination with denatured, fragmented salmon sperm DNA (or any other heterologous DNA of high complexity) and a detergent, such as SDS. Often a very high concentration of SDS is used as a blocking agent. Temperatures may be between 42 and 65° C. or higher, and buffers may be 3 × SSC, 25 mM HEPES, pH 7.0, 0.25% SDS final.
The RNA-cleaving enzyme cleaves the hybridized region formed between the first portion of the sensed RNA and the anti-repeat portion of the non-natural tracrRNA, or it processes the other single-stranded end of the ncrRNA. This enzyme is preferably RNase III from E. coli but can be RNase III from other bacteria or archaea as well as other dsRNA-cleaving enzymes, such as Drosha, Dicer, DCL1, Rntp1, or Pac1p. The RNA-cleaving enzyme can also cleave ssRNA, such as RNase A.
Another aspect of the present invention then relates to a method for transcriptional recording, in particular in a multiplexed setting, in vitro or in vivo (see, for example,
Previous examples of transcriptional recording sense environmental signals or non-specifically record the transcriptional profile of the cell. Environmental recording relies on inducible promoters that express CRISPR acquisition proteins or guide RNAs that drive editing. In contrast, transcriptional recording with non-natural tracrRNAs can record one or multiple specific transcripts in a cell. The recording does not require inducible promoters, and the non-natural tracrRNA dictates which transcript is recorded.
Multiple non-natural tracrRNAs can also be combined with corresponding DNA targets for multiplexed recording.
Perli SD (in: 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) disclose a self-contained analog memory device for longitudinal recording of molecular stimuli into DNA mutations in human cells. This device consists of a self-targeting guide RNA (stgRNA) that repeatedly directs Streptococcus pyogenes Cas9 nuclease activity toward the DNA that encodes the stgRNA, enabling localized, continuous DNA mutagenesis as a function of stgRNA expression. We demonstrate programmable and multiplexed memory storage in human cells triggered by exogenous inducers or inflammation, both in vitro and in vivo. This tool, Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE), provides a distinct strategy for investigating cell biology in vivo and enables continuous evolution of targeted DNA sequences.
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 scalable platform for encoding logic and memory in bacterial and eukaryotic cells. Using an efficient single-nucleotide-resolution Read-Write head for DNA manipulation, DOMINO converts the living cells’ DNA into an addressable, readable, and writable medium for computation and storage. DOMINO operators enable analog and digital molecular recording for long-term monitoring of signaling dynamics and cellular events. Furthermore, multiple operators can be layered and interconnected to encode order-independent, sequential, and temporal logic, allowing recording and control over the combination, order, and timing of molecular events in cells. They envision that DOMINO will lay the foundation for building robust and sophisticated computation-and-memory gene circuits for numerous biotechnological and biomedical applications.
Tang W, and Liu DR. (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) disclose that Cas9 nucleases and base editors can be used to record amplitude, duration, and order of stimuli as stable changes in both genomic and extrachromosomal DNA content (see the Perspective by Ho and Bennett). The recording of multiple stimuli— including exposure to antibiotics, nutrients, viruses, and light, as well as Wnt signaling— was achieved in living bacterial and human cells. Recorded memories could be erased and re-recorded over multiple cycles.
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) use CRISPR spacer acquisition to capture and convert intracellular RNAs into DNA, enabling DNA-based storage of transcriptional information. In Escherichia coli, we show that defined stimuli, such as an RNA virus or arbitrary sequences, as well as complex stimuli, such as oxidative stress, result in quantifiable transcriptional records that are stored within a population of cells. They demonstrate that the transcriptional records enabled classifying and describing complex cellular behaviors and identifying the precise genes that orchestrate differential cellular responses. In the future, CRISPR spacer acquisition-mediated recording of RNA followed by deep sequencing (Record-seq) could be used to reconstruct transcriptional histories that describe complex cell behaviors or pathological states.
Preferred is a method for transcriptional recording, in particular in a multiplexed setting, in vitro or in vivo of at least one target DNA in a cell, tissue and/or sample, said method comprising: a) contacting said sample, cell or tissue with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with a first portion of a sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with said at least one target DNA; wherein said least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed 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; and b) nicking or cleaving of said at least one target nucleic acid in said sample by said nuclease, c) detectably editing said at least one target DNA comprising, for example, non-homologous end joining (NHEJ) repair, microhomology-mediated end joining (MMEJ), homologous-directed repair (HDR), a detectable marker, a detectable modification, base editing, prime editing or RNA editing, and d) optionally, suitably detecting said editing of said at least one target DNA comprising, for example, at least one method selected from detecting the presence and/or length of indels or gene edits, the activation/repression of a gene or other genetic element as encoded by said at least one target nucleic acid, and/or the detection of said prime or base editing; e) wherein detecting said editing of said at least one target DNA records transcription of said at least one sensed RNA in said cell, tissue and/or sample. Preferred embodiments and components are similar to the other methods as described herein.
Preferred uses of the transcriptional recording according to the present invention relate to the analysis of microbiomes and/or microbial sentinels (i.e. commensal bacteria that serve 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 viral or bacterial spread in vivo, such as whole organisms, or based on samples, for example taken from the environment (e.g. spread of viruses or resistant bacteria detected in waste water samples, etc.).
During work in the context of the present invention using the 84-21 strain of Campylobacterjejuni (Cje), the inventors assessed RNAs that co-immunoprecipitated with the CjeCas9. While one set shared complementarity to the crRNA2 in that strain, another set shared complementarity to the anti-repeat portion of the trans-acting CRISPR RNA (tracrRNA), responsible for crRNA maturation. This binding paralleled how tracrRNAs process crRNAs from the repeat to form the mature crRNA. These insights provided the first indication that mature crRNAs (which guide Cas9 to their target) could be derived from mRNAs and other RNAs besides those encoded by CRISPR arrays, although it was thought that crRNAs can only be derived from within CRISPR arrays and CRISPR-Cas systems.
It was surprisingly found in the context of the present invention, that the tracrRNA-dependent CRISPR nuclease enzyme system can be designed in certain ways in order to provide a highly versatile and specific diagnostic tool. An essential element of this tool is the design of non-naturally occurring tracrRNAs. The specific modification and thus “reprogramming” of the tracrRNAs allow for the generation of purpose-specific ncrRNAs. Interestingly, the tracrRNA-dependent CRISPR nuclease enzyme system was found to be still functional, despite the expectation that reprogramming the tracrRNA would potentially disrupt Cas9 recognition and activity.
In the context of the present invention, RNAs as identified with the motif complementary to the tracrRNA anti-repeat had the approximate size of a processed crRNA (36 - 37 nts). In one specific example (derived from the fliF gene), the ncrRNA matched to an architecture of a crRNA (~24 nt guide + ~12 nt processed repeat). Preferred is a method according to the present invention, wherein said portion of said at least one non-naturally occurring tracrRNA that specifically hybridizes with said first portion of said sensed RNA hybridizes with 10 or more nucleotides, preferably with 11 and more nucleotides, and more preferred with about 12 nucleotides or more. Preferred ranges are between 9 and 15 nucleotides, more preferred 10 to 14 nucleotides, and most preferred 12 nucleotides. As mentioned above, extensions to the hybridizing portions in the complex (5′ extension of tracrRNA, 5′-extensions to ncrRNA) are possible and preferred, and provide the advantage of a more stable formation the complex.
The non-naturally occurring tracrRNA as used in the present invention comprises a sequence that specifically hybridizes with a first portion of said sensed RNA, forming a stem-like secondary structure basically mimicking the original repeat:anti-repeat stem formed between a crRNA and a tracrRNA. Preferred is a method according to the present invention, wherein said portion of said at least one non-naturally occurring tracrRNA that specifically hybridizes with said first portion of said sensed RNA hybridizes with 10 or more nucleotides, preferably with 11 and more nucleotides, and more preferred with about 12 nucleotides or more.
Preferred is a method according to the present invention, wherein said first portion of said sensed RNA comprises a PAM (see
In a preferred embodiment of the methods according to the present invention, the nuclease can be modified in order to recognize a broader panel of PAM sites, e.g. by replacing the key region in the PAM interaction (PI) domain of Cas9 with the corresponding region in a panel of related 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 broadens the possible genomic targets in cells.
As mentioned above, the non-naturally occurring tracrRNA as used in the present invention comprises a sequence that specifically hybridizes with a first portion of said sensed RNA, forming a stem-like secondary structure basically mimicking the original repeat:anti-repeat stem. In a preferred embodiment of the methods 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, creating a dsRNA, and thus an RNase III recognition site or domain. This structure can be used in certain assays, e.g. when attaching detectable groups to the stem extension that could be cleaved off by the RNase activity. The inventors evaluated mismatches in the repeat-anti-repeat stem recognized and cleaved by RNase III. ncrRNA variants were expressed with a tracrRNA. Mutations were made in the ncrRNA to preserve the tracrRNA. Overall, even large mutations could be tolerated. Without wanting to be bound by theory, this suggests that RNase III might be dispensable for Cas9 activity.
It was furthermore analyzed by the inventors, how far ncrRNAs can deviate from traditional crRNAs, indicating the “robustness” of the system with respect to the stem-like secondary structure basically mimicking the original repeat:anti-repeat stem in the natural duplex (see above). A particular focus was put on the base stem bound by Cas9. Bulges were created by mutating the repeat, thereby preserving the tracrRNA. The results show that bulges can be tolerated to quite some degree, especially when the bulge is smaller, away from the base of the stem, or on the repeat side. A very similar result was found when accommodating some mismatches in the stem. See
The inventors further analyzed how far ncrRNAs can deviate from traditional crRNAs, indicating the “robustness” of the system with respect to the stem-like secondary structure basically mimicking the original repeat:anti-repeat stem in the natural duplex (see above). A second particular focus was put on the base stem bound by Cas12. Bulges were created by mutating the repeat, thereby preserving the tracrRNA. The results show that bulges can be tolerated to quite some degree, especially when the bulge is smaller, away from the base of the stem, or on the repeat side. A very similar result was found when accommodating some mismatches in the stem. See
Therefore, as shown with the two examples Cas 9 and Cas12, ncrRNAs can deviate from traditional crRNAs, indicating the “robustness” of the system with respect to the stem-like secondary structure basically mimicking the original repeat:anti-repeat stem in the natural duplex (see above).
Furthermore, the effect of reprogramming tracrRNA to convert different RNAs into crRNAs was tested. Changes to the repeat:anti-repeat duplex using sgRNAs and CjeCas9 in TXTL were evaluated, and it was found that all changes could be accommodated, with little impact on target cleavage (see
Similar rules apply to Cas12 and other enzymes as disclosed herein (see
It was furthermore shown that non-natural tracrRNAs converting a sensed RNA into an active crRNA function well with CjeCas9 (
The second portion of the sensed RNA specifically hybridizes with at least one target nucleic acid, defining the nucleic acid target of the Cas protein, creating the crRNA-like ncrRNA. The at least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme.
The non-naturally occurring tracrRNA can be produced in accordance with standard methods, e.g. synthetically produced, generated by in vitro transcription, and/or cloned into a plasmid. The tracrRNA can be derived from naturally occurring sequences that are then modified in order to generate sequences in the molecule as desired. The tracrRNA can further comprise additional modifications, such as labels or modified nucleotides, e.g. inosines, or the like.
In the second aspect thereof, the object of the present invention is solved by providing a method for detecting at least one sensed RNA in a cell and/or sample, said method comprising: a) contacting said sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with a first portion of said sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with at least one target nucleic acid; wherein said at least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of an ssRNA and/or dsRNA cleaving enzyme, such as RNase III enzyme; b) detecting cleavage of said at least one target nucleic acid in said sample by said nuclease enzyme; c) wherein detecting said cleavage of at least one target nucleic acid detects said at least one sensed RNA in said cell, tissue and/or sample.
While the basic principles of this second aspect regarding the components as used are as above, in the aspect of the present invention, the detection of said at least one sensed RNA in said cell, tissue and/or sample relies on the detection of a cleaving of said at least one target nucleic acid in said sample by said nuclease enzyme, and detecting said cleaving of said at least one target nucleic acid detects said at least one sensed RNA in said cell, tissue and/or sample.
The detection of the binding and/or cleavage product based on the target nucleic acid can be direct (e.g. detecting a size change of the cleaved nucleic acid) or indirect using suitable fluorophores with or without quenchers. Cleaving the nucleic acid may release a fluorophore that can be detected, passing the cleaved nucleic acid through a nanopore sequencer detects changes, a fluorescently labeled nuclease could be localized, and/or the cleaving affects electrical currents that can be detected. The detection of the cleaved product can also include a quantitative detection, i.e. the amount of fragment(s) as detected is used to determine the amount of sensed RNA in said cell, tissue and/or sample.
Detection of binding can be indirect based on modulating the expression of a reporter gene. An at least one catalytically-dead nuclease can directly repress transcription by binding the promoter or coding region of the reporter gene. Alternatively, the nuclease can recruit regulatory domains responsible for the local modulation of gene expression, such as domains that upregulate expression (e.g. VP16, VPR) or downregulate expression (e.g. KRAB). The domains can be operably linked to the at least one catalytically-dead nuclease. The domains can be recruited through interactions with binding domains fused to the at least one catalytically-dead nuclease and/or the tracrRNA.
In the third aspect thereof, the object of the present invention is solved by providing a method for detecting at least one sensed RNA in a cell, tissue and/or sample, said method comprising: a) contacting said sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with a first portion of said sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with at least one target nucleic acid; wherein said least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of an ssRNA and/or dsRNA cleaving enzyme, such as RNase III enzyme; and b) nicking or cleaving of said at least one target nucleic acid in said cell, tissue and/or sample by said nuclease, c) detectably editing said at least one target nucleic acid comprising, for example, non-homologous end joining (NHEJ) repair, microhomology-mediated end joining (MMEJ), homologous-directed repair (HDR), prime editing, base editing or RNA editing, and d) suitably detecting said editing of said at least one target nucleic acid comprising, for example, at least one method selected from detecting the presence and/or length of indels or gene edits, the activation/repression of a gene or other genetic element as encoded by said at least one target nucleic acid, and/or the detection of said prime or base editing; e) wherein detecting at least one target nucleic acid detects said at least one sensed RNA in said cell, tissue and/or sample.
While the basic principles of this third aspect regarding the components as used are as above, in this aspect of the present invention, the detection of said at least one sensed RNA in said cell, tissue and/or sample relies on the detection of a cleaving of said at least one target nucleic acid in said sample by said nuclease enzyme, detectably editing said cleaved at least one target nucleic acid, and furthermore suitably detecting said editing of said at least one target nucleic acid, wherein detecting said editing of said at least one target nucleic acid detects said at least one sensed RNA in said cell, tissue and/or sample. Therefore, this aspect relies on detecting changes to the target nucleic acid that are controlled or caused (“driven”) by the sensed RNA.
Said detectably editing said cleaved at least one target nucleic acid can preferably comprise, non-homologous end joining (NHEJ) repair, microhomology-mediated end joining (MMEJ), homologous-directed repair (HDR), and/or base editing. For example, cells or single cell zygotes commonly repair Cas9 cleaved DNA using the error-prone non-homologous end joining (NHEJ) repair pathway, which randomly inserts or deletes DNA bases to repair double-strand breaks. The most common indels from NHEJ repair range from 1 to 15, preferably 1 to 9 nucleotides. There are multiple repair pathways known to the person of skill that can be engaged depending on the cell type and conditions.
Detecting said editing of said at least one target nucleic acid can comprise, for example, at least one method selected from detecting the presence and/or length of indels or gene edits, the activation/repression of a gene or other genetic element as encoded by said at least one target nucleic acid, and/or the detection of base editing, e.g. through sequencing, restriction digest and or detection of length changes of the target nucleic acid. As above, detection of the cleavage product based on the target nucleic acid can be direct (e.g. detecting a size change of the cleaved nucleic acid) or indirect using suitable fluorophores. Cleaving the nucleic acid may release a fluorophore that can be detected, passing the cleaved nucleic acid through a nanopore sequencer detects changes, a fluorescently labeled nuclease could be localized, and/or the cleaving affects electrical currents that can be detected. The detection of the cleaved product can also include a quantitative detection, i.e. the amount of fragment(s) as detected is used to determine the amount of sensed RNA in said cell, tissue and/or sample.
Preferred is a method according to the present invention, wherein the length of said indels as detected is between about 1 to 15, and more preferable between about 1 to 9 nucleotides, and wherein optionally said indels can be detected through sequencing, restriction digest and or detection of length changes of the target nucleic acid, as well as qPCR. In one preferred aspect, this will allow an improvement of the efficiency of gene editing by so-called iterative self-targeting. The presence of indels in most instances allows a re-targeting of the sequence, hence leading to a re-attack. The approach can then lead to larger sized indels and/or a higher frequency of homologous recombination, since some indels don’t disrupt said re-attack. In practice, this approach advantageously allows for a follow-up of lineage tracing.
Base editing that can be detected represents a distinct means of editing. In general, there are two classes of base editors–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 like APOBEC. Base editors are targeted to a specific locus by the present system, and they can convert cytidine to uridine within a small editing window near the PAM site. Uridine is subsequently converted to thymidine through base excision repair, creating a C to T change (or a G to A on the opposite strand). Adenosine base editors have been engineered to convert adenosine to inosine, which is treated like guanosine by the cell, creating an A to G (or T to C) change. Adenine DNA deaminases do not exist in nature, but have been created by directed evolution of the Escherichia coli TadA, a tRNA adenine deaminase. Like cytosine base editors, the evolved TadA domain is fused to a Cas9 protein to create the adenine base editor.
Both types of base editors are available with multiple Cas9 variants including high fidelity Cas9′s (see below). Further advancements have been made by optimizing expression of the fusions, modifying the linker region between Cas variant and deaminase to adjust the editing window, or adding fusions that increase product purity such as the DNA glycosylase inhibitor (UGI) or the bacteriophage Mu-derived Gam protein (Mu-GAM). While many base editors are designed to work in a very narrow window proximal to the PAM sequence, some useful base editing systems create a wide spectrum of single-nucleotide variants (somatic hypermutation) in a wider editing window, and are thus well suited to directed evolution applications. Examples of these base editing systems include targeted AID-mediated mutagenesis (TAM) and CRISPR-X, in which Cas9 is fused to activation-induced cytidine deaminase (AID).
In a preferred embodiment of the methods according to the present invention, said method is performed in vivo, for example in a cell, tissue or in a bacterium, fungus, plant or animal, or in vitro, in a sample. The sample can be a solid or liquid sample, and can be selected from a sample comprising cells, and an acellular in vitro sample. The cells are preferably plant cells or animal cells, such as mammalian cells, preferably human cells.
The sample can be a biological sample, preferably obtained from a tissue sample, saliva, blood, plasma, sera, stool, urine, sputum, mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, or swab of skin or a mucosal membrane surface. In one aspect, the cells, tissues and/or samples can be crude samples and/or wherein the one or more nucleic acid molecules are not purified or amplified from the sample prior to application of the methods. In another aspect, the cells, tissues and/or samples can be purified or partially purified (enriched) samples and/or the one or more nucleic acid molecules are purified or amplified from the sample prior to application of the methods. In another aspect, the cells can be part of an environmental sample such as air, a natural body of water (e.g. river, lake, ocean), wastewater, or soil.
The methods according to the present invention can be partially or fully automated, e.g. performed fully or in part by robots. The methods according to the present invention can involve the use of computers and respective databases for performing and/or analysis of the results as obtained.
In one aspect, several or even a multitude of target nucleic acids are analyzed in one or more samples, tissues and/or cells, and preferably in a multitude of samples, tissues and/or cells. In one aspect, several or even a multitude of sensed RNAs are detected in one or more samples, tissues and/or cells, and preferably in several or even a multitude of samples, tissues and/or cells.
In a preferred embodiment of the methods according to the present invention, more than one non-naturally occurring tracrRNAs are selected, designed, generated (produced, see above) and used, each specifically hybridizing with a different portion of said sensed RNA in one or more samples, tissues and/or cells, and preferably in several or even a multitude of samples, tissues and/or cells.
The present system could, for example, target anywhere from 2 to 7 genetic loci by cloning multiple tracrRNAs into a single plasmid. These multiplex tracrRNA vectors can conceivably be suitably combined with any of the aforementioned CRISPR nucleases in order be used in the aspects of the invention.
In a preferred embodiment of the methods according to the present invention, said at least one sensed RNA is single or initially double stranded. In the context of the present invention, a sensed RNA is any RNA of interest to be detected using the methods according to the present invention. Usually and preferably, the sensed RNA is a single-stranded RNA molecule, such as an mRNA or non-coding RNAs. The RNA can be of natural origin or artificially produced. The single stranded sensed RNA can be from a human cell, an animal cell, a plant cell, an immune cell, a cancerous cell, an infected cell, or a diseased cell, and/or can be derived from a virus, a parasite, a helminth, a fungus, a protozoan, a bacterium, or a pathogenic bacterium.
In a preferred embodiment of the methods according to the present invention, said at least one sensed RNA is derived from a virus selected from Zika virus, human immunodeficiency virus (HIV), hepatitis B virus, hepatitis C virus, herpes virus, coronavirus, influenza, herpes simplex virus I, herpes simplex virus II, papillomavirus, rabies virus, cytomegalovirus, human serum parvo-like virus, respiratory syncytial virus, varicella-zoster virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, west Nile virus, and yellow fever virus.
In a preferred embodiment of the methods according to the present invention, said at least one sensed RNA is derived from a pathogenic bacterium selected from Mycobacteriumtuberculosis, Streptococcusagalactiae, methicillin-resistantStaphylococcusaureus, Legionellapneumophila, Streptococcuspyogenes, Escherichiacoli, Neisseriagonorrhoeae, Neisseriameningitidis, Pneumococcus, Cryptococcusneoformans, Treponemapallidum, Lymediseasespirochetes, Pseudomonasaeruginosa, Mycobacteriumleprae, and Brucellaabortus.
In a preferred embodiment of the methods according to the present invention, the at least one sensed RNA is derived from a gene the transcription and/or expression thereof is modified in response to external factors, such as, for example, metabolic factors or signals, hormones, pathogens, toxins, drugs, ageing and/or biotic or abiotic stress.
In a preferred embodiment of the methods according to the present invention, the sensed RNAs are selected to be environment, species, strain, disease, cell- and/or tissue specific. In this aspect, the methods of the invention help to identify and/or to group cells or organisms based on the sensed RNAs as selected. The at least one sensed RNA is preferably related to a condition selected from a viral infection, such as, for example, coronavirus infection, infection with a pathogen, a metabolic disease, cancer, neurodegenerative diseases, ageing, a drug, and biotic or abiotic stress.
In a preferred embodiment of the methods according to the present invention, the at least one sensed RNA can be added to said cell, tissue and/or sample prior to step a), and/or wherein said method further comprises at least one step selected from in vitro transcription of DNA into RNA, reverse transcription of RNA into DNA, and – optimally – subsequent in vitro transcription of said DNA into RNA. This can be done in order to provide suitable or desired signal amplification. Usually, the sensed RNA in said cell, tissue or sample is present in a range of from about 500 fM to about 1 uM, e.g. from about 500 fM to about 1 nM, preferably is present in a range of from about 1 pM to about 1 nM. Optimally, the method can detect a single molecule per cell, tissue and/or sample.
In another preferred embodiment of the methods according to the present invention, any tracrRNA-dependent CRISPR nuclease enzyme can be used, as long as it requires a tracrRNA for the binding function thereof as listed, for example, in Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants (Nat Rev Microbiol. 2020;18(2):67-83), the listing of which is incorporated herein by reference. Preferably, the nuclease is selected from type II Cas9 and type V Cas12 nuclease enzymes, as well as 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 nuclease selected from the group consisting of II-A, -B, and -C, and type Cas12 nuclease selected from the group consisting of type V-B, -C, -D, -E, -F, -G, and -K. Usually, suitable plasmid systems are used in order to introduce and express Cas nucleases. Type V-A, which was identified as the first known type V system in the human pathogenic bacterium Francisellatularensis in 2013, has the signature effector protein Cas12a (formerly designated Cpf1). Shmakov et al. identified type V-B and type V-C in 2015, Harrington et al. identified type V-F in 2018, and Yan et al. identified type V-G, type V-H, and type V-Iin 2019. By querying uncultivated bacteria in groundwater and sediment samples via genome-resolved metagenomics, Burstein and colleagues identified type V-D and type V-E in 2017 (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).
Deltcheva et al. (in: CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011; 471(7340):602-607. doi:10.1038/nature09886) report that differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24 nucleotide complementarity to the repeat regions of crRNA precursor transcripts. They show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous endoribonuclease RNase III and the CRISPR-associated Cas9 (Csn1) protein; all these components are found to be essential to protect S. pyogenes against prophage-derived DNA. The 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 sgRNAs are effectively relying on the tracrRNA. The cleaved RNA duplex is then bound by Cas9, and the crRNA portion directs the DNA targeting. Also, tracrRNAs have been identified in subtype V-B systems, although in this case, the cleavage enzyme remains uncharacterized (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).
In yet another preferred embodiment of the methods according to the present invention, the at least one tracrRNA-dependent CRISPR nuclease enzyme is modified, for example mutated, and/or a recombinant fusion protein. A given RNA targeting sequence will have additional sites throughout the genome where partial homology exists, or there could be multiple target nucleic acids, e.g. as part of an in vitro diagnostic, that resemble each other. These sites are called off-targets and need to be considered when designing an ncrRNA. In addition to optimizing RNA design, CRISPR specificity can also be increased through modifications to Cas9. If high specificity is crucial using the dual nickase approach to create a double nick-induced double strand break can be used, which can also be combined with HDR-mediated gene editing for specific gene edits. Another approach is the use of mutated Cas nucleases, called high fidelity enzymes. For example, phage-assisted continuous evolution (PACE) methods resulted in xCas9 3.7 which has 7 mutations found in the REC2, REC3, and PAM interacting domains and allows for expanded PAM recognition as well as increased specificity and lower off-target activity. Therefore, the tracrRNA-dependent CRISPR nuclease enzyme can be mutated in the PAM interacting domains in order to alter PAM-recognition. As another example, by screening E. coli cells transformed with a pooled library of SpCas9 variants, mutated variants can be identified, like the so-called Sniper-Cas, which has less off-target activity than wild type Cas9 (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)).
Cas9, for example, can be fused with transcriptional repressors or activators, and targeting these dCas9 fusion proteins to the promoter region results in robust transcriptional repression (CRISPR interference, or CRISPRi) or activation (CRISPRa) of downstream target genes. The simplest dCas9-based activators and repressors consist of dCas9 fused directly to a single transcriptional activator (e.g. VP64) or repressor (e.g. KRAB). Additionally, activation strategies have been developed for more potent activation of target genes in mammalian cell, such as co-expression of epitope-tagged dCas9 and antibody-activator effector proteins (e.g. SunTag® system); dCas9 fused to several different activation domains in series (e.g. dCas9-VPR; or co-expression of dCas9-VP64 with a modified scaffold sgRNA and additional RNA-binding helper activators (e.g. SAM activators). Importantly, unlike the genome modifications induced by Cas9 or Cas9 nickase, dCas9-mediated gene activation or repression is reversible, since it does not permanently modify the genomic DNA. dCas9 has also been used for genome-wide screens to activate or repress gene expression in mice and human cells. Synthetic CRISPR-Cas gene activators have been developed for bacteria by using a scaffold RNA that contains the gRNA and an RNA hairpin to recruit activation proteins. CRISPRi can also been adapted for 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.
In another aspect of the methods according to the present invention, said first portion of said sensed RNA comprises a protospacer adjacent motif (PAM). As an example, for Cas12 nucleases, the PAM is present within the repeat portion upstream of the guide portion of the resulting ncrRNA, whereas for Cas9, the PAM is present in the repeat portion downstream of the guide portion of the ncrRNA. By selecting targets with the appropriate PAM, the generated crRNA can target its own site. This allows editing only if the gene is transcribed. This strategy greatly limits editing, for example to specific tissues, depending on the expression profile of the gene to be targeted.
As mentioned above, in a preferred embodiment of the methods according to the present invention, the sensed RNA and the non-naturally occurring tracrRNA create a dsRNA, and thus a dsRNA cleaving enzyme, such as RNase III enzyme recognition site or domain. An ssRNA cleaving enzyme, such as RNase A, can also be applied to promote further processing or clear RNAs not converted into ncrRNAs. Preferably, said dsRNA cleaving enzyme substrate in said sensed RNA has a length of 10 or more nucleotides, such as up to about 50 nucleotides or even more, preferably of 11 and more nucleotides, and more preferred of about 12 nucleotides or more. Preferred ranges are between 9 and 15 nucleotides, more preferred 10 to 14 nucleotides, and most preferred 12 nucleotides.
In another aspect, the portions of the nucleic acid molecules as used in the methods of the present invention that hybridize are complementary to at least 80%, preferably complementary to more than 90%, more preferably to more than 95%, and most preferably are 100% complementary to each other. As discussed above, it was surprisingly found that ncrRNAs can deviate from traditional crRNAs, indicating the “robustness” of the system with respect to the stem-like secondary structure basically mimicking the original repeat:anti-repeat stem, and a particular focus was put on the base stem bound by Cas9 and Cas12 (see
In another aspect of the methods according to the present invention, said methods, at least in part, comprise a quantitative analysis. Preferred is therefore a step comprising detecting the amount of the at least one target nucleic acid as modified, e.g. the products cleaved and/or edited and/or said at least one sensed RNA, as well as the bound target nucleic acid in said sample, tissue and/or cell. Preferably the amount per sample, tissue and/or cell is determined, when compared to a control. Quantification assays are known to the person of skill, and may include absorbance (e.g. UV, spectrophotometry) and/or fluorescence assays, and real-time PCR. The assays can quantify the amount(s) and/or ratios of the nucleic acid(s) as quantified (modified at least one target nucleic acid and/or said at least one sensed RNA) as a single value (e.g. as the result or at the “end” of the assay as used) or can monitor changes over time in the nucleic acids, i.e. preferably further comprising detecting a change in the amount of said modification of said at least one target nucleic acid (e.g. the products cleaved and/or edited) and/or said at least one sensed RNA in said sample, tissue and/or cell, in particular when compared to a control.
In yet another aspect of the methods according to the present invention, multiple labels and/or markers are used. Markers can be used both for the nucleic acid molecules that form part of the assays, as well as the protein components (e.g. nuclease and/or fusions). Labels and markers can be included into the components of the assays (in particular the nucleic acids and/or the proteins), as well as constitute moieties that are attached, either covalently or non-covalently.
Another aspect of the present invention then relates to a method for detecting a medical condition in a cell, tissue or organism, such as a mammal, preferably a human, wherein said condition is related to the presence of, expression of and/or mutation(s) in at least one sensed RNA. The method comprises performing the method according to the invention as above, and detecting said medical condition based on the presence of, expression of and/or mutation(s) in said at least one sensed RNA as detected.
The medical conditions that can be detected using the present invention are those that are related to the at least one sensed RNA molecule. As explained above, the sensed RNA can either itself constitute the origin of the condition or disease, for example in case of infections, e.g. viral, such as, for example, coronavirus infection, bacterial and/or fungal infections of the cells, tissues and/or samples as tested. Other conditions may relate more indirectly to the at least one sensed RNA molecule, for example in case of an RNA that is aberrantly transcribed (present or found), expressed, processed (e.g. splicing) and/or mutated. The sensed RNA molecule can be present in an increased or decreased amount when compared to a healthy control (e.g. a control based on a group of healthy or diseased samples).
In a preferred embodiment of the methods according to the present invention, said at least one sensed RNA is single or initially double stranded. The single stranded sensed RNA can be/relates to from a human cell, an animal cell, a plant cell, an immune cell, a cancerous cell, an infected cell, or a diseased cell, and/or can be derived from a virus (see above), a parasite, a helminth, a fungus, a protozoan, a bacterium, or a pathogenic bacterium (see above). In a preferred embodiment of the methods according to the present invention, the at least one sensed RNA is derived from/related to a gene the transcription and/or expression thereof is modified in response to external factors, such as, for example, metabolic factors or signals, hormones, pathogens, toxins, drugs, ageing and/or biotic or abiotic stress. Consequently, the at least one sensed RNA is preferably related to a condition selected from a viral infection, such as, for example, coronavirus infection, infection with a pathogen, a metabolic disease, cancer, neurodegenerative diseases, ageing, a drug, and biotic or abiotic stress). Usually, the presence of or the increased or decreased amount of the sensed RNA is indicative for the presence of said disease or condition. Another aspect of the method relates to a monitoring of the amount or presence of said sensed RNA during a treatment of said individual, patient or organism, in particular the individual, patient or organism from which the cell, tissue and/or sample has been obtained.
In yet another aspect of the present invention, the methods of the present invention comprise a step wherein said one or more sensed RNAs are detected as (part of) a set of RNAs constituting a gene signature (i.e. a set of sensed RNAs that is specific for an organism, a cell and/or tissue). The method thus detects one or more gene signatures, wherein each gene signature identifies an organism, a cell and/or tissue, such as a plant, animal or microbial species, one or more plant, animal or microbial phenotypes, or both. In turn, the methods of the present invention can be used to identify a set of RNAs constituting a gene signature, e.g. an expression profile for one or more sensed RNAs, wherein said signature is specific for an organism, a cell and/or tissue, such as a plant, animal or microbial species, one or more plant, animal or microbial phenotypes, or both (see also above for transcriptional recording).
Examples for preferred in vitro diagnostic formats for methods of the present invention are microarrays, in particular with double-stranded DNA to monitor many DNAs at once, and lateral flow assays. Lateral flow assays are known to the person of skill, and operate on the same principles as enzyme-linked immunosorbent assays (ELISA). In essence, these tests run the liquid sample along the surface of a pad with reactive molecules that show a visual positive or negative result.
As mentioned above, in a preferred embodiment of the methods according to the present invention, the at least one sensed RNA can be added to said cell, tissue and/or sample prior to step a), and/or wherein said method further comprises at least one step selected from in vitro transcription of DNA into RNA, reverse transcription of RNA into DNA, and – optimally – subsequent in vitro transcription of said DNA into RNA. This can be done in order to provide suitable or desired signal amplification. Usually, the sensed RNA in said cell, tissue or sample is present in a range of from about 500 fM to about 1 uM, e.g. from about 500 fM to about 1 nM, preferably is present in a range of from about 1 pM to about 1 nM. Optimally, the method can detect a single molecule per cell, tissue and/or sample.
Yet another aspect of the present invention then relates to a method for treating a disease or medical condition in a cell, tissue or organism, such as a mammal, preferably a human, wherein said condition is related to the presence of, expression of and/or mutation(s) in at least one sensed RNA. The method comprises providing a suitable treatment to said cell, tissue or organism, in particular a specific medical treatment, performing the method according to the invention as above, and modifying said treatment of said disease or medical condition based on the presence of, expression of and/or mutation(s) in said at least one sensed RNA as detected. The medical conditions that can be detected using the present invention are those that are related to the at least one sensed RNA molecule. As explained above, the sensed RNA can either itself constitute the origin of the condition or disease, for example in case of infections, e.g. viral, such as, for example, coronavirus infection, bacterial and/or fungal infections of the cells, tissues and/or samples as tested. Other conditions may relate more indirectly to the at least one sensed RNA molecule, for example in case of an RNA that is aberrantly transcribed (present or found), expressed, processed and/or mutated. The sensed RNA molecule can be present in an increased or decreased amount when compared to a healthy control (e.g. a control based on a group of healthy or diseased samples).
In a preferred embodiment of the methods according to the present invention, said at least one sensed RNA is single or initially double stranded. The single stranded sensed RNA can be/relates to from a human cell, an animal cell, a plant cell, an immune cell, a cancerous cell, an infected cell, or a diseased cell, and/or can be derived from a virus (see above), a parasite, a helminth, a fungus, a protozoan, a bacterium, or a pathogenic bacterium (see above). In a preferred embodiment of the methods according to the present invention, the at least one sensed RNA is derived from/related to a gene the transcription and/or expression thereof is modified in response to external factors, such as, for example, metabolic factors or signals, hormones, pathogens, toxins, drugs, ageing and/or biotic or abiotic stress. Consequently, the at least one sensed RNA is preferably related to a condition selected from a viral infection, such as, for example, coronavirus infection, infection with a pathogen, a metabolic disease, cancer, neurodegenerative diseases, ageing, a drug, and biotic or abiotic stress). Usually, the presence of or the increased or decreased amount of the sensed RNA is indicative for the presence of said disease or condition. Another aspect of the method relates to a monitoring of the amount or presence of said sensed RNA during a treatment of said individual, patient or organism, in particular the individual, patient or organism from which the cell, tissue and/or sample has been obtained.
For medical treatments, reprogrammed tracrRNAs would provide a quantitative readout of specific RNAs of interest in a patient sample that would directly inform the course of treatment. For instance, reprogrammed tracrRNAs can be used to detect viral sequences in a blood draw or sputum to determine the specific virus infecting the patient such as, for example, coronavirus infection. The identified virus would directly inform the course of treatment. For instance, identifying rhinovirus would result in a more modest treatment plan involving fluids and rest, whereas identifying coronavirus would require close monitoring and containment, while identifying ebola would require immediate medical intervention. Similarly, reprogrammed tracrRNAs can be used to detect specific RNAs in a cancer biopsy to identify key markers of cancer progression and drug susceptibilities. This information would in turn inform the doctor how to proceed with the course of treatment. Finally, reprogrammed tracrRNAs could be used with a sample containing an infecting bacterial or fungal pathogen (e.g. Clostridioides difficile in a stool sample, Pseudomonas aeruginosa in a sputum sample) to identify specific markers of antibiotic resistance to determine the best regime of antibiotics to administer to a patient.
An embodiment of the above described method for treating a disease or medical condition in a cell, tissue or organism, such as a mammal, preferably a human, wherein said condition is related to the presence of, expression of and/or mutation(s) in at least one sensed RNA, in particular a viral RNA or DNA, reprogrammed (e.g. specific for a virus target nucleic acid) tracrRNAs according to the invention are be used to detect viral sequences in a blood draw or sputum to determine the specific virus, such as, for example, coronavirus, infecting the patient. The attending physician then treats the viral infection as detected with antiviral chemotherapeutics and/or biologics. In the course of the treatment, the reprogrammed tracrRNAs provides a quantitative readout of specific RNAs of interest in the patient sample that would directly inform about the course and effect/success of the treatment. The attending physician will then adjust the treatment accordingly, i.e. provide more with antiviral chemotherapeutics and/or biologics, if required. This treatment schedule can be repeated, if required.
Also, identifying a virus like rhinovirus can result in a more modest treatment plan involving fluids and rest, whereas identifying coronavirus would require close monitoring and containment, while identifying ebola would require immediate medical intervention.
Similarly, similarly as above, reprogrammed tracrRNAs (e.g. specific for a cancer target nucleic acid) can be used to detect specific RNAs in a cancer biopsy to identify key markers of cancer progression and drug susceptibilities. This information would in turn inform the doctor how to proceed with the course of treatment.
Finally, again similarly as above, reprogrammed tracrRNAs (e.g. specific for a bacterial or fungal target nucleic acid) could be used with a sample containing an infecting bacterial or fungal pathogen (e.g. Clostridioides difficile in a stool sample, Pseudomonas aeruginosa in a sputum sample) to identify specific markers of antibiotic resistance to determine the best regime of antibiotics to administer to a patient.
In another preferred aspect thereof, the object of the present invention is solved by providing a non-naturally occurring tracrRNA nucleic acid molecule, comprising an anti-repeat region sequence that is designed to specifically hybridize to a preselected sensed RNA sequence through forming a complex that mimics the natural crRNA:tracrRNA duplex. The non-naturally occurring tracrRNA nucleic acid molecule may be part of a complex comprising the non-naturally occurring tracrRNA nucleic acid molecule according to the present invention, at least one tracrRNA-dependent CRISPR nuclease enzyme, and at least one sensed RNA., and may be further bound to a target DNA nucleic acid molecule comprising a sequence based or designed based on the at least one sensed RNA sequence.
Thus, the non-naturally occurring tracrRNA nucleic acid molecule may be part of a complex comprising the non-naturally occurring tracrRNA nucleic acid molecule according to the present invention, at least one tracrRNA-dependent CRISPR nuclease enzyme, and at least one sensed RNA, and may be further bound to a target DNA nucleic acid molecule comprising a sequence based or designed based on the at least one sensed RNA sequence.
In the context of the present invention, the non-naturally occurring tracrRNA nucleic acid molecule comprises an anti-repeat region sequence that is designed to specifically hybridize to a preselected sensed RNA sequence through forming a complex that mimics the natural crRNA:tracrRNA duplex. This shall mean that the non-naturally occurring tracrRNA nucleic acid molecule and the preselected sensed RNA sequence molecule form a duplex that structurally resembles the consensus structure of crRNA:tracrRNA duplexes as disclosed in the literature. For example, the crRNA:tracrRNA duplex may form a lower stem, bulge, and upper stem modules for Cas9, lower stem and upper stem modules for Cas12b, and a stem and triple helix for Cas12e. The crRNA contains the spacer module, and the tracrRNA contains the nexus and terminal hairpins (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).
In another preferred aspect thereof, the object of the present invention is solved by providing a detection system for a sensed RNA comprising a) a non-naturally occurring tracrRNA designed to bind to at least one portion of said sensed RNA, said sensed RNA further comprising a portion that specifically hybridizes with a first portion of a target nucleic acid, and b) at least one tracrRNA-dependent CRISPR nuclease enzyme. Preferred is a detection system for the parallel detection of several sensed RNAs in parallel, comprising a set of several non-naturally occurring tracrRNAs for said several sensed RNAs. Another detection system comprises several non-naturally occurring tracrRNAs that hybridize at several positions on one sensed RNA.
This aspect of the present invention provides the components, e.g. non-naturally occurring tracrRNA nucleic acid molecule according to the invention as above, for performing the methods according to the invention as a detection system, for example as a part of a diagnostic kit. Preferably, said detection system is provided in one or more containers, and comprises suitable enzymes, buffers, and excipients, as well as instructions for use. The components can be – at least in part – immobilized on a substrate, wherein said substrate can be exposed to said cell, tissue and/or sample. The detection system can be applied to multiple discrete locations on said substrate, such as a flexible materials substrate, for example a chip. The flexible materials substrate can be a paper substrate, a fabric substrate, or a flexible polymer-based substrate.
Examples for preferred in vitro diagnostic formats for methods of the present invention are microarrays, in particular with double-stranded DNA to monitor many DNAs at once, and lateral flow assays. Lateral flow assays are known to the person of skill, and operate on the same principles as enzyme-linked immunosorbent assays (ELISA). In essence, these tests run the liquid sample along the surface of a pad with reactive molecules that show a visual positive or negative result.
In another preferred aspect thereof, the object of the present invention is solved by providing the use of the non-naturally occurring tracrRNA nucleic acid molecule or the sensed RNA detection system according to the aspect as above for performing a method according to any one of the aspects as above, in particular for detecting a sensed RNA, a viral sensed RNA, a sensed RNA transcribed from a disease marker, and/or for generating an expression profile for one or more sensed RNAs as mentioned above.
The examples described herein can be used for wide-ranging applications, such as diagnosing medical conditions to inform the course of treatment, identifying SNPs associated with health outcomes or disease, such as in acute sepsis, determining the identity of pathogen, virulence factors, resistance markers, SNPs, viral detection, i.e. the identity, and/or viral variants (see, for example, SARS CoV-2 as disclosed herein), cancer diagnostics, in cancer samples, such as biopsies, determining mutations, and/or SNPs, identifying microbial contaminants in potable water, identifying viral or microbial contaminants in fermentations or cell cultures, identifying plant or insect variants, or identifying key microbial members in mixed communities (e.g. in the gut, soil, water), such as analysis of microbiomes and/or microbial sentinels (i.e. commensal bacteria that serve as reporters for non-invasive measurements), in particular the identity, relative abundance, resistance markers, metabolic genes, phylum/genus/species/strain-specific genes, and the like, and the tracking of viral or bacterial spread in vivo, such as in whole organisms, or based on samples, for example taken from the environment (e.g. spread of viruses or resistant bacteria detected in waste water samples, etc.).
In the context of the present invention, unless explicitly mentioned otherwise, the term “about” shall mean a value as given +/- 10%.
Starting from the characterization of a native CRISPR-Cas9 system in the bacterial pathogen C. jejuni, the inventors found that cellular transcripts can be converted into non-canonical crRNAs through hybridization with the tracrRNA. This discovery adds ncrRNAs to the list of RNA guides found in nature, including crRNAs, small CRISPR-Cas associated RNAs (scaRNAs) that similarly pair with the tracrRNA anti-repeat, and “natural” sgRNAs formed through upstream transcription of the tracrRNA. Importantly, these prior examples are all encoded within CRISPR-Cas loci.
The inventors further demonstrated that non-natural tracrRNAs can link the presence of an RNA-of-interest to sequence-specific DNA targeting by Cas9. The RNA-of-interest otherwise had no association with the Cas9 or the CRISPR-Cas system. Thereby, non-natural tracrRNAs enable in vivo applications with Cas9 such as multiplexed transcriptional recording or transcription-dependent editing. The most immediate application involves multiplexed RNA detection in vitro through LEOPARD, as disclosed herein. In a preferred embodiment, LEOPARD adds to the existing CRISPR diagnostic platforms based on Cas12a or Cas13 while offering scalable multiplexing in a single reaction and the first diagnostic platform involving Cas9.
The inventors’ reliance on gel electrophoresis provided a proof-of-principle demonstration of multiplexed detection. More advanced implementations are also possible though, such as existing technologies based on microarrays or next-generation sequencing chips associate DNA sequences with specific spatial locations, potentially scaling up to millions of targets monitored in parallel. Either approach will enhance assay sensitivity due to the limited number of DNA molecules in a given cluster, with the detection of sensed RNAs well below the 40 nM used in the exemplary gel-based assays. DNA targets linked to fluorophores with distinct spectra and cleavable quenchers can also be employed as sequence-specific and scalable readouts.
Incorporating isothermal pre-amplification similar to existing CRISPR diagnostics will also offer a means to enhance sensitivity. With further development, LEOPARD will become a powerful diagnostic tool not only for viral detection but also for other applications requiring multiplexed readouts, such as screening for cancer mutations, identifying pathogens and antibiotic-resistance markers, or determining gene expression profiles for drug susceptibility, as also discussed herein.
Herein, non-natural tracrRNAs were exemplary established with Cas9, and Cas12 nucleases that also rely on tracrRNAs (see also
While systematically perturbing the standard crRNA:tracrRNA duplex, the inventors found that many deviations -particularly outside of the 5′ end of the repeat- were tolerated by Cas9 and Cas12 and still led to targeting of designed DNA targets. However, despite this promiscuity, targeting is still determined by the upstream guide sequence and the requirement for a flanking PAM. Thus, both anti-repeat hybridization and guide-dependent DNA targeting limit the potential of off-targeting. Correspondingly, the inventors did not observe any detectable off-targeting when assessing multiple viral RNAs or introducing yeast total RNA with LEOPARD (
As stated herein, the present invention particularly relates to the following items.
Item 1. A non-naturally occurring tracrRNA nucleic acid molecule, comprising an anti-repeat region sequence that is designed to specifically hybridize to a preselected sensed RNA sequence through forming a complex that mimics the natural crRNA:tracrRNA duplex.
Item 2. A complex comprising the non-naturally occurring tracrRNA nucleic acid molecule according to Item 1, at least one tracrRNA-dependent CRISPR nuclease enzyme, and at least one sensed RNA.
Item 3. The non-naturally occurring tracrRNA nucleic acid molecule according to Item 2, further bound to a target DNA nucleic acid molecule comprising a sequence based or designed based on the at least one sensed RNA sequence.
Item 4. A method for detecting at least one sensed RNA in a cell, tissue, and/or sample, said method comprising: a) contacting said sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with a first portion of said sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with at least one target nucleic acid, in particular DNA, RNA, or ssDNA; wherein said at least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed 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 binding of said at least one tracrRNA-dependent CRISPR nuclease enzyme to said at least one nucleic acid DNA; c) wherein binding detects said at least one sensed RNA in said cell, tissue and/or sample.
Item 5. The method according to Item 4, wherein said nuclease is inactivated in its cleaving and/or nicking activity.
Item 6. A method for detecting at least one sensed RNA in a cell and/or sample, said method comprising: a) contacting said sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with a first portion of said sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with at least one target nucleic acid; wherein said at least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed 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 said at least one target nucleic acid in said sample by said nuclease enzyme; c) wherein detecting said cleavage of at least one target nucleic acid detects said at least one sensed RNA in said cell, tissue and/or sample.
Item 7. The method according to any one of Items 4 to 6, wherein detecting said 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, a fluorophore, or electrical conductivity, and/or detecting the said cleaved at least one target nucleic acid fragment itself.
Item 8. A method for detecting at least one sensed RNA in a cell, tissue and/or sample, said method comprising: a) contacting said sample with at least one non-naturally occurring tracrRNA comprising a portion that specifically hybridizes with a first portion of said sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with at least one target nucleic acid; wherein said least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed 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; and b) nicking or cleaving of said at least one target nucleic acid in said sample by said nuclease, c) detectably editing said at least one target nucleic acid comprising, for example, non-homologous end joining (NHEJ) repair, microhomology-mediated end joining (MMEJ), homologous-directed repair (HDR), base editing, or prime editing and d) suitably detecting said editing of said at least one target nucleic acid comprising, for example, at least one method selected from detecting the presence and/or length of indels or gene edits, the activation/repression of a gene or other genetic element as encoded by said at least one target nucleic acid, and/or the detection of said prime or base editing; e) wherein detecting said editing of said at least one target nucleic acid detects said at least one sensed RNA in said cell, tissue and/or sample.
Item 9. The method according to any one of Items 4 to 8, wherein said method is performed in vivo or in vitro.
Item 10. The method according to any one of Items 4 to 9, wherein said sample is selected from a sample comprising cells, and an acellular in vitro sample, wherein said cells are preferably plant cells or animal cells, such as mammalian cells, preferably human cells.
Item 11. The method according to any one of Items 4 to 10, wherein several or a multitude of sensed RNAs are detected in one or more samples, tissues and/or cells.
Item 12. The method according to any one of Items 4 to 11, wherein said at least one sensed RNA is single stranded.
Item 13. The method according to any one of Items 4 to 12, wherein said at least one sensed RNA is environment, species, strain, disease, cell- and/or tissue specific or related to a condition selected from a viral infection, such as, for example, coronavirus infection, infection with a pathogen, a metabolic disease, cancer, neurodegenerative diseases, ageing, a drug, and biotic or abiotic stress.
Item 14. The method according to any one of Items 4 to 13, wherein said at least one sensed RNA is added to said cell, tissue and/or sample prior to step a), and/or further comprising a step of transcribing DNA into sensed RNA prior to step a).
Item 15. The method according to any one of Items 4 to 14, wherein said at least one tracrRNA-dependent CRISPR nuclease enzyme is selected from type II Cas9 and type V Cas12 nuclease enzymes, such as type II Cas9 nuclease selected from the group consisting of II-A, -B, and -C, and type Cas12 nuclease selected from the group consisting of type V-B, -C, -D, -E, -F, -G, and -K.
Item 16. The method according to any one of Items 4 to 15, wherein said at least one tracrRNA-dependent CRISPR nuclease enzyme is a recombinant fusion protein.
Item 17. The method according to any one of Items 4 to 16, wherein said portion of said at least one non-naturally occurring tracrRNA that specifically hybridizes with said first portion of said sensed RNA hybridizes with 10 or more nucleotides, preferably with 11 and more nucleotides, and more preferred with about 12 nucleotides or more.
Item 18. The method according to any one of Items 4 to 17, wherein said first portion of said sensed RNA comprises a protospacer adjacent motif (PAM).
Item 19. The method according to any one of Items 4 to 18, wherein said non-naturally occurring tracrRNA furthermore comprises at least one portion hybridizing with said sensed RNA providing a dsRNA cleaving enzyme substrate.
Item 20. The method according to Item 19, wherein said substrate of a dsRNA-cleaving enzyme in said sensed RNA has a length of 10 or more nucleotides, preferably of 11 and more nucleotides, and more preferred of about 12 nucleotides or more.
Item 21. The method according to any one of Items 4 to 20, wherein said portions that hybridize are complementary to at least 80%, preferably complementary to more than 90%, more preferably to more than 95%, and most preferably are 100% complementary.
Item 22. The method according to any one of Items 4 to 21, wherein the nucleotide sequence of said at least one portion of said tracrRNA that specifically hybridizes with a first portion of said sensed RNA is produced and/or modified in order to be complementary to at least 80%, preferably complementary to more than 90%, more preferably to more than 95%, and most preferably are 100% complementary to said first portion to said sensed RNA.
Item 23. The method according to any one of Items 4 to 22, wherein more than one non-naturally occurring tracrRNA are generated and used that each specifically hybridize with a different portion of said sensed RNA.
Item 24. The method according to any one of Items 4 to 23, further comprising detecting the amount of said at least one target nucleic acid and/or said at least one sensed mRNA in said sample, tissue and/or cell, preferably the amount per sample, tissue and/or cell.
Item 25. The method according to Item 24, further comprising detecting a change in the amount of said at least one target nucleic acid and/or said at least one sensed mRNA in said sample, tissue and/or cell when compared to a control.
Item 26. The method according to any one of Items 8 to 25, wherein the length of said indels as detected is between 1 to 14, and more preferable between 5 to 9 nucleotides, and wherein optionally said indels can be detected through sequencing and/or qPCR.
Item 27. The method according to any one of Items 4 to 26, wherein multiple labels and/or markers are used.
Item 28. A method for detecting a medical condition in a mammal, wherein said condition is related to the presence of, expression of and/or mutation(s) in at least one sensed RNA, comprising performing the method according to any one of Items 4 to 27, and detecting said medical condition based on the presence of, expression of and/or mutation(s) in said at least one sensed RNA as detected.
Item 29. The method according to Item 25, wherein said at least one sensed RNA is environment, species, strain, disease, cell- and/or tissue specific or related to a condition selected from a viral infection, such as, for example, coronavirus infection, infection with a pathogen, a metabolic disease, cancer, neurodegenerative diseases, ageing, a drug, and biotic or abiotic stress.
Item 27. The method according to Item 25 or 26, wherein said at least one sensed RNA is added to said cell, tissue and/or sample prior to step a), and/or further comprising a step of transcribing DNA into sensed RNA prior to step a).
Item 28. A sensed RNA detection system comprising a) a non-naturally occurring tracrRNA designed to bind to at least one portion of said sensed RNA, said sensed RNA further comprising a portion that specifically hybridizes with a first portion of a target nucleic acid, and b) at least one tracrRNA-dependent CRISPR nuclease enzyme.
Item 29. The sensed RNA detection system according to Item 28, further comprising c) at least one target nucleic acid molecule comprising a label for detecting cleavage of said target nucleic acid.
Item 30. The sensed RNA detection system according to Item 28 or 29, further comprising d) at least one dsRNA cleaving enzyme, such as RNase III enzyme.
Item 31. The sensed RNA detection system according to any one of Items 28 to 30, wherein at least one of said components a) to d) is encoded on a nucleic acid vector system.
Item 32. Use of the sensed RNA detection system according to any one of Items 28 to 31 for performing a method according to any one of Items 1 to 26, in particular for detecting a sensed RNA, a viral sensed RNA, a sensed RNA transcribed from a disease marker, or for generating an expression profile for one or more sensed RNAs.
The present invention will now be further described in the following examples with reference to the accompanying Figures, nevertheless, without wanting to be limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. The present disclosure includes as sequence listing comprising SEQ ID Nos: 1 to 128 as part of the description, which is also incorporated by reference in its entirety.
In the figures, the non-naturally occurring tracrRNA according to the present invention is sometimes referred to as “rptr”.
C.
jejuni mutant strains (deletion, chromosomal 3xFLAG-tagging) were constructed using double-crossover homologous recombination (High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacterjejuni isolates. PLoS Genet. 2013;9(5):e1003495). Briefly, PCR products with 500 bp homologous ends or genomic DNA with desired mutation were introduced into C.jejuni using electroporation or natural transformation, respectively, as described previously (High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacterjejuni isolates. PLoS Genet. 2013;9(5):e1003495). C.jejuni strains were routinely grown on Müller-Hinton agar plates or with shaking in Brucella broth (BB), both supplemented with 10 µg/ml vancomycin, at 37° C. under microaerobic (10% CO2, 5% O2) conditions.
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 (CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacterjejuni Cas9. Mol Cell. 2018;69(5):893-905.e7).
Briefly, Co-IP of chromosomally tagged Cas9-3xFLAG was performed using anti-FLAG antibody and Protein A-Sepharose beads from the lysates of C.jejuni CG8421 cas9-3 x FLAG and WT (untagged, control) strains grown to mid-exponential phase (OD600 = 0.6) at 37° C. Cells were harvested by centrifugation, and pellets were resuspended in 1 mL Buffer A (20 mM Tris-HCl, pH 8.0, 150 mM KCl, 1 mM MgCl2, 1 mM DTT), and subsequently centrifuged (3 min, 11,000 x g, 4° C.). The pellets were shock-frozen in liquid nitrogen and stored at -80° C. Frozen pellets were thawed on ice and resuspended in 0.8 mL Buffer A. An equal volume of glass beads was then added to the cell suspension. Cells were lysed using a Retsch MM40 ball mill (30 s-1, 10 min) in pre-cooled blocks (4° C.), and centrifuged for 2 min at 15,200 x g, 4° C. The supernatant was transferred to a new tube, and an additional 0.4 mL of Buffer A was added to the remaining unlysed cells with beads. Lysis of the remaining cells was achieved by a second round of lysis at 30 s-1 for 5 min. Centrifugation was repeated and this second supernatant was combined with the first one. The combined supernatant was centrifuged again for 30 min at 15,200 x g, 4° C. for clarification and the resulting supernatant (lysate fraction) was transferred to a new tube. The lysate was incubated with 35 µL anti-FLAG antibody (Monoclonal ANTI-FLAG M2, Sigma, #F 1804, RRID: AB_262044) for 30 min at 4° C. on a rocker. Next, 75 µL of Protein A-Sepharose (Sigma, #P6649), prewashed with Buffer A, was added and the mixture was rocked for another 30 min at 4° C. After centrifugation, the supernatant was removed and the pelleted beads were washed 5 times with 0.5 mL Buffer A. Finally, 500 µL Buffer A was added to the beads and RNA and proteins were separated using phenol-chloroform-isoamyl alcohol. From each CoIP, ~1000 ng of RNA was recovered from the aqueous phase. 100 µL of 1 x protein loading buffer was added to the final protein sample precipitated along with beads (CoIP sample). For verification of a successful CoIP, protein samples obtained during different stages of the coIP (culture, lysate, supernatant, wash, and CoIP) were analysed using western blot.
Residual gDNA was removed using DNase I treatment on the coIP RNA obtained above. cDNA libraries for Illumina sequencing were constructed by vertis Biotechnologie AG, Germany (http://www.vertis-biotech.com) in a strand-specific manner as described previously (High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacterjejuni isolates. PLoS Genet. 2013;9(5):e1003495). In brief, the RNA samples were poly(A)-tailed using poly(A) polymerase. Then, 5′-triphosphates were removed using tobacco acid pyrophosphatase (TAP), and an RNA adaptor was then ligated to the resulting 5′-monophosphate. First-strand cDNA was synthesized with an oligo(dT)-adaptor primer using M-MLV reverse transcriptase. In a PCR-based amplification step, using a high-fidelity DNA polymerase, the cDNA concentration was increased to 10-20 ng/µL. For all libraries, the Agencourt AMPure XP kit (Beckman Coulter Genomics) was used to purify the DNA, which was subsequently analyzed by capillary electrophoresis.
A library-specific barcode for multiplex sequencing was included as part of a 3′-sequencing adaptor. The cDNA inserts were flanked by TruSeq_Sense_primer (SEQ ID NO: 1) and TruSeq_Antisense_NNNNNN_primer (NNNNNN = 6 nt barcode for multiplexing) (SEQ ID NO: 2). The samples were run on an Illumina HiSeq instrument with ~100 cycles in single-read mode.
To assure high sequence quality, the Illumina reads in FASTQ were trimmed with a cut-off phred score of 20 by the program fastq_quality_trimmer from FASTX toolkit version 0.0.13. After trimming, we applied the pipeline READemption version 0.4.3 for the following analysis steps: poly(A)-tail sequences were clipped from the 3′ ends of reads, size filtering was applied to eliminate read sequences shorter than 12 nt and the collections of remaining reads were mapped to the self-curated C. jejuni CG8421 genome using segemehl version 0.2.0 with an accuracy cut-off of 95%.
READemption was used to generate coverage plots representing the numbers of mapped reads per nucleotide. Reads that mapped to multiple locations contributed a fraction to the coverage value. Each read with a minimum overlap of 10 nt was counted with a value based on the number of locations where the read was mapped. If the read overlapped more than one annotation, the value was divided by the number of regions and counted separately for each region (e.g., 1/3 for a read mapped to 3 locations).
For visualization in the Integrated Genome Browser (IGB), the coverage files were normalized to the number of reads that could be mapped from the respective library. To restore the original data range, each graph was then multiplied by the minimum number of mapped reads over the two libraries.
In order to automatically define Cas9-bound RNA regions or peaks from the Cas9-3xFLAG coIP datasets, we applied an in-house tool “sliding_window_peak_calling_script” based on a sliding window approach that was originally developed to detect binding regions of CsrA in C.jejuni (The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacterjejuni. Nat Commun. 2016;7:11667). The script has been deposited at Zenodo under https://doi.org/10.5281/zenodo.49292. The script is written in Python 3 and requires installation of the Python 3 packages numpy and scipy for execution.
In brief, the “sliding_window_peak_calling_script” software uses rRNA normalized wiggle files of the Cas9-3xFLAG and control coIP libraries as input to determine sites showing a continuous enrichment of the Cas9-3xFLAG-tagged library compared to the control. The identification of enriched regions is based on four parameters: a minimum required fold-change (FC) for the enrichment, a factor multiplied by the 90th percentile of the wiggle graph which reflects the minimum required expression (MRE) in the tagged library, a window size in nt (WS) for which the previous two values are calculated in a sliding window approach, and a nucleotide step size (SS) which defines the steps in which the window is moved along the genomic axis. All consecutive windows that fulfill the enrichment requirements are assembled into a single peak region. The peak detection is performed separately for the forward and reverse strand of each replicon. For the Cas9-3xFLAG coIP datasets, the following parameters were used: FC = 5, MRE = 3, WS = 25 and SS = 5. MEME was then used for the prediction of consensus motifs based on the peak sequences. The two significantly enriched motifs were then compared to the crRNA and tracrRNA sequences to deduce their probable mode of binding.
For Northern Blot analysis, 100 µg of DNaseI treated total RNA sample was loaded on each lane. After separation on 6% polyacrylamide (PAA) gels containing 7 M urea, RNA was transferred to Hybond-XL membranes (GE-Healthcare) by electroblotting. After blotting, the RNA was UV cross-linked to the membrane and hybridized with y32P-ATP end-labeled DNA oligonucleotides.
All primers, gBlocks used in this work were ordered from Integrated DNA Technologies (IDT, Coralville, US). NEBuilder® HiFi DNA Assembly Cloning Kit was used for plasmid construction by Gibson assembly. The associated SEQ ID numbers (SEQ ID NOS: 1 to 96) can be found in the attached sequence listing, which is incorporated by reference in its entirety. Q5 site-directed mutagenesis kit was used for small insertion and nucleotides substitution. Unless otherwise specified, all nucleases used were expressed in plasmids with Cm selective marker and p15A origin of replication (ORI). All sgRNA or tracrRNA-crRNA or tracrRNA-ncrRNA plasmids were expressed in plasmids with Amp selective marker and ColE1 ORI, and all targeted plasmids were expressed in plasmids with Kan selective marker and pSC101 ORI.
For Cas9 and Cas12 plasmid construction, SpyCas9 and E.coli codon-optimized CjeCas9 genes were PCR-amplified from plasmids pCB843 and pCB588, then Gibson-assembled into a PCR-linearized vector pCBS583, respectively.
To construct plasmids encoding sgRNAs with different lengths of 5′-overhangs, gBlocks encoding fliF- or 1093-targeting sgRNAs and their variants with different 5′-overhangs were Gibson-assembled into PCR-linearized vector pCSM180, respectively. For crRNA with 5′-overhangs in the dual-RNA-guided (tracrRNA/crRNA) system, the tracrRNA expressing cassette was obtained by PCR-amplification with the template of gBlock CJfr0018 and primer pair of CJpr0304/CJpr0305 (SEQ ID NOs: 3-4) in order to introduce homologous arms with vector and crRNA cassettes. fliF-targeting crRNA and its 5′ overhang variants expressing cassettes were obtained by PCR-amplification with the templates of CJfr0022, CJfr0070 and CJfr0071, and primer pair of CJpr0110/CJpr0291 (SEQ ID NOs: 5-9) to introduce homologous arms with vector and tracrRNA cassette. TracrRNA expressing cassette and crRNA with 5′-overhangs expressing cassettes were Gibson-assembled into PCR-linearized vector pCSM180, respectively.
Similar methods were performed in order to construct plasmids encoding crRNA with 3′-overhangs in the dual-RNA system, the only difference was to obtain fliF-targeting crRNA and its 3′-overhang variants expressing cassettes by PCR-amplification with the templates of CJfr0080, CJfr0081 and CJfr0176 (SEQ ID NOs: 10-12).
In order to construct reprogrammed ncrRNA plasmids for CjeCas9, SpyCas9, and Sth1Cas9, mRNA harboring peak 113 (spanning genes CJ8421_04970 & 04975 & 04980) was used as the template for modified tracrRNAs to target. This peak was one of the RNAs identified as significantly enriched as part of the CjeCas9 co-immunoprecipitation. gBlocks encoding modified tracrRNAs, and the peak 113-harbored mRNA was PCR-amplified from plasmid pCJ174 and Gibson-assembled into PCR-linearized vector pCSM180.
In order to construct a self-targeting ncrRNAs for Cj eCas9, the peak 113-harbored mRNA and reporter gene GFP were arranged on one plasmid, and the modified tracrRNA on another plasmid. A PAM sequence was introduced into the base of the repeat-anti-repeat stem of tracrRNA-ncrRNA using Q5 site-directed mutagenesis.
For all targeted plasmids construction, target sequences were inserted upstream of promoter OR2-OR1 or between promoter OR2-OR1 and GFP CDS using Q5 site-directed mutagenesis with the template of pCSM168.
TXTL systems were used to characterize the ncrRNAs, and how the tracrRNA can be reprogrammed/modified. The system works by introducing linear or plasmid DNA encoding each component into E.coli. The E.coli. lysate then transcribes and translates the components based on E.coli. signals (e.g. promoters, RBS). By using a GFP reporter containing the PAM-flanked target of the crRNA (or ncrRNA), the biogenesis and activity of the crRNA/ncrRNA can be monitored over time. The 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 be free of nucleases (DNases, RNases) and inhibitors of the TXTL machinery. Cas9, sgRNA/dual-RNA, and deGFP-encoding reporter plasmids were added into myTXTL Sigma 70 Master Mix (Arbor Biosciences) with a final concentration of 1.5 nM, 0.5 nM and 1 nM, respectively. When expressing linear fragments, GamS (Arbor Biosciences) at a final concentration of 5 uM was supplemented, and a fliF-targeted plasmid (pCJ002) with target sequence inserted between promoter and deGFP coding sequence was used. T7 RNA polymerase-encoding plasmid at a final concentration of 0.5 nM was supplemented when expressing T7 promoter-driven genes. Fluorescence was measured on a BioTek Synergy Neo2 plate reader using a 96-well V-bottom plate (Corning Costar 3357) with Excitation filter at 485 nm and Emission filter at 528 nm. Time-course measurements were run for 16 hours at 29° C. with an interval of 3 minutes between reads.
Peak 113-harbored mRNA was used as the template for reprogram tracrRNAs to the target. Regions containing AT-enriched sequences preferentially recognized by the E. coli Rnase III were chosen as targets for reprogrammed tracrRNAs (In vivo cleavage rules and target repertoire of RNase III in Escherichiacoli.Nucleic Acids Res. 2018;46(19):10380-10394). These regions contained an AT-enriched motif, and were 25 bp in length for CjeCas9, and 38 bp for SpyCas9 and Sth1Cas9. For CjeCas9, the anti-repeat part of native tracrRNA was replaced with the sequences complementary to the chosen targets, respectively. For SpyCas9 and Sth1Cas9, an extra four or three nucleotides were introduced between the 6th and 7th, or between the 9th and 10th nucleotides (counting from bottom to top in the duplex) in the anti-repeat part of tracrRNA to create a bulge, which is necessary to maintain SpyCas9 and Sth1Cas9activity (Guide RNA functional modules direct Cas9 activity and orthogonality. Mol Cell. 2014;56(2):333-339). Correspondingly, the 20-bp to 24-bp sequences upstream of these base-pairing regions were treated as spacers.
The plasmid clearance assay was performed as previously described (CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacterjejuni Cas9. Mol Cell. 2018;69(5):893-905.e7). 40 nM sgRNA, ncrRNA plasmids were electroporated into E. coli strains containing Cas9 and targeted or non-targeted plasmids. After 1 h recovery in SOC medium, 3 ul of 10-fold serial dilutions of these transformants were spotted onto LB plates with Cm+Amp+Kan antibiotics for overnight incubation at 37° C. Colonies from countable spots were used to calculate the fold-reduction of transformation efficiency by dividing non-targeting CFUs by targeting CFUs.
RNA was in vitro-transcribed for 4 h at 37° C. using HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs) according to the products’ manual. The transcribed RNA was purified using RNA Clean & Concentrator-25 (Zymo Research) with residual DNA removed by in-column DNase I treatment according to the products’ manual.
Sensed RNAs and non-natural tracrRNAs were encoded in synthetic gBlocks beginning with a T7 promoter and were transcribed using the HiScribe T7 in vitro transcription kit (New England Biolabs, cat. #E2040S). The transcribed RNAs were purified using RNA Clean & Concentrator-25 (Zymo Research) with residual DNA removed by In-Column DNase I treatment. A Cy5-labeled forward primer paired with different reverse primers were used to PCR-amplify the associated DNA targets from targeted plasmids.
Sensed RNAs and their associated non-natural tracrRNAs were annealed at a 1:1 molar ratio in 1X NEB3.1 buffer (New England Biolabs, cat. #B7203S) by heating at 95° C. for 2 minutes and gradually cooling to 25° C. over 45 min using a thermocycler. SpyCas9 (New England Biolabs, cat. #M0386M) was then added, and the mixture was incubated at room temperature for 25 min to promote RNP assembly. DNA targets were then added, and the reaction was incubated at 37° C. for 1 h to promote DNA cleavage.
To explore the impact of adding RNases on ncrRNA, Shortcut® RNase III (New England Biolabs, cat. #M0245L) at a final concentration of 33.3 U/ml and/or RNase A (Thermo Fisher Scientific, cat. #EN0531) at a final concentration of 166.7 µg/ml were added before or during the DNA cleavage step. Proteinase K (New England Biolabs, cat. #P8107S) was then added at a final concentration of 25.8 U/ml, and the reaction was incubated at room temperature for 15 min to digest Cas9. For reactions with unlabeled DNA, the final concentration of the DNA target was 10 nM, while the final concentrations of the sensed RNAs, non-natural tracrRNAs and SpyCas9 were all 200 nM. The unlabeled DNA products were separated on a 2% agarose gel and visualized by Intas GelStick Imager. For reactions with Cy5-labeled DNA, the final concentration of the DNA target was 2 nM, while the final concentration of each sensed 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 for detecting virus RNAs individually or in a set of three or five.
The resulting products for individual or multiplexed RNA detection were loaded along with Novex® TBE HiDensity buffer without tracking dyes, separated on a 4-20% Novex™ TBE gel (Invitrogen, cat. #EC6225BOX).
In vitro RNA transcription and detection using LEOPARD was performed as indicated above. For multiplexed RNA detection, all non-natural tracrRNAs and DNA targets were present in a single reaction tube for detecting virus RNAs individually or in a set of three or five. The resulting products for individual or multiplexed RNA detection were loaded along with Novex® TBE HiDensity buffer without tracking dyes, and separated on a 4-20% Novex™ TBE gel (Invitrogen, cat. #EC6225BOX). The individual products for specific detection of the D614G mutation in the SARS-CoV-2 Spike protein and the experiment with yeast total RNA were loaded on a 2% agarose gel. Images for gels with Cy5 labels were captured on an Amersham Typhoon™ Biomolecular Imager (GE Healthcare).
The D614G mutation in the spike protein of SARS-CoV-2 serves as a proof of function-example, as this mutation results from a single nt change (A23403G) and has been associated with increased infectivity and its global spread. By placing this nt change within the seed region of the target, the inventors could detect the WT or D614G RNA using one non-natural tracrRNA combined with either the WT or D614G target (
As part of designing the non-natural tracrRNAs, the sequence of the anti-repeat region of the tracrRNA is changed to be complementary to a region of the sensed RNA. Identifying these regions is relatively straightforward and can be achieved by evaluating the base-pairing potential of the tracrRNA with one of the repeats in the CRISPR array, such as by using common RNA folding algorithms such as NUPACK or mfold. Alternatively, this region can be determined experimentally by hybridizing purified repeat and purified tracrRNA and then evaluating RNA structure through common probing approaches (e.g. using DMS, SHAPE). The anti-repeat region will be the region either partially or fully base paired with the repeat region and should begin around the 5′ end of the tracrRNA sequence. See
A collection of RNAs are extracted from a sample using common approaches to purify RNA, such as GenEasy RNA extraction kit sold by Sigma-Aldrich or the RNeasy RNA extraction kit sold by Qiagen. The sample can be from a human or animal (e.g. blood, sputum, biopsy), a plant (e.g. clipped root, clipped leaf), soil, water, or air.
A set of RNAs to be detected within each sample are selected based on the specific needs of the user. An accompanying set of non-natural tracrRNAs are designed by changing the sequence of the anti-repeat portion of the tracrRNA to be complementary to a portion of the RNA.
A dsDNA target is then designed for each ncrRNA. The target comprises the guide sequence of the ncrRNA flanked by an appropriate PAM recognized by the CRISPR nuclease (e.g. NNNNACAC for CjeCas9, NGG for SpyCas9, NNAGAA for Sth1Cas9). See
The reaction also involves purified tracrRNA-dependent nuclease (e.g. SpyCas9) and optionally the dsRNA-cleaving enzyme (e.g. RNase III). The nuclease can be catalytically-dead or catalytically-active depending on whether the detection step involves target nucleic acid binding or cleavage.
As part of representative detection assays, the purified RNAs from the sample are mixed with the set of non-natural tracrRNAs in a binding buffer that promotes RNA hybridization (e.g. 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2 and 0.05 % Tween-20, pH 7.8). and incubated at 37° C. for 10 minutes to form duplexes between the RNAs. The tracrRNA-dependent CRISPR nuclease and dsRNA-cleaving enzyme are then added, and the mixture is incubated at the same temperature for an additional 20 minutes. During this step, the dsRNA-cleaving enzymes cleaves RNA duplexes, such as those formed between a sensed RNA and a non-natural tracrRNA, and ribonucleoprotein complexes form between the tracrRNA-dependent nuclease and the cleaved RNA duplex. The resulting mixture is then exposed to the DNA targets, allowing ribonucleoprotein complexes to target their respective DNA targets. The DNA targets can be added as soluble molecules or they can be affixed to a solid surface (e.g. magnetic beads, on a silicon chip). A wash step can then be added (e.g. with the binding buffer) to remove RNAs, the dsRNA-cleaving enzyme, unbound ribonucleoprotein complexes, and any cleaved and unaffixed portions of DNA targets.
Finally, the DNA targets are assessed to measure the extent to which they were recognized by the ribonucleoprotein complexes (see
In all of these examples, the measured output is linked to specific DNA targets, thus serving as an indirect readout of the presence of the sensed RNA. The readout can be rendered quantitative to specify the concentration of each sensed RNA in the sample based on the fluorescence intensity. For instance, if a cluster of molecules encoding the same DNA target sequence are present, then the fluorescence intensity would scale with the number of DNA molecules recognized by riboncleoprotein complexes encoding the appropriate ncrRNA—and thus the number of sensed RNA molecules that produced the ncrRNAs.
Beyond using dsDNA targets, ssDNA and RNA targets can also be used as part of the diagnostic platform. Multiple Cas9 and Cas12 nucleases (e.g. CjeCas9, NmeCas9, SauCas9) have been shown to recognize ssDNA and RNA, where recognition involves base pairing to the guide region of the guide RNA but does not involve the PAM. Similar setups for detection could be employed, such attaching a fluorophore to one end and a quencher to the other end of the target so target cleavage releases the quencher, or a fluorophore-conjugated nuclease could localize fluorescence upon target binding.
Some Cas9 nucleases (e.g. SpyCas9) are unable to recognize ssDNA or RNA unless a double-stranded PAM sequence is present next to the target. This could be incorporated into the target design by using a ssDNA target with a fixed sequence for the PAM and flanking sequence. Another ssDNA oligo could then be hybridized to the fixed sequence, providing a double-stranded PAM and flanking sequence with a single-stranded target.
The examples described herein can be used for wide-ranging applications, such as diagnosing medical conditions to inform the course of treatment, identifying SNPs associated with health outcomes or disease, identifying microbial contaminants in potable water, identifying viral or microbial contaminants in fermentations or cell cultures, identifying plant or insect variants, or identifying key microbial members in mixed communities (e.g. in the gut, soil, water).
For this application, the non-natural tracrRNAs are employed to record the presence of one or multiple sensed RNAs in a cell in the form of edited DNA or the altered expression of a measurable reporter gene.
The tracrRNA-dependent nuclease and the non-natural tracrRNAs are encoded in linear DNA, a plasmid or a viral construct for delivery and cellular expression. The tracrRNAs can be expressed separately as individual expression constructs or in a single construct with intervening RNA cleavage sequences (e.g. tRNAs, ribozymes).
DNA representing the targets of generated ncrRNAs can be encoded in the same vector or a separate vector, where its composition will depend on how the nucleases are employed to record a detected RNA. See
The constructs can express the dsRNA-cleaving enzyme (e.g. the E.coli RNase III) for ncrRNA biogenesis. However, virtually all cells express a dsRNA-cleaving enzyme that can be employed instead. The dsRNA-cleaving enzyme can also be entirely dispensable.
To carry out detection and recording, the DNA constructs are introduced into cells through standard means (e.g. electroporation, chemical transformation, conjugation, transfection, viral delivery), and the DNA can be present only transiently (e.g. through transient transfection) or can be stably maintained (e.g. under selection or integrated into chromosomal DNA). The components are then expressed from the constructs to actively detect the presence of the sensed RNA. If the RNA is present, then it will be converted into an ncrRNA and direct the tracrRNA-dependent nuclease to the target DNA reporter. For reporters that rely on expression, standard techniques can be used to measure reporter activity (e.g. fluorescence by flow cytometry or a fluorescence microtiter plate reader). For reporters that rely on DNA editing, the target could be probed through standard approaches that directly or indirectly output DNA sequences (e.g. Sanger sequencing, next-generation sequencing).
As one example, non-natural tracrRNAs were combined with a base editor derived from the SpyCas9 (e.g. BE3) to sense the expression of a multidrug resistance gene (e.g. encoding NMD-1) or a stress-response gene (e.g. encoding the iron-starvation induced sRNA RyhB) in E. coli. The non-natural tracrRNA were designed to hybridize to a portion of the sensed RNA as indicated in
Paralleling the above example, non-natural tracrRNAs could be combined with the CRISPR transposon based on Cas12k for transcription-dependent recording. Cas12k, the transposon components (TnsB, TnsC, TnsQ), an insertion template flanked by the transposon repeats (e.g. encoding a reporter gene or an antibiotic resistance marker), and the non-natural tracrRNAs could be encoded within expression plasmids along with plasmid DNA encoding the corresponding target sites. Using this setup, the presence of the sensed RNA would drive template insertion proximal to the target sequence. The insertion could then be detected through PCR or sequencing.
Another application of non-natural tracrRNAs involves editing DNA targets in cells only when the gene is actively transcribed. As part of this application, the non-natural tracrRNA is designed to hybridize to a region of a sensed RNA encoding a PAM sequence recognized by the tracrRNA-dependent CRISPR nuclease. The PAM would be specifically located at the 5′ end or 3′ end of the hybridizing portion of the sensed RNA for Type II or V CRISPR nucleases, respectively. By encoding the PAM in this location, the resulting ncrRNA can direct the tracrRNA-dependent CRISPR nuclease to the DNA locus encoding the sensed RNA. Because the sensed RNA is an essential component in this process, the DNA locus would only undergo targeting when the sensed RNA is present, and thus when the DNA locus is transcribed.
To achieve transcription-dependent targeting in cells, the tracrRNA-dependent nuclease and the non-natural tracrRNAs are encoded in a plasmid or viral construct for delivery and cellular expression. The tracrRNAs can be expressed separately as individual expression constructs or in a single construct with intervening RNA cleavage sequences (e.g. tRNAs, ribozymes). The nuclease can drive indel formation or homologous recombination in the presence of a DNA repair template, or the nuclease can generate other edits if modified to function as a base editor or as a prime editor. Alternatively, a catalytically-dead nuclease can modulate expression of the sensed gene, such as blocking transcription or by recruiting the KRAB to drive transcriptional repression.
The constructs can express the dsRNA-cleaving enzyme (e.g. the E.coli RNase III) for ncrRNA biogenesis. However, virtually all cells express a dsRNA-cleaving enzyme that can be employed instead.
For example, transcription-dependent editing could be implemented in the context of gene drives in mosquitos only when the target locus is actively transcribed. The non-natural tracrRNA is designed to generate an ncrRNA from the relevant target locus (e.g. the doublesex gene), and the hybridizing region in the sensed RNA encodes a PAM (e.g. NGG at the 5′ end of this region when using the SpyCas9). The tracrRNA-dependent CRISPR nuclease, the non-natural tracrRNA, and any genetic cargo are encoded in an expression construct that is inserted into the target locus through standard gene-manipulation techniques for mosquitos. When the resulting mosquitos mate and produce off-spring, the gene drive would be expected to transfer only in cells in which the locus is actively transcribed. The end effect is limited to the tissue type in which the gene drive is transferred and introducing an extra layer of control depending on the expression profile of the locus.
Multiple non-natural tracrRNAs can be designed against the doublesex gene and can be encoded in the gene-drive locus to improve the frequency of transfer and reduce the frequency of drive failure due to the formation of disruptive indels at the target site.
The conversion of a cellular RNA into an ncrRNA was based on sequences bearing extensive complementarity to the tracrRNA anti-repeat, analogous to the natural biogenesis of crRNAs. If the tracrRNA anti-repeat sequence can be changed to hybridize to other RNAs while maintaining the appropriate structure for Cas9 recognition, then the resulting non-natural tracrRNA could drive the conversion of any expressed cellular RNA into an ncrRNA, which guides Cas9 to matching DNA targets (
Naturally, the CjeCas9 recognizes a perfect RNA duplex formed between the crRNA repeat and tracrRNA anti-repeat. Based on mutational analysis of the crRNA repeat, the inventors already observed that the crRNA repeat:tracrRNA anti-repeat duplex can accommodate several mutations. Therefore, the inventors evaluated GFP silencing in TXTL after mutating both sides of the duplex in the fliF sgRNA while preserving the secondary structure (s26-s31,
Given the functionality of CjeCas9 non-natural tracrRNAs, the inventors asked whether tracrRNAs for other Cas9 homologs can be similarly reprogrammed. The inventors selected the well-characterized Streptococcuspyogenes Cas9 (SpyCas9) and the Streptococcus thermophilus CRISPR1 Cas9 (Sth1Cas9) as examples. In both cases, the inventors devised design rules for non-natural tracrRNAs based on the known secondary structure of the crRNA:tracrRNA duplex and the preference of RNase III to cleave double stranded RNA with AT-rich sequences. All ten tested non-natural tracrRNAs significantly reduced GFP levels compared to the non-targeting crRNA control (p = 1x 10-7 - 1 x 10-4). As before, GFP expression was restored by disrupting the seed sequence in the ncrRNA guide, scrambling the tracrRNA anti-repeat, or swapping either of the Cas9´s for FnCas12a. In many cases, the extent of GFP silencing approached that of the targeting crRNA control (
By linking DNA targeting to an RNA-of-interest, non-natural tracrRNAs offer a unique opportunity for RNA detection and a different paradigm for CRISPR diagnostics. Current CRISPR diagnostics rely on Cas12a or Cas13 searching for double-stranded DNA or RNA targets in a sample, where target recognition elicits non-specific single-stranded DNA or RNA cleavage of a fluorescent reporter. The non-specific readout effectively limits one test to one target sequence. In contrast, non-natural tracrRNAs would convert sensed RNAs into ncrRNAs, and the ncrRNAs would direct Cas9 to bind or cleave matching DNA. Cas9 binding or cleavage of a DNA sequence would then indicate whether the associated RNA is present in the sample. Because the sequence of each DNA target is unique, large numbers of target sequences could be monitored in parallel in one test. The inventors designate the resulting diagnostic platform as LEOPARD, for Leveraging Engineering tracrRNAs and On-target DNAs for PArallel RNA Detection (
To begin assessing LEOPARD, the inventors performed a simplified in vitro reaction using T7-transcribed RNAs, commercially available Cas9 protein, and linear DNA targets. The inventors began with RNA corresponding to one of the synthetic ncrRNA loci within CJ8421_04970 (
Realizing the full multiplexing potential of LEOPARD requires monitoring many DNA targets at once. The inventors therefore devised a readout scheme based on resolving distinct cleavage products from pooled DNA targets by gel electrophoresis (
| Number | Date | Country | Kind |
|---|---|---|---|
| 20160004.6 | Feb 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/055060 | 3/1/2021 | WO |
