Method of identifying cleavage sites of RNA-cleaving molecules

Abstract

The present invention relates to a method for identifying one or more RNA cleavage sites of an RNA-cleaving molecule, the method comprising: (a) incubating the RNA-cleaving molecule with a target RNA under conditions suitable for cleavage of the target RNA by the RNA- cleaving molecule to form cleaved RNA fragments; (b) processing the 5' and 3' ends of the cleaved RNA fragments into 5' phosphate and 3'-hydroxyl ends respectively; (c) ligating the processed cleaved RNA fragments with adapter sequences to generate a sequencing library; (d) sequencing the sequencing library to obtain a plurality of sequenced reads; (e) aligning the plurality of sequenced reads to a DNA sequence of the target RNA and identifying a 5' base position and a 3' base position for each sequencing read; (f) measuring the frequency of 5' base positions and of 3' base positions of the plurality of sequencing reads as respective percentages against a total number of aligned sequencing reads; and (g) identifying the one or more RNA cleavage sites based on the measurement results of step (f), wherein an increased frequency of 5' base positions is indicative of an RNA cleavage site and wherein an increased frequency of 3' base positions is indicative of an RNA cleavage site.

Description

DESCRIPTIONTITLE OF THE INVENTION:METHOD OF IDENTIFYING CLEAVAGE SITES OF RNA-CLEAVING MOLECULESFIELD OF THE INVENTION

[0001] The invention generally relates to the field of molecular biology. In particular, the invention relates to a method of identifying one or more RNA cleavage sites of an RNA- cleaving molecule.BACKGROUND OF THE INVENTION

[0002] The CRISPR-Cas13 systems are RNA-guided RNA-targeting endonucleases. The Cas13 enzymes have exhibited tremendous value for biotechnological and clinical applications, ranging from next-generation diagnostics, RNA base-editing, RNA editing, RNA modifications, RNA therapeutics, and antiviral therapeutics. CRISPR-Cas13 has enabled a wide range of applications that are not addressable with the DNA-targeting CRISPR-Cas9 and CRISPR-Cas12 systems.

[0003] An important feature of CRISPR-Cas13-gRNA is that it cuts the target RNA molecule in more than 1 site (i.e. in addition to the target sequence complementary to the gRNA) and other bystander non-target RNA molecules, in what has been termed “collateral damage”. Identifying the RNA sequences, and the respective frequency, that are cut by CRISPR-Cas13-gRNAs is hence critical in determining the safety and efficacy profiles of these new RNA editors.

[0004] Existing studies therefore systematically investigated the preferred site / motif for RNA cleavage in different Cas13b variants. Most of these existing studies, however, focused on the collateral cleavage activity (non-specific cleavage of any RNAs that are not targeted by the gRNA), which is a unique characteristic of Cas13b that enables the development of diagnostic tools for viral RNA detection. Meanwhile, there is a limited number of publications profiling the exact cleavage sites on target RNAs themselves. Furthermore, none of these studies have provided quantitative and single-nucleotide resolution of cleavage patterns.

[0005] Understanding how HEPN domain in Cas13b cleaves target RNAs could be helpful to facilitate the manipulation of its cleavage mechanism for RNA editing without inactivating the HEPN nuclease activity fully. However, existing studies are conducted with single-base RNA editors that have been created with nuclease-dead versions of Cas13b. Such adeactivation of Cas13b’s cleavage function precludes the development of editors that perform a wider range of edits that are more complex.

[0006] There is thus a need for a method of identifying the cleavage sites of RNA-cleaving molecules that overcome the drawbacks of the prior art. Furthermore, other desirable features and characteristics will become apparent from the subsequent detailed description and the appended claims, taken in conjunction with the accompanying drawings and this background of the disclosure.SUMMARY OF THE INVENTION

[0007] In one aspect, the present invention provides a method for identifying one or more RNA cleavage sites of an RNA-cleaving molecule, the method comprising: (a) incubating the RNA-cleaving molecule with a target RNA under conditions suitable for cleavage of the target RNA by the RNA-cleaving molecule to form cleaved RNA fragments; (b) processing the 5’ and 3’ ends of the cleaved RNA fragments into 5’ phosphate and 3’-hydroxyl ends respectively; (c) ligating the processed cleaved RNA fragments with adapter sequences to generate a sequencing library; (d) sequencing the sequencing library to obtain a plurality of sequenced reads; (e) aligning the plurality of sequenced reads to a DNA sequence of the target RNA and identifying a 5’ base position and a 3’ base position for each sequencing read, wherein the 5’ base position is the nucleotide in the DNA sequence of the target RNA that is aligned with the 5’ end of each sequencing read and wherein the 3’ base position is the nucleotide in the DNA sequence of the target RNA that is aligned with the 3’ end of each sequencing read; (f) measuring the frequency of 5’ base positions and of 3’ base positions of the plurality of sequencing reads as respective percentages against a total number of aligned sequencing reads; and (g) identifying the one or more RNA cleavage sites based on the measurement results of step (f), wherein an increased frequency of 5’ base positions is indicative of an RNA cleavage site and wherein an increased frequency of 3’ base positions is indicative of an RNA cleavage site.

[0008] In one embodiment, step (a) comprises incubating the RNA-cleaving molecule with a guide RNA (gRNA) to form an RNA-cleaving molecule:gRNA complex and incubating the RNA-cleaving molecule:gRNA complex with the target RNA under conditions suitable for cleavage of the target RNA by the RNA-cleaving molecule:gRNA complex to form cleaved RNA fragments.

[0009] In one embodiment, step (a) is carried out in vitro.

[0010] In one embodiment, the RNA-cleaving molecule is Cas13 protein.

[0011] In one embodiment, the Cas13 protein is Cas13b.

[0012] In one embodiment, the RNA-cleaving molecule is a ribozyme.

[0013] In one embodiment, step (a) comprises incubating the RNA-cleaving molecule with the gRNA for 15 minutes at 37°C.

[0014] In one embodiment, step (a) comprises incubating the RNA-cleaving molecule:gRNA complex with the target RNA for 15 minutes at 37°C.

[0015] In one embodiment, after step (a), a stop solution is added to terminate the enzymatic activity of the RNA-cleaving molecule.

[0016] In one embodiment, the cleaved RNA fragments are separated on a denaturing polyacrylamide gel electrophoresis (PAGE) for visualization of the cleaved RNA fragments.

[0017] In one embodiment, step (b) comprises a first dephosphorylation reaction to dephosphorylate 5’-triphosphate ends into 5’-hydroxyl RNA ends, a second dephosphorylation reaction to dephosphorylate 2’-3’ cyclic phosphate into 3’-hydroxyl RNA ends, and a phosphorylation reaction to phosphorylate the 5’-hydroxyl RNA ends into 5’-phosphate ends.

[0018] In one embodiment, step (d) comprises converting the adapter-ligated RNA fragments into a cDNA library via reverse transcription and sequencing the cDNA library.

[0019] In one embodiment, after step (d), the method as described herein further comprises the step of checking the plurality of sequenced reads for sequencing quality and for the presence of adapter sequences at the ends of the sequenced reads, followed by trimming the ends having low sequencing quality and having adapter sequences.

[0020] In one embodiment, step (f) further comprises normalizing the respective percentages by subtracting a control percentage corresponding to each base position from the respective percentages at each base position.

[0021] In one embodiment, the control percentage is derived from (i) a first control condition in which the RNA-cleaving molecule and gRNA are omitted; or (ii) a second control condition in which the gRNA is omitted.

[0022] In one embodiment, step (f) further comprises normalizing the respective percentages by dividing the respective percentages at each base position by a control percentage corresponding to each base position.

[0023] In one embodiment, step (f) further comprises adding a pseudocount at each base position prior to the step of dividing the respective percentages at each base position by the control percentage corresponding to each base position.

[0024] In one embodiment, step (g) comprises determining a reference level, wherein any percentage from step (f) that is above the reference level is indicative of an RNA cleavage site.

[0025] In one embodiment, step (g) comprises visualizing the frequencies of the 5’ base positions and 3’ base positions on a line chart in which a first axis of the line chart shows the nucleotide position of the DNA sequence of the target RNA and a second axis of the line chart shows the respective percentages, wherein any peak on the line chart having a value on the second axis that is higher than the reference level is indicative of an RNA cleavage site.

[0026] In another aspect, the present invention provides a use of the method as described herein for quantifying the number of cleavage sites in the target RNA and the frequency of cleavage at each cleavage site, wherein the number of cleavage sites in the target RNA is quantified based on the identification results of step (g) and wherein the frequency of cleavage at each cleavage site is quantified based on the measurement results of step (f).BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0028] Fig. 1 shows an in vitro cleavage assay workflow: Target RNA and gRNA were produced via T7 in vitro transcription system and purified using silica columns. Cas13b proteins were isolated and purified from bacteria expressing a cloned Cas13b plasmid. The purified gRNAs and Cas13b proteins were then pre-incubated to assemble gRNA-Cas13b complexes before adding target RNAs to initiate cleavage. The in vitro cleavage reactions were quenched before performing PAGE analysis.

[0029] Fig. 2 shows the downstream workflow to identify Cas13b cleavage sites. Target RNAs produced via in vitro transcription carry 5’-triphosphates (5’-PPP) and 3’-OH, while cleaved RNA products contain a mixture of 5’-PPP and 5’OH, as well as 3’-OH and 2’,3’-cyclic phosphate. All RNA fragment ends must be converted to 5’-phosphate and 3’-OH before library preparation for single-ended sequencing (SE-seq). After SE-seq, the reads were processed with Fastqc and Trimmomatic before they were aligned using Bowtie2 to a reference sequence. Pysam was then used to identify start and end positions of the RNA fragments. Nucleotide positions at which there was an enriched number of reads starting, or ending, indicate potential Cas13b cleavage sites.

[0030] Fig. 3 shows the in vitro cleavage assay of Figure 1 , performed for 2 different Cas13b orthologs isolated from Prevotella sp. (Psp) and Riemerella anatipestifer (Ran) bacteria. For each Cas13b ortholog, 3 gRNAs were tested. (A) Positions of gRNA spacers aligned to the common 129-bp target RNA sequence. (B) Denaturing PAGE analysis revealsdistinct RNA cleavage products that differ based on gRNA sequences and Cas13b orthologs, indicating that programming Cas13b with different gRNAs induces different cleavage patterns on the same RNA molecule.

[0031] Fig. 4 shows the identification of Cas13b cleavage sites based on the workflow described. Incubating 129-bp target RNA with either (A) PspCas13b or (B) RanCas13b, along with their respective gRNA 1 , produces distinct cleavage patterns. Cleavage sites can be identified at single-nucleotide resolution.

[0032] Fig. 5 shows the distribution of gRNAs across the target RNA.

[0033] Fig. 6 shows a PAGE gel showing cleavage patterns induced by other gRNAs. gRNA 5 is non-complementary to the target RNA and serves as a non-targeting guide (negative control).

[0034] Fig. 7 shows plots showing cleavage patterns of the additional gRNAs.

[0035] Fig. 8 shows the workflow to identify protospacer flanking sequence (PFS) requirements of Psp and Ran Cas13b orthologs. The 3 nucleotides upstream and downstream of gRNA1 protospacer were replaced with random nucleotides. Subsequently, uncleaved target RNAs were sequenced to identify PFS that inhibit Cas13 cleavage activity.

[0036] Fig. 9 shows a heatmap showing depleted bases at PFS. As only full-length target RNAs are sequenced, target RNAs that originally contained PFS that promote cleavage would be depleted. PspCas13b has a 5’ NGC preference, while RanCas13b has a 5’ NGC and a 3’ CCN preference.DETAILED DESCRIPTION OF THE INVENTION

[0037] The present disclosure provides an in vitro workflow that identifies RNA cleavage site(s) in its native sequence context down to single base resolution. As a proof-of-principle, the present embodiment was demonstrated with two different Cas13b, Prevotella sp. (PspCas13b) and Riemerella anatipestifer Cas13b (RanCas13b).

[0038] The present disclosure provides an in vitro method of identifying and quantifying RNA cleavage site(s) and motifs of Cas13b at nucleotide resolution and potentially other RNases with specific cleavage activity via Next Generation Sequencing (NGS).

[0039] The method of the present disclosure achieves unbiased, single-nucleotide resolution mapping of Cas13 cleavage patterns not achieved by prior art.

[0040] The method of the present disclosure may be applied in determining the safety and efficiency of RNA editing therapeutics and in developing Cas13-based diagnostics.

[0041] In one embodiment, the present disclosure provides a method to identify and quantify cleavage sites of RNA-cleaving molecules (e.g. CRISPR-Cas13-gRNA) across RNA molecules. The method of the present disclosure measures cleaved RNA ends at singlenucleotide resolution, with sensitivity conferred by next-generation sequencing:1 . Design and synthesize target and guide RNAs through in-vitro transcription (IVT), and purify RNA-cleaving molecules (e.g. Cas13b proteins).2. Perform an in vitro cleavage assay and validating cleavage through PAGE analysis.3. Perform a specific sequence of enzymatic treatments and clean-up of RNA fragments to convert their 5’ and 3’ ends to ends that are compatible for next generation sequencing (NGS) library prep.4. NGS library prep and Illumina short-read sequencing.5. Data processing involving Fastqc, Trimmomatic, Bowtie2, Pysam to identify Cas13b cleavage sites at single-nucleotide resolution.

[0042] In one aspect, the present disclosure provides a method for identifying one or more RNA cleavage sites of an RNA-cleaving molecule, the method comprising: (a) incubating the RNA-cleaving molecule with a target RNA under conditions suitable for cleavage of the target RNA by the RNA-cleaving molecule to form cleaved RNA fragments; (b) processing the 5’ and 3’ ends of the cleaved RNA fragments into 5’ phosphate and 3’-hydroxyl ends respectively; (c) ligating the processed cleaved RNA fragments with adapter sequences to generate a sequencing library; (d) sequencing the sequencing library to obtain a plurality of sequenced reads; (e) aligning the plurality of sequenced reads to a DNA sequence of the target RNA and identifying a 5’ base position and a 3’ base position for each sequencing read, wherein the 5’ base position is the nucleotide in the DNA sequence of the target RNA that is aligned with the 5’ end of each sequencing read and wherein the 3’ base position is the nucleotide in the DNA sequence of the target RNA that is aligned with the 3’ end of each sequencing read; (f) measuring the frequency of 5’ base positions and of 3’ base positions of the plurality of sequencing reads as respective percentages against a total number of aligned sequencing reads; and (g) identifying the one or more RNA cleavage sites based on the measurement results of step (f), wherein an increased frequency of 5’ base positions is indicative of an RNA cleavage site and wherein an increased frequency of 3’ base positions is indicative of an RNA cleavage site.

[0043] As used herein, the term “RNA-cleaving molecules” includes any molecules or complexes capable of cleaving RNA. This includes but is not limited to protein enzymes (e.g. RNases such as Cas13 proteins) and ribozymes which are capable of cleaving RNA. Examples of RNases include RNase A, RNase H, Cas13 proteins and Cas13 orthologues. Inone embodiment, the RNA-cleaving molecule is Cas13b protein. As used herein, the term “RNA-guided” in the context of an RNA-cleaving molecule includes either complementary base-pairing via an inherent sequence of the RNA-cleaving molecule (e g. a ribozyme recognizing a target RNA substrate via a complementary RNA sequence on the ribozyme), or the use of complementarity between an external RNA (e g. a guide RNA) and target nucleic acid sequences for recognition of genetic elements by the RNA-cleaving molecule. In various embodiments, the term “RNA-guided” may refer to the use of a specific RNA sequence (whether an additional entity such as gRNA, or an inherent entity such as RNA sequence on ribozyme itself) to targetan RNA substrate for cleavage. In various embodiments, RNA-guided RNA-cleaving molecules may form a complex with a gRNA. RNA-guided RNA-cleaving molecules include RNA-cleaving entities that are homed to specific target RNA sequence to perform RNA cleavage with the help of a gRNA, whereby the RNA-cleaving entity forms a complex with the gRNA prior to performing RNA cleavage. The gRNA facilitates recognition of targeted RNA for cleavage via complementary base pairing of the gRNA spacer sequence and the target RNA sequence. The term “RNA-guided RNA-cleaving molecules” also includes ribozymes which are guided by the complementarity within the ribozyme to its RNA target and that have the ability to perform RNA cleavage. Such ribozymes may also be known as “RNA- cleaving ribozymes” or “RNA-guided RNA-cleaving ribozymes”. In RNA-guided RNA-cleaving ribozymes, complementary base pairing forms the secondary structure of RNA necessary to facilitate cleavage. Such sequence that promotes base pairing can exist in nature, or be engineered into or appended to the DNA sequence that encodes the ribozyme.

[0044] In various embodiments, the incubation of the RNA-cleaving molecule with a target RNA under conditions suitable for cleavage of the target RNA by the RNA-cleaving molecule may comprise incubation of the RNA-cleaving molecule with the target RNA to form a RNA- cleaving molecule:target RNA complex, or incubation of the RNA-cleaving molecule and / or the target RNA with further entities such as gRNA.

[0045] In one embodiment, step (a) comprises incubating the RNA-cleaving molecule with a guide RNA (gRNA) to form an RNA-cleaving molecule:gRNA complex and incubating the RNA-cleaving molecule:gRNA complex with the target RNA under conditions suitable for cleavage of the target RNA by the RNA-cleaving molecule:gRNA complex to form cleaved RNA fragments.

[0046] In one embodiment, step (a) is carried out in vitro.

[0047] In one embodiment, the target RNA is synthesized using a T7 in vitro transcription system.

[0048] In one embodiment, the gRNA is synthesized using a T7 in vitro transcription system.

[0049] In one embodiment, the RNA-cleaving molecule is Cas13 protein.

[0050] In one embodiment, the Cas13 protein is Cas13b.

[0051] In one embodiment, the RNA-cleaving molecule is a ribozyme.

[0052] In one embodiment, the ribozyme is selected from the group consisting of a hairpin ribozyme and a hammerhead ribozyme.

[0053] As the method of the present disclosure would allow the mapping of cleaved RNA ends, it would be generally understood by the skilled person that the method of the present disclosure can also be applied on non-Cas13 modalities, such as on other RNases or RNA- cleaving ribozymes. The reaction buffer conditions for RNA-cleavage would be generally known to a person skilled in the art. For example, in the embodiment where the RNA-cleaving molecule is an RNA-cleaving ribozyme, the standard reaction conditions would include high Mg2+ concentrations and the temperatures may range from room temperature to 37°C.

[0054] In one embodiment, step (a) comprises incubating the RNA-cleaving molecule with the gRNA for 15 minutes at 37°C.

[0055] In one embodiment, step (a) comprises incubating the RNA-cleaving molecule:gRNA complex with the target RNA for 15 minutes at 37°C.

[0056] In one embodiment, after step (a), a stop solution is added to terminate the enzymatic activity of the RNA-cleaving molecule.

[0057] In one embodiment, the cleaved RNA fragments are separated on a denaturing polyacrylamide gel electrophoresis (PAGE) for visualization of the cleaved RNA fragments.

[0058] As used herein, the term “quantify cleavage sites” is meant to include quantifying the number of cleavage sites in a target RNA as well as the frequency / amount of cleavage at each cleavage site. The amount of cleavage at each cleavage site may be visualized as peaks in a line chart.

[0059] In one embodiment, the PAGE analysis serves as a quality control step before committing to a more costly procedure of Next Generation Sequencing in the following step. In one embodiment, as a routine quality control, the steps of library preparation and sequencing may not be carried out if no cleavage were observed in the PAGE analysis. This serves as a resource-conserving checkpoint. However, this checkpoint is optional.

[0060] The prevalent thinking in the field is that there is non-specific collateral cleavage once Cas13 is activated - this is an extrapolation from Cas13a, which is more well- characterized to date. Due to this prevalent thought, the skilled person would not consider it useful to sequence for cleavage sites, because doing so would generate randomly locatedcleavage patterns. Furthermore, the skilled person would expect RNAs to be cleaved so extensively that they are not retrievable for downstream processing. It is thus surprising and non-obvious that the cleavage patterns produced by Cas13b as disclosed in the present application are not randomly located. This prompted the present inventors to determine the RNA cleavage pattern of several Cas13b proteins, using the novel method involving end repair and ligation as presently disclosed.

[0061] In one embodiment, step (b) comprises a first dephosphorylation reaction to dephosphorylate 5’-triphosphate ends into 5’-hydroxyl RNA ends, a second dephosphorylation reaction to dephosphorylate 2’-3’ cyclic phosphate into 3’-hydroxyl RNA ends, and a phosphorylation reaction to phosphorylate the 5’-hydroxyl RNA ends into 5’-phosphate ends.

[0062] In one embodiment, the first dephosphorylation reaction is effected by adding Quick calf intestinal alkaline phosphatase (CIP) in CutSmart Buffer.

[0063] In one embodiment, the second dephosphorylation reaction is effected by adding T4 polynucleotide kinase (T4 PNK).

[0064] In one embodiment, the phosphorylation reaction is effected by adding ATP.

[0065] In one embodiment, step (d) comprises converting the adapter-ligated RNA fragments into a cDNA library via reverse transcription and sequencing the cDNA library.

[0066] In one embodiment, after step (d), the method as described herein further comprises the step of checking the plurality of sequenced reads for sequencing quality and for the presence of adapter sequences at the ends of the sequenced reads, followed by trimming the ends having low sequencing quality and having adapter sequences.

[0067] In one embodiment, step (f) further comprises normalizing the respective percentages by subtracting a control percentage corresponding to each base position from the respective percentages at each base position.

[0068] In one embodiment, the control percentage is derived from (i) a first control condition in which the RNA-cleaving molecule and gRNA are omitted; or (ii) a second control condition in which the gRNA is omitted.

[0069] In one embodiment, step (f) further comprises normalizing the respective percentages by dividing the respective percentages at each base position by a control percentage corresponding to each base position.

[0070] In one embodiment, step (f) further comprises adding a pseudocount at each base position prior to the step of dividing the respective percentages at each base position by the control percentage corresponding to each base position.

[0071] In one embodiment, step (g) comprises determining a reference level, wherein any percentage from step (f) that is above the reference level is indicative of an RNA cleavage site.

[0072] In one embodiment, step (g) comprises visualizing the frequencies of the 5’ base positions and 3’ base positions on a line chart in which a first axis of the line chart shows the nucleotide position of the DNA sequence of the target RNA and a second axis of the line chart shows the respective percentages, wherein any peak on the line chart having a value on the second axis that is higher than the reference level is indicative of an RNA cleavage site.

[0073] In another aspect, the present disclosure provides a use of the method as described herein for quantifying the number of cleavage sites in the target RNA and the frequency of cleavage at each cleavage site, wherein the number of cleavage sites in the target RNA is quantified based on the identification results of step (g) and wherein the frequency of cleavage at each cleavage site is quantified based on the measurement results of step (f).

[0074] In various embodiments, the frequencies of the 5’ base positions and 3’ base positions can be visualized on a line chart or a histogram format. In one embodiment, there could be a reference level of frequency above which the frequency would be indicative of an RNA cleavage site. The factors to be considered when determining the reference level include the variance of each sequencing run, the baseline sequencing error, and differences in sample preparation steps (e.g. the ratio of RNA-cleaving enzymes:gRNA:RNA substrate, the incubation time for RNA cleavage and the sequence of RNA substrate). For example, a noisier signal may have a higher variance and hence require a higher reference level to be statistically significant above the average baseline.

[0075] In one embodiment, a cut-off normalized frequency or average baseline (i.e. reference level) can be derived by averaging the frequencies at every base position across the target RNA sequence. Base positions with normalized frequencies exceeding the cut-off line (i.e. peaks) may be considered cleavage sites and the number of cleavage sites can be counted thereafter. As an alternative to using the average baseline described above as a cutoff (i.e. reference level), the reference level could be 1 standard deviation above the mean. Given the above, a person skilled in the art would be able to determine a reference level without undue experimentation.

[0076] The following is an example on how the average baseline (with and without the inclusion of standard deviation) can be derived as a reference level. Consider a scenario where the target RNA is 10 nucleotides long and the normalized read percentages at all 10 base positions are 0, 0, 2, 0, 0, 0, 4, 0, 0, 0. That will give rise to an average baseline (reference level) of (0+0+2+0+0+0+4+0+0+0)% / 10 = 0.6%. With a reference cut-off of 0.6%, both peaksat 2% and 4% will be considered cleavage sites. Alternatively, a higher reference cut-off level could be adjusted with the inclusion of 1 standard deviation (1 .35 in this case), therefore giving a stricter cleavage site determination (peaks with normalized reads > 1.95%).

[0077] The term “3’ end repair process” as used herein is meant to include the processing of the 3’ ends of the cleaved RNA fragments into 3’-hydroxyl (i.e. 3’ OH) ends. The term “5’ end repair process” as used herein is meant to include the processing of the 5’ ends of the cleaved RNA fragments into 5’ phosphate ends. The terms “5’ phosphate” and “5’ monophosphate” are used interchangeably.

[0078] In various embodiments, the stop solution functions to prevent the continued enzymatic cleaving action of the gRNA-Cas13 complex and may prevent the unintended cleavage of RNA / adaptors following the ligation step. It would be generally understood by the skilled person that other ways of terminating the enzymatic reaction such as temperature or pH can be used. However, extreme temperatures or pH might induce hydrolysis and affect the integrity of the small RNA fragments and thereby affect the downstream identification of cleavage sites. The skilled person would be capable of adjusting the composition of the stop solution to achieve the inactivation of the enzymatic reaction.

[0079] In one embodiment, the step of checking the plurality of sequenced reads for sequencing quality and for the presence of adapter sequences is effected using Fastqc software. In one embodiment, the step of trimming the ends having low sequencing quality and having adapter sequences is effected using Trimmomatic software. In one embodiment, the alignment in step (f) of the method as described herein is effected using Bowtie2 alignment software. In one embodiment, in step (f) of the method as described herein, the 5’ and 3’ ends of each sequencing read are identified using Pysam software.

[0080] Advantageously, the method of the present disclosure allows the identification of RNA cleavage site(s) down to single nucleotide resolution and also provides a quantitative measurement of cleavage patterns.

[0081] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features,modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0082] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0083] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0084] MATERIAL AND METHODS

[0085] Protocol for Quantification

[0086] Sequenced reads (presumably cleaved by a Cas13 protein such as Cas13b) were aligned to the full length target RNA sequence to determine the base positions on the target RNA where the 5’ (starting) and 3’ (ending) ends of sequenced reads aligned to. For every base position in the target RNA sequence, the number of 5’- and 3'-aligned reads were counted (with Pysam) and converted into percentages of the total number of aligned reads.

[0087] The step of incubating the Cas13 protein with a gRNA under conditions suitable for formation of a gRNA-Cas13 complex was performed for sequenced reads in the test sample (with Cas13, gRNA and target RNA) and a control sample (with the same Cas13 and target RNA only). To normalize the frequencies (%) of aligned reads (for 5’ and 3’) at every base position, the frequencies in the test sample were subtracted from the frequencies in the control sample.

[0088] The normalized frequencies (%) were then plotted on a line chart at the y-axes with the base positions of the target RNA sequence forming the x-axes.

[0089] A cut-off normalized frequency can be derived by averaging the frequencies at every base position across the target RNA sequence. Base positions with normalized frequencies exceeding the cut-off line (peaks) will be considered cleavage sites and the number of cleavage sites can be counted thereafter.

[0090] EXAMPLES

[0091] Non-limiting examples of the invention and comparative examples will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

[0092] Example 1 : General Methodology

[0093] Target RNA of interest (<150 nucleotides long) to be cleaved and guide RNA (gRNA) are synthesized and purified using T7 polymerase from DNA templates with an upstream T7 promoter each. RNA-cleaving Cas13b were expressed in E.coli and purified with affinity columns (Nickel and Streptavidin agarose columns). The Cas13b proteins expressed are fused with 6xHis and Streptavidin tags at terminal ends. To facilitate effective RNA cleavage of target RNA by Cas13b, the Cas13b is pre-incubated with gRNA for 15 mins at 37oC before adding in target RNA for cleavage for another 15 mins of incubation at 37°C for target RNA cleavage. Upon RNA cleavage, the enzymatic activity is terminated with the enzyme stop solution and the mixture of cleaved RNA fragments is separated on a denaturing acrylamide gel, which is subsequently stained with SYBR Gold for RNA fragment visualization. The stained gel shows the cleaved RNA fragments of different sizes as an indication of Cas13b-mediated RNA cleavage.

[0094] Cleaved RNA fragments are then enzymatically-treated to process both 5’ and 3’ ends of cleaved RNA fragments that are not 5’-phosphate (5’-P) and 3’-hydroxyl (3’-OH) ends. The processed RNA fragments (5’-P and 3’-OH ends) can then be ligated with adapter sequences for library preparation of short RNA sequencing (NEBNext Small RNA Library Prep for Illumina Sequencing). Sequenced reads are then quality-checked with Fastqc. Ends with low sequencing quality and adaptor sequences are then trimmed with Trimmomatic. Trimmed reads are subsequently aligned (global alignment) to the full length sequence of target RNA of interest via Bowtie2 with the default settings. The base positions of RNA fragment ends (5’ and 3’) relative to the target RNA of interest are identified for each sequenced read. The number of corresponding reads with its 5’ end (start) aligned at each base position of the target RNA of interest is quantified. The same is determined for the 3’ end (end) base alignment. The quantified number of reads is converted into percentages of the total number of reads. A control condition where no gRNA is added in the mixture (hence no target RNA cleavage expected) is also prepared, forming the baseline for normalization. The quantified number of reads (%) for test conditions, where positive RNA cleavage is detected (gRNA added), is normalized against the control condition to minimize background noise. Normalization is performed through subtraction of the percentages of quantified reads (test - control) at each base position.

[0095] Alternatively, normalization could also be performed using division (test I control) at each base position. However, to circumvent the potential issue of the undefined outcome of division when the control percentage at a specific base position is 0, the normalization may be performed using pseudocount before division (i.e. adding a pseudocount to every base position in the control used for normalization and subsequently dividing the percentages at each base position by the respective percentages from the control). For example, consider a scenario where the target RNA is 10 nucleotides long, and the counts at the 10 base positions are 0, 0, 20, 0, 0, 0, 10, 0, 0, 0 in the test sample and 0, 0, 1 , 0, 0, 0, 4, 0, 0, 0 in the control sample. Using the subtraction approach, the normalized count will be 0, 0, 19, 0, 0, 0, 6, 0, 0, 0. Using the division approach, and a pseudocount of 1 , the normalized count will be 0 / (0+1), 0 / (0+1), 20 / (1 +1), 0 / (0+ 1), 0 / (0+1), 0 / (0+1), 10 / (4+1), 0 / (0+ 1), 0 / (0+1), 0 / (0+ 1), which evaluates to 0, 0, 10, 0, 0, 0, 2, 0, 0, 0.

[0096] A line chart of normalized read percentage against the base position with respect to the target RNA of interest is subsequently plotted to identify and quantify the cleavage sites and sequence motifs specific to different variants of Cas13b and / or gRNAs.

[0097] Example 2: Detailed Methodology

[0098] In one embodiment, the method to map RNA cleavage by Cas13b begins with the design and synthesis of target RNA and gRNA using T7 in vitro transcription system (Figure 1). The DNA templates to be transcribed consist of the T7 promoter (5’ - TAATACGACTCACTATA - 3’) (SEQ ID NO: 1), followed by ‘GG’ and the respective desired sequences. The ‘GG’ dinucleotides help to improve the in vitro RNA yield in a T7 transcription system.

[0099] Here, the target RNA sequence is 129 nucleotides long (129 bp in DNA template) and spans across exons 2 and 3 (spliced) of the KRAS gene (Transcript ID: ENST00000311936.8). The size of the transcribed target RNA was deliberately limited to within 150 nucleotides as this is the recommended maximum length of RNAs that could be processed using the NEBNext® Small RNA Library Prep for Illumina (NEB). Meanwhile, the gRNAs were made up of a 30-nucleotide spacer sequence targeting different regions of the target RNA, followed by a gRNA scaffold sequence specific to the Cas13b variants.

[0100] To purify the in vitro transcribed RNAs, the in vitro transcription reaction products were run on a denaturing PAGE gel containing 7M urea, the gel was stained with SYBR Gold, and RNA fragments of the right sizes were extracted using the Zymoclean Gel RNA Recovery Kit (Zymo Research).

[0101] To generate sufficient Cas13b proteins, recombinant PspCas13b and RanCas13b proteins were expressed separately in Rosetta2 (DE3) competent cells in the presence of 200pM IPTG at 18°C for 20 hours once the bacterial growth reached mid-log phase (OD600nm~0.5). Bacterial cells were lysed chemically with a Cell Lysis Buffer (50 nM HEPES, 500 mM NaCI, 2 mM MgCl2, 1 % v / v Triton X-100, 1 mM DTT, 1 mg / ml lysozyme, 2.5 U / ml DNase protease inhibitor cocktail (1 tablet per 10 ml buffer), 20 mM imidazole), followed by sonication on ice (20 mins; 30s ON, 20s OFF). The cell lysates were filtered (0.45 m PVDF filter) before being passed through Nickel and Streptavidin affinity columns (recombinant proteins were tagged with His- and Strep-Tag at terminal ends) to purify the Cas13b proteins.

[0102] To perform RNA cleavage in vitro, an 18 pl reaction was set up by first mixing and incubating 200 nM Cas13b protein with 100 nM gRNA in RNA Cleavage Buffer (40 mM Tris- HCI pH 7.4, 60 mM NaCI, 6 mM MgCl2, 1 U / ul RNase inhibitor, Murine) at 37°C for 15 mins. This step assembles Cas13b-gRNA complexes that are ready to engage the target RNA for cleavage. Thereafter, purified target RNA was added into this reaction mixture, to a final concentration of 500 nM. The reaction was then incubated at 37°C for 15 mins to allow RNA cleavage. Finally, the nuclease activity from Cas13b was terminated by adding 1 pl of Enzyme Stop Solution (10 mg / ml Proteinase K, 4M Urea, 20 mM Tris Buffer pH 8.0, 80 mM EDTA) into the mixture before a final incubation at 37°C for 15 mins. 10 pl (half) of the in vitro cleavage reaction mixture was removed to perform denaturing (7M urea) PAGE analysis. The presence of smaller RNA fragments (<129 nt long) indicates successful RNA cleavage by Cas13b (Figure 3).

[0103] The remaining half of the in vitro cleavage reaction mixture was then cleaned up with the Monarch® RNA Cleanup Kit (NEB) to remove proteins and other contaminants that could potentially interfere with downstream library preparation for RNA sequencing (Figure 2).

[0104] As the detailed biochemical mechanism of Cas13b-mediated RNA cleavage remains unknown, it was hypothesized that Cas13b work similarly to other HEPN RNases and would generate 2’-3’ cyclic phosphate and 5’-OH RNA ends. On the other hand, T7 polymerase-transcribed RNAs have 5’-PPP ends. Such RNA ends are not amenable for adaptor ligation in the library preparation for Illumina sequencing wherein 3’-OH and 5’- phosphate RNA ends are required (using NEBNext® Small RNA Library Prep for Illumina). Therefore, the purified RNA fragments were first treated with QuickClP in CutSmart Buffer at 37°C for 20 mins to dephosphorylate 5’-PPP ends. The dephosphorylated RNAs were then cleaned up with the Monarch® RNA Cleanup Kit. This is followed by a second dephosphorylation reaction by adding 10 U of T4 Polynucleotide Kinase (T4 PNK), in the absence of ATP, to the RNAs and incubating at 37°C for 30 mins. As a bifunctional enzyme, T4 PNK has a 3’-terminal phosphatase activity that could remove 2’-3’ cyclic phosphate at the HEPN-mediated RNA cleavage sites. Subsequently, ATP was added, to a final concentrationof 1 mM, to the reaction before incubating at 37°C for 30 mins to phosphorylate all the 5’-OH RNA ends (both generated from CIP-mediated dephosphorylation of 5’-PPP and from HEPN- mediated cleavage). Monarch® RNA Cleanup Kit was then used to purify and concentrate treated RNA fragments after this last phosphorylation step. Thereafter, the RNA fragments should possess 5’-P and 3’-OH ends, ready for adaptor ligation in library preparation.

[0105] To prepare the processed RNA fragments for sequencing, the NEBNext® Small RNA Library Prep for Illumina kit was used to directly join adapter and barcode sequences to both ends of the RNA fragments while converting the RNA into cDNA via reverse transcription. The cDNAs were further PCR-amplified for 15 cycles according to manufacturer’s protocol before performing DNA column purification. A 6 pl aliquot of the purified cDNA mixture was separated on a TBE 6% polyacrylamide gel before staining with SYBR Gold to visually check whether the cDNA library was successfully generated. The concentration of cDNA library was quantified with Qubit™ dsDNA High Sensitivity Kit before multiplexing and diluting the multiplexed cDNA library according to iSeqIOO manufacturer’s protocol. The cDNA library was sequenced on an iSeqIOO (Illumina) on a single-end read setting with a spike-in of Phi-X at 5 - 20% while ensuring 100,000 - 200,000 read counts per sample.

[0106] The sequencing reads were preliminarily checked for sequencing quality and the presence of adapter sequences at the terminal ends of reads with Fastqc. Reads shorter than 10 nucleotides were removed and read ends with low base calling quality (average base calling quality value in a 5-nucleotide window <20) were trimmed with Trimmomatic (with the following parameters - LEADING:20; TRAILING:3; SLIDINGWINDOW:5:20; MINLEN:10).

[0107] To identify RNA cleavage site(s) on the target RNA generated by the Cas13b-gRNA complex, sequencing reads from the fastq file were aligned to the full-length target RNA sequence (in DNA template format) as a reference using Bowtie2 with global alignment default setting. Any reads that do not entirely align to the target RNA sequence (e.g., sequence of the gRNA scaffold) were excluded. The starting (5’) and ending (3’) positions where each sequenced read aligned to (numbered nucleotide from 5’ to 3’ relative to the target RNA sequence) were extracted from the SAM output file with Pysam. The frequency of corresponding reads having their 5’-end nucleotides aligned to each nucleotide position of the target RNA sequence were quantified as a percentage against the total number of aligned reads. Similarly, the frequency at each position with 3’-end nucleotide read’s alignment was determined. Two control samples - (1) Target RNA only, (2) Target RNA + Cas13b (without gRNA) - were analyzed in parallel with the target RNA + Cas13b-gRNA in vitro cleavage reaction products. The controls were used as a control baseline for normalization (subtraction of alignment frequencies at each position). Control (2) was initially verified to be non-significantly different against control (1) before using it to normalize subsequent test conditions (samples with added gRNA). Once normalized, the frequency of the start (5’) and end (3’) of read alignment (normalized read count) at each position was visualized on a line chart where the x-axis denotes the nucleotide position of the target RNA sequence, and the y-axis shows the normalized read counts (%). Any peak on the plot represents an RNA cleavage site: an light grey peak indicates an increased proportion of RNAs terminating at this position (3’ ends of RNA fragments), while a dark grey peak indicates an increased proportion of RNAs beginning at this position (5’ ends). Positions containing adjacent light grey and dark grey peaks (in particular, an light grey peak followed by a dark grey peak) indicate high-confidence RNA cleavage sites. The identified cleavage site(s) could be further compared against the denaturing PAGE to validate the cleavage activity of specific Cas13b, with the caveat that PAGE electrophoresis does not give single nucleotide resolution that the present invention offers. Importantly, the line chart provides cleavage data at single-nucleotide resolution to identify and quantify RNA cleavage sites specific to each Cas13b and gRNA complex (Figure 4).

[0108] Analyzing gRNA 1 alone, PspCas13b cleaves RNAs after nucleotide A and not before nucleotide C. On the other hand, RanCas13b cleaves after nucleotide U and not before nucleotide C. Hence, the preferred RNA cleavage dinucleotide motifs are AD and UD (D: A / U / G) respectively.

[0109] Using the above workflow, 5 additional gRNAs were designed and tested (Table 1 ; Figures 5, 6 and 7).

[0110] Table 1 : Target RNA sequence and gRNA sequences. For the gRNA sequences, the spacer sequences are in non-bold font, while the respective hairpin sequences for Psp Cas13b and Ran Cas13b are in bold.Table 1.

[0111] The method of the present disclosure enables the high-resolution mapping of sequence contexts that could affect Cas13b cleavage activity. It was previously reported that another Cas13 ortholog, Leptotrichia shahii Cas13a (LshCas13a), had specific protospacer flanking sequence (PFS) requirements for in vitro cleavage activity and in bacterial cells. The PFS is the RNA sequence directly flanking the on-target protospacer sequence. In addition, PFS requirements were also previously observed for some Cas13b orthologs’ cleavage activity in bacterial cells. Noticing that some of the gRNAs did not result in cleavage of the target RNA (Figure 3), the present inventors investigated if there might be specific protospacer flanking sites (PFS) that alter PspCas13b and RanCas13b cleavage activity.

[0112] A 129-bp target RNA was created, where the sequences flanking gRNA 1 protospacer were replaced with random 3-nt sequences. This target RNA was then subjected to the same in vitro cleavage assay workflow as described above. However, instead of analyzing all RNA fragments including the cleavage products, the present inventors sequenced only full-length uncleaved target RNAs to identify potential PFSs that enhance / inhibit Cas13b cleavage activity (Figures 8 and 9). Here it was determined that PspCas13b has a 5’ NGC preference (where “N” can be any nucleotide), while RanCas13b has a 5’ NGC and a 3’ CCN preference. This indicates that targeting RNA sequences harboring these PFS sequences will lead to higher editing efficiency. The method of the present disclosure hence enables mapping Cas13 targeting requirements (PFS), quantifies on-target cleavage efficiency, identifies off-target collateral cleavage sites in cis, and quantifies the off-target cleavage frequencies on these off-target sites.

[0113] While embodiments of the invention have been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims. The scope of the invention is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.

Claims

CLAIMS1 . A method for identifying one or more RNA cleavage sites of an RNA-cleaving molecule, the method comprising:(a) incubating the RNA-cleaving molecule with a target RNA under conditions suitable for cleavage of the target RNA by the RNA-cleaving molecule to form cleaved RNA fragments;(b) processing the 5’ and 3’ ends of the cleaved RNA fragments into 5’ phosphate and 3’-hydroxyl ends respectively;(c) ligating the processed cleaved RNA fragments with adapter sequences to generate a sequencing library;(d) sequencing the sequencing library to obtain a plurality of sequenced reads;(e) aligning the plurality of sequenced reads to a DNA sequence of the target RNA and identifying a 5’ base position and a 3’ base position for each sequencing read, wherein the 5’ base position is the nucleotide in the DNA sequence of the target RNA that is aligned with the 5’ end of each sequencing read and wherein the 3’ base position is the nucleotide in the DNA sequence of the target RNA that is aligned with the 3’ end of each sequencing read;(f) measuring the frequency of 5’ base positions and of 3’ base positions of the plurality of sequencing reads as respective percentages against a total number of aligned sequencing reads; and(g) identifying the one or more RNA cleavage sites based on the measurement results of step (f), wherein an increased frequency of 5’ base positions is indicative of an RNA cleavage site and wherein an increased frequency of 3’ base positions is indicative of an RNA cleavage site.

2. The method of claim 1, wherein step (a) comprises incubating the RNA-cleaving molecule with a guide RNA (gRNA) to form an RNA-cleaving molecule:gRNA complex and incubating the RNA-cleaving molecule:gRNA complex with the target RNA under conditions suitable for cleavage of the target RNA by the RNA-cleaving molecule:gRNA complex to form cleaved RNA fragments.

3. The method of claim 1 or 2, wherein step (a) is carried out in vitro.

4. The method of any one of claims 1 to 3, wherein the RNA-cleaving molecule is Cas13 protein.

5. The method of claim 4, wherein the Cas13 protein is Cas13b.

6. The method of any one of claims 1 to 3, wherein the RNA-cleaving molecule is a ribozyme.

7. The method of any one of claims 2 to 6, wherein step (a) comprises incubating the RNA-cleaving molecule with the gRNA for 15 minutes at 37°C.

8. The method of any one of claims 2 to 7, wherein step (a) comprises incubating the RNA-cleaving molecule:gRNA complex with the target RNA for 15 minutes at 37°C.

9. The method of any one of claims 1 to 8, wherein after step (a), a stop solution is added to terminate the enzymatic activity of the RNA-cleaving molecule.

10. The method of any one of claims 1 to 9, wherein the cleaved RNA fragments are separated on a denaturing polyacrylamide gel electrophoresis (PAGE) for visualization of the cleaved RNA fragments.

11. The method of any one of claims 1 to 10, wherein step (b) comprises a first dephosphorylation reaction to dephosphorylate 5’-triphosphate ends into 5’-hydroxyl RNA ends, a second dephosphorylation reaction to dephosphorylate 2’-3’ cyclic phosphate into 3’-hydroxyl RNA ends, and a phosphorylation reaction to phosphorylate the 5’-hydroxyl RNA ends into 5’-phosphate ends.

12. The method of any one of claims 1 to 11 , wherein step (d) comprises converting the adapter-ligated RNA fragments into a cDNA library via reverse transcription and sequencing the cDNA library.

13. The method of any one of claims 1 to 12, wherein after step (d), the method further comprises the step of checking the plurality of sequenced reads for sequencing quality and for the presence of adapter sequences at the ends of the sequenced reads,followed by trimming the ends having low sequencing quality and having adapter sequences.

14. The method of any one of claims 1 to 13, wherein step (f) further comprises normalizing the respective percentages by subtracting a control percentage corresponding to each base position from the respective percentages at each base position.

15. The method of claim 14, wherein the control percentage is derived from (i) a first control condition in which the RNA-cleaving molecule and gRNA are omitted; or (ii) a second control condition in which the gRNA is omitted.

16. The method of any one of claims 1 to 13, wherein step (f) further comprises normalizing the respective percentages by dividing the respective percentages at each base position by a control percentage corresponding to each base position.

17. The method of claim 16, wherein step (f) further comprises adding a pseudocount at each base position prior to the step of dividing the respective percentages at each base position by the control percentage corresponding to each base position.

18. The method of any one of claims 1 to 17, wherein step (g) comprises determining a reference level, wherein any percentage from step (f) that is above the reference level is indicative of an RNA cleavage site.

19. The method of claim 18, wherein step (g) comprises visualizing the frequencies of the 5’ base positions and 3’ base positions on a line chart in which a first axis of the line chart shows the nucleotide position of the DNA sequence of the target RNA and a second axis of the line chart shows the respective percentages, wherein any peak on the line chart having a value on the second axis that is higher than the reference level is indicative of an RNA cleavage site.

20. Use of the method of any one of claims 1 to 19 for quantifying the number of cleavage sites in the target RNA and the frequency of cleavage at each cleavage site, wherein the number of cleavage sites in the target RNA is quantified based on the identification results of step (g) and wherein the frequency of cleavage at each cleavage site is quantified based on the measurement results of step (f).

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