WO2024259135A1

WO2024259135A1 - Assays for analysis of ribonucleic acid (rna) molecules

Info

Publication number: WO2024259135A1
Application number: PCT/US2024/033854
Authority: WO
Inventors: Christine Kitsos; Yan Tiffany LAI; Kutralanathan Renganathan; Jing Yang; Nicholas NISBETT
Original assignee: Intellia Therapeutics Inc
Current assignee: Intellia Therapeutics Inc
Priority date: 2023-06-13
Filing date: 2024-06-13
Publication date: 2024-12-19
Anticipated expiration: 2025-12-13

Abstract

In certain aspects, the disclosure relates to a method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, e.g., a sample comprising lipid nanoparticles, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises an RNA region and a DNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (b) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting the presence or absence of at least one of the fragments of the RNA molecule. In some embodiments, the RNA molecule is a guide RNA, e.g., a sgRNA. In some embodiments, the method can be used to confirm the identity of the RNA molecule in the sample. In certain aspects, the disclosure relates to methods of quantifying an RNA molecule in a sample.

Description

ASSAYS FOR ANALYSIS OF RIBONUCLEIC ACID (RNA) MOLECULES

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/472,686 filed on June 13, 2023, the contents of which are incorporated by reference herein in their entirety.

BACKGROUND

[0002] The number of ribonucleic acid- (RNA)- based therapeutics has grown significantly in the past 20 years, and the number is expected to further increase as many RNA therapeutics are being tested in late-stage clinical trials. See Kim, 2022, Experimental and Molecular Medicine 54: 455-465. Accordingly, a need exists for methods of confirming the identity of an RNA molecule in a sample (e.g., for quality control of RNA-based therapeutics), and for methods of quantifying an RNA molecule in a sample e.g., for confirmation of sgRNA activity when compared to reference standard).

SUMMARY

[0003] As the number of RNA-based therapeutics in clinical trials and approved for clinical use continues to increase, a need exists for improved methods of confirming the identity of these therapeutics during manufacturing and clinical evaluation. The present disclosure meets this need by providing novel methods of detecting the presence of an RNA molecule in a sample that may be used, for example, to confirm the identity of an RNA-based therapeutic for clinical use. The present disclosure also provides novel methods of quantifying RNA molecules using an oligonucleotide probe.

[0004] For example, in certain aspects, the present disclosure relates to a method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample. In one embodiment, the method comprises: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (b) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule. In one embodiment, the contacting in step (a) allows the oligonucleotide to anneal to the RNA molecule.

[0005] In certain aspects, the disclosure relates to a method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (b) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

[0006] In some embodiments, the oligonucleotide further comprises an upstream RNA region, wherein a 3’ end of the upstream RNA region is covalently attached to a 5’ end of the DNA region. In some embodiments, the oligonucleotide is from about 10 to about 30 nucleotides in length. In some embodiments, the DNA region of the oligonucleotide is from about 2 to about 10 nucleotides in length. In some embodiments, the DNA region of the oligonucleotide is about 4 nucleotides in length. In some embodiments, the downstream RNA region of the oligonucleotide is from about 10 to about 25 nucleotides in length. In some embodiments, the upstream RNA region of the oligonucleotide is from about 1 to about 10 nucleotides in length. In some embodiments, at least one ribonucleotide in the oligonucleotide comprises at least one chemical modification. In some embodiments, the chemical modification is a 2’-O-methyl modification. In some embodiments, the at least two fragments of the RNA molecule are each at least about 10 nucleotides in length. In some embodiments, the at least two fragments of the RNA molecule are different sizes. In some embodiments, the at least two fragments differ in size by at least about 20 nucleotides. In some embodiments, the at least two fragments differ in size by about 50 to about 120 nucleotides. In some embodiments, the at least two fragments differ in size by at least about 100 nucleotides. In some embodiments, detecting the absence of at least one of the fragments of the RNA molecule indicates that the sample does not comprise the RNA molecule.

[0007] In some embodiments, the detecting step is performed using liquid chromatography-mass spectrometry (LC-MS), or liquid chromatography-ultraviolet (LC- UV). In some embodiments, the detecting step is performed using liquid chromatographymass spectrometry (LC-MS). In some embodiments, the method further comprises isolating the RNA molecule from a lipid nanoparticle (LNP) in the sample before step (a).

[0008] In some embodiments, isolating the RNA molecule from the LNP comprises deformulating the LNP in ethanol. In some embodiments, the method further comprises separating the at least two fragments of the RNA molecule from each other before the detecting step. In some embodiments, step (a) comprises heating at a temperature and for a time sufficient to denature secondary structure in the RNA molecule. In some embodiments, step (a) comprises heating at about 75°C for about 1 to about 10 minutes and cooling at room temperature. In some embodiments, step (b) comprises incubating the sample with RNase H for about 30 minutes to about three hours at about 37°C. In some embodiments, the RNA molecule is from about 40 to about 200 nucleotides in length. In some embodiments, the RNA molecule comprises a crRNA molecule.

[0009] In some embodiments, the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence. In some embodiments, the downstream RNA region of the oligonucleotide is complementary to the targeting sequence of the sgRNA. In some embodiments, the DNA region of the oligonucleotide is complementary to the targeting sequence of the sgRNA. In some embodiments, when the sgRNA is present in the sample, the RNase H binds to the duplex and cleaves the sgRNA within 5 nucleotides of a 3’ end of the targeting sequence. In some embodiments, when the sgRNA is present in the sample, the RNase H binds to the duplex and cleaves the sgRNA within 2 nucleotides of a 3’ end of the targeting sequence. In some embodiments, when the sgRNA is present in the sample, the RNase H binds to the duplex and cleaves the sgRNA at a 3’ end of the targeting sequence. In some embodiments, when the sgRNA is present in the sample, the RNase H binds to the duplex and cleaves the sgRNA at a position from about 10 to about 30 nucleotides from the 5’ end of the sgRNA. In some embodiments, when the sgRNA is present in the sample, the RNase H binds to the duplex and cleaves the sgRNA at a position from about 20 to about 24 nucleotides from the 5’ end of the sgRNA. In some embodiments, the DNA region of the oligonucleotide is complementary to a region of the sgRNA that is from about 10 to about 30 nucleotides from the 5’ end of the sgRNA. In some embodiments, the at least two fragments of the sgRNA comprise a first fragment of the sgRNA and a second fragment of the sgRNA, and wherein the first fragment of the sgRNA is about 10 to about 30 nucleotides in length, and wherein the second fragment of the sgRNA is about 70 to about 90 nucleotides in length. [0010] In some embodiments, the at least two fragments of the sgRNA comprise a first fragment comprising the targeting sequence and a second fragment, and detecting the absence of the first fragment comprising the targeting sequence indicates that the sample does not comprise the RNA molecule.

[0011] In some embodiments, the sample further comprises an mRNA molecule. In some embodiments, the mRNA molecule encodes an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is a Cas nuclease. In some embodiments, the method further comprises removing the mRNA molecule from the sample before the contacting step (a).

[0012] In some embodiments, the RNA molecule is from about 40 to about 6000 nucleotides in length. In some embodiments, the RNA molecule is from about 1000 to about 6000 nucleotides in length. In some embodiments, the RNA molecule is a messenger RNA (mRNA).

[0013] In some embodiments, the sample does not comprise a cell. In some embodiments, the RNA molecule is not comprised within a cell.

[0014] In certain aspects, the disclosure relates to a method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence encapsulated in a lipid nanoparticle (LNP), the method comprising: (a) deformulating the LNP; (b) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (c) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (d) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

[0015] In certain aspects, the disclosure relates to a method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, an upstream RNA region, a deoxyribonucleic acid (DNA) region, and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (b) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

[0016] In certain aspects, the disclosure relates to a method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) about 80 nucleotides to about 130 nucleotides in length and comprises a targeting sequence, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, an upstream RNA region, a deoxyribonucleic acid (DNA) region, and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (b) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the oligonucleotide binds to at least a portion of the targeting sequence of the RNA molecule, and wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

[0017] In certain aspects, the disclosure relates to a method of quantifying a ribonucleic acid (RNA) molecule in a sample, the method comprising:

(a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region, thereby forming a duplex comprising the RNA molecule and the oligonucleotide;

(b) contacting the sample with RNase H; wherein the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and

(c) detecting at least one of the fragments of the RNA molecule to generate a measurement of the at least one fragment of the RNA molecule; and

(d) comparing the measurement of the at least one fragment of the RNA molecule to a standard curve generated from serially diluted solutions of a reference standard containing known quantities of the RNA molecule, thereby quantifying the RNA molecule in the sample.

[0018] In some embodiments, the method further comprises generating the standard curve by:

(i) subjecting the serially diluted solutions of the reference standard containing known quantities of the RNA molecule to steps (a), (b) and (c); and

(ii) calculating a correlation between measurements obtained in step (c) for the serially diluted solutions of the reference standard and the known quantities of the RNA molecules in the serially diluted solutions of the reference standard.

[0019] In some embodiments, the standard curve is generated for only one of the fragments of the RNA molecule. In some embodiments, standard curves are generated for each of at least two fragments of the RNA molecule.

[0020] In certain aspects, the disclosure relates to a method of quantifying a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence encapsulated in a lipid nanoparticle (LNP), the method comprising:

(a) deformulating the LNP;

(b) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region, thereby forming a duplex comprising the RNA molecule and the oligonucleotide;

(c) contacting the sample with RNase H; wherein the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and

(d) detecting at least one of the fragments of the RNA molecule to generate a measurement of the at least one fragment of the RNA molecule; and

(e) comparing the measurement of the at least one fragment of the RNA molecule to a standard curve generated from serially diluted solutions of a reference standard containing known quantities of the RNA molecule, thereby quantifying the RNA molecule in the sample.

[0021] In certain aspects, the disclosure relates to a method of quantifying a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence, the method comprising:

[0022] In certain aspects, the disclosure relates to a method of quantifying a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) about 80 nucleotides to about 130 nucleotides in length and comprises a targeting sequence, the method comprising:

(a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region, thereby forming a duplex comprising the RNA molecule and the oligonucleotide, wherein the oligonucleotide binds to at least a portion of the targeting sequence of the RNA molecule;

BRIEF DESCRIPTION OF THE FIGURES

[0023] Figure 1 shows a general overview of an exemplary assay for detecting the presence of a guide RNA in a sample.

[0024] Figure 2 shows a separation chromatogram for an assay for detecting gRNA-A and gRNA-B in a sample using oligonucleotide probes OP-A and OP-B, respectively.

Samples were analyzed using Liquid Chromatography-Ultraviolet (LC-UV). [0025] Figure 3 shows a separation chromatogram for an assay for detecting gRNA-A and gRNA-B in samples comprising a gRNA and Cas9 mRNA, using oligonucleotide probes OP-A and OP-B, respectively. Samples were analyzed using Liquid Chromatography- Ultraviolet (LC-UV) and Liquid Chromatography-Mass spectrometry (LC-MS).

[0026] Figure 4 shows a separation chromatogram for demonstrating the specificity of an assay for detecting gRNA-A and gRNA-B in samples comprising mRNA, using oligonucleotide probes OP-A and OP-B, respectively. Samples were analyzed using Liquid Chromatography-Ultraviolet (LC-UV) and Liquid Chromatography-Mass spectrometry (LC- MS).

[0027] Figure 5 shows a separation chromatogram for an assay for detecting gRNA-A and gRNA-B in LNP-A and LNP-B in a sample using oligonucleotide probes OP-A and OP- B, respectively. Samples were analyzed using Liquid Chromatography-Ultraviolet (LC-UV) and Liquid Chromatography-Mass spectrometry (LC-MS).

[0028] Figure 6 shows an overview of an exemplary process for generating a standard curve for quantifying an sgRNA molecule in a sample.

[0029] Figure 7 shows chromatograms generated by IP-RP UPLC for a reference standard (top panel) and a sample containing gRNA-B (bottom panel).

DETAILED DESCRIPTION

[0030] In certain aspects, the present disclosure relates to a method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (b) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

[0031] In certain aspects, the present disclosure relates to a method of quantifying a ribonucleic acid (RNA) molecule in a sample, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region, thereby forming a duplex comprising the RNA molecule and the oligonucleotide;

(d) comparing the measurement of the at least one fragment of the RNA molecule to a standard curve generated from_serially diluted solutions of a reference standard containing known quantities of the RNA molecule, thereby quantifying the RNA molecule in the sample.

I. Definitions

[0032] The terms “oligonucleotide” and “oligonucleotide probe” are used herein interchangeably to refer to a single-stranded multimer of nucleotides from 10 to 500 nucleotides in length, e.g., 10 to 100 nucleotides in length. Oligonucleotides may be synthetic or may be made enzymatically. In some embodiments, the oligonucleotide contains a combination of ribonucleotides and deoxyribonucleotides. In some embodiments, the oligonucleotide comprises at least one DNA region and at least one RNA region.

[0033] The term “DNA region” as used herein with respect to an oligonucleotide refers to a portion of the oligonucleotide comprising contiguous deoxyribonucleotides. In some embodiments, the DNA region can comprise from 2 to 50 deoxyribonucleotides.

[0034] The term “RNA region” as used herein with respect to an oligonucleotide refers to a portion of the oligonucleotide comprising contiguous ribonucleotides. In some embodiments, the RNA region can comprise from 2 to 50 ribonucleotides.

[0035] In some embodiments, the RNA region is an upstream RNA region. The term “upstream RNA region” as used herein refers to an RNA region in an oligonucleotide that is upstream of a DNA region in the oligonucleotide. In some embodiments, the upstream RNA region is directly upstream of the DNA region, i.e., a 3’ end of the upstream RNA region is covalently attached to a 5’ end of the DNA region.

[0036] In some embodiments, the RNA region is a downstream RNA region. The term “downstream RNA region” as used herein refers to an RNA region in an oligonucleotide that is downstream of a DNA region in the oligonucleotide. In some embodiments, the downstream RNA region is directed downstream of the DNA region, i.e., a 5’ end of the downstream RNA region is covalently attached to a 3’ end of the DNA region.

[0037] The term “complementary” as used herein refers to a nucleotide sequence (e.g, an oligonucleotide) that base-pairs by non-covalent bonds to a region of a target nucleic acid, e.g, an RNA molecule. In the canonical Watson-Crick base pairing, adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA. As such, A is complementary to T and G is complementary to C. In RNA, thymine is replaced by uracil (U). In RNA, A is complementary to U and vice versa. Typically, “complementary” refers to a nucleotide sequence that is at least partially complementary. This term also encompasses duplexes that are fully complementary such that every nucleotide in one strand is complementary to every nucleotide in the other strand in corresponding positions. In certain cases, a nucleotide sequence may be partially complementary to a target, in which not all nucleotides are complementary to every nucleotide in the target nucleic acid in all the corresponding positions. For example, the oligonucleotide may be completely (i.e., 100%) complementary to the target RNA molecule, or the oligonucleotide may share some degree of complementarity to the target RNA molecule which is less than 100% (e.g., 80, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence complementarity). For example, in some embodiments the oligonucleotide and the RNA molecule may contain at least one mismatch, e.g., 1 , 2 , 3 , or 4 mismatches. The percent complementarity of two nucleotide sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment). The nucleotides at corresponding positions are then compared, and the percent sequence complementarity between the two sequences is a function of the number of complementary positions shared by the sequences (i.e., % complementarity = # of complementary positions/total # of positions* 100). When a position in one sequence is occupied by a complementary nucleotide as the corresponding position in the other sequence, then the molecules are complementary at that position. A non-limiting example of such a mathematical algorithm is described in Karlin et aL, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul et aL, Nucleic Acids Res. 25:389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. In one aspect, parameters for sequence comparison can be set at score=100, wordlength=12, or can be varied (e.g., wordlength=5 or wordlength=20). [0038] The terms “Guanine-Cytosine content” and “GC content” are used herein interchangeably to refer to the percentage of nitrogenous bases in a polynucleotide (e.g., an oligonucleotide or RNA molecule) that are either guanine (G) or cytosine (C).

[0039] The term “duplex” as used herein refers to the structure formed when two nucleic acid molecules, e.g., an RNA molecule and an oligonucleotide, are non-covalently bound together through Watson and Crick base pairing.

[0040] The terms “single guide RNA” and “sgRNA” are used herein interchangeably to refer to an RNA molecule comprising a targeting sequence and a sequence that interacts with an RNA-guided DNA binding agent. In some embodiments, the sgRNA comprises a crRNA (or a portion thereof) comprising a targeting sequence covalently linked to a tracrRNA. In some embodiments, the crRNA and the tracrRNA are covalently linked via a linker.

[0041] The terms “CRISPR RNA” and “crRNA” are used herein interchangeably to refer to an RNA molecule comprising a targeting sequence and a repeat region. The targeting sequence can form a gRNA:DNA heteroduplex through Watson and Crick base pairing with a DNA target site, while the repeat region binds to the anti-repeat region of a tracrRNA also through Watson and Crick base pairing (Jinek et al., 2012, Science 337, 816-821; and Nishimasu c/ a/., 2014, Cell 156, 935-949).

[0042] As used herein the term “tracrRNA” refers to a nucleic acid sequence that recruits a Cas endonuclease to a target nucleic acid bound (hybridized) to a complementary guide RNA sequence. In some embodiments, the tracrRNA comprises at least one stem loop structure. In some embodiments, the tracrRNA comprises two, three, or four stem loop structures. In some embodiments, the tracrRNA comprises at least one anti-repeat region that binds to the repeat region of a crRNA. Exemplary tracrRNAs are described, for example, in Jinek, et al. Science (2012) 337(6096):816-821, Ran, et al. Nature Protocols (2013) 8:2281- 2308, WO2014/093694, and WO2013/176772.

[0043] The term “targeting sequence” as used herein refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent. In some embodiments, the target sequence is in a gene or on a chromosome and is complementary to the targeting sequence.

[0044] As used herein, an “RNA-guided DNA binding agent” means a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binding agents include Cas cleavases/nickases and inactivated forms thereof (“dCas DNA binding agents”). “Cas nuclease”, also called “Cas protein”, as used herein, encompasses Cas cleavases, Cas nickases, and dCas DNA binding agents. Cas cleavases/nickases and dCas DNA binding agents include a Csm or Cmr complex of a type III CRISPR system, the CaslO, Csml, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases. As used herein, a “Class 2 Cas nuclease” is a single-chain polypeptide with RNA-guided DNA binding activity, such as a Cas9 nuclease or a Cpfl nuclease. Class 2 Cas nucleases include Class 2 Cas cleavases and Class 2 Cas nickases (e.g., H840A, D10A, or N863 A variants), which further have RNA-guided DNA cleavases or nickase activity, and Class 2 dCas DNA binding agents, in which cleavase/nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926 A variants), HypaCas9 (e.g, N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g, K810A, K1003A, R1060 A variants), and eSPCas9(l. l) (e.g, K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpfl protein, Zetsche etal., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain. Cpfl sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Zetsche, Tables SI and S3. “Cas9” encompasses Spy Cas9, the variants of Cas9 listed herein, and equivalents thereof. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0045] “mRNA” is used herein to refer to a polynucleotide that is RNA, modified RNA, or a combination thereof, and comprises an open reading frame that can be translated into a polypeptide (z.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2'-methoxy ribose residues. In some embodiments, the sugars of a nucleic acid phosphate-sugar backbone consist essentially of ribose residues, 2'-methoxy ribose residues, or a combination thereof.

[0046] As used herein, the term “lipid nanoparticle” (LNP) refers to a particle that comprises a plurality of (i.e., more than one) lipid molecules physically associated with each other by interm olecular forces. The LNPs may be, e.g., microspheres (including unilamellar and multilamellar vesicles, e.g., “liposomes”-lamellar phase lipid bilayers that, in some embodiments, are substantially spherical — and, in more particular embodiments, can comprise an aqueous core, e.g., comprising a substantial portion of RNA molecules), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension. See also, e.g., WO2017173054A1 and WO2019067992A1, the contents of which are hereby incorporated by reference in their entirety.

[0047] The term “reference standard” as used herein refers to a sample comprising a known concentration of an RNA molecule, e.g., an sgRNA. A series of dilutions of a reference standard, comprising different concentrations of the RNA molecule, may be used to generate a standard curve for quantifying the RNA molecule in a sample. For example, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 different dilutions of the reference standard, each containing a different concentration of the RNA molecule, may be used. The concentrations of the RNA molecule in the serially diluted solutions of reference standard may span at least 1-log, at least 2-logs or at least 3-logs from highest to lowest.

[0048] The term “fluorescent probe” as used herein refers to a molecule comprising a dye which, after excitation at a certain wavelength, emits light of a higher wavelength. In some embodiments, the dye comprised within the fluorescent probe is selected from the group consisting of 6-FAM, 6-JOE, Alexa Fluor 568, Alexa Fluor 633, Alexa Fluor 680, Bodipy, CAL Fluor, CAL Fluor Red 610, TAMRA, HEX, Oregon Green, TET, Texas Red, Marina Blue, Edans Bothell Blue, Fluorescein, Yakima Yellow, Glod 540, Cy3.5 and Cy5.

II. RNA Molecules

[0049] The methods described herein may be used to confirm the identity of or quantify a range of different types of RNA molecules in a sample, including but not limited to, sgRNAs, crRNAs, mRNAs, siRNAs and antisense oligonucleotides (ASO’s).

[0050] In some embodiments, the RNA molecule identified in the sample or quantified is about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 140, about 150, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000 or about 10,000 nucleotides in length. In some embodiments, the RNA molecule is less than about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 140, about 150, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000 or about 10,000 nucleotides in length. In some embodiments, the RNA molecule is greater than about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 140, about 150, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000 or about 10,000 nucleotides in length. Any of these values may be used to define a range for the length of the RNA molecule. For example, in some embodiments, the RNA molecule is from about 15 to about 10,000 nucleotides in length, from about 1000 to about 6000 nucleotides in length, from about 40 to about 200 nucleotides in length, from about 80 to about 120 nucleotides in length, from about 80 to about 140 nucleotides in length, from about 80 to about 150 nucleotides in length, from about 90 to about 125 nucleotides in length, from about 90 to about 110 nucleotides in length, from about 95 to about 105 nucleotides in length, or from about 15 to about 50 nucleotides in length.

[0051] In some embodiments, the RNA molecule identified or quantified in the sample comprises a targeting sequence (also referred to as a spacer sequence). The terms “guide sequence” and “spacer sequence” as used herein refer to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent. In some embodiments, the target sequence is in a gene or on a chromosome and is complementary to the targeting sequence. In some embodiments, the targeting sequence is at the 5’ end of the RNA molecule.

[0052] For example, in some embodiments, the RNA molecule identified or quantified in the sample is a single guide RNA (sgRNA). The terms “single guide RNA” and “sgRNA” are used herein interchangeably to refer to an RNA molecule comprising a targeting sequence and a sequence that interacts with an RNA-guided DNA binding agent. In some embodiments, the sgRNA comprises a crRNA (or a portion thereof) comprising a targeting sequence covalently linked to a tracrRNA. In some embodiments, the crRNA and the tracrRNA are covalently linked via a linker.

[0053] In certain aspects, the disclosure relates to a method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence, the method comprising: (a) contacting the sample with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (b) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

[0054] In certain aspects, the disclosure relates to a method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) about 80 nucleotides to about 150 nucleotides in length and comprising a targeting sequence, the method comprising: (a) contacting the sample with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (b) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the oligonucleotide binds to at least a portion of the guide region of the RNA molecule, and wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

[0055] In certain aspects, the disclosure relates to a method of quantifying a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region, thereby forming a duplex comprising the RNA molecule and the oligonucleotide; (b) contacting the sample with RNase H; wherein the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting at least one of the fragments of the RNA molecule to generate a measurement of the at least one fragment of the RNA molecule; and (d) comparing the measurement of the at least one fragment of the RNA molecule to a standard curve generated from a series of dilutions of a reference standard containing known quantities of the RNA molecule, thereby quantifying the RNA molecule in the sample. [0056] In certain aspects, the disclosure relates to a method of quantifying a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) about 80 nucleotides to about 130 nucleotides in length and comprises a targeting sequence, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region, thereby forming a duplex comprising the RNA molecule and the oligonucleotide, wherein the oligonucleotide binds to at least a portion of the targeting sequence of the RNA molecule; (b) contacting the sample with RNase H; wherein the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting at least one of the fragments of the RNA molecule to generate a measurement of the at least one fragment of the RNA molecule; and (d) comparing the measurement of the at least one fragment of the RNA molecule to a standard curve generated from a series of dilutions of a reference standard containing known quantities of the RNA molecule, thereby quantifying the RNA molecule in the sample.

[0057] In some embodiments, the sgRNA is about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, or about 200 nucleotides in length. In some embodiments, the sgRNA is less than about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, or about 200 nucleotides in length. In some embodiments, the sgRNA is greater than about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, or about 200 nucleotides in length. Any of these values may be used to define a range for the length of the sgRNA. For example, in some embodiments, the sgRNA is from about 40 to about 200 nucleotides in length, from about 80 to about 150 nucleotides in length, from about 90 to about 130 nucleotides in length, from about 90 to about 120 nucleotides in length, or from about 95 to about 105 nucleotides in length. In some embodiments, the sgRNA is about 100 nucleotides in length. [0058] In some embodiments, the sgRNA may interact with a Cas nuclease e.g., Cas9) from a variety of different species. In some embodiments, the Cas9 protein is derived from a Streptococcus pyogenes Cas9 protein, e.g., a S. pyogenes Cas9 (SpyCas9). In some embodiments, the Cas9 protein is derived from a Staphylococcus aureus Cas9 protein, e.g., a SaCas9. In some embodiments, the Cas9 protein is derived from Neisseria meningitidis Cas9 protein, e.g., Nme2Cas9.

[0059] In some embodiments, the RNA molecule identified or quantified in the sample comprises a crRNA. The terms “CRISPR RNA” and “crRNA” are used herein interchangeably to refer to an RNA molecule comprising a targeting sequence and a repeat region. The targeting sequence forms a gRNA:DNA heteroduplex through Watson and Crick base pairing with a DNA target site, while the repeat region binds to the anti-repeat region of a tracrRNA, also through Watson and Crick base pairing (Jinek et al., 2012, Science 337, 816-821; and Nishimasu c/ a/., 2014, Cell 156, 935-949).

[0060] In some embodiments, the crRNA is about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 nucleotides in length. In some embodiments, the crRNA is less than about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 nucleotides in length. In some embodiments, the crRNA is greater than about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 nucleotides in length. Any of these values may be used to define a range for the length of the crRNA. For example, in some embodiments, the crRNA is from about 20 to about 150 nucleotides in length, from about 40 to about 100 nucleotides in length, or from about 30 to about 80 nucleotides in length.

[0061] In some embodiments, the RNA molecule identified or quantified in the sample is a messenger RNA (mRNA). mRNA-based therapies exert their therapeutic effect by exploiting the fact that even exogenous mRNAs can be translated into functional proteins. These mRNAs are typically synthesized using in vitro transcription, and a cap analog can be attached to their 5' end to facilitate their recognition by the translational machinery in the cell. See Jani, 2012, J Vis Exp, doi.org/10.3791/3702. mRNA-based therapies can be divided into two broad subcategories based on their purpose. In the first category, exogenous mRNAs are introduced into cells to replace or supplement endogenous proteins. One example of this is the treatment of patients with a genetic deficiency in an essential enzyme, and mRNA therapy can be applied to replenish the levels of this enzyme and rescue the deficiency. In the second category, the mRNA transcript is designed to act as a vaccine against infectious diseases or cancer antigens. For example, the utility of mRNA-based vaccines has been convincingly demonstrated in their application as vaccines against COVID-19.

[0062] In some embodiments, the mRNA is about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000 or about 10,000 nucleotides in length. In some embodiments, the mRNA is less than about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000 or about 10,000 nucleotides in length. In some embodiments, the mRNA is greater than about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000 or about 10,000 nucleotides in length. Any of these values may be used to define a range for the length of the mRNA. For example, in some embodiments, the mRNA is from about 200 to about 10,000 nucleotides in length, from about 500 to about 6000 nucleotides in length, or from about 1000 to about 6000 nucleotides in length.

[0063] In some embodiments, the RNA molecule identified or quantified in the sample is an antisense oligonucleotide (ASO). Antisense oligonucleotides modulate the expression of target RNAs via sequence-specific binding, and although the structure of these antisense oligonucleotides is determined primarily by their specific sequence, their chemistry can be modulated to produce novel effects, such as increased specificity and stability. See Kim, 2022, Experimental and Molecule Medicine 54: 455-465. Antisense oligonucleotides use several diverse mechanisms of action, but approved antisense oligonucleotide drugs can be divided into two broad categories based on their mechanism. The first group induces the cleavage of a target mRNA by binding to the target sequence. These antisense oligonucleotides are often modified to include DNA-based central sequences surrounded by chemically modified RNA. Once these antisense oligonucleotides form a duplex with their target RNA, their central region produces a DNA-RNA hybrid that is recognized by RNase H. RNase H then cleaves the RNA sequence between the DNA and RNA duplex, inducing the degradation of the target RNA. The second group of antisense oligonucleotide drugs is primarily used to regulate the splicing of pre-mRNAs via a steric hindrance-based mechanism. Diverse RNA binding proteins affect splicing via their binding of specific sequences within the pre-mRNA transcripts, where they modulate other splicing factors to produce numerous modes of alternative splicing. This second group of antisense oligonucleotides drugs targets these sequences in pre-mRNAs, where alternative splicing may result in the inhibition of disease.

[0064] In some embodiments, the ASO is about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, or about 50 nucleotides in length. In some embodiments, the ASO is less than about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, or about 50 nucleotides in length. In some embodiments, the ASO is greater than about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, or about 50 nucleotides in length. Any of these values may be used to define a range for the length of the ASO. For example, in some embodiments, the ASO is from about 15 to about 50 nucleotides in length, from about 18 to about 30 nucleotides in length, or from about 20 to about 25 nucleotides in length.

[0065] In some embodiments, the RNA molecule (e.g., an sgRNA, mRNA, etc.) is encapsulated within a lipid nanoparticle (LNP). In some embodiments, the methods described herein can further comprise a step of deformulating the LNP encapsulating the RNA molecule (e.g., an sgRNA and/or a mRNA). In some embodiments, the LNP is deformulated in ethanol. In some embodiments, the methods described herein further comprise a step of isolating the RNA molecule (e.g., an sgRNA and/or mRNA) from a lipid nanoparticle (LNP) encapsulating the RNA molecule.

[0066] For example, in some aspects, the disclosure relates to a method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence and encapsulated in a lipid nanoparticle (LNP), the method comprising: (a) deformulating the LNP; (b) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (c) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (d) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

[0067] In some aspects, the disclosure relates to a method of quantifying a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence encapsulated in a lipid nanoparticle (LNP), the method comprising: (a) deformulating the LNP; (b) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region, thereby forming a duplex comprising the RNA molecule and the oligonucleotide;

(d) detecting at least one of the fragments of the RNA molecule to generate a measurement of at least one fragment of the RNA molecule; and (e) comparing the measurement of the at least one fragment of the RNA molecule to a standard curve generated from a series of dilutions of a reference standard containing known quantities of the RNA molecule, thereby quantifying the RNA molecule in the sample.

[0068] In some embodiments, the RNA molecule for identification or quantification in a sample is not comprised within a cell.

[0069] An RNA molecule as described herein may comprise at least one chemical modification, e.g., two, three, four, or more chemical modifications. A modified ribonucleotide can have a modified sugar and/or a modified nucleobase. In some embodiments, every base of the RNA molecule is chemically modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all, or substantially all, of the phosphate groups of the RNA molecule are replaced with phosphorothioate groups. In some embodiments, every sugar of the RNA molecule is chemically modified, e.g., every sugar of the RNA region has a 2'-O-Me modification. In some embodiments, all of the ribonucleotides in the RNA molecule are chemically modified. For example, in some embodiments, all ribonucleotides in the RNA molecule have a modified phosphate group, such as a phosphorothioate group. In some embodiments, all ribonucleotides in the RNA molecule have a modified sugar, such as a 2'-O-Me modification. In some embodiments, modified RNA molecules comprise at least one modified ribonucleotide at or near the 5' end of the RNA molecule. In some embodiments, modified RNA molecules comprise at least one modified ribonucleotide at or near the 3' end of the RNA molecule.

[0070] In some embodiments, an RNA molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more modified residues. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the ribonucleotides in an RNA molecule are chemically modified. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the ribonucleotides in the RNA molecule have a 2'-O-Me modification. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the ribonucleotides in the RNA molecule have a phosphorothioate modification.

III. Oligonucleotides

[0071] In some embodiments, the oligonucleotides for use in the methods described herein comprise, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream ribonucleic acid (RNA) region. In some embodiments, the oligonucleotide consists of, in 5’ to 3’ order, a DNA region, and a downstream RNA region. In some embodiments, the oligonucleotide may further comprise an upstream RNA region. For example, in some embodiments, the oligonucleotide comprises, in 5’ to 3’ order, an upstream RNA region, a DNA region, and a downstream RNA region. In some embodiments, the oligonucleotide consists of, in 5’ to 3’ order, an upstream RNA region, a DNA region, and a downstream RNA region.

[0072] In some embodiments, the oligonucleotide is about 10, about 11, about 12, about

13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 nucleotides in length. In some embodiments, the oligonucleotide is less than about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 nucleotides in length. In some embodiments, the oligonucleotide is greater than about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 nucleotides in length. Any of these values may be used to define a range for the length of the oligonucleotide. For example, in some embodiments, the oligonucleotide is about 10 to about 50 nucleotides in length, about 10 to about 30 nucleotides in length, about 20 to about 30 nucleotides in length, or about 20 to about 24 nucleotides in length. In some embodiments, the oligonucleotide is about 17 nucleotides in length.

[0073] In some embodiments, the oligonucleotide has at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence complementarity to the target RNA molecule (z.e., the RNA molecule to be identified in the sample) or a portion thereof. In some embodiments, the oligonucleotide has 100% sequence complementarity to the target RNA molecule or a portion thereof.

[0074] In some embodiments, the upstream RNA region is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 ribonucleotides in length. In some embodiments, the upstream RNA region is less than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 ribonucleotides in length. In some embodiments, the upstream RNA region is greater than about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 ribonucleotides in length. Any of these values may be used to define the length of the upstream RNA region of the oligonucleotide. For example, in some embodiments, the upstream RNA region of the oligonucleotide is about 0 to about 10 nucleotides in length, about 1 to about 10 nucleotides in length, about 1 to about 20 nucleotides in length, about 2 to about 50 nucleotides in length, about 5 to about 25 nucleotides in length, about 10 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 15 to about 20 nucleotides in length, or about 10 to about 20 nucleotides in length. In some embodiments, the upstream RNA region is about 4 nucleotides in length. In some embodiments, the upstream RNA region is about 8 nucleotides in length.

[0075] In some embodiments, the upstream RNA region of the oligonucleotide has at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence complementarity to the target RNA molecule (z.e., the RNA molecule that is being detected using the methods set forth herein) or a portion thereof. In some embodiments, the upstream RNA region of the oligonucleotide has 100% sequence complementarity to the target RNA molecule or a portion thereof.

[0076] In some embodiments, the downstream RNA region is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 ribonucleotides in length. In some embodiments, the downstream RNA region is less than about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 ribonucleotides in length. In some embodiments, the downstream RNA region is greater than about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 ribonucleotides in length. Any of these values may be used to define the length of the downstream RNA region of the oligonucleotide. For example, in some embodiments, the downstream RNA region of the oligonucleotide is about 2 to about 50 nucleotides in length, about 5 to about 50 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotide in length, about 5 to about 15 nucleotides in length, or about 5 to about 10 nucleotides in length. In some embodiments, the downstream RNA region is about 5 nucleotides in length. In some embodiments, the downstream RNA region is about 9 nucleotides in length.

[0077] In some embodiments, the downstream RNA region of the oligonucleotide has at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence complementarity to the target RNA molecule or a portion thereof. In some embodiments, the downstream RNA region of the oligonucleotide has 100% sequence complementarity to the target RNA molecule or a portion thereof.

[0078] In some embodiments, the DNA region is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 deoxyribonucleotides in length. In some embodiments, the DNA region is less than about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 deoxyribonucleotides in length. In some embodiments, the DNA region is greater than about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 deoxyribonucleotides in length. Any of these values may be used to define the length of the DNA region of the oligonucleotide. For example, in some embodiments, the DNA region of the oligonucleotide is about 2 to about 50 deoxyribonucleotides in length, about 2 to about 40 deoxyribonucleotides in length, about 2 to about 30 deoxyribonucleotides in length, about 2 to about 20 deoxyribonucleotides in length, about 2 to about 10 deoxyribonucleotides in length, about 2 to about 5 deoxyribonucleotides in length, about 5 to about 10 deoxyribonucleotides in length, about 1 to about 20 deoxyribonucleotides in length, or about 1 to about 10 deoxyribonucleotides in length. In some embodiments, the DNA region is about 4 deoxyribonucleotides in length. [0079] In some embodiments, the DNA region of the oligonucleotide has at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence complementarity to the target RNA molecule or a portion thereof. In some embodiments, the DNA region of the oligonucleotide has 100% sequence complementarity to the target RNA molecule or a portion thereof.

[0080] In some embodiments, the upstream RNA region is about 0 to about 10 ribonucleotides in length, the DNA region is about 1 to about 10 deoxyribonucleotides in length, and the downstream RNA region is about 1 to about 10 ribonucleotides in length. In some embodiments, the upstream RNA region is about 0 to about 20 ribonucleotides in length, the DNA region is about 1 to about 20 deoxyribonucleotides in length, and the downstream RNA region is about 1 to about 20 ribonucleotides in length. In some embodiments, the upstream RNA region is about 0 to about 20 ribonucleotides in length, the DNA region is about 1 to about 50 deoxyribonucleotides in length, and the downstream RNA region is about 1 to about 50 ribonucleotides in length. In some embodiments, the upstream RNA region is about 1 to about 10 ribonucleotides in length, the DNA region is about 2 to about 10 deoxyribonucleotides in length, and the downstream RNA region is about 10 to about 25 ribonucleotides in length.

[0081] In some embodiments, the guanine-cytosine (GC) content of the oligonucleotide is about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59% or about 60%. In some embodiments, the GC content of the oligonucleotide is less than 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59% or about 60%. In some embodiments, the GC content of the oligonucleotide is greater than 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59% or about 60%. Any of these values may be used to define a range for the GC content of the oligonucleotide. For example, in some embodiments, the GC content of the oligonucleotide is about 30% to about 60%, about 40% to about 55%, or about 40% to about 50%. In some embodiments, the GC content of the oligonucleotide is about 47%. In some embodiments, the GC content of the oligonucleotide is about 41%.

[0082] The oligonucleotide may be designed to bind to a particular region of the target RNA molecule to direct cleavage of the target RNA molecule by RNase H at a particular site. RNase H is a family of non-sequence-specific endonuclease enzymes that catalyze the cleavage of RNA in an RNA/DNA duplex via a hydrolytic mechanism. Accordingly, the target RNA molecule may be cleaved at a particular site by directing the DNA region of the oligonucleotide to hybridize to a particular region of the target RNA molecule. For example, in some embodiments, the RNAse H cleaves the target RNA molecule in the RNA/DNA duplex with the oligonucleotide directly after the ribonucleotide that is complementary to the deoxyribonucleotide at the 5’ end of the DNA region of the oligonucleotide.

[0083] In some embodiments, the downstream RNA region of the oligonucleotide binds to a targeting sequence of an RNA molecule (e.g., the targeting sequence of an sgRNA or crRNA), or a portion thereof. For example, in some embodiments, the entire downstream RNA region of the oligonucleotide is complementary to the targeting sequence of the RNA molecule (e.g., an sgRNA or crRNA) or a portion thereof. In some embodiments, at least a portion of the downstream RNA region of the oligonucleotide is complementary to the targeting sequence of the RNA molecule or a portion thereof. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 16, 17, 18, 19 or 20 ribonucleotides of the downstream RNA region of the oligonucleotide are complementary to the targeting sequence of the RNA molecule or a portion thereof. [0084] In some embodiments, the downstream RNA region of the oligonucleotide has at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence complementarity to the targeting sequence of the RNA molecule or a portion thereof. In some embodiments, the downstream RNA region of the oligonucleotide has 100% sequence complementarity to the targeting sequence of the RNA molecule or a portion thereof.

[0085] In some embodiments, the DNA region of the oligonucleotide binds to a targeting sequence of an RNA molecule (e.g., the targeting sequence of an sgRNA or crRNA), or a portion thereof. For example, in some embodiments, the entire DNA region of the oligonucleotide is complementary to the targeting sequence of the RNA molecule (e.g., an sgRNA or crRNA) or a portion thereof. In some embodiments, at least a portion of the DNA region of the oligonucleotide is complementary to the targeting sequence of the RNA molecule or a portion thereof. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 16, 17, 18, 19 or 20 deoxyribonucleotides of the DNA region of the oligonucleotide are complementary to the targeting sequence of the RNA molecule or a portion thereof.

[0086] In some embodiments, the DNA region of the oligonucleotide has at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence complementarity to the targeting sequence of the RNA molecule or a portion thereof. In some embodiments, the DNA region of the oligonucleotide has 100% sequence complementarity to the targeting sequence of the RNA molecule or a portion thereof.

[0087] The oligonucleotide may be designed to direct cleavage of the target RNA molecule by RNase H at a particular site by controlling where the DNA region of the oligonucleotide hybridizes to the target RNA molecule. For example, in some embodiments, when the oligonucleotide hybridizes to the target RNA molecule, the deoxyribonucleotide at the 5’ end of the DNA region of the oligonucleotide is complementary to (e.g., hybridizes to) a ribonucleotide of the RNA molecule that is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides from the 5’ end of the RNA molecule. In some embodiments, the deoxyribonucleotide at the 5’ end of the DNA region of the oligonucleotide is complementary to (e.g., hybridizes to) a ribonucleotide of the RNA molecule that is from about 10 to about 30 ribonucleotides from the 5’ end of the RNA molecule. In some embodiments, the deoxyribonucleotide at the 5’ end of the DNA region of the oligonucleotide is complementary to (e.g., hybridizes to) a ribonucleotide of the RNA molecule that is about 15 to about 25 ribonucleotides from the 5’ end of the RNA molecule. In some embodiments, the deoxyribonucleotide at the 5’ end of the DNA region of the oligonucleotide is complementary to (e.g., hybridizes to) a ribonucleotide of the RNA molecule that is about 20 ribonucleotides from the 5’ end of the RNA molecule.

[0088] In some embodiments, the upstream RNA region of the oligonucleotide binds to a target RNA molecule (e.g., an sgRNA) or a portion thereof that interacts with an RNA- guided DNA binding agent. For example, in some embodiments, the entire upstream RNA region of the oligonucleotide is complementary to a sequence of an RNA molecule (e.g., an sgRNA) or a portion thereof that interacts with an RNA-guided DNA binding agent. In some embodiments, at least a portion of the upstream RNA region of the oligonucleotide is complementary to a sequence of an RNA molecule (e.g., an sgRNA) or a portion thereof that interacts with an RNA-guided DNA binding agent. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 16, 17, 18, 19 or 20 ribonucleotides of the upstream RNA region of the oligonucleotide are complementary to a sequence of an RNA molecule (e.g., an sgRNA) or a portion thereof that interacts with an RNA-guided DNA binding agent. In some embodiments, the 3’ end of the upstream RNA region of the oligonucleotide is complementary to (e.g., hybridizes to) the 5’ end of the sequence of the RNA molecule (e.g., an sgRNA) that interacts with an RNA-guided DNA binding agent.

[0089] In some embodiments, the upstream RNA region of the oligonucleotide has at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence complementarity to a sequence of an RNA molecule (e.g., an sgRNA) or a portion thereof that interacts with an RNA-guided DNA binding agent. In some embodiments, the upstream RNA region of the oligonucleotide has 100% sequence complementarity to a sequence of an RNA molecule (e.g., an sgRNA) or a portion thereof that interacts with an RNA-guided DNA binding agent.

[0090] In some embodiments, the oligonucleotide is designed such that the RNase H cuts at or near the 3’ end of the targeting sequence of an sgRNA or crRNA after hybridization of the oligonucleotide to the sgRNA or crRNA. For example, in some embodiments, the 5’ end of the DNA region of the oligonucleotide is complementary to (e.g., hybridizes to) the 3’ end of the targeting sequence. In some embodiments, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule at or near a 3’ end of the targeting sequence. In some embodiments, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule within 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of a 3’ end of the targeting sequence.

[0091] The oligonucleotide may be designed to direct cleavage of the target RNA molecule by RNase H at a particular site relative to the 5’ end of the RNA molecule. For example, in some embodiments, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule at a position 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides from the 5’ end of the RNA molecule. In some embodiments, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule at a position from about 10 to about 30 nucleotides from the 5’ end of the RNA molecule. In some embodiments, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule at a position from about 20 to about 24 nucleotides from the 5’ end of the RNA molecule.

[0092] In some embodiments, the oligonucleotide is designed to direct cleavage of the target RNA molecule by RNase H at a particular site relative to the 3’ end of the RNA molecule. For example, in some embodiments, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule at a position 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides from the 3’ end of the RNA molecule. In some embodiments, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule at a position from about 70 to about 90 nucleotides from the 3’ end of the RNA molecule.

[0093] An RNA region in the oligonucleotide (e.g., the upstream RNA region and/or the downstream RNA region) may comprise at least one chemical modification, e.g., two, three, four, or more chemical modifications. Chemical modifications of ribonucleotides are known in the art and are described, for example, in US2020/0248180, which is incorporated by reference herein in its entirety. A modified ribonucleotide can have a modified sugar, a modified phosphate backbone, and/or a modified nucleobase. In some embodiments, every base of the RNA region is chemically modified. For example, in some embodiments each phosphate group in the phosphate backbone is a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all, or substantially all, of the phosphate groups of the RNA region of the oligonucleotide are replaced with phosphorothioate groups. In some embodiments, every sugar of the RNA region is chemically modified, e.g., every sugar of the RNA region has a 2'-0-Me modification. In some embodiments, all of the ribonucleotides in the oligonucleotide are chemically modified. For example, in some embodiments, all ribonucleotides in the oligonucleotide have a modified phosphate group, such as a phosphorothioate group. In some embodiments, all ribonucleotides in the oligonucleotide have a modified sugar, such as a 2'-0-Me modification. In some embodiments, modified oligonucleotides comprise at least one modified ribonucleotide at or near the 5' end of the oligonucleotide. In some embodiments, modified oligonucleotides comprise at least one modified ribonucleotide at or near the 3' end of the oligonucleotide. [0094] In some embodiments, an RNA region (e.g., an upstream RNA region and/or a downstream RNA region) of the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified residues. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the ribonucleotides in an RNA region of the oligonucleotide are chemically modified. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the ribonucleotides in an RNA region of the oligonucleotide have a 2'-O-Me modification. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the ribonucleotides in an RNA region of the oligonucleotide have a phosphorothioate modification.

IV. Chemical Modifications

[0095] The RNA molecules for identification in a sample and RNA regions of the oligonucleotides described herein may comprise any one or more of the chemical modifications described herein.

[0096] For example, in some embodiments, the phosphate group of a modified ribonucleotide can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified ribonucleotide, e.g., modified ribonucleotide present in a modified oligonucleotide, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution. [0097] Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the nonbridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (z.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.

[0098] The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methyl enedimethylhydrazo and methyleneoxymethylimino.

[0099] Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

[00100] The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, z.e., a sugar modification. For example, the 2' hydroxyl group (OH) can be modified, e.g., replaced with a number of different “oxy” or “deoxy” substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'- alkoxide ion.

[00101] Examples of 2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g, from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the 2' hydroxyl group modification can be 2'-0-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluoro modification, which replaces the 2' hydroxyl group with a fluoride. In some embodiments, the 2' hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a Cl-6 alkylene or Cl-6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; 0-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH2)n-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2' hydroxyl group modification can included “unlocked” nucleic acids (UNA) in which the ribose ring lacks the C2'-C3' bond. In some embodiments, the 2' hydroxyl group modification can include the methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative).

[00102] “Deoxy” 2' modifications can include hydrogen (z.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid);

NH(CH2CH2NH)nCH2CH2 — amino (wherein amino can be, e.g., as described herein), — NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein. [00103] The sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g., L-nucleosides.

[00104] The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified oligonucleotide, can include a modified base, also called a nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified oligonucleotides. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.

[00105] In some embodiments, both the upstream RNA region and the downstream RNA region of the oligonucleotide contain chemical modifications. Such chemical modifications may be at one or both ends of the oligonucleotide. In some embodiments, the entire RNA region may be chemically modified, e.g., the entire upstream RNA region and the entire downstream RNA region may be chemically modified. In some embodiments, the 5' end of the oligonucleotide is chemically modified. In some embodiments, the 3' end of the oligonucleotide is chemically modified.

[00106] In some embodiments, the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) bond between nucleotides. The terms “mA,” “mC,” “mil,” or “mG” may be used to denote a nucleotide that has been modified with 2'-O-Me. Modification of 2'-O-methyl can be depicted as follows:

RNA 2'-O-Me

[00107] Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2'-fluoro (2'-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability. The terms “fA,” “fC,” “fU,” or “fG” may be used to denote a nucleotide that has been substituted with 2'-F. Substitution of 2'-F can be depicted as follows:

RNA 2'F-RNA

Natural composition of 2'F substitution RNA

[00108] Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos. A may be used to depict a PS modification. The terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3') nucleotide with a PS bond. The terms “mA*,” “mC*,” “mil*,” or “mG*” may be used to denote a nucleotide that has been substituted with 2'-0-Me and that is linked to the next (e.g., 3') nucleotide with a PS bond. The diagram below shows the substitution of S — into a nonbridging phosphate oxygen, generating a PS bond in lieu of a phosphodiester bond:

Phosphodiester Phosphorothioate (PS)

Natural phosphodiester Modified phosphorothioate linkage of RNA (PS) bond

[00109] Abasic nucleotides refer to those which lack nitrogenous bases. The figure below depicts an oligonucleotide with an abasic (also known as apurinic) site that lacks a base:

[00110] Inverted bases refer to those with linkages that are inverted from the normal 5' to 3' linkage (z.e., either a 5' to 5' linkage or a 3' to 3' linkage). For example:

Normal oligonucleotide Inverted oligonucleotide linkage linkage

[00111] An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5' nucleotide via a 5' to 5' linkage, or an abasic nucleotide may be attached to the terminal 3 ' nucleotide via a 3 ' to 3 ' linkage. An inverted abasic nucleotide at either the terminal 5' or 3' nucleotide may also be called an inverted abasic end cap.

[00112] In some embodiments, one or more of the first three, four, or five nucleotides at the 5' terminus of the oligonucleotide, and one or more of the last three, four, or five nucleotides at the 3' terminus of the oligonucleotide are chemically modified. In some embodiments, the modification is a 2'-0-Me, 2'-F, inverted abasic nucleotide, PS bond, or other nucleotide modification known in the art to increase stability and/or performance. In some embodiments, the first four nucleotides at the 5' terminus of the oligonucleotide, and the last four nucleotides at the 3 ' terminus of the oligonucleotide are linked with phosphorothioate (PS) bonds.

[00113] In some embodiments, the first three nucleotides at the 5' terminus of the oligonucleotide, and the last three nucleotides at the 3 ' terminus of the oligonucleotide comprise a 2'-O-methyl (2'-0-Me) modified nucleotide.

V. Methods of Detecting the Presence of an RNA Molecule in a Sample

[00114] In certain aspects, the disclosure relates to a method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream ribonucleic acid (RNA) region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (b) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

[00115] In certain aspects, the disclosure relates to a method of confirming the identity of a ribonucleic acid (RNA) molecule in a sample, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream ribonucleic acid (RNA) region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (b) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

[00116] In some embodiments, the method comprises contacting the sample in vitro with two or more different oligonucleotides that are complementary to the RNA molecule. For example, in some embodiments the method comprises contacting the sample with 2, 3, 4 or 5 different oligonucleotides that are complementary to the RNA molecule. Two or more different oligonucleotides may be used to cleave the RNA molecule into more than two fragments. For example, two different oligonucleotides may be used to cleave the RNA molecule into three fragments, three different oligonucleotides may be used to cleave the RNA molecule into four fragments, etc.

A, Preparing the Sample

[00117] In some embodiments, the sample analyzed in the methods described herein comprises a pharmaceutical composition prepared for administration to a subject, e.g., a human subject. In some embodiments, the RNA molecule in the sample is an sgRNA or crRNA. In some embodiments, the sample further comprises an mRNA molecule, e.g., an mRNA molecule encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is a Cas nuclease. In some embodiments, the sample comprises an LNP comprising an sgRNA or crRNA. In some embodiments, the LNP further comprises an mRNA molecule, e.g., an mRNA molecule encoding an RNA-guided DNA binding agent. In some embodiments, the sample comprises an LNP comprising an sgRNA and an mRNA molecule encoding an RNA-guided DNA binding agent, e.g., a Cas nuclease. The presence of multiple RNAs of varying lengths (e.g., mRNA and sgRNA) in the sample does not alter the ability to detect the presence of an RNA (e.g., mRNA or sgRNA) using the methods set forth herein. Notwithstanding, in some embodiments, the methods described herein may optionally further comprise a step of removing the mRNA molecule from the sample before contacting the sample with an oligonucleotide.

[00118] In some embodiments, the LNP comprising the sgRNA or crRNA and optionally further comprising an mRNA molecule is deformulated. The LNP may be deformulated by, for example, adding ethanol to the LNP, centrifuging (e.g., centrifuging at 12000 g for 15 min. at 4°C), removing the ethanol by pipetting, quickly spinning the sample, optionally removing any residual ethanol by pipetting, decanting and/or using a centrifugal vacuum concentrator, and resuspending the pellet in molecular grade water by vortexing.

[00119] In some embodiments, the mRNA molecule is not removed from the sample before contacting the sample with RNAse H and detecting the presence or absence of at least one of the fragments of the RNA molecule.

[00120] In some embodiments, the mRNA molecule is removed from the sample before contacting the sample with RNAse H and detecting the presence or absence of at least one of the fragments of the RNA molecule. The mRNA molecule may be removed from the sample using known methods such as commercially available kits for separating large and small RNA molecules (e.g., Qiagen miRNeasy Mini Kit, Cat. No. / ID 217084)

[00121] For example, in certain aspects, the disclosure relates to a method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, wherein the sample comprises an sgRNA and an mRNA encoding an RNA-guided DNA binding agent, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, an upstream RNA region, a deoxyribonucleic acid (DNA) region, and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample; (b) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and c) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule. In some embodiments the method comprises, before step (a) removing the mRNA molecule encoding the RNA-guided DNA binding agent from the sample.

[00122] In some embodiments, the sample analyzed in the methods described herein does not comprise a cell.

B, Contacting the Sample with an Oligonucleotide

[00123] In some embodiments, the methods described herein comprise a step of contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule. This contacting step may comprise heating the sample at a temperature and for a time sufficient to denature secondary structure in the RNA molecule. For example, in some embodiments, the sample is heated to a temperature of about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 °C. In some embodiments, the sample is heated to a temperature of at least about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 °C. In some embodiments, the sample is heated to a temperature of less than about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 °C. Any of these values may be used to define a range for the temperature to which the sample is heated. For example, in some embodiments, the sample is heated to a temperature of about 50 to about 80 °C, about 60 to about 80 °C, or about 70 to about 80 °C. In some embodiments, the sample is heated to a temperature of about 75 °C.

[00124] In some embodiments, the sample is heated for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25 or about 30 minutes. In some embodiments, the sample is heated for less than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25 or about 30 minutes. In some embodiments, the sample is heated for greater than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25 or about 30 minutes. Any of these values may be used to define a range for the time for which the sample is heated. For example, in some embodiments, the sample is heated for about 1 to about 10 minutes, for about 5 to about 10 minutes, or for about 1 to about 30 minutes.

[00125] In some embodiments, the sample is cooled to room temperature after heating. In some embodiments, the sample is heated to about 75 °C for about 1 to about 10 minutes and then cooled to room temperature.

C. Hydrolyzing the RNA molecule

[00126] In some embodiments, the methods described herein comprise a step of contacting a sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex of the oligonucleotide and the RNA molecule and cleaves the RNA molecule to produce at least two fragments of the RNA molecule. RNase H is a family of non-sequence-specific endonuclease enzymes that catalyze the cleavage of RNA in an RNA/DNA duplex via a hydrolytic mechanism. Accordingly, the size of the fragments of the RNA molecule may be controlled by directing the DNA region of the oligonucleotide to a particular site on the RNA molecule.

[00127] In some embodiments, one or more fragments of the RNA molecule are about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000 or about 10,000 nucleotides in length. In some embodiments, one or more fragments of the RNA molecule are less than about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000 or about 10,000 nucleotides in length. In some embodiments, one or more fragments of the RNA molecule are greater than about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000 or about 10,000 nucleotides in length. Any of these values may be used to define a range for the size of a fragment of the RNA molecule. For example, in some embodiments, the size of a fragment of the RNA molecule is about 10 to about 30 nucleotides in length, about 70 to about 90 nucleotides in length, about 200 to about 6000 nucleotides in length, or about 10 to about 10,000 nucleotides in length. In some embodiments, the at least two fragments of the RNA molecule are each at least about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 nucleotides in length.

[00128] The at least two fragments of the RNA molecule may be different sizes, e.g., to allow for separation of the two fragments based on size. For example, in some embodiments, the at least two fragments differ in size by at least about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, or about 150 nucleotides. In some embodiments, the at least two fragments of the RNA molecule comprise a first fragment of the RNA molecule and a second fragment of the RNA molecule, wherein the first fragment of the RNA molecule is about 10 to about 30 nucleotides in length, and wherein the second fragment of the RNA molecule is about 70 to about 90 nucleotides in length. In some embodiments, the at least two fragments of the RNA molecule comprise a first fragment of the RNA molecule and a second fragment of the RNA molecule, wherein the first fragment of the RNA molecule is about 15 to 35 nucleotides in length, and wherein the second fragment of the RNA molecule is about 70 to about 135 nucleotides in length. [00129] In some embodiments, the sample is incubated with the RNase H for about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, or about 180 minutes. In some embodiments, the sample is incubated with the RNase H for less than about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, or about 180 minutes. In some embodiments, the sample is incubated with the RNase H for at least about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, or about 180 minutes. In some embodiments, the sample is incubated with the RNase H for at least about 60 minutes. Any of these values may be used to define a range for the amount of time for which the sample is incubated with the RNase H. For example, in some embodiments, the sample is incubated with the RNase H for about 30 minutes to about 90 minutes, about 60 minutes to about 180 minutes, about 60 minutes to about 120 minutes, about 60 minutes to about 90 minutes, about 30 minutes to about 60 minutes, about 30 minutes to about 120 minutes, or about 30 minutes to about 180 minutes. The sample may be incubated with the RNase H at a temperature of about 37°C.

[00130] When the RNA molecule is present in the sample and forms a duplex with the oligonucleotide, incubation of the sample with the RNase H results in the production of at least two fragments of the RNA molecule. In some embodiments, the methods described herein further comprise a step of separating the at least two fragments of the RNA molecule from each other before the detecting step.

D. Detecting Fragments of the RNA molecule

[00131] Methods of detecting fragments of RNA molecules are known in the art and are described, for example, in US 2022/0243263 and US 2022/0220552, which are incorporated by reference herein in their entirety. In some embodiments, the step of detecting the presence or absence of at least one of the fragments of the RNA molecule comprises one or more of the following methods: mass spectrometry, liquid chromatography-mass spectrometry (LC- MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography with ultraviolet detection (LC-UV), high performance liquid chromatography (HPLC), ion mobility spectrometry (e.g., ion mobility spectrometry coupled with mass spectrometry), capillary electrophoresis (e.g., capillary electrophoresis coupled with mass spectrometry); gel electrophoresis, quantitative real-time PCR (qPCR), reverse transcriptase-polym erase chain reaction (RT-PCR), and RNA sequencing. In some embodiments, the step of detecting the presence or absence of at least one of the fragments of the RNA molecule comprises LC-UV. In some embodiments, the step of detecting the presence or absence of at least one of the fragments of the RNA molecule comprises LC-MS. In some embodiments, the step of detecting the presence or absence of at least one of the fragments of the RNA molecule comprises mass spectrometry.

[00132] In some embodiments, mass spectrometer formats include continuous or pulsed electrospray (ESI) and related methods or other mass spectrometer that can detect RNA fragments like MALDI-MS. HPLC-MS measurements can be performed using high resolution time-of-flight or Orbitrap mass spectrometers that have a mass accuracy of less than 5 ppm. The use of such mass spectrometers facilitates accurate discernment between cytosine and uridine bases in the RNA sequence.

[00133] In some embodiments, Mobile Phase A for liquid chromatography (LC) comprises HFIP (l,l,l,3,3,3-Hexafluoro-2-propanol) (e.g., 0.1-10% HFIP, or 0.5-2% HFIP), DIPEA (Diisopropylethylamine) (e.g., 0.01%-10% DIPEA, or 0.05%-l% DIPEA, or water, or a combination thereof (e.g., 0.1-10% HFIP, 0.01%-10% DIPEA in water). In some embodiments, Mobile Phase B for LC comprises HFIP (e.g., 0.01-5% HFIP), DIPEA (e.g., 0.01-5% DIPEA), Acetonitrile (e.g., 40-80% ACN), and/or water (e.g., 20-60% water). In some embodiments, Mobile Phase C for LC comprises methanol (e.g., 25-75% methanol) and water (e.g., 25-75% water). In some embodiments, Mobile Phase C can additionally contain Formic Acid, e.g., 0.01-3% Formic Acid). In some embodiments, the LC column comprises a hydrophobic surface chemistry. In some embodiments, the column is a C8 column, a Cl 8 column, a polyphenyl column, or a DNA-Pac column. In some embodiments, the column is an ACQUIT Y UPLC BEH Cl 8 Column, an XTERRA MS C18 column, or a DNAPac RP column. In some embodiments, the column temperature is 40-75°C. In some embodiments, the column temperature is 70°C. In some embodiments, the injection volume is 5 pl.

[00134] The step of detecting the presence or absence of at least one of the fragments of the RNA molecule in the sample may be used to determine whether the sample contains the RNA molecule. For example, in some embodiments, the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule. In some embodiments, detecting the absence of at least one of the fragments of the RNA molecule indicates that the sample does not comprise the RNA molecule. [00135] In some embodiments, the methods described herein are used to determine whether a sample contains an RNA molecule comprising a targeting sequence, e.g., an sgRNA or a crRNA. For example, in some embodiments, the at least two fragments of the RNA molecule comprise a first fragment comprising the targeting sequence of the guide RNA, and a second fragment that comprises the scaffold portion of the guide RNA, that interacts with an RNA-guided DNA binding agent (e.g., a Cas nuclease). In some embodiments, the first fragment comprising the targeting sequence is detected. Detection of the first fragment comprising the targeting sequence may be used, for example, to differentiate between two sgRNAs that contain different targeting sequences, but the same sequence that interacts with an RNA-guided DNA binding agent (e.g., a Cas nuclease). For example, in some embodiments, detecting the first fragment comprising the targeting sequence indicates that the sample comprises the RNA molecule. In some embodiments, detecting the absence of the first fragment comprising the targeting sequence indicates that the sample does not comprise the RNA molecule.

[00136] In some embodiments, the methods described herein are used to confirm the identity of an RNA molecule (e.g., an sgRNA, a crRNA, or an mRNA molecule) contained within lipid nanoparticles (LNPs). The methods described herein are compatible with samples containing, e.g, residual lipid following deformulation. Accordingly, the methods described herein can be used, in some embodiments, to quickly and effectively confirm the identity of RNA cargo present in LNPs. For example, the methods described herein can be used to confirm the identity of the sgRNA and/or mRNA molecules present in a pharmaceutical composition comprising LNPs encapsulating sgRNA and/or mRNA. Examples of LNPs that can encapsulate RNA molecules are known in the art, and can include, for example, those described in PCT/US2017/024973, PCT/US22/025076, PCT/US22/025074, PCT/US20/29812, or PCT/US2019/54240, the entire contents of each of which are hereby incorporated by reference herein.

VI. Methods of Quantifying an RNA Molecule in a Sample

[00137] In certain aspects, the disclosure relates to a method of quantifying a ribonucleic acid (RNA) molecule in a sample, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region, thereby forming a duplex comprising the RNA molecule and the oligonucleotide; (b) contacting the sample with RNase H; wherein the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (c) detecting at least one of the fragments of the RNA molecule to generate a measurement of the at least one fragment of the RNA molecule; and (d) comparing the measurement of the at least one fragment of the RNA molecule to a standard curve generated from a series of dilutions of a reference standard containing known quantities of the RNA molecule, thereby quantifying the RNA molecule in the sample.

[00138] The standard curve for quantifying the RNA molecule in the sample may be generated by processing a series of dilutions of a reference standard containing different known concentrations of the RNA molecule through steps (a) to (c) described above. The term “reference standard” as used herein refers to a sample comprising a known concentration of an RNA molecule, e.g., an sgRNA. A series of dilutions of a reference standard comprising different concentrations of the RNA molecule are used to generate a standard curve for quantifying the RNA molecule in a sample. For example, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 different dilutions of the reference standard, each containing a different concentration of the RNA molecule, may be used. The concentrations of the RNA molecule in the serially diluted solutions of the reference standard may span at least 1-log, at least 2-logs or at least 3 -logs from highest to lowest.

[00139] For example, in some embodiments, the standard curve is generated by:

(a) contacting a series of dilutions of a reference standard of an RNA molecule in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region, thereby forming a duplex comprising the RNA molecule and the oligonucleotide;

(b) contacting the each of the dilutions of the reference standard with RNase H; wherein the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule;

(c) detecting at least one of the fragments of the RNA molecule to generate measurements of the at least one fragment of the RNA molecule for each dilution of the reference standard; and

(d) calculating a correlation between the measurements obtained in step (c) for each dilution of the reference standard and the known quantity of the RNA molecule in each dilution of the reference standard.

[00140] In some embodiments, the methods of quantifying the RNA molecule further comprise generating a standard curve for the at least one fragment of the RNA molecule. A separate standard curve may be generated for each fragment of the RNA molecule. For example, in some embodiments, a separate standard curve is generated for each of the RNA molecule fragments that is detected.

[00141] The measurements generated in the detecting step (c) described above will depend on the method used to detect the at least one fragment of the RNA molecule. In some embodiments, the detecting of the at least one fragment of the RNA molecule is performed using liquid chromatography-mass spectrometry (LC-MS), or liquid chromatographyultraviolet (LC-UV). When LC-MS or LC-UV is used for detection, the measurement generated by the detecting step may be the area of the peak generated by LC-MS or LC-UV for the at least one fragment of the RNA molecule in the reference standard. The standard curve may be generated by calculating the correlation between the area of the peak for the least one fragment of the RNA molecule in each dilution of the reference standard with the concentration of the RNA molecule in each dilution of the reference standard. Once the standard curve for a particular RNA molecule fragment is generated, the concentration of the RNA molecule in a sample may be determined by measuring the peak area for an RNA molecule fragment from the sample using the methods described herein, and comparing the peak area for the RNA molecule fragment from the sample to the standard curve.

[00142] In some embodiments, the detecting of the RNA molecule is performed using one or more fluorescent probes that hybridize to the RNA molecule. The fluorescent probe can be a molecular beacon with a fluorophore and a quencher attached and placed in close proximity so that the fluorescence is quenched. Hybridization of the molecular beacon to the target RNA molecule separates the fluorophore from the quencher and restores the fluorescence from the probe. An analyzer which is capable of emitting light of the excitation wavelength of the fluorescent dye and also of detecting light of the emission wavelength of the fluorescent dye may be used to measure the fluorescence signal of the probe. In some embodiments, the analyzer used to detect the fluorescent probe is a spectrophotometer. In some embodiments, the analyzer used to detect the fluorescent probe is a fluorometer. In some embodiments, the fluorescent probe is detected using a non-denaturing gel. When fluorescent probes are used for detection, the measurement generated by the detecting step may be an amount of fluorescence as measured by the analyzer.

[00143] In some embodiments, only one fragment of the RNA molecule is detected for quantifying the RNA molecule in the sample. When only one fragment of the RNA molecule is detected, one standard curve is generated for this fragment. In some embodiments, two or more fragments of the RNA molecule are detected for quantifying the RNA molecule in the sample. When two or more fragments of the RNA molecule are detected, a separate standard curve is generated for each of the fragments detected. In some embodiments, only two fragments of the RNA molecule are detected. When only two fragments of the RNA molecule are detected, two standard curves are generated, i.e., one for each fragment.

[00144] The methods of quantifying an RNA molecule may be used to quantify any of the RNA molecules described herein. For example, in some embodiments, the RNA molecule to be quantified is an sgRNA. In some embodiments, the sgRNA is about 80 nucleotides to about 130 nucleotides in length. In some embodiments, the RNA molecule to be quantified is from about 1000 to about 6000 nucleotides in length. In some embodiments, the RNA molecule to be quantified is an mRNA molecule.

[00145] Any of the oligonucleotides described herein may be used in the methods of quantifying RNA molecules. For example, in some embodiments, the oligonucleotide used for quantifying the RNA molecule further comprises an upstream RNA region, wherein a 3’ end of the upstream RNA region is covalently attached to a 5’ end of the DNA region. In some embodiments, the oligonucleotide is from about 10 to about 30 nucleotides in length. In some embodiments, the DNA region of the oligonucleotide is from about 2 to about 10 nucleotides in length. In some embodiments, the DNA region of the oligonucleotide is about 4 nucleotides in length. In some embodiments, the downstream RNA region of the oligonucleotide is from about 10 to about 25 nucleotides in length. In some embodiments, the upstream RNA region of the oligonucleotide is from about 1 to about 10 nucleotides in length. In some embodiments, at least one ribonucleotide in the oligonucleotide comprises at least one chemical modification. In some embodiments, the chemical modification is a 2’-O- methyl modification.

[00146] In certain aspects, the disclosure relates to a method of quantifying a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence encapsulated in a lipid nanoparticle (LNP), the method comprising:

(a) deformulating the LNP;

(c) contacting the sample with RNase H; wherein the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and (d) detecting at least one of the fragments of the RNA molecule to generate a measurement of the at least one fragment of the RNA molecule; and

(e) comparing the measurement of the at least one fragment of the RNA molecule to a standard curve generated from a series of dilutions of a reference standard containing known quantities of the RNA molecule, thereby quantifying the RNA molecule in the sample.

[00147] In certain aspects, the disclosure relates to a method of quantifying a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence, the method comprising:

(d) comparing the measurement of the at least one fragment of the RNA molecule to a standard curve generated from a series of dilutions of a reference standard containing known quantities of the RNA molecule, thereby quantifying the RNA molecule in the sample.

[00148] In certain aspects, the disclosure relates to a method of quantifying a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) about 80 nucleotides to about 130 nucleotides in length and comprises a targeting sequence, the method comprising:

(c) detecting at least one of the fragments of the RNA molecule to generate a measurement of the at least one fragment of the RNA molecule; and (d) comparing the measurement of the at least one fragment of the RNA molecule to a standard curve generated from a series of dilutions of a reference standard containing known quantities of the RNA molecule, thereby quantifying the RNA molecule in the sample.

EXAMPLES

[00149] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references and published patents and patent applications cited throughout the application are hereby incorporated by reference in their entirety. Those skilled in the art will recognize that the invention may be practiced with variations on the disclosed structures, materials, compositions and methods, and such variations are regarded as within the ambit of the invention.

Example 1 - Materials and Methods

1.1 Preparation of RNA

[00150] Messenger RNAs were produced by in vitro transcription. Guide RNAs were chemically synthesized by commercial vendors or using standard in vitro synthesis techniques with modified nucleotides.

1.2 Lipid Nanoparticle Formulation

[00151] In general, ^e lipid nanoparticle (LNP) components were dissolved in ethanol at various molar ratios. The RNA cargo (e.g., Cas9 mRNA and sgRNA) was dissolved in a citrate buffer. The LNPs used contained ionizable lipid ((9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-di enoate), also called herein Lipid A, cholesterol, distearoylphosphatidyl choline (DSPC), and 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2K-DMG) (catalog # GM- 020 from NOF, Tokyo, Japan). The LNPs used comprised one sgRNA and one mRNA (e.g., Cas9 mRNA).

[00152] The LNPs were prepared using standard cross-flow techniques (for example, see WO20 16010840 FIG. 2). Alternatively, the LNPs were formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems NanoAssemblr™ Benchtop Instrument, according ^t0 the manufacturer's protocol. The LNPs were held for at least 1 hour at room temperature, and further diluted with water (approximately 1 : 1 v/v). Diluted LNPs were buffer exchanged into a Tris-sucrose buffer, then concentrated and filtered as needed by methods known in the art. The final LNPs were characterized to determine the encapsulation efficiency, polydispersity index, and average particle size. The LNPs were stored at 4°C or - 80°C until further use.

Example 2 - Oligonucleotide probe design for the detection of guide RNAs in a sample [00153] Oligonucleotide probe A (OP-A) and oligonucleotide probe B (OP-B) specific for the detection of guide RNA A (gRNA-A) and guide RNA B (gRNA-B) respectively, were designed as follows. An RNase H cleavage site on each of the guide RNAs was identified 20 nucleotides from the 5’ end of the targeting sequence (i.e., between nucleotide N20 and nucleotide N21 in the targeting sequence). A probe target region surrounding the RNase H cleavage site in the targeting sequence was selected, and the sequence complementary to the probe target region (also referred to as the “probe target region complementary sequence”) was determined, as shown in Table 1 below. The probe target region complementary sequence is the reverse complement of the underlined regions in Table 1 below.

[00154] Table 1. Sequence analyzer tool data for the probe target region surrounding the

RNase H cleavage site in gRNA-A and gRNA-B.

[00155] The oligonucleotide probe sequence specific to each guide RNA was designed based on the probe target region complementary sequence, with DNA nucleotides replacing RNA nucleotides around the RNase cleavage site. Specifically, Oligonucleotide Probe A (OP -A) was designed using the 17 nucleotide probe target region complementary sequence for gRNA-A as indicated in Table 1, wherein the RNA nucleotides complementary to nucleotides N17 to N20 of the gRNA-A sequence are replaced with corresponding deoxyribonucleotides to form the DNA region of the oligonucleotide probe, and wherein all other nucleotides in OP-A are 2’-O-methyloxylated ribonucleotides. Similarly, Oligonucleotide Probe B (OP-B) was designed using the 17 nucleotide probe target region complementary sequence for gRNA-B as indicated in Table 1, wherein the RNA nucleotides complementary to nucleotides N17 to N20 of the gRNA-B sequence are replaced with corresponding deoxyribonucleotides to form the DNA region of the oligonucleotide, and wherein all other nucleotides in OP-B are 2’-O-methyloxylated ribonucleotides. The sequence length for each of OP-A and OP-B was adjusted to target a melting temperature range of 40 - 65 °C, while ensuring that any added nucleotide was complementary to the corresponding nucleotide in the guide RNA sequence. Without being bound by theory, the higher the melting temperature, the higher the oligonucleotide probe sequence specificity in binding to the probe target region in the correctly matched guide RNA. The target oligonucleotide probe sequence length was initially between 10 and 30 nucleotides, and the oligonucleotide probe sequence was further shortened or lengthened to reach a target GC content of 40-60%. A sequence analyzer tool (IDT, Coralville, IA) was used to calculate the melting temperature, GC content, and length of the final oligonucleotide probe sequences, as indicated in Table 2.

[00156] Table 2. Sequence analyzer tool data for the final oligonucleotide probes specific to gRNA-A and gRNA-B.

Example 3 - Guide RNA ID determination in guide RNA samples

[00157] An assay was developed to confirm the identity of guide RNAs gRNA-A and/or gRNA-B in a sample by detecting the presence of the guide RNAs using oligonucleotide probes OP -A and OP-B respectively, as described in Example 2. A general overview of this assay is provided in Figure 1.

[00158] Solution samples comprising gRNA-A or gRNA-B were prepared by diluting the guide RNA in RNase-free water to a final concentration of 2.5 pM. Water was used as a negative control.

[00159] OP-A and OP-B were designed as described in Example 2 and synthesized by Integrated DNA Technology, Inc. (IDT, Coralville, IA). The oligonucleotide probes were reconstituted in RNase-free water to oligonucleotide probe solutions with a final concentration of lOOpM, and aliquoted and stored at -20°C until further use.

[00160] Aliquots of each oligonucleotide probe solution were thawed at room temperature for at least 15 minutes. 4pL of oligonucleotide probe solution (lOOpM) were added to 40pL of guide RNA solution sample or water. Each sample was mixed, spun down and incubated at 75 ± 1 °C for 5 minutes in a heating block. The samples were cooled down to room temperature for at least 15 minutes. 5pL of 10X RNase H buffer and IpL of RNase H enzyme (New England Biolabs, Cat. M0297S) were added to each sample and mixed well. The samples were incubated at 37 ± 1 °C for 1 hour and cooled on ice for 1-3 hours until further analysis.

[00161] Samples were analyzed using Liquid Chromatography-Ultraviolet (LC-UV). 5 pL of each sample was injected onto the analytical column (Waters, Acquity UPLC BEH Cl 8) which was held at 75 °C for analysis and determination of the presence of the correctly matched guide RNA sequence. The separated analytes were detected by Ultraviolet (UV) detection at 260 nm wavelength. Results from the separation chromatograms are shown in Figure 2.

[00162] The identity of the guide RNA was determined by the presence or absence of the 20-mer cleavage peak in the separation chromatograms as shown in Figure 2. Without being bound by theory, when an oligonucleotide probe was incubated with samples comprising a complementary guide RNA, the guide RNA hybridized to the oligonucleotide probe and was cleaved by RNase H into two distinct fragments of approximately 20 nucleotides (20-mer) and 80 nucleotides (80-mer), which were chromatographically separable from the oligonucleotide probe. When the oligonucleotide probe was incubated with samples containing a non-complementary guide RNA, no cleavage peak was formed or detected on the chromatograms. When OP -A was incubated with gRNA-A, 20-mer and 80-mer cleavage peaks were observed. These cleavage peaks were absent when OP -A was incubated with non-complementary gRNA-B, and a single 100-mer peak corresponding to uncleaved gRNA- B was observed. When OP-B was incubated with gRNA-B, three cleavage peaks were observed due to inefficient cleavage site recognition, with major cleavage peaks corresponding to the 20-mer and 80/81-mer cleavage products, and a minor cleavage peak corresponding to a 19-mer. When OP-B was incubated with non-complementary gRNA-A, a single 100-mer peak corresponding to uncleaved gRNA-A was observed.

Example 4 - Guide RNA identification in mixtures of guide RNA and mRNA

[00163] An assay was conducted to detect and identify guide RNAs gRNA-A and gRNA- B using oligonucleotide probes OP-A and OP-B respectively, as described in Example 2, in samples that additionally contained mRNA encoding a Cas9 enzyme.

[00164] Sample solutions were prepared by mixing the Cas9 mRNA with one guide RNA (gRNA-A or gRNA-B) to a total RNA concentration of 0.5 mg/mL. Water was used as a negative control.

[00165] OP-A and OP-B were designed as described in Example 2 and purchased from Integrated DNA Technology, Inc. (IDT, Coralville, IA). The oligonucleotide probes were reconstituted in RNase-free water to oligonucleotide probe solutions with a final concentration of 100 pM, and aliquoted and stored at -20°C until further use.

[00166] Aliquots of the oligonucleotide probe solution were thawed at room temperature for at least 15 minutes. 4pL of oligonucleotide probe solution (100 pM) was added to 40 pL of sample solution or water. Each sample was mixed, spun down and incubated at 75 ± 1°C for 5 minutes in a heating block. The samples were cooled down to room temperature for at least 15 minutes. 5 pL of 10X RNase H buffer and IpL of RNase H enzyme (New England Biolabs, Cat. M0297S) were added to each sample and mixed well. The samples were incubated at 37 ± 1 °C for 1 hour and cooled on ice for 1-3 hours until further analysis.

[00167] Samples were analyzed using Liquid Chromatography-Ultraviolet (LC-UV). 5pL of each sample was injected onto the analytical column (Waters Acquity UPLC BEH Cl 8) which was held at 75 °C for analysis and determination of the presence of gRNA cleavage products, and in particular, the 20 nucleotide cleavage peak. The separated analytes were detected by Ultraviolet (UV) detection at 260nm wavelength. Samples were further analyzed with Liquid Chromatography -Mass spectrometry (LC-MS) using the same liquid chromatography conditions as in the LC-UV protocol. The separated analytes were detected by Mass Spectrometry performed at full scan mode operated in negative ion mode.

[00168] The identity of the guide RNA in the solution samples was determined by detecting the presence of the 20-mer cleavage peak in the separation chromatograms as shown in Figure 3.

[00169] The specificity of the analytical method was demonstrated by using oligonucleotide probes in solution samples that contained non-complementary guide RNA. In such case, no cleavage peaks were observed on the chromatograms (Figure 4). In each case, a 100-mer peak was observed, corresponding to full-length (uncleaved) guide RNA (data not shown).

[00170] The reproducibility of the method was demonstrated by repeating the analysis on samples prepared from 4 different lots of each guide RNA, and comprising a Cas9 mRNA. The identity of the major peaks in the chromatograms was confirmed by LC-MS as described above. For samples comprising gRNA-A tested with OP-A, two cleavage peaks corresponding to the 20-mer and 80-mer cleavage products were observed. For samples comprising gRNA-B tested with OP-B, three cleavage peaks were observed with two major peaks corresponding to the 20-mer and 80/81-mer cleavage products, and another minor peak corresponding to a 19-mer. The Relative Retention Times (RRT) for the 20-mer corresponding to each guide RNA lot are indicated in Tables 3 and 4.

[00171] Table 3. Relative Retention Times (RRT) corresponding to the 20-mer obtained from the analysis of samples comprising a Cas9 mRNA and 4 different lots of gRNA-A.

[00172] Table 4. Relative Retention Times (RRT) corresponding to the 20-mer obtained from the analysis of samples comprising a Cas9 mRNA and 4 different lots of gRNA-B.

Example 5 - Guide RNA identification in LNP samples

[00173] An assay was conducted to detect and identify guide RNAs gRNA-A and gRNA- B encapsulated within Lipid Nanoparticles (LNPs) using oligonucleotide probes OP -A and OP-B respectively, as described in Example 2.

[00174] LNPs comprising a guide RNA (gRNA-A or gRNA-B) and a mRNA encoding a Cas9 enzyme as listed in Table 5 were prepared as described in Example 1. lOOpL of LNPs were mixed with 1000 pL of ethanol (Fisher Scientific, Cat. # BP2818-500) and vortexed for 3 seconds, then centrifuged at 12,000 RCF for 15 minutes at 4°C to precipitate the encapsulated RNA. The supernatant ethanol solution was removed, and the pelleted RNA was dried in a vacufuge at 25-30 °C for 10 ± 5 minutes. The RNA pellet was redissolved in 300uL of RNase-free water, thereby producing a RNA sample solution extracted from each LNP.

[00175] Oligonucleotide probes OP -A and OP-B were designed as described in Example 2 and purchased from Integrated DNA Technology, Inc. (IDT, Coralville, IA). The oligonucleotide probes were reconstituted in RNase-free water to oligonucleotide probe solutions with a final concentration of lOOpM, and aliquoted and stored at -20°C until further use.

[00176] Aliquots of the oligonucleotide probe solution were thawed at room temperature for at least 15 minutes. 4 pL of oligonucleotide probe solution (lOOpM) were added to 40 pL of RNA sample solution extracted from each LNP. Each sample was mixed, spun down and incubated at 75 ± 1 °C for 5 minutes in a heating block. The samples were cooled down to room temperature for at least 15 minutes. 5pL of 10X RNase H buffer and 1 pL of RNase H enzyme (New England Biolabs, Cat. M0297S) were added to each sample and mixed well. The samples were incubated at 37 ± 1 °C for 1 hour and cooled on ice for 1-3 hours until further analysis.

[00177] Table 5. LNP contents and corresponding oligonucleotide probes used in this experiment

[00178] Samples were analyzed using Liquid Chromatography-Ultraviolet (LC-UV). 5pL of each sample was injected onto the analytical column (Waters, Acquity UPLC BEH Cl 8, 130 A, 1.7 pm, 2.1 x 150 mm, cat. 186005516) which was held at 75 °C for analysis and determination of the presence of the correctly matched guide RNA sequence. The separated analytes were detected by Ultraviolet (UV) detection at 260 nm wavelength. Samples were further analyzed with Liquid Chromatography-Mass spectrometry (LC-MS) using the same liquid chromatography conditions as in the LC-UV protocol. The separated analytes were detected by Mass Spectrometry performed at full scan mode operated in negative ion mode. [00179] The identity of the guide RNA in each LNP sample was determined by detecting the presence of the 20-mer cleavage peak in the separation chromatograms as shown in Figure 5. When the RNA sample solution extracted from a LNP was contacted with an oligonucleotide probe that was complementary to the guide RNA, the guide RNA was cleaved into two distinct fragments consisting of approximately 20 nucleotides (20-mer) and 80 nucleotides (80-mer), which were chromatographically separated from the oligonucleotide probe and the coextracted mRNA (Fig. 4, upper left panel and lower right panel). The specificity of the analytical method was demonstrated by using an oligonucleotide probe with RNA sample solutions extracted from a LNP that contained a non-complementary guide RNA. In such case, no cleavage peak was formed or detected on the chromatograms (Fig. 4, upper right panel and lower left panel). In this manner, the oligonucleotide probe was used to identify the guide RNA present in each LNP sample. Example 6 - Quantification of an sgRNA molecule in a sample

[00180] An assay was developed to quantify the amount of the guide RNA gRNA-B (described in Example 2 above) in a sample by detecting the guide RNA using oligonucleotide probe OP-C. A custom-synthesized probe containing DNA/RNA bases that bind specifically between the variable region and the conserved region of the guide RNA was prepared. After binding, the DNA/RNA complex was cleaved using an RNase-H enzyme. A well characterized reference standard of gRNA-B was used to prepare serially diluted solutions which underwent similar digestion procedures, and the resulting 20-mer and 80-mer peak areas were utilized to generate individual standard curves for each of the 20-mer and 80- mer fragments. A general overview of this assay is provided in Figure 6.

[00181] The oligonucleotide probe C (OP-C) specific for the detection of guide RNA B (gRNA-B) was designed as follows. An RNase H cleavage site on the guide RNA was identified 20 nucleotides from the 5’ end of the targeting sequence (i.e., between nucleotide N20 and nucleotide N21 in the targeting sequence). A probe target region surrounding the RNase H cleavage site in the targeting sequence was selected, and the sequence complementary to the probe target region (also referred to as the “probe target region complementary sequence”) was determined, as shown in Table 6 below. The probe target region complementary sequence is the reverse complement of the underlined region in Table 6 below.

Table 6. Sequence analyzer tool data for the probe target region surrounding the RNase H cleavage site in gRNA-B.

[00182] The oligonucleotide probe sequence specific to gRNA-B was designed based on the probe target region complementary sequence, with DNA nucleotides replacing RNA nucleotides around the RNase cleavage site. Specifically, Oligonucleotide Probe C (OP-C) was designed using the 30 nucleotide probe target region complementary sequence for gRNA-B as indicated in Table 6, wherein the RNA nucleotides complementary to nucleotides N17 to N20 of the gRNA-B sequence are replaced with corresponding deoxyribonucleotides to form the DNA region of the oligonucleotide probe, and wherein all other nucleotides in OP-B are 2’-O-methyloxylated ribonucleotides. The sequence length for OP-C was adjusted to target a melting temperature range of 40 - 65 °C, while ensuring that any added nucleotide was complementary to the corresponding nucleotide in the guide RNA sequence. A sequence analyzer tool (IDT, Coralville, IA) was used to calculate the melting temperature, GC content, and length of the oligonucleotide probe sequence, as indicated in Table 7.

Table 7. Sequence analyzer tool data for an oligonucleotide probe specific to gRNA-B.

[00183] Five serially diluted solutions of reference material at different concentrations for generation of the standard curve, samples for quantification, and nuclease-free water (negative control) were combined with oligonucleotide probe OP-C at a 1 :2 ratio. The sample/probe combinations were gently mixed and incubated at 75 ± 1 °C for 5 minutes in a heating block to facilitate binding. The samples were cooled, RNase H enzyme solution was added, and the samples were incubated at 37 ± 1 °C for 1 hour.

[00184] The cleaved samples were injected into an IP-RP UPLC system with a gradient optimized to provide the best resolution between the 20-mer fragment of the sgRNA, the 80- mer fragment of the sgRNA, and the OP-C oligonucleotide probe. Peaks of interests were integrated based on the reference control. Using a Reference Standard, relative retention time (RRT) for all cleavage peaks before the probe peak with the probe peak set as RRT = 1 was established. A major 20-mer cleavage peak was expected with RRT -0.94 for gRNA-B. The standard was utilized to quantitate 20-mer and 80-mer peaks. Chromatograms for the gRNA- B sample and the gRNA-B reference material used to generate the standard curve are shown in Figure 7.

[00185] The peak areas for the 20-mer and 80-mer fragments were each used to generate standard curves for the concentration of each sgRNA fragment in the sample. The correlation between the peak area of each fragment (20-mer or 80-mer) and the concentration of the RNA molecule in each diluted solution of the reference standard was calculated. The linear regression equation was generated with correlation of determination (R²) determined for the standard curve of each fragment. Standard curves for each fragment are shown in Figure 6, bottom right panel. The concentration of gRNA-B in the sample was determined by comparing the peak area of each fragment in the sample to the corresponding standard curve for that fragment.

Claims

1. A method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, the method comprising:

(a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample;

(b) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and

(c) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

2. The method of claim 1, wherein detecting the absence of at least one of the fragments of the RNA molecule indicates that the sample does not comprise the RNA molecule.

3. A method of quantifying a ribonucleic acid (RNA) molecule in a sample, the method comprising:

4. The method of claim 3, further comprising generating the standard curve by:

5. The method of any one of claims 1 to 4, wherein the detecting comprises detecting only one of the fragments of the RNA molecule.

6. The method of any one of claims 1 to 4, wherein the detecting comprises detecting at least two fragments of the RNA molecule.

7. The method of claim 4 or 5, wherein the standard curve is generated for only one of the fragments of the RNA molecule.

8. The method of claim 4 or 6, wherein standard curves are generated for each of at least two fragments of the RNA molecule.

9. The method of any one of the previous claims, wherein the oligonucleotide further comprises an upstream RNA region, wherein a 3’ end of the upstream RNA region is covalently attached to a 5’ end of the DNA region.

10. The method of any one of the previous claims, wherein the oligonucleotide is from about 10 to about 30 nucleotides in length.

11. The method of any one of the previous claims, wherein the DNA region of the oligonucleotide is from about 2 to about 10 nucleotides in length.

12. The method of any one of the previous claims, wherein the DNA region of the oligonucleotide is about 4 nucleotides in length.

13. The method of any one of the previous claims, wherein the downstream RNA region of the oligonucleotide is from about 10 to about 25 nucleotides in length.

14. The method of any one of the previous claims, wherein the upstream RNA region of the oligonucleotide is from about 1 to about 10 nucleotides in length.

15. The method of any one the previous claims, wherein at least one ribonucleotide in the oligonucleotide comprises at least one chemical modification.

16. The method of claim 15, wherein the chemical modification is a 2’-O-methyl modification.

17. The method of any one of the previous claims, wherein the at least two fragments of the RNA molecule are each at least about 10 nucleotides in length.

18. The method of any one of the previous claims, wherein the at least two fragments of the RNA molecule are different sizes.

19. The method of any one of the previous claims, wherein the at least two fragments differ in size by at least about 20 nucleotides.

20. The method of any one of the previous claims, wherein the at least two fragments differ in size by about 50 to about 120 nucleotides.

21. The method of any one of the previous claims, wherein the at least two fragments differ in size by at least about 100 nucleotides.

22. The method of any one of the previous claims, wherein the detecting step is performed using liquid chromatography-mass spectrometry (LC-MS), or liquid chromatography-ultravi ol et (LC -U V) .

23. The method of any one of the previous claims, wherein the detecting step is performed using liquid chromatography-mass spectrometry (LC-MS).

24. The method of any one of claims 1 to 21, wherein the detecting step is performed using one or more fluorescent probes that hybridize to a fragment of the RNA molecule.

25. The method of any one of the previous claims, further comprising isolating the RNA molecule from a lipid nanoparticle (LNP) in the sample before step (a).

26. The method of claim 25, wherein isolating the RNA molecule from the LNP comprises deformulating the LNP in ethanol.

27. The method of any one of the previous claims, further comprising separating the at least two fragments of the RNA molecule from each other before the detecting step.

28. The method of any one of the previous claims, wherein step (a) comprises heating at a temperature and for a time sufficient to denature secondary structure in the RNA molecule.

29. The method of any one of the previous claims, wherein step (a) comprises heating at about 75°C for about 1 to about 10 minutes and cooling at room temperature.

30. The method of any one of the previous claims, wherein step (b) comprises incubating the sample with RNase H for about 30 minutes to about three hours at about 37°C.

31. The method of any one of the previous claims, wherein the RNA molecule is from about 40 to about 200 nucleotides in length.

32. The method of any one of the previous claims, wherein the RNA molecule comprises a crRNA molecule.

33. The method of any one of the previous claims, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence.

34. The method of claim 33, wherein the downstream RNA region of the oligonucleotide is complementary to the targeting sequence of the sgRNA.

35. The method of claim 33 or 34, wherein the DNA region of the oligonucleotide is complementary to the targeting sequence of the sgRNA.

36. The method of any one of claims 33 to 35, wherein, when the sgRNA is present in the sample, the RNase H binds to the duplex and cleaves the sgRNA within 5 nucleotides of a 3’ end of the targeting sequence.

37. The method of any one of claims 33 to 35, wherein, when the sgRNA is present in the sample, the RNase H binds to the duplex and cleaves the sgRNA within 2 nucleotides of a 3’ end of the targeting sequence.

38. The method of any one of claims 33 to 35, wherein, when the sgRNA is present in the sample, the RNase H binds to the duplex and cleaves the sgRNA at a 3’ end of the targeting sequence.

39. The method of any one of claims 33 to 35, wherein, when the sgRNA is present in the sample, the RNase H binds to the duplex and cleaves the sgRNA at a position from about 10 to about 30 nucleotides from the 5’ end of the sgRNA.

40. The method of any one of claims 33 to 35, wherein, when the sgRNA is present in the sample, the RNase H binds to the duplex and cleaves the sgRNA at a position from about 20 to about 24 nucleotides from the 5’ end of the sgRNA.

41. The method of any one of claims 33 to 35, wherein the DNA region of the oligonucleotide is complementary to a region of the sgRNA that is from about 10 to about 30 nucleotides from the 5’ end of the sgRNA.

42. The method of any one of claims 33 to 41, wherein the at least two fragments of the sgRNA comprise a first fragment of the sgRNA and a second fragment of the sgRNA, and wherein the first fragment of the sgRNA is about 10 to about 30 nucleotides in length, and wherein the second fragment of the sgRNA is about 70 to about 90 nucleotides in length.

43. The method of any one of claims 33 to 41, wherein the at least two fragments of the sgRNA comprise a first fragment comprising the targeting sequence and a second fragment, and detecting the absence of the first fragment comprising the targeting sequence indicates that the sample does not comprise the RNA molecule.

44. The method of any one of claims 33 to 43, wherein the sample further comprises an mRNA molecule.

45. The method of claim 44, wherein the mRNA molecule encodes an RNA-guided DNA binding agent.

46. The method of claim 45, wherein the RNA-guided DNA binding agent is a Cas nuclease.

47. The method of any one of claims 44 to 46, wherein the method further comprises removing the mRNA molecule from the sample before the contacting step (a).

48. The method of any one of claims 1 to 30, wherein the RNA molecule is from about 40 to about 6000 nucleotides in length.

49. The method of any one of claims 1 to 30, wherein the RNA molecule is from about 1000 to about 6000 nucleotides in length.

50. The method of any one of claims 1 to 30 and 49, wherein the RNA molecule is a messenger RNA (mRNA).

51. The method of any one of the previous claims, wherein the sample does not comprise a cell.

52. The method of any one of claims 1 to 50, wherein the RNA molecule is not comprised within a cell.

53. A method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence encapsulated in a lipid nanoparticle (LNP), the method comprising:

(a) deformulating the LNP; (b) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample;

(c) contacting the sample with RNase H; wherein, when the RNA molecule is present in the sample, the RNase H binds to the duplex and cleaves the RNA molecule to produce at least two fragments of the RNA molecule; and

(d) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

54. A method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence, the method comprising:

(a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, an upstream RNA region, a deoxyribonucleic acid (DNA) region, and a downstream RNA region; and wherein a duplex comprising the RNA molecule and the oligonucleotide is formed if the RNA molecule is present in the sample;

55. A method of detecting the presence of a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) about 80 nucleotides to about 130 nucleotides in length and comprises a targeting sequence, the method comprising:

(c) detecting the presence or absence of at least one of the fragments of the RNA molecule; wherein the oligonucleotide binds to at least a portion of the targeting sequence of the RNA molecule, and wherein the presence of at least one of the fragments of the RNA molecule indicates that the sample comprises the RNA molecule.

56. A method of quantifying a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence encapsulated in a lipid nanoparticle (LNP), the method comprising:

(a) deformulating the LNP;

57. A method of quantifying a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) comprising a targeting sequence, the method comprising: (a) contacting the sample in vitro with an oligonucleotide that is complementary to the RNA molecule; wherein the oligonucleotide comprises, in 5’ to 3’ order, a deoxyribonucleic acid (DNA) region, and a downstream RNA region, thereby forming a duplex comprising the RNA molecule and the oligonucleotide;

58. A method of quantifying a ribonucleic acid (RNA) molecule in a sample, wherein the RNA molecule is a single guide RNA (sgRNA) about 80 nucleotides to about 130 nucleotides in length and comprises a targeting sequence, the method comprising: