Quantification of free guide RNA in crispr complexes

Abstract

The present invention relates to a novel analytical method for quantifying the concentration of uncomplexed ("free") gRNA present in a Cas-gRNA ribonucleoprotein complex produced for genome editing.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Publication Number (43) Publication Date (21) Application Number 202480023499.X (22) Application Date 2024.04.01 (30) Priority Data 63 / 493,225 2023.03.30 US (85) PCT International Application Entering National Phase Date 2025.09.29 (86) PCT International Application Application Data PCT / US2024 / 022511 2024.04.01 (87) PCT International Application Publication Data WO2024 / 206997 EN 2024.10.03 (71) Applicant Alderflon LLC Address North Dakota, USA (72) Inventors Jonathan W. Cooper Alexander Statz Chunchano Coton (74) Patent Agency Shenzhen Eagle Wing Intellectual Property Agency Co., Ltd. 44658 Patent Attorney Wang Yijin Ye Huanbiao (51) Int.Cl. C12Q 1 / 6806 (2006.01) C12Q 1 / 6816 (2006.01) G01N 27 / 447 (2006.01) G01N 27 / 26 (2006.01) (54) Invention Title Quantification of Free Guide RNA in CRISPR Complexes (57) Abstract This invention relates to a novel analytical method for quantifying the concentration of uncomplexed (“free”) gRNA present in Cas-gRNA ribonucleoprotein complexes manufactured for genome editing. Claims 2 pages, Description 10 pages, Drawings 14 pages, CN 121285636 A 2026.01.06 CN 1 21 28 56 36 A 1. A method for quantifying free RNA present in a composition by capillary zone electrophoresis (CZE), the composition comprising a ribonucleoprotein (RNP) complex formed by a gene-editing endonuclease and one or more related RNAs, the method comprising: (i) providing a CZE system comprising: a source reservoir containing a separation buffer and a target reservoir containing a separation buffer, wherein the source reservoir further contains an RNA staining dye, an anode disposed in the source reservoir and a cathode disposed in the target reservoir, wherein the anode and the cathode are connected to a power source, a fused silica capillary connecting the two reservoirs, and a detector adjacent to the cathode for detecting a signal from the capillary; (ii) introducing the composition into the capillary adjacent to the anode; (iii) Perform electrophoresis on the system; and (iv) quantify the signal from the RNA-binding dye using the detector, wherein the amount of the signal indicates the concentration of free RNA in the composition.2. The method of claim 1, wherein the RNA is guide RNA (gRNA). 3. The method of claim 1 or 2, wherein the gene-editing endonuclease is a CRISPR endonuclease. 4. The method of any one of claims 1 to 3, wherein the separation buffer is formulated to maintain the native conformation of the RNP. 5. The method of any one of claims 1 to 4, wherein the separation buffer is a borate buffer. 6. The method of any one of the preceding claims, wherein the RNA staining dye is a fluorescent dye. 7. The method of claim 6, wherein the fluorescent dye comprises a chromophore unit with the following chemical structure: 8. The method of any one of the preceding claims, wherein the detector is a laser-induced fluorescence (LIF) detector. 9. The method of any one of claims 2 to 8, wherein the gRNA is at least 40 or at least 100 nucleotides in length. 10. The method of any one of claims 3 to 9, wherein the CRISPR endonuclease is selected from *Streptococcus pyogenes* Cas9 (SpCas9), *Cpf1*, and *Eureca-V*. 11. The method of any of the preceding claims, wherein free RNA at concentrations as low as 0.2 μM or 6.8 μg / ml can be detected. 12. A composition comprising a ribonuclease and one or more associated RNAs forming a ribonucleoprotein (RNP) complex, the composition being obtained by a method for separating any free RNA from the RNP complex, wherein the method comprises: (i) providing a CZE system comprising: a source reservoir containing a separation buffer and a target reservoir containing a separation buffer, wherein the source reservoir further contains an RNA staining dye; an anode disposed in the source reservoir and a cathode disposed in the target reservoir, wherein the anode and the cathode are connected to a power source; a fused silica capillary connecting the two reservoirs; and a detector adjacent to the cathode for detecting a signal from the capillary; (ii) introducing the composition into the capillary adjacent to the anode; (iii) performing electrophoresis on the system to separate the free RNA from the RNP complex; and (iv) quantifying the signal from the RNA-binding dye using the detector, wherein the amount of the signal indicates the concentration of free RNA in the RNP complex. 13. The composition of claim 12, wherein the RNA is guide RNA (gRNA). 14. The composition of claim 12 or 13, wherein the gene-editing endonuclease is a CRISPR endonuclease.15. The composition of any one of claims 12 to 14, wherein the separation buffer is formulated to maintain the native conformation of the RNP. 16. The composition of any one of claims 12 to 15, wherein the separation buffer is a borate buffer. 17. The composition of any one of the preceding claims, wherein the RNA staining dye is a fluorescent dye. 18. The composition of claim 17, wherein the fluorescent dye comprises a chromophore unit with the following chemical structure: 19. The composition of any one of the preceding claims, wherein the detector is a laser-induced fluorescence (LIF) detector. 20. The composition of any one of claims 13-19, wherein the gRNA is at least 40 or at least 100 nucleotides in length. 21. The composition of any one of claims 14-20, wherein the CRISPR endonuclease is selected from *Streptococcus pyogenes* Cas9 (SpCas9), *C. nigra* Cpf1, and *Eureca-V*. 22. The composition of any one of the preceding claims, wherein the concentration of free RNA is at least 0.2 μM or 6.8 μg / ml. Claims 2 / 2 Page 3 CN 121285636 A Quantification of Free Guide RNA in CRISPR Complex Cross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application 63 / 493,225, filed March 30, 2023, the contents of which are incorporated herein by reference in their entirety. Background Art

[0002] Gene editing is being developed as a therapeutic tool for treating a variety of diseases. One method of gene editing involves the use of CRISPR (clustered regularly spaced short palindromic repeats) technology. This technology utilizes RNA-guided endonucleases (such as Cas (CRISPR-associated) enzymes) to target specific genomic sequences complementary to the guide RNA. The guide RNA (gRNA) is a type of RNA molecule that binds to a Cas endonuclease, and the target sequence of the gRNA specifies the location where the Cas nuclease will cleave the DNA strand. The most commonly used Cas endonuclease is Streptococcus pyogenes Cas9 (SpCas9). The Cas9 protein consists of a recognition (REC) leaflet and a nuclease (NUC) leaflet. The NUC leaflet contains a domain that interacts with the adjacent motif (PAM) of the prototype spacer and two distinct nuclease domains, HNH and RuvC.

[0003] Cas-gRNA ribonucleoproteins (RNPs) are complexes of Cas nucleases with one or more guide RNAs (gRNAs) and are used in gene editing therapies. When these RNPs are manufactured for intracellular delivery, the FDA requires quantification of the concentration of unreinforced (“free”) gRNA present in these manufactured RNPs.However, no method has yet been described in the current scientific literature to meet this regulatory requirement. Therefore, an accurate and reproducible analytical method is still needed to determine the amount of free gRNA present in the Cas-gRNA RNP complex manufactured for intracellular delivery. Summary of the Invention

[0004] This disclosure provides a method for quantifying free RNA present in a composition comprising a ribonucleoprotein (RNP) complex formed by a gene-editing endonuclease and one or more related RNAs by capillary zone electrophoresis (CZE). The method includes the following steps: (i) providing a CZE system comprising a source reservoir containing a separation buffer and a target reservoir containing a separation buffer, wherein the source reservoir further contains an RNA staining dye; an anode disposed in the source reservoir and a cathode disposed in the target reservoir, wherein the anode and the cathode are connected to a power source; a fused silica capillary connecting the two reservoirs; and a detector adjacent to the cathode for detecting a signal from the capillary; (ii) introducing the composition into the capillary adjacent to the anode; (iii) performing electrophoresis on the system; and (iv) quantifying the signal from the RNA-binding dye using the detector, wherein the amount of the signal indicates the level of free RNA in the composition.

[0005] In some embodiments, the RNA is guide RNA (gRNA), such as gRNA having a length of at least 40 or at least 100 nucleotides. In some embodiments, the gene-editing endonuclease is a CRISPR endonuclease, such as Streptococcus pyogenes Cas9 (SpCas9), Acidaminococcus Cpf1, and Eureca-V. In some embodiments, a separation buffer (e.g., borate buffer) is prepared to maintain the native conformation of the RNP. In some embodiments, the RNA staining dye is a fluorescent dye, such as a dye comprising a chromophore unit with the chemical structure shown below: [Specification 1 / 10 page 4 CN 121285636 A] In some embodiments, the detector is a laser-induced fluorescence (LIF) detector. In some embodiments, free RNA at concentrations as low as 0.2 μM or 6.8 μg / ml can be detected.

[0006] This disclosure also provides a composition comprising a ribonuclease (RNP) complex formed by a gene-editing endonuclease and one or more associated RNAs, the composition being obtained by a method of separating any free RNA from the RNP complex, wherein the method comprises (i) providing a CZE system comprising a source reservoir containing a separation buffer and a target reservoir containing a separation buffer, wherein the source reservoir further contains an RNA staining dye, an anode disposed in the source reservoir and a cathode disposed in the target reservoir, wherein the anode and the cathode are connected to a power source; a fused silica capillary connecting the two reservoirs, and a detector adjacent to the cathode for detecting a signal from the capillary; (ii) introducing the composition into the capillary adjacent to the anode; (iii) performing electrophoresis on the system to separate the free RNA from the RNP complex; and (iv) quantifying the signal from the RNA-binding dye using the detector, wherein the amount of the signal indicates the concentration of free RNA in the RNP complex.

[0007] In some embodiments, the RNA is guide RNA (gRNA), such as gRNA with a length of at least 40 or at least 100 nucleotides. In some embodiments, the gene-editing endonuclease is a CRISPR endonuclease, such as Streptococcus pyogenes Cas9 (SpCas9), Cpf1 of Aminococcus spp., and Eureca-V. In some embodiments, a separation buffer (e.g., borate buffer) is prepared to maintain the native conformation of the RNP. In some embodiments, the RNA staining dye is a fluorescent dye, such as a dye comprising a chromophore unit with the chemical structure shown below: In some embodiments, the detector is a laser-induced fluorescence (LIF) detector. In some embodiments, the concentration of free RNA is at least 0.2 μM or 6.8 μg / ml.

[0008] Other features, objects, and advantages of the invention will become apparent in the following detailed description. However, it should be understood that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only and not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description. Specification 2 / 10 Page 5 CN 121285636 A Description of Drawings

[0009] Figure 1 is an electrophoresis diagram showing the peak corresponding to the 100-nucleotide-long unreinforced gRNA. The migration time is shown on the X-axis, and the fluorescence signal is shown on the Y-axis.

[0010] Figure 2 is an electrophoresis diagram showing the peak corresponding to the 100-nucleotide-long unreinforced gRNA and the peak corresponding to the 100-nucleotide-long gRNA (Cas9-gRNA ribonucleoprotein (RNP) complex) complexed with Cas9 protein.Migration time is shown on the X-axis, and fluorescence signal is shown on the Y-axis. Earlier peaks correspond to the Cas9-gRNA RNP complex, and later peaks correspond to unreintegrated (“free”) gRNA.

[0011] Figures 3A and 3B are gRNA standard calibration curves. gRNA concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 μM) are shown on the X-axis, and peak areas are shown on the Y-axis.

[0012] The graph in Figure 4 shows the amount and percentage (%) of unreintegrated (“free”) gRNA present in the RNP complex at five concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 μM). RNP concentration (μM) is shown on the X-axis, the percentage (%) of unreintegrated (“free”) gRNA present in the RNP complex (shown as orange vertical bars) is shown on the right Y-axis, and the peak area of ​​free gRNA (shown as blue circles) is shown on the left Y-axis.

[0013] The graph in Figure 5 shows the percentage (%) of unreintegrated (“free”) gRNA present in two different RNP complexes. The first and second RNP complexes contain gRNA and CRISPR spCas9 endonuclease at concentration ratios of 1:2 and 1:4, respectively. gRNA concentration (μM) is shown on the X-axis, peak area (shown as orange vertical bars) is shown on the left Y-axis, and the percentage of free gRNA (shown as yellow circles) is shown on the right Y-axis.

[0014] The graph in Figure 6 shows the percentage (%) of spiked recovery of unreintegrated (“free”) gRNA present in the spiked gRNA RNP sample. The concentrations of RNP (0.4 μM), free gRNA (0.2, 0.4, 0.6, 0.8, or 1.0 μM), and (RNP + free gRNA) are shown on the X-axis; peak area (shown as blue vertical bars) is shown on the left Y-axis, and the percentage of spiked recovery (shown as orange circles) is shown on the right Y-axis. The standard gRNA calibration curve is shown in the upper left corner of the graph.

[0015] Figures 7A, 7B, and 7C are the standard gRNA calibration curves for runs 1-3, as described in Example 2. gRNA concentrations (0.4, 0.8, 1.2, 1.6, 2.0, and 2.2 μM) are shown on the X-axis and peak areas are shown on the Y-axis.

[0016] Figure 8 is an electrophoresis diagram of the preparation buffer (negative control). Migration time is shown on the X-axis and fluorescence signal is shown on the Y-axis.

[0017] Figure 9 is an electrophoresis diagram showing the peaks corresponding to unreinforced gRNA and the peaks corresponding to the Cas9-gRNA RNP complex. Migration time is shown on the X-axis and fluorescence signal is shown on the Y-axis. Earlier peaks correspond to gRNA bound to the RNP complex (“bound” gRNA) and later peaks correspond to unreinforced (“free”) gRNA.

[0018] Figure 10 shows a linear graph of the expected free gRNA content relative to the theoretical free gRNA content. Known amounts of gRNA were spiked into RNP samples and the corresponding concentrations were measured. The expected results were plotted against the theoretical results. The measured gRNA concentration (μM) is shown on the X-axis, and the expected gRNA concentration (μM) is shown on the Y-axis.

[0019] Figure 11 is a set of overlapping electrophoresis plots showing the peaks of unreintegrated gRNA and the Cas9-gRNARNP complex in RNP samples (all at 0.3 μM) corresponding to different concentrations (0, 0.4, 0.6, 0.8, 1.0, and 1.2 μM) of gRNA. Migration times are shown on the X-axis, and fluorescence signals are shown on the Y-axis. The earlier set of peaks corresponds to gRNA bound to the RNP complex (“bound” gRNA), and the later set of peaks corresponds to unreintegrated (“free”) gRNA. Detailed Description

[0020] This disclosure provides a novel capillary zone electrophoresis (CZE) assay for separating, detecting, and / or quantifying the concentration of free intact ribonucleic acid (RNA) (e.g., guide RNA (gRNA)) in ribonucleoprotein (RNP) complexes (e.g., Cas9-gRNARNP complexes). "Free" RNA in the RNP complex refers to any RNA not bound to an RNA-binding protein (RBP) (e.g., Cas9 nuclease). In capillary zone electrophoresis, molecular separation occurs inside a capillary filled with separation buffer. Molecules are separated according to their mass-to-charge ratio. In this disclosure, one or more intact RNA molecules are separated from any unbound RBPs and RNP complexes based on their mass-to-charge ratio. The separated RNA molecules are then stained with a fluorescent dye and detected using a laser-induced fluorescence (LIF) detector, as indicated by the time it takes for RBP-free RNA to migrate to the detection window. The isolated RNA appears as peaks in an electrophoresis pattern that depicts the relationship between time and detector signal and determines the area under each peak. The concentration of free intact RNA in the RNP sample is calculated using an RNA standard calibration curve and the peak area of ​​free RNA in the RNP sample.

[0021] The analytical method described in this disclosure is advantageous because the fluorescence detection of free RNA is linear, accurate, precise, repeatable, sensitive, and reproducible. This assay is suitable for isolating, detecting, and / or quantifying free RNA present in RNP complexes containing various ratios of RNA and RBP. This assay is also suitable for isolating RNA molecules of different sizes from a variety of RBPs.

[0022] Due to these improvements, this analytical method will allow for the widespread use of CZE-based quantification of free intact RNA in the field of biochemical analysis, for biopharmaceutical research, medical diagnostics, and environmental research.This will apply to Cas9, Cas12a, and other nucleases or proteins with different charge densities compared to the oligonucleotides in the separation buffer. I. Capillary Zone Electrophoresis

[0023] In capillary zone electrophoresis (CZE) (also known as capillary electrophoresis (CE)), in a buffer-filled capillary, under the influence of an electric field, the target analyte is separated from other components in the mixture based on its charge-to-mass ratio (Sun et al., Proteomics (2014) 14(0):622–8). A typical CZE system contains some or all of the following components: (i) Capillary: The capillary is used to separate the target analyte from one or more contaminants. (ii) Sample vial: A container for holding the source sample. (iii) Syringe: Also known as a sample manager or autosampler, the syringe is used to introduce the source sample into the capillary. (iv) Separation buffer: A buffer that allows the target analyte ions to migrate electrophoretically in an electric field. (v) Source reservoir: A first reservoir at the injection end of the capillary, which contains the separation buffer and an anode. (vi) Target reservoir: A second reservoir at the detector end of the capillary, the second reservoir containing a separation buffer and a cathode. (vii) Power supply: A high-voltage power supply (e.g., 10 kV to 30 kV) to drive the separation of the target analyte. (viii) Detector: A fluorescence detector, such as a laser-induced fluorescence (LIF) detector or a UV detector. (ix) Detection window: The capillary region where the polyimide coating is absent. A. Capillary

[0024] In some embodiments, the capillary is a coated or uncoated fused silica capillary. Fused silica (silica) (silicon dioxide, SiO2) is a non-crystalline glass form of silica manufactured by melting and crystallizing silica. In some embodiments, the capillary is an uncoated (bare) fused silica capillary. In some embodiments, the capillary is about 10 to about 100 cm long and has an inner diameter of about 10 to about 100 μm. In some embodiments, the capillary is about 67 cm long and has an inner diameter of about 50 μm. In some embodiments, the capillary may be a capillary with a narrow inner diameter.Instruction manual, page 4 / 10, 7 CN 121285636 A B. Separation buffer

[0025] In some embodiments, the separation buffer may contain ammonium acetate, ammonium formate, ammonium carbonate, ammonium bicarbonate, succinate, acetic acid, boric acid, borate, trifluoroacetic acid, formic acid, oxalic acid, phosphoric acid, iminodiacetic acid, aspartic acid, malonic acid, citric acid, citrate, succinic acid, MES (2-(N-morpholino)ethanesulfonic acid), ADA (N-2-acetamidoiminodiacetic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), BIS-TRIS propane, The buffer contains imidazole, MOPS (3-(N-morpholino)propanesulfonic acid), MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid), morpholine, TES (2-(tris(hydroxymethyl)methyl)amino)ethanesulfonic acid), hydrochloride, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), HEPPS (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), CHAPSO ((dimethylammonium)-2-hydroxy-1-propanesulfonic acid cholamidopropyl ester), CHES (2-(cyclohexylamino)ethanesulfonic acid), CAPS (N-cyclohexyl-3-aminopropanesulfonic acid), hydrazine, TRICINE (N-tris(hydroxymethyl)methylglycine), TRIS (tris(hydroxymethyl)aminomethane), BICINE (bis(2-hydroxyethyl)amine), glycine, bisglycine peptide, ammonia, trimethylamine, imidazole, phosphate, succinate, and triethylamine. In some embodiments, the separation buffer contains boric acid.

[0026] In some embodiments, the separation buffer may contain one or more additives, such as salts, surfactants, chelating agents, glycerol, PEG (polyethylene glycol), sucrose, glucose, amino acids, guanidine HCl, and urea. In some embodiments, the salt is selected from ammonium sulfate ((NH4)2SO4), sodium chloride (NaCl), sodium citrate, sodium sulfate (Na2SO4), sodium tetraborate (Na2B4O7), potassium chloride (KCl), calcium chloride (CaCl2), and magnesium chloride (MgCl2). In some embodiments, the surfactant is selected from polysorbate 80, Triton X-100, Tween-80, CHAPS, and sodium dodecyl sulfate (SDS). In some embodiments, the chelating agent is EDTA (ethylenediaminetetraacetic acid). In some embodiments, the amino acid is selected from glycine, proline, and L-arginine. In some embodiments, the PEG is PEG 3350. C. Fluorescent Dye

[0027] In some embodiments, the separation buffer may contain a fluorescent dye. In some embodiments, the fluorescent dye is a nucleic acid binding dye, such as an RNA binding dye.In some embodiments, the fluorescent nucleic acid binding dye is selected from Invitrogen™ Molecular Probes™ SYTO™ RNASelect green fluorescent cell staining agent, YOYOTM-1, SYTOX™ blue staining agent, and SYBR™ Green II RNA gel staining agent. In some embodiments, the fluorescent nucleic acid binding dye is SYBR™ Green II RNA gel staining agent, and the chemical structure of its chromophore unit (Saarnio et al., Dyes and Pigments (2020) 177:108282) is as follows: II. Target Analyte

[0028] In some embodiments, the target analyte is a free intact RNA molecule. In some embodiments, the length of the RNA molecule is greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides. In some embodiments, the length of the RNA molecule is less than 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides. In some embodiments, the length of the RNA molecule is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides. In some embodiments, the length of the RNA molecule is at least 40 and no more than 100 nucleotides.

[0029] The RNA molecules disclosed herein may be naturally occurring or artificially designed. In some embodiments, the RNA molecule is a protein-coding mRNA molecule. In some embodiments, the RNA molecule is a non-protein-coding or non-coding RNA molecule (ncRNA). In some embodiments, the RNA molecule contains chemically modified nucleic acids. In some embodiments, the ncRNA is a long non-coding RNA (lncRNA; >200 nucleotides) or a short non-coding RNA (sncRNA; <200 nucleotides). In some embodiments, ncRNA includes, but is not limited to, transfer RNA (tRNA), tRNA-derived small RNA (tsRNA), ribosomal RNA (rRNA), microRNA, short interfering RNA (siRNA), PIWI-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), snoRNA-derived small RNA (sdRNA), extracellular RNA (exRNA), small cahar body-specific RNA (scaRNA), Xist, and HOTAIR.A. Guide RNA

[0030] In some embodiments, an RNA molecule (e.g., a guide RNA (gRNA) molecule) is associated with a gene-editing endonuclease. The gRNA molecule as described herein may comprise two parts: 1) a nucleotide sequence containing a “target sequence” complementary to an RNA or DNA target site and capable of hybridizing with a target site nucleic acid sequence, and 2) a nucleotide sequence binding a polynucleotide-guided RNA or DNA-binding domain (e.g., a CRISPR-Cas protein domain). The target site may be a double-stranded DNA sequence containing a PAM sequence and a target sequence, the target sequence being on the same strand as and directly adjacent to the PAM sequence. The nucleotide sequence in 1) may be referred to, for example, crispr RNA or crRNA. The nucleotide sequence in 2) may be referred to as a scaffold sequence for guiding nucleic acids, such as tracrRNA, or an activation region for guiding nucleic acids, and may contain stem-loop structures. Parts 1) and 2) as described above may be fused to form a single guide (e.g., a single guide RNA or sgRNA), or may be on two separate nucleic acid molecules. In some embodiments, the gRNA molecule comprises parts 1) and 2) connected by a linker. In some embodiments, the gRNA molecule comprises portions 1) and 2) linked by non-nucleic acid adapters (e.g., peptide adapters or chemical adapters).

[0031] The RNA molecules disclosed herein may be single-stranded or double-stranded. RNA may contain naturally occurring ribonucleotides and / or their chemically modified counterparts. As used herein, the term “RNA” is not limited to oligonucleotides having only ribose sugar. RNA as used herein may encompass polynucleotide molecules in which the ribose portion of some or all of its nucleotides has been replaced by another portion. The RNA disclosed herein may contain one or more modifications. Modifications may include any modifications known in the art, including, for example, terminal modifications, base modifications, sugar modifications / replacements, and backbone modifications. Terminal modifications may include, for example, 5' terminal modifications (e.g., phosphorylation, conjugation, and reverse bonding) and 3' terminal modifications (e.g., conjugation, DNA nucleotides, and reverse bonding). Base modifications may include, for example, substitution with a stable base, an unstable base, or a base paired with a base in an amplified partner library; base removal (base-free modification of a nucleotide); or conjugation of a base. Sugar modifications or substitutions may include, for example, modifications at the 2' or 4' positions of the sugar moiety, or substitution of the sugar moiety itself. Skeletal modifications may include, for example, modifications or substitutions of phosphodiester bonds, such as modifications or substitutions with one or more thiophosphates, dithiophosphates, triphosphates, methyl phosphonates and other alkyl phosphonates, hypophosphonates, and aminophosphates. Additional modifications may include conjugation of RNA with fluorescent dyes and other tracer molecules.B. RNA-binding protein (RBP) complex

[0032] In some embodiments, the target analyte is separated from the uncomplexed RNA-binding protein (RBP). The RBP contains one or more RNA-binding domains (RBDs). Exemplary RBDs include, but are not limited to, RNA recognition motifs (RRMs), K-homology domains (KHs), RGG (Arg-Gly-Gly) boxes, zinc fingers, double-stranded RNA-binding domains (dsRBDs), target recognition (REC) valves, Pumilio / PUF domains, and Piwi / Argonaute / Zwille (PAZ) domains. In some embodiments, the RBP is a gene-editing endonuclease, such as a CRISPR-associated (Cas) endonuclease. In some embodiments, the Cas endonuclease is a Cas3, Cas9, or Cas10 endonuclease. The Cas endonuclease may be enzymatically active or inactivated (inactivated Cas9 or dCas9). In some embodiments, the Cas endonuclease is a Cas12a (Cpf1) endonuclease. The Cpf1 endonuclease may be enzymatically active or inactivated (inactivated Cpf1 or dCpf1). C. Ribonucleoprotein (RNP) complex

[0033] In some embodiments, the target analyte is separated from the ribonucleoprotein (RNP) complex (i.e., a complex of RNA and RNA-binding proteins). In some embodiments, the RNP complex is a gRNA-CRISPR / Cas endonuclease RNP complex suitable for direct intracellular delivery. In some embodiments, the RNP complex is a gRNA-Cas3 RNP complex. In some embodiments, the RNP complex is a gRNA-Cas9 RNP complex. In some embodiments, the RNP complex is a gRNA-Cas10 RNP complex. In some embodiments, the RNP complex is a gRNA-Cas12 RNP complex. In some embodiments, the RNP complex is a gRNA-Eureca-V RNP complex.

[0034] In some embodiments, gRNA and RBP are compounded at a ratio greater than 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, gRNA and RBP are compounded at a ratio less than 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, gRNA and RBP are compounded at a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.III. Applications

[0035] The novel CZE assay disclosed herein can be used to analyze (e.g., detect and / or quantify) various free intact RNA molecules, including gRNA molecules, in samples containing RNP complexes (e.g., Cas3-gRNARNP complex, Cas9-gRNA RNP complex, Cas10-gRNA RNP complex, Cas12-gRNA RNP complex, and Eureca-V-gRNA RNP complex). In some embodiments, the gRNA molecule is an sgRNA molecule. In some embodiments, the CZE assay described herein is used to quantify the concentration of free intact gRNA or sgRNA present in Cas3-gRNA or Cas3-sgRNA RNP complexes, respectively. In some embodiments, the CZE assay described herein is used to quantify the concentration of free intact gRNA or sgRNA present in Cas9-gRNA or Cas9-sgRNARNP complexes, respectively. In some embodiments, the CZE assay described herein is used to quantify the concentration of free intact gRNA or sgRNA present in Cas10-gRNA or Cas10-sgRNARNP complexes, respectively. In some embodiments, the CZE assay described herein is used to quantify the concentration of free intact gRNA or sgRNA present in the Cas12-gRNA or Cas12-sgRNA RNP complex, respectively. In some embodiments, the CZE assay described herein is used to quantify the concentration of free intact gRNA or sgRNA present in the Eureca-V-gRNA or Eureca-V-sgRNA RNP complex, respectively.

[0036] As further illustrated in the following working examples, the novel CZE assay disclosed herein comprises the following steps: i) before each run, the capillary is first rinsed with capillary conditioning buffer and then rinsed with separation buffer. ii) The capillary is filled with separation buffer that is always maintained at a specific temperature (e.g., 25°C). iii) A sample from a sample vial (the sample is diluted in CE grade water and stored at a specific temperature (e.g., 4°C)) is injected into the capillary. iv) The target analyte is separated from the sample according to its charge-to-mass ratio by applying a potential difference along the capillary, thereby generating an electric field. v) The separated target analytes are then stained with fluorescent dyes and detected using a laser-induced fluorescence (LIF) detector in the detection window. vi) The separated target analytes appear as different peaks in the electrophoresis diagram. vii) The area under each peak is measured. viii) The concentration of the target analytes is calculated using the peak area of ​​the target analytes and the standard calibration curve. Specification 7 / 10 pages 10 CN 121285636 A

[0037] To better understand the present invention, the following examples are described.These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way. Example 1: Quantification of free gRNA using capillary zone electrophoresis (CZE) assay

[0038] This example describes the quantification of free intact gRNA that does not associate with any nucleases in ribonucleoprotein (RNP) products using the novel capillary zone electrophoresis (CZE) assay described in this disclosure. Capillary Zone Electrophoresis (CZE)

[0039] Capillary zone electrophoresis (CZE) was performed using a PA800Plus drug analysis system (Sciex, Massachusetts) with laser-induced fluorescence (LIF) detection. Fluoresceins were excited using a 497 nm argon ion laser source (Sciex, Massachusetts), and their fluorescence was detected using a 520 nm filter. Data were acquired and analyzed using 32Karat software (Sciex, Massachusetts). Electrophoresis was performed using a bare fused silica capillary with a total length of 67 cm and an effective length of 50 cm from the injection point to the detection window. The capillary was pre-rinsed sequentially with 0.1N HCl, 0.1N NaOH, and water at 20 psi for 2 minutes each time. The capillary was then rinsed with separation buffer at 20 psi for 4 minutes. The sample (stored at 4°C) was injected into the capillary at 20 psi for 5 seconds. Analytes were separated from the sample by generating an electric field of 424 V cm⁻¹ by applying a potential difference of 25.1 kV along the capillary for 15 minutes. The capillary was maintained at 25°C throughout. The separation buffer used for all analyses was borate buffer, density 1.0000 g / mL, pH 9.2.

[0040] In the CZE assay, two different sizes of gRNA (40 or 100 nucleotides) were isolated from three different nucleases (Streptococcus pyogenes Cas9 (SpCas9), Cpf1, and Eureca-VTM) within a capillary filled with a separation buffer containing a fluorescent RNA staining dye. The gRNA was separated from the conjugated nuclease and RNP based on their mass-to-charge ratio and detected using a LIF detector, as indicated by the time taken for the unconjugated RNA and RNP to migrate to the detection window, as shown in Figures 1 and 2. For each integration peak in the resulting electrophoresis pattern, the peak area was determined using 32Karat software. The concentration of free gRNA in the sample was calculated by comparing the peak area of ​​gRNA in the RNP sample with the peak area of ​​a gRNA standard of known concentration using a gRNA standard calibration curve. Standard curve preparation involved diluting the gRNA (typically the same sequence used in the RNP complex) to a working concentration of 10 μM with molecular biology grade (MBG) water and mixing thoroughly.The gRNA was then serially diluted with MBG water to final concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 μM and thoroughly mixed. The gRNA dilutions were then measured to plot a standard calibration curve (Figure 3). The upper and lower limits of the gRNA concentration used to plot the standard curve can be changed based on the mass of the gRNA. Table 1: gRNA Standard Curve Preparation Sample Preparation Instructions 8 / 10 pages 11 CN 121285636 A

[0041] Each RNP sample was diluted with MBG water to a final concentration of 1 μM and thoroughly mixed. Separation Buffer

[0042] Borate buffer (pH 9.2) was used as the separation buffer. Fluorescent Dye

[0043] Sybr™ Green II RNA Gel Staining Agent (Thermo Fisher Scientific) was used as the fluorescent dye. Results

[0044] In this novel analytical method, the concentration and percentage of unassociated, free, intact gRNA in the RNP samples were determined. Exemplary methods can detect free RNA (e.g., gRNA) at concentrations as low as the minimum reference standard of 0.2 μM or 6.8 μg / ml. Example 2: Evaluation of Specificity, Linearity, Accuracy, and Reproducibility of CZE Assay

[0045] This example uses an RNP formed by gRNA and SpCas9 at a 3:1 ratio to evaluate the specificity, linearity, accuracy, and reproducibility of the CZE assay described in Example 1. The target acceptance criteria for the parameters being tested are summarized in Table 2 below. Table 2: Study Parameters and Target Standard Methods

[0046] Before performing the CZE assay, preliminary experiments were run to determine the appropriate range of gRNA standard curves, covering five levels from 0.4 mM to 2.0 mM in increments of 0.4 mM. A sixth point of 2.2 mM was added to broaden the range of the standard curve. Next, five gRNA spiking levels (samples S1 to S5) were selected, ranging from 0.4 mM to 1.2 mM in increments of 0.2 mM. Unspecified RNP samples (SO) were analyzed as a control to capture the baseline amount of free gRNA present in the samples. Each sample was then tested with a single injection in three runs to assess the specificity, reproducibility, accuracy, and linearity of the assay. Results

[0047] The results of the three runs were analyzed separately by integrating each result into the instrument software. Linear regression was performed on six points within each gRNA standard curve (Figure 7A-C) and then used to quantify the amount of free gRNA present in the unspecified RNP samples and each spiked sample. Specificity

[0048] In each run, the prepared buffer showed no interference with the free gRNA peak (Figure 8). The bound gRNA peak dissociated from the free gRNA peak. Results are shown in Figure 9.

[0049] In each run, the measured amount of free gRNA in the unspiked RNP sample was subtracted from the total observations for each spiked sample to quantify the measured spiking amount at that level. The average measured spiking amount of free gRNA across all three runs was then plotted against the theoretical amount of free gRNA spiked at each level, and linear regression was performed. The results are shown in Table 3 and Figure 10.

[0050] The percentage of recovery (recovery%) at each level within each run was calculated as a percentage of the measured spiking amount of free gRNA relative to the theoretical spiking amount. The average percentage of recovery at each level was evaluated against an initial range criterion of 100% ± 10%. Although one individual S1 spiking level was outside this range, it was determined to have no effect because the sensitivity of this metric increased at lower levels and the average percentage of recovery at that level was acceptable. The results are shown in Table 3 and Figure 11. Reproducibility

[0051] Reproducibility was calculated by obtaining the percentage of relative standard deviation (RSD%) of the total measured amount of free gRNA in the unspiked RNP sample and each spiked sample over three runs. Results were assessed based on an initial target of RSD% ≤ 5%. The RSD% of the unspiked RNP sample was more sensitive due to the relatively small amount. For this reason, the RSD% results for the S0 control were acceptable with a target of ≤ 10%. Results are shown in Table 3. Table 3: Results Summary Conclusion

[0052] The above data indicate that the CZE assay described in this disclosure exhibits significant specificity, linearity, accuracy, and reproducibility for quantifying free gRNA in the concentration range of 0.2 to 1.0 μM.Instruction manual page 10 / 10, 13 CN 121285636 A, Figure 1; Instruction manual figure 1 / 14, 14 CN 121285636 A, Figure 2; Instruction manual figure 2 / 14, 15 CN 121285636 A, Figure 3A; Instruction manual figure 3 / 14, 16 CN 121285636 A, Figure 3B; Instruction manual figure 4 / 14, 17 CN 121285636 A, Figure 4; Instruction manual figure 5 / 14, 18 CN 121285636 A, Figure 5; Instruction manual figure 6 / 14, 19 CN 121285636 A, Figure 6; Instruction manual figure 7 / 14, 20 CN 121285636 A, Figure 7A; Instruction manual figure 8 / 14, 21 CN 121285636 A, Figure 7B; Instruction manual figure 9 / 14, 22 CN 121285636 A, Figure 7C. Figure 8 of the instruction manual, page 10 / 14, CN 121285636 A; Figure 9 of the instruction manual, page 11 / 14, CN 121285636 A; Figure 10 of the instruction manual, page 12 / 14, CN 121285636 A; Figure 11 of the instruction manual, page 14 / 14, CN 121285636 A.

Claims

1. A method of quantifying free RNA present in a composition comprising a ribonucleoprotein (RNP) complex formed by a gene editing endonuclease and one or more associated RNAs by capillary zone electrophoresis (CZE), the method comprising: (i) providing a CZE system comprising: a source reservoir containing a separation buffer and a destination reservoir containing a separation buffer, wherein the source reservoir further contains an RNA staining dye, an anode placed in the source reservoir and a cathode placed in the destination reservoir, wherein the anode and the cathode are connected to a power supply, a fused silica capillary connecting the two reservoirs, and a detector adjacent to the cathode for detecting signals from the capillary, (ii) introducing the composition into the capillary adjacent to the anode; (iii) performing electrophoresis on the system; and (iv) quantifying the signal from the RNA binding dye by using the detector, wherein the amount of signal is indicative of the concentration of free RNA in the composition.

2. The method of claim 1, wherein the RNA is a guide RNA (gRNA).

3. The method of claim 1 or 2, wherein the gene editing endonuclease is a CRISPR endonuclease.

4. The method of any one of claims 1 to 3, wherein the separation buffer is formulated to maintain the native conformation of the RNP.

5. The method of any one of claims 1 to 4, wherein the separation buffer is a borate buffer.

6. The method of any one of the preceding claims, wherein the RNA staining dye is a fluorescent dye.

7. The method of claim 6, wherein the fluorescent dye comprises a chromophoric unit having a chemical structure as shown below:

8. The method of any one of the preceding claims, wherein the detector is a laser induced fluorescence (LIF) detector.

9. The method of any one of claims 2 to 8, wherein the gRNA is at least 40 or at least 100 nucleotides in length.

10. The method of any one of claims 3 to 9, wherein the CRISPR endonuclease is selected from Streptococcus pyogenes Cas9 (SpCas9), Lachnospiraceae Cpf1, and Eureca-V.

11. The method of any one of the preceding claims, wherein free RNA is detectable at a concentration as low as 0.2 mM or 6.8 pg / ml.

12. A composition comprising a ribonucleoprotein (RNP) complex formed by a gene editing endonuclease and one or more associated RNAs, the composition obtained by a method of separating any free RNA from the RNP complex, wherein the method comprises: (i) providing a CZE system comprising: a source reservoir containing a separation buffer and a destination reservoir containing a separation buffer, wherein the source reservoir further contains an RNA staining dye, an anode placed in the source reservoir and a cathode placed in the destination reservoir, wherein the anode and the cathode are connected to a power supply, a fused silica capillary connecting the two reservoirs, and a detector adjacent to the cathode for detecting signals from the capillary, (ii) introducing the composition into the capillary adjacent to the anode; (iii) performing electrophoresis on the system; and (iv) quantifying the signal from the RNA binding dye by using the detector, wherein the amount of signal is indicative of the concentration of free RNA in the composition. an anode placed in the source reservoir and a cathode placed in the target reservoir, wherein the anode and the cathode are connected to a power supply, a fused silica capillary connecting the two reservoirs, and a detector adjacent to the cathode, the detector for detecting a signal from the capillary, (ii) introducing the composition into the capillary adjacent to the anode; (iii) performing electrophoresis on the system to separate free RNA from the RNP complex; and (iv) quantifying the signal from the RNA binding dye by using the detector, wherein the amount of the signal indicates the concentration of free RNA in the RNP complex.

13. The composition of claim 12, wherein the RNA is a guide RNA (gRNA).

14. The composition of claim 12 or 13, wherein the gene editing endonuclease is a CRISPR endonuclease.

15. The composition of any one of claims 12-14, wherein the separation buffer is formulated to maintain the native conformation of the RNP.

16. The composition of any one of claims 12-15, wherein the separation buffer is a borate buffer.

17. The composition of any one of the preceding claims, wherein the RNA staining dye is a fluorescent dye.

18. The composition of claim 17, wherein the fluorescent dye comprises a chromophoric unit having a chemical structure as shown below:

19. The composition of any one of the preceding claims, wherein the detector is a laser induced fluorescence (LIF) detector.

20. The composition of any one of claims 13-19, wherein the gRNA has a length of at least 40 or at least 100 nucleotides.

21. The composition of any one of claims 14-20, wherein the CRISPR endonuclease is selected from Streptococcus pyogenes Cas9 (SpCas9), Lachnospiraceae Cpf1, and Eureca-V.

22. The composition of any one of the preceding claims, wherein the concentration of free RNA is at least 0.2 mM or 6.8 pg / ml.

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