Quantification of free guide RNA in the CRISPR complex

Abstract

This invention relates to a novel analytical method for quantifying the concentration of non-complexed ("free") gRNA present in Cas-gRNA ribonucleoprotein complexes manufactured for genome editing.

Description

Technical Field

[0001] Cross-references to related applications This application claims priority based on U.S. Provisional Application No. 63 / 493,225, filed on March 30, 2023, the content of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Gene editing has been developed as a treatment tool for various diseases. As one approach to gene editing, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology is used. This technology uses an RNA-guided endonuclease such as a Cas (CRISPR-associated) enzyme to target a specific genomic sequence complementary to a guide RNA. A guide RNA (gRNA) is a type of RNA molecule that binds to a Cas endonuclease and identifies the position where the Cas nuclease cleaves a DNA strand based on the target sequence of the gRNA. The most frequently used Cas endonuclease is Streptococcus pyogenes Cas9 (SpCas9). The Cas9 protein is composed of a recognition (REC) lobe and a nuclease (NUC) lobe. The NUC lobe contains a domain that interacts with a protospacer adjacent motif (PAM) and two different nuclease domains, HNH and RuvC.

[0003] Cas-gRNA ribonucleoproteins (RNPs) are complexes of Cas nucleases and one or more guide RNAs (gRNAs) used in gene editing therapies. When these RNPs are manufactured for intracellular delivery, the FDA requires the quantification of the concentration of gRNAs that are not part of the complex ("free") present in the manufactured RNPs. However, currently, there is no scientific literature describing a methodology that meets this regulatory requirement. Therefore, there is still a need for accurate and reproducible analytical methods to measure the amount of free gRNA present in Cas-gRNA RNP complexes manufactured for intracellular delivery. [Overview of the project]

[0004] This disclosure provides a method for quantifying free RNA present in a composition comprising a ribonucleoprotein (RNP) complex formed between a gene-editing endonuclease and one or more associated RNAs, by capillary zone electrophoresis (CZE). This method is (i) Below: Source reservoir containing separation buffer and target reservoir (source reservoir also contains RNA staining dye), The anode is located in the source reservoir and the cathode is located in the target reservoir (both the anode and cathode are connected to the power supply). A fused silica capillary tube connecting the two reservoirs, and A cathode-near detector for detecting signals from the capillary tube. A step of providing a CZE system including, (ii) The step of introducing the composition into a capillary tube near the anode, (iii) the step of performing electrophoresis on the system, and (iv) A step of quantifying the signal from an RNA-binding dye using a detector, wherein the amount of the signal indicates the level of free RNA in the composition. Includes.

[0005] In some embodiments, the RNA is a guide RNA (gRNA), for example, a gRNA of at least 40 nucleotides or at least 100 nucleotides in length. In some embodiments, the gene editing endonuclease is a CRISPR endonuclease, for example, Streptococcus pyogenes Cas9 (SpCas9), Acidaminococcus Cpf1, and Eureca-V. In some embodiments, the isolation buffer, for example, borate buffer, is prepared to maintain the natural conformation of the RNP. In some embodiments, the RNA staining dye is a fluorescent dye, for example, a dye containing a chromophore unit having the chemical structure shown below. [ka]

[0006] In some embodiments, the detector is a laser-induced fluorescence (LIF) detector. In some embodiments, free RNA can be detected at low concentrations of 0.2 μM or 6.8 μg / ml.

[0007] This disclosure also provides compositions comprising a ribonucleoprotein (RNP) complex formed between a gene-editing endonuclease and one or more associated RNAs. The compositions are obtained by a process of separating free RNA from the RNP complex, the process being: (i) Below: Source reservoir containing separation buffer and target reservoir (source reservoir also contains RNA staining dye), The anode is located in the source reservoir and the cathode is located in the target reservoir (both the anode and cathode are connected to the power supply). A fused silica capillary tube connecting the two reservoirs, and A cathode-near detector for detecting signals from the capillary tube. A step of providing a CZE system including, (ii) The step of introducing the composition into a capillary tube near the anode, (iii) The step of performing electrophoresis on the system to separate free RNA from the RNP complex, and (iv) A step of quantifying the signal from an RNA-binding dye using a detector, wherein the amount of the signal indicates the level of free RNA in the RNP complex. Includes.

[0008] In some embodiments, the RNA is a guide RNA (gRNA), for example, a gRNA of at least 40 nucleotides or at least 100 nucleotides in length. In some embodiments, the gene editing endonuclease is a CRISPR endonuclease, for example, Streptococcus pyogenes Cas9 (SpCas9), Acidaminococcus Cpf1, and Eureca-V. In some embodiments, the isolation buffer, for example, borate buffer, is prepared to maintain the natural conformation of the RNP. In some embodiments, the RNA staining dye is a fluorescent dye, for example, a dye containing a chromophore unit whose chemical structure is shown below. [ka]

[0009] 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.

[0010] Other features, purposes, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that while the detailed description illustrates embodiments and aspects of the present invention, it is for illustrative purposes only and not limiting. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawing]

[0011] [Figure 1]Electrophoretic graph showing peaks corresponding to 100-nucleotide gRNAs that do not form a complex. Migration time is shown on the X-axis, and fluorescence signals are shown on the Y-axis. [Figure 2] This electrophoresis diagram shows peaks corresponding to 100-nucleotide gRNA that does not form a complex and 100-nucleotide gRNA that has formed a complex with the Cas9 protein (Cas9-gRNA ribonucleoprotein (RNP) complex). Migration time is shown on the X axis, and fluorescence signals are shown on the Y axis. The first peak corresponds to the Cas9-gRNA RNP complex, and the second peak corresponds to gRNA that does not form a complex ("free" gRNA). [Figure 3A] This is a gRNA standard calibration curve. The gRNA concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 μM) are shown on the X-axis, and the peak areas are shown on the Y-axis. [Figure 3B] This is a gRNA standard calibration curve. The gRNA concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 μM) are shown on the X-axis, and the peak areas are shown on the Y-axis. [Figure 4] This graph shows the amount and percentage (%) of uncomplexed ("free") gRNA present in RNP complexes at five concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 μM). The RNP concentration (μM) is shown on the X-axis, the percentage (%) of uncomplexed ("free") gRNA present in the RNP complex (shown by orange vertical bars) is shown on the right Y-axis, and the peak area of ​​free gRNA (shown by blue circles) is shown on the left Y-axis. [Figure 5] This graph shows the percentage of gRNA that is not complexed ("free") in two different RNP complexes. The first and second RNP complexes contain gRNA and CRISPR spCas9 endonuclease in concentration ratios of 1:2 and 1:4, respectively. The gRNA concentration (μM) is shown on the X-axis, the peak area (orange vertical bars) is shown on the left Y-axis, and the percentage of free gRNA (yellow circles) is shown on the right Y-axis. [Figure 6]A graph showing the spike recovery rate (%) of uncomplexed ( "free") gRNA present in the RNP sample spiked with gRNA. 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. The peak area (blue vertical bars) is shown on the left Y-axis, and the spike recovery rate (orange circles) is shown on the right Y-axis. The calibration curve of the standard gRNA is shown in the upper left corner of the graph. [Figure 7A] It is the gRNA standard calibration curve in Experiments 1 to 3 described in Example 2. The gRNA concentrations (0.4, 0.8, 1.2, 1.6, 2.0, and 2.2 μM) are shown on the X-axis, and the peak area is shown on the Y-axis. [Figure 7B] It is the gRNA standard calibration curve in Experiments 1 to 3 described in Example 2. The gRNA concentrations (0.4, 0.8, 1.2, 1.6, 2.0, and 2.2 μM) are shown on the X-axis, and the peak area is shown on the Y-axis. [Figure 7C] It is the gRNA standard calibration curve in Experiments 1 to 3 described in Example 2. The gRNA concentrations (0.4, 0.8, 1.2, 1.6, 2.0, and 2.2 μM) are shown on the X-axis, and the peak area is shown on the Y-axis. [Figure 8] It is an electrophoresis diagram of the formulation buffer (negative control). The migration time is shown on the X-axis, and the fluorescence signal is shown on the Y-axis. [Figure 9] [Figure 10] A graph showing a linearity plot of the expected value and the theoretical value of the free gRNA content. Known amounts of gRNA were spiked into the RNP sample, and the corresponding concentrations were measured. The expected value is plotted against the theoretical value. The measured gRNA concentration (μM) is shown on the X-axis, and the expected value of the gRNA concentration (μM) is shown on the Y-axis. [Figure 11] ​This is a superimposed electrophoresis map showing peaks corresponding to uncomplexed gRNAs and Cas9-gRNA RNP complexes for RNP samples (0.3 μM each) spiked with gRNAs of various concentrations (0, 0.4, 0.6, 0.8, 1.0, and 1.2 μM). Migration time is shown on the X-axis, and fluorescence signals are shown on the Y-axis. Earlier sets of peaks correspond to gRNAs bound to the RNP complex ("bound" gRNAs), while later sets of peaks correspond to uncomplexed ("free") gRNAs. [Modes for carrying out the invention]

[0012] This disclosure provides a novel capillary zone electrophoresis (CZE) assay for separating, detecting, and / or quantifying the concentration of free, intact ribonucleic acid (RNA), such as guide RNA (gRNA), in ribonucleoprotein (RNP) complexes, such as Cas9-gRNA RNP complexes. “Free” RNA in an RNP complex refers to RNA that is not bound to RNA-binding protein (RBP), such as Cas9 nuclease. In capillary zone electrophoresis, molecules are separated in a capillary tube filled with separation buffer. Molecules are separated based on their charge-mass ratio. In this disclosure, one or more intact RNA molecules are separated from unbound RBP and RNP complexes based on their charge-mass ratio. The separated RNA molecules are stained with a fluorescent dye and detected using a laser-induced fluorescence (LIF) detector, indicated by the time it takes for RBP-free RNA to reach the detection window. The separated RNA appears as peaks in an electrophoresis diagram plotting time and detector signals, and the area under each peak is measured. The concentration of free intact RNA in an RNP sample is calculated using the RNA standard calibration curve and the peak area of ​​free RNA in the RNP sample.

[0013] The analytical method described herein offers advantages in that the fluorescence detection of free RNA is linear, accurate, precise, repeatable, sensitive, and reproducible. This assay is suitable for the isolation, detection, and / or quantification of free RNA present in RNP complexes containing various ratios of RNA and RBP. Furthermore, this assay is also suitable for the isolation of RNA molecules of different sizes from various RBPs.

[0014] These improvements enable the CZE method for quantifying free intact RNA to be widely used in biochemical analysis fields such as biopharmaceutical research, medical diagnostics, and environmental research. It is applicable to Cas9, Cas12a, and other nucleases and proteins that have different charge densities than oligonucleotides in the separation buffer.

[0015] I. Capillary Zone Electrophoresis Capillary zone electrophoresis (CZE), also known as capillary electrophoresis (CE), separates a target analyte from other components in a mixture based on their charge-to-mass ratio under the influence of an electric field within a buffer-filled capillary (Sun et al., Proteomics (2014) 14(0):622-8). A typical CZE system consists of the following components: (i) Capillary: A capillary tube is used to separate the target analyte from one or more contaminants. (ii) Sample vial: A container for holding the source sample. (iii) Injector: Also known as a sample manager or autosampler, it is used to introduce a source sample into a capillary tube. (iv) Separation buffer: A buffer used to electrophores target analyte ions in an electric field. (v) Source reservoir: The first reservoir located at the injection end of the capillary tube, containing the separation buffer and anode. (vi) Target reservoir: A second reservoir located at the detector end of the capillary tube, containing the separation buffer and cathode. (vii) Power supply: A high-voltage power supply (e.g., 10-30kV) to drive the separation of the target analyte. (viii) Detector: Fluorescence detector, e.g., laser-induced fluorescence (LIF) detector, or UV detector. (ix) Detection window: Capillary region not coated with polyimide. It consists of some or all of the following.

[0016] A. Capillary In some embodiments, the capillary tube is a coated or uncoated fused silica capillary tube. Fused silica (fused silicon dioxide (SiO2)) is silicon dioxide in an amorphous glass form produced by melting crystalline silica. In some embodiments, the capillary tube is an uncoated (bare) fused silica capillary tube. In some embodiments, the length of the capillary tube is about 10 to about 100 cm and the inner diameter is about 10 to about 100 μm. In some embodiments, the length of the capillary tube is about 67 cm and the inner diameter is about 50 μm. In some embodiments, the capillary tube may be a capillary tube with a narrower bore diameter.

[0017] B. Separation buffer In some embodiments, the separation buffer is ammonium acetate, ammonium formate, ammonium carbonate, ammonium bicarbonate, succinic acid, 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-acetamide)-2-aminoethanesulfonic acid), BIS-TRIS propane, imidazole, MOPS (3-(N-morpholino)propanesulfonic acid), MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid), morpholine, TES (2-(tris(hydroxymethyl)methyl)amino The separation buffer may include ethanesulfonic acid, hydrochloride, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), HEPPS (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), CHAPSO (coramidopropyldimethylammonio)-2-hydroxy-1-propanesulfonic acid), 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), glycinamide, glycylglycine, ammonia, trimethylamine, imidazole, phosphate, succinate, and triethylamine. In some embodiments, the separation buffer contains boric acid.

[0018] 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, PEG is PEG 3350.

[0019] C. Fluorescent dyes 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 stain, RiboGreen®, YOYO®-1, SYTOX® Blue stain, and SYBR® Green II RNA gel stain. In some embodiments, the fluorescent nucleic acid-binding dye is SYBR® Green II RNA gel stain, and the chemical structure of its chromophore unit (Saarnio et al., Dyes and Pigments (2020) 177:108282) is shown below. [ka]

[0020] II. Target analyte In some embodiments, the target analyte is a free, intact RNA molecule. In some embodiments, the RNA molecule is greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides in length. In some embodiments, the RNA molecule is less than 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides in length. In some embodiments, 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 length. In certain embodiments, the RNA molecule is at least 40 nucleotides long and 100 nucleotides or less in length.

[0021] The RNA molecules of this disclosure 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-coding RNA molecule (ncRNA) that does not code for proteins. In some embodiments, the RNA molecule includes 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, ncRNAs include, but are not limited to, transfer RNA (tRNA), tRNA-derived small RNA (tsRNA), ribosomal RNA (rRNA), microRNA, small interfering RNA (siRNA), PIWI-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), snoRNA-derived small RNA (sdRNA), extracellular RNA (exRNA), small Cajal body-specific RNA (scaRNA), Xist, and HOTAIR.

[0022] A. Guide RNA In some embodiments, an RNA molecule, such as a guide RNA (gRNA) molecule, is bound to a gene-editing endonuclease. The gRNA molecule described herein may comprise two parts: 1) a nucleotide sequence containing a “targeting sequence” that is complementary to the RNA or DNA target site and can hybridize with the nucleic acid sequence of the target site; and 2) a nucleotide sequence that binds to a polynucleotide-induced 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 located directly adjacent to the PAM sequence on the same strand. The nucleotide sequence in 1) may be called, for example, crispr RNA or crRNA. The nucleotide sequence in 2) may be called the guide nucleic acid scaffold sequence (e.g., tracrRNA) or the guide nucleic acid activation region and may comprise a stem-loop structure. The above parts 1) and 2) may 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 includes portions 1) and 2) linked by a linker. In some embodiments, the gRNA molecule includes portions 1) and 2) linked by a non-nucleic acid linker, such as a peptide linker or a chemical linker.

[0023] The RNA molecules of this disclosure may be single-stranded or double-stranded. RNA may include naturally occurring ribonucleotides and / or chemically modified analogs thereof. The term “RNA” as used herein is not limited to oligoribonucleotides containing only ribose-containing nucleotides. RNA as used herein may include polynucleotide molecules in which some or all of the ribose portions of a nucleotide are replaced by other portions. The RNA of this disclosure may include one or more modifications. Modifications may include any modifications known in the art, such as end modifications, base modifications, sugar modifications / substitutions, and back-chain modifications. End modifications may include, for example, 5' end modifications (e.g., phosphorylation, conjugation, and reverse bonding) and 3' end modifications (e.g., conjugation, DNA nucleotides, and reverse bonding). Base modifications may include, for example, substitution with stabilizing bases, destabilizing bases, or bases that form base pairs with an extended partner repertoire, base removal (nucleotide base modifications), or conjugated bases. Sugar modifications or substitutions may include, for example, modifications at the 2' or 4' position of the sugar moiety, or substitution of the sugar moiety. Backbone modifications may include, for example, modifications or substitutions of phosphodiester bonds (e.g., by one or more phosphorothioates, phosphorodithioates, phosphotryesters, methylphosphonates and other alkylphosphonates, phosphinates and phosphoramidites). Additional modifications include conjugation of RNA with fluorescent dyes and other tracking molecules.

[0024] B. RNA-binding protein (RBP) complex In some embodiments, the target analyte is isolated from an RNA-binding protein (RBP) that does not form a complex. The RBP contains one or more RNA-binding domains (RBDs). Exemplary RBDs include, but are not limited to, an RNA recognition motif (RRM), a K homology domain (KH), an RGG (Arg-Gly-Gly) box, a zinc finger, a double-stranded RNA-binding domain (dsRBD), a target recognition (REC) lobe, a Pumilio / PUF domain, and a Piwi / Argonaute / Zwille (PAZ) domain. 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 in an enzyme-active or inactive (dead) form (inactive Cas9 or dCas9). In some embodiments, the Cas endonuclease is Cas12a(Cpf1) endonuclease. The Cpf1 endonuclease may be in an enzyme-active or inactive form (inactive Cpf1 or dCpf1).

[0025] C. Ribonucleoprotein (RNP) complex In some embodiments, the target analyte is isolated from a ribonucleoprotein (RNP) complex, i.e., a complex of RNA and RNA-binding protein. 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.

[0026] In some embodiments, gRNA complexes with RBP in 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 complexes with RBP in 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 complexes with RBP in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0027] III.Applications The novel CZE assays described herein can be used to analyze (e.g., detect and / or quantify) various free intact RNA molecules, including gRNA molecules, in samples of RNP complexes (e.g., Cas3-gRNA RNP 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 assays described herein are 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 assays described herein are used to quantify the concentration of free intact gRNA or sgRNA present in Cas9-gRNA or Cas9-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 Cas10-gRNA or Cas10-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 Cas12-gRNA or Cas12-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 Eureca-V-gRNA or Eureca-V-sgRNA RNP complexes, respectively.

[0028] As further illustrated in the following examples, the novel CZE assay of this disclosure comprises the following steps: i) Before each experiment, wash the capillaries first with capillary conditioning buffer, and then with separation buffer. ii) Fill the capillary with separation buffer that is always maintained at a specific temperature (e.g., 25°C). iii) Inject the sample from the sample vial (the sample is diluted with CE-grade water and stored at a specific temperature (e.g., 4°C)) into the capillary. iv) A potential difference is applied along the capillary to generate an electric field, and the target analytes in the sample are separated based on their charge-mass ratio. v) The separated target analytes are stained with a fluorescent dye and detected using a laser-induced fluorescence (LIF) detector on the detection window. vi) The separated target analytes appear as distinct peaks in the electrophoresis diagram. vii) Measure the area under each peak. viii) Calculate the concentration of the target analyte using the peak area and standard calibration curve.

[0029] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. [Examples]

[0030] Example 1: Quantification of free gRNA using capillary zone electrophoresis (CZE) assay This example describes a method for quantifying unbound, free intact gRNA in ribonucleoprotein (RNP) products using the novel capillary zone electrophoresis (CZE) assay described herein.

[0031] Capillary zone electrophoresis (CZE) A capillary zone electrophoresis (CZE) assay using laser-induced fluorescence (LIF) detection was performed using a PA 800 Plus Pharmaceutical Analysis System (Sciex, Massachusetts). Fluorescent dyes 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 32 Karat 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-washed with 0.1 N HCl, 0.1 N NaOH, and water in that order at a pressure of 20 psi for 2 minutes each time. Next, the capillary was washed with separation buffer at a pressure of 20 psi for 4 minutes. Samples (stored at 4°C) were injected into the capillary at a pressure of 20 psi over 5 seconds. The analyte in the sample was subjected to a potential difference of 25.1 kV along the capillary for 15 minutes, resulting in a measurement of 424 Vcm². -1 The separation was achieved by generating an electric field. The capillary was always maintained at 25°C. The separation buffer used for all analyses was borate buffer (density 1.0000 g / mL, pH 9.2).

[0032] In the CZE assay, two different sizes of gRNA (40 or 100 nucleotides) were separated from three nucleases (Streptococcus pyogenes Cas9 (SpCas9), Acidaminococcus Cpf1, and Eureca-V®) in a capillary filled with separation buffer containing a fluorescent RNA stain. The gRNA was separated from the complexed nuclease and RNP based on the charge-to-mass ratio and detected using a LIF detector, as shown in Figures 1 and 2, by the time it took for uncomplexed RNA and RNP to reach the detection window. For each integrated peak in the resulting electrophoresis, the peak area was determined using 32 Karat software. The concentration of free gRNA in the RNP sample was calculated by comparing the peak area of ​​gRNA in the sample with the peak area of ​​a gRNA standard at a known concentration using a gRNA standard calibration curve.

[0033] Create a standard curve gRNA (typically the same sequence used in RNP complexes) was diluted to a working concentration of 10 μM with molecular biology grade (MBG) water and thoroughly mixed. Next, the gRNA was 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 resulting gRNA dilutions were assayed to create a standard calibration curve (Figure 3). The upper and lower limits of the gRNA concentration used in creating the standard curve may be modified depending on the quality of the gRNA.

[0034] [Table 1]

[0035] Sample preparation Each RNP sample was diluted to a final concentration of 1 μM with MBG water and thoroughly mixed. separation buffer Borate buffer (pH 9.2) was used as the separation buffer. fluorescent dyes Sybr® Green II RNA gel stain (Thermo Fisher Scientific) was used as the fluorescent dye.

[0036] result This novel analytical method was used to measure the concentration and percentage of free intact gRNA in RNP samples. This method can detect free RNA (e.g., gRNA) at low concentrations, such as the minimum reference standard of 0.2 μM or 6.8 μg / ml.

[0037] Example 2: Evaluation of the specificity, linearity, accuracy, and repeatability of the CZE assay. In this example, the specificity, linearity, accuracy, and reproducibility of the CZE assay described in Example 1 are evaluated using an RNP with a gRNA-to-SpCas9 ratio of 3:1. The target acceptance criteria for the parameters under test are summarized in Table 2 below. [Table 2]

[0038] method Prior to performing the CZE assay, preliminary experiments were conducted to determine an appropriate range for the gRNA standard curve, setting five levels from 0.4 mM to 2.0 mM in 0.4 mM increments. To broaden the range of the standard curve, a sixth point of 2.2 mM was added. Next, five gRNA spike levels (samples S1 to S5) were selected from 0.4 mM to 1.2 mM in 0.2 mM increments. To determine the baseline amount of free gRNA present in the samples, a spike-free RNP sample (S0) was analyzed as a control. Each sample was tested in three experiments with single injections to evaluate the specificity, reproducibility, precision, and linearity of the assay method.

[0039] result The results from each of the three experiments were analyzed by integrating them within the instrument software. Linear regression was performed on the six points within each gRNA standard curve (Figures 7A-C), and this was used to quantify the amount of free gRNA present in the RNP sample without spikes and in each spiked sample.

[0040] specificity In each experiment, the prepared buffer did not interfere with the free gRNA peak (Figure 8). The bound gRNA peak was separated from the free gRNA peak. See Figure 9 for results.

[0041] linearity In each experiment, the measured amount of free gRNA in the unspiked RNP sample was subtracted from the total measured amount of spiked samples to quantify the measured spike at that concentration. The average measured amount of free gRNA spikes across all three experiments was plotted against the theoretical amount of spiked free gRNA at each concentration, and linear regression was performed. See Table 3 and Figure 10 for the results.

[0042] accuracy The recovery rate (recovery %) was calculated for each concentration in each experiment as the percentage of the ratio of the measured spike amount to the theoretical spike amount of free gRNA. The average recovery rate for each concentration was evaluated against the initial range standard of 100 ± 10%. Although one of the individual S1 spike concentrations fell outside this range, the sensitivity of this indicator improved at lower concentrations, and the average recovery rate at that concentration was acceptable, so it was judged that there was no impact. See Table 3 and Figure 11 for the results.

[0043] reproducibility Reproducibility was calculated by determining the total amount of free gRNA in each RNP sample (both unspiked and spiked) as the relative standard deviation percentage (%RSD) over three experiments. The results were evaluated against the initial target value of %RSD ≤ 5%. Due to the relatively small sample size, the %RSD of the unspiked RNP sample was more sensitive. Therefore, the %RSD result for the S0 control was acceptable against the target value of ≤ 10%. See Table 3 for results.

[0044] [Table 3]

[0045] conclusion The data above demonstrates that the CZE assay method described in this disclosure exhibits excellent specificity, linearity, accuracy, and reproducibility in the quantification of free gRNA in the concentration range of 0.2–1.0 μM.

Claims

1. A method for quantifying free RNA present in a composition comprising a ribonucleoprotein (RNP) complex formed between a gene-editing endonuclease and one or more associated RNAs by capillary zone electrophoresis (CZE), comprising the following steps: (i) Below: A source reservoir containing separation buffer and a target reservoir, wherein the source reservoir further contains an RNA staining dye, an anode located in a source reservoir and a cathode located in a target reservoir, the anode and cathode connected to a power supply, A fused silica capillary tube connecting the two reservoirs, and A cathode-near detector for detecting signals from the capillary tube. A step of providing a CZE system including, (ii) A step of introducing the composition into the capillary tube near the anode, (iii) The step of performing electrophoresis on the system, and (iv) A step of quantifying the signal from an RNA-binding dye using a detector, wherein the amount of the signal indicates the concentration of free RNA in the composition. Methods that include...

2. The method according to claim 1, wherein the RNA is guide RNA (gRNA).

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

4. The method according to any one of claims 1 to 3, wherein the isolation buffer is prepared to maintain the natural conformation of the RNP.

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

6. The method according to any one of claims 1 to 5, wherein the RNA staining dye is a fluorescent dye.

7. The method according to claim 6, wherein the fluorescent dye comprises a chromophore unit having the following chemical structure. 【Chemistry 1】

8. The method according to any one of claims 1 to 7, wherein the detector is a laser-induced fluorescence (LIF) detector.

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

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

11. The method according to any one of claims 1 to 10, wherein free RNA can be detected even at low concentrations of 0.2 μM or 6.8 μg / ml.

12. A composition comprising a ribonucleoprotein (RNP) complex formed between a gene-editing endonuclease and one or more associated RNAs, wherein the composition is obtained by a process of separating free RNA from the RNP complex, the process comprising the following steps: (i) Below: A source reservoir containing separation buffer and a target reservoir, wherein the source reservoir further contains an RNA staining dye, an anode located in a source reservoir and a cathode located in a target reservoir, the anode and cathode connected to a power supply, A fused silica capillary tube connecting the two reservoirs, and A cathode-near detector for detecting signals from the capillary tube. A step of providing a CZE system including, (ii) A step of introducing the composition into the capillary tube near the anode, (iii) The step of performing electrophoresis on the system to separate free RNA from the RNP complex, and (iv) A step of quantifying the signal from an RNA-binding dye using a detector, wherein the amount of the signal indicates the concentration of free RNA in the RNP complex. A composition containing the following:

13. The composition according to claim 12, wherein the RNA is guide RNA (gRNA).

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

15. The composition according to any one of claims 12 to 14, wherein the isolation buffer is prepared to maintain the natural conformation of the RNP.

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

17. The composition according to any one of claims 12 to 16, wherein the RNA staining dye is a fluorescent dye.

18. The composition according to claim 17, wherein the fluorescent dye comprises a chromophore unit having the following chemical structure. 【Chemistry 2】

19. The composition according to any one of claims 12 to 18, wherein the detector is a laser-induced fluorescence (LIF) detector.

20. The composition according to any one of claims 13 to 19, wherein the gRNA is at least 40 nucleotides long or at least 100 nucleotides long.

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

22. The composition according to any one of claims 12 to 21, wherein the concentration of free RNA is at least 0.2 μM or 6.8 μg / ml.

Images