Methods for detecting sgRNA in cells

Abstract

A method for detecting sgRNA in cells is provided, which mainly includes performing quantitative real-time PCR on samples using specific forward and reverse primers, and also includes using probes linked to fluorescent and quenching groups. This method can achieve high specificity, high sensitivity, and convenient operation for detecting intracellular sgRNA. A composition comprising the forward primer, reverse primer, and probe, a kit comprising the composition, and the application of the forward and reverse primers, the composition, or the kit in detecting sgRNA in cells are also provided.

Description

Methods for detecting sgRNA in cells

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311868911.1 filed on December 29, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to biological detection technology, and specifically to the technology of detecting sgRNA in cells. Background Art

[0004] The CRISPR / Cas system is an acquired immune system found in most archaea and bacteria. It is used to defend against foreign genetic material (such as plasmids and bacteriophages) and retain foreign gene fragments in its own loci as "memory" to prevent further invasion. Currently, the CRISPR / Cas system has been adapted by scientists as a powerful tool for gene editing, relying on a single guide RNA (sgRNA) and Cas enzymes for genome editing. The specificity of the targeted genome is determined by the small guide RNA (sgRNA), and the sequence of the targeted genome has a three-base prototype adjacent motif (PAM) at the 3' or 5' end.

[0005] In recent years, with the widespread application of CRISPR / Cas systems in cell therapy, the intracellular retention and decay of sgRNA, a key raw material, after transfection into cells has remained largely unstudied. According to the SFDA's "Technical Guidelines for Pharmaceutical Research and Evaluation of Gene Transduction and Modification Systems," when using nucleic acid vectors for gene transduction or modification, continued attention should be paid to the residual presence of the vector system in the cell product, such as residual RNA. Advanced technologies are encouraged to detect residual RNA to ensure that the risk of residual sgRNA in the product is manageable. Established limits must be scientifically and rationally calculated.

[0006] For this type of external testing, NGS or qPCR (quantitative PCR) methods are generally used. Although the second-generation sequencing method (NGS) can accurately determine the nucleic acid sequence, it has a long experimental cycle, high cost, and lacks quantitative capabilities. In contrast, qPCR can more conveniently and accurately quantify residual nucleic acids. In addition, it has a short experimental cycle and low cost. In the future, it can be applied to the quality research and release of cell therapy products, helping to better control the quality and risk of cell therapy products.

[0007] Taqman probe detection, a real-time quantitative PCR technique, is a rapid, high-throughput detection method. It incorporates a specific fluorescent probe during the PCR amplification process, reflecting the increase in product through changes in the fluorescent signal, enabling quantitative analysis of the initial template in the sample. In recent years, due to its unique advantages in specificity, sensitivity, and accuracy, Taqman probe technology has gained widespread recognition and application in disease-related testing fields such as detecting gene mutations and gene quantification. Summary of the Invention

[0008] In one aspect, the present application provides a method for detecting sgRNA in cells, comprising: (1) pretreating the cells, extracting RNA from the cells, and obtaining a sample to be detected; (2) performing fluorescent quantitative PCR on the sample to be detected using a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2.

[0009] In some embodiments, step (2) further comprises using a probe connected to a fluorescent group and a quencher group, wherein the nucleotide sequence of the probe is shown in SEQ ID NO: 3.

[0010] In some embodiments, the fluorescent quantitative PCR is RT-qPCR.

[0011] In some embodiments, the fluorescent group is one or more of FAM, JOE, JA270, TET, Cal Fluor Gold 540, HEX, VIC, Cal Fluor Orang 560, TAMRA, Cyanine 3, Quasar 570, Cal Fluor Red 590, Rox, Texas Red, Cyanine 5, Quasar 670, and Cyanine 5.5, preferably FAM.

[0012] In some embodiments, the quencher group is one or more of MGB, TAMRA, DABCYL, BHQl-3, and Eclipse, preferably MGB.

[0013] In some embodiments, the molar ratio of the forward primer, reverse primer, and probe is in the range of 1:1:1 to 2:2:1. In some embodiments, the molar ratio of the forward primer, reverse primer, and probe is about 2:2:1.

[0014] In some embodiments, the concentrations of the forward primer, reverse primer, and probe are each in the range of 0.1-1.0 μM. In some embodiments, the concentrations of the forward primer, reverse primer, and probe are each in the range of 0.1-0.4 μM.

[0015] In some embodiments, the enzymes used in the fluorescent quantitative PCR include reverse transcriptase, amplification polymerase, and auxiliary enzymes, wherein the amplification polymerase includes Taq DNA polymerase, and the auxiliary enzyme includes a ribonuclease inhibitor and / or a thermolabile UDG enzyme.

[0016] In some embodiments, the pretreatment in step (1) comprises lysing cells and extracting RNA through an RNA column, wherein extracting RNA through the RNA column comprises adding preheated ddH2O without ribozyme to the center of the membrane of the RNA column, and the preheating temperature is 40-50° C. In some embodiments, the preheating temperature is about 45° C.

[0017] In some embodiments, the sgRNA comprises the nucleotide sequence shown in SEQ ID NO:4, or a nucleotide sequence having at least about 95% sequence identity to SEQ ID NO:4.

[0018] In another aspect, the present application provides the use of the forward primer shown in SEQ ID NO: 1 and the reverse primer shown in SEQ ID NO: 2 in detecting sgRNA in cells.

[0019] In some embodiments, the sgRNA comprises the nucleotide sequence shown in SEQ ID NO:4, or a nucleotide sequence having at least about 95% sequence identity to SEQ ID NO:4.

[0020] In yet another aspect, the present application provides a composition comprising a forward primer, a reverse primer and a probe, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO: 1, the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2, the nucleotide sequence of the probe is shown in SEQ ID NO: 3, and the probe is connected to a fluorescent group and a quenching group.

[0021] In some embodiments, the fluorescent group is one or more of FAM, JOE, JA270, TET, Cal Fluor Gold 540, HEX, VIC, Cal Fluor Orang 560, TAMRA, Cyanine 3, Quasar 570, Cal Fluor Red 590, Rox, Texas Red, Cyanine 5, Quasar 670, and Cyanine 5.5, preferably FAM.

[0022] In some embodiments, the quencher group is one or more of MGB, TAMRA, DABCYL, BHQl-3, and Eclipse, preferably MGB.

[0023] In some embodiments, the molar ratio of the forward primer, reverse primer, and probe is in the range of 1:1:1 to 2:2:1. In some embodiments, the molar ratio of the forward primer, reverse primer, and probe is about 2:2:1.

[0024] In some embodiments, the concentrations of the forward primer, reverse primer, and probe are each in the range of 0.1-1.0 μM. In some embodiments, the concentrations of the forward primer, reverse primer, and probe are each in the range of 0.1-0.4 μM.

[0025] In yet another aspect, the present application provides a kit comprising the composition of any one of claims 16-22.

[0026] In some embodiments, the kit further comprises one or more of an sgRNA standard, an RNA amplification enzyme, and a ROX reference dye. In some embodiments, the RNA amplification enzyme comprises a reverse transcriptase, an amplification polymerase, and an auxiliary enzyme, wherein the amplification polymerase comprises Taq DNA polymerase, and the auxiliary enzyme comprises a ribonuclease inhibitor and / or a thermolabile UDG enzyme.

[0027] In yet another aspect, the present application provides use of the composition or the kit in detecting sgRNA in cells.

[0028] In some embodiments, the sgRNA comprises the nucleotide sequence shown in SEQ ID NO:4, or a nucleotide sequence having at least about 95% sequence identity to SEQ ID NO:4.

[0029] This application provides methods, compositions, and kits for detecting intracellular sgRNA with good specificity, high sensitivity, and convenient operation. These methods, compositions, and kits are particularly suitable for practical application scenarios of sgRNA in gene-edited cells using the CRISPR / Cas system, and can quantitatively characterize intracellular sgRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 shows the distribution of sgRNA amplification primers and probes.

[0031] Figure 2 shows the results of the amplification system screening, where the upper figure is the amplification curve of the sgRNA standard amplified by the Q231 system, and the lower figure is the amplification curve of the sgRNA standard amplified by the Q222 system.

[0032] FIG3 shows the amplification curve of the standard detected in Example 4.

[0033] Figure 4 shows the linear relationship of the standards (standard curve).

[0034] FIG5 shows the amplification curve obtained from the specificity verification experiment in Example 5.

[0035] FIG6 shows the standard curve obtained from the specificity verification experiment in Example 5.

[0036] Figure 7 shows a comparison of the attenuation curves of sgRNA (percentage) modified with the Fam fluorescent group in HEK293T cells under different detection methods in Example 6.

[0037] FIG8 shows the time-dependent curve of the intracellular residual sgRNA concentration in three parallel experiments in Example 6.

[0038] Figure 9 shows the curve of sgRNA residue in T cells changing over time in Example 7. DETAILED DESCRIPTION

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0040] The term "or" refers to a single element of the listed alternative elements, unless the context clearly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more or all of the listed alternative elements.

[0041] The terms "comprising", "including" and "having" mean "including but not limited to", but also consist of only the listed elements.

[0042] The term "about," when used with a numerical variable, means that the value of the variable is within ±5% of the stated value.

[0043] The term "detection" refers to determining whether a target substance (e.g., sgRNA) is present or its content. The content can be an absolute content or a relative content.

[0044] The term "fluorescent quantitative PCR" refers to a PCR technique that introduces a fluorescent substance into the PCR amplification reaction system and detects the fluorescent signal of the amplification reaction cycle product to detect the target nucleic acid (such as sgRNA). When detecting RNA, "fluorescent quantitative PCR" can be reverse transcription PCR (RT-PCR). "Fluorescent quantitative PCR" can be non-real-time PCR or real-time quantitative PCR. The fluorescent substance can be a fluorescent dye (such as TB Green), or a fluorescent probe (such as the fluorescent probe provided in this application). Although the fluorescent probe method is used for real-time quantitative PCR in the specific embodiments given in this application, those skilled in the art should understand that it is merely exemplary and not restrictive. Based on the forward primer and reverse primer provided in this application, non-real-time or fluorescent quantitative PCR performed with a fluorescent dye method can also achieve the technical effect of detecting sgRNA (for example, sgRNA in cells).

[0045] The term "sgRNA" herein may include "small guide RNA" and / or "single guide RNA," and refers to an RNA that is complementary or substantially complementary to a region of a target nucleic acid sequence and guides the Cas protein to that region during gene editing using the CRISPR / Cas system. sgRNA generally comprises crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA), both of which may exist in a fused single-stranded form connected by a linker (i.e., a "single guide RNA") or in a non-fused, separate form.

[0046] An exemplary sgRNA universal sequence is shown in SEQ ID NO: 4. In some embodiments, the sgRNA being tested comprises the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the sgRNA being tested comprises a nucleotide sequence having at least about 95% sequence identity to SEQ ID NO: 4.

[0047] The term "sequence identity" refers to the similarity between nucleic acid or amino acid sequences, which is typically measured as a percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods of alignment of sequences for comparison are well known. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, details sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from a variety of sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions for determining sequence identity using this program are available on the NCBI website on the internet.

[0048] The present application provides a method for detecting a target sgRNA, comprising detecting the target sgRNA using a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is as shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO: 2. The detection can be performed by PCR. In some embodiments, the PCR is fluorescent quantitative PCR.

[0049] The target sgRNA may be a target sgRNA present in any location or region. For example (but not limited to), it may be an sgRNA present in a biological product (e.g., a blood product, etc.). For another example, it may be an sgRNA present in the environment, such as the environment in which sgRNA is produced, stored, transported, or used as a raw material and / or products involving sgRNA are produced. For another example, it may be an sgRNA present in a cell, such as a cell that is gene-edited using the CRISPR / Cas system. In some embodiments, the location or region where the target sgRNA may be present may be sampled and / or pretreated so that the target sgRNA is in a detectable state. In some embodiments, the sample obtained after sampling and / or pretreatment may also be diluted so that the concentration of the sample reaches a range that is most suitable for detection.

[0050] In some embodiments, the detection further comprises using a probe connected to a fluorescent group and a quencher group, wherein the nucleotide sequence of the probe is shown in SEQ ID NO: 3. In some embodiments, the PCR is RT-qPCR.

[0051] In some embodiments, the fluorescent group is one or more of FAM, JOE, JA270, TET, Cal Fluor Gold 540, HEX, VIC, Cal Fluor Orange 560, TAMRA, Cyanine 3, Quasar 570, Cal Fluor Red 590, Rox, Texas Red, Cyanine 5, Quasar 670, and Cyanine 5.5. In some embodiments, the fluorescent group is FAM.

[0052] In some embodiments, the quencher group is one or more of MGB, TAMRA, DABCYL, BHQ1-3, and Eclipse. In some embodiments, the quencher group is MGB.

[0053] In some embodiments, the molar ratio of the forward primer, reverse primer, and probe is in the range of 1:1:1 to 2:2:1. In some embodiments, the molar ratio of the forward primer, reverse primer, and probe is about 2:2:1.

[0054] In some embodiments, the concentrations of the forward primer, reverse primer, and probe are each in the range of 0.1-1.0 μM. In some embodiments, the concentrations of the forward primer, reverse primer, and probe are each in the range of 0.1-0.4 μM.

[0055] In some embodiments, the target sgRNA comprises a nucleotide sequence as shown in SEQ ID NO: 4. In some embodiments, the target sgRNA comprises a nucleotide sequence having at least about 95% sequence identity to SEQ ID NO: 4.

[0056] The forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 provided herein, a composition comprising the primers and a probe of SEQ ID NO: 3 linked to a fluorescent group and a quenching group, and a kit comprising the composition can be used to detect target sgRNA in any form, for example (but not limited to) detecting sgRNA in cells in the context of gene editing using the CRISPR / Cas system. The cells can be HEK293T cells or other cell types including but not limited to T cells.

[0057] When referring to "detecting sgRNA in cells", the "pre-treating the cells and extracting RNA from the cells" described in this application refers to treating the cells in which it is desired to detect the presence of sgRNA or to determine the (absolute or relative) content of sgRNA so that the sgRNA (if any) in the cells is in a detectable state. This may include lysing the cells and recovering the lysate (in which the RNA is in a detectable state), and optionally further extracting RNA using, for example, an RNA column. The "sample to be tested" may be a cell lysate, or a product after further RNA extraction using, for example, an RNA column, or a diluted version of the above product.

[0058] The “probe connected to a fluorescent group and a quencher group” described in the present application is a TaqMan fluorescent probe, which is connected to a fluorescent group at the 5' end and a quencher group at the 3' end. The nucleotide sequence designed in the present application, such as the SEQ ID NO: 3 probe, has sufficient complementarity with the target sgRNA fragment (for example, it contains a nucleotide sequence as shown in SEQ ID NO: 4 or a nucleotide sequence with at least about 95% sequence identity with SEQ ID NO: 4). When the probe is intact, the fluorescent signal emitted by the fluorescent group is absorbed by the quencher group. When the primer is extended, under the action of Taq DNA polymerase, the probe bound to the DNA template will be cut off, the fluorescent group will be away from the quencher group, and the energy can no longer be absorbed, and the RT-qPCR instrument can detect the fluorescent signal. Based on this, real-time quantitative detection of the target sgRNA can be achieved.

[0059] In this article, “RNase” and “ribonuclease” are used interchangeably, “RNase-free” and “ribonuclease-free” are used interchangeably, “sgRNA universal” and “sgRNA-Universal” are used interchangeably, and “Primer & Probe Mix” and “Primer Probe Mix” are used interchangeably.

[0060] The present invention is further illustrated below by means of specific examples.

[0061] Example 1. qPCR primer probe design

[0062] Based on the sgRNA-Universal sequence, the preferred primer-probe combination was designed and screened (see Figure 1). The obtained primer pairs (including forward primer F and reverse primer R2) with good specificity for sgRNA and the nucleotide sequences of the probe (P) are shown in Table 1:

[0063] Table 1: sgRNA primer and probe sequences

[0064] Example 2. Preparation of standard products

[0065] 1. Given the sequence of the standard (see Table 2), obtain the sgRNA standard (dry powder) (purchased from Nanjing GenScript, sgRNA-RUO inner single). Dilute with RNase-free water according to the instructions, and use NanoDrop to accurately measure the concentration of the sgRNA standard.

[0066] 2. Dilution and storage: Dilute the precisely quantified sgRNA standard to 160 ng / μL using low TE (purchased from Nanjing Ruibeixi). After vortexing and mixing, aliquot into 0.5 mL shipping tubes, 50 μL / tube, and store in a -30°C refrigerator (-80°C is recommended for long-term storage).

[0067] Table 2: sgRNA-Universal sequence information Note: m indicates 2'OMe modification, * indicates phosphorothioate modification, and r indicates RNA.

[0068] Example 3. Screening of amplification reagents in the kit

[0069] Two amplification detection systems, Q222 and Q231 (purchased from Nanjing Vazyme), were selected respectively, and the sgRNA standards were amplified simultaneously. The standard curves (400 ng / ul-40 fg / ul) and the detection Ct values ​​(10 replicate wells) at a template amount of 2 fg / ul (quantification limit concentration) were compared. The detection Ct values ​​were substituted into the standard curves to calculate the detection concentration of each well, and the coefficient of variation (CV%) of the detection concentration of 10 replicate wells was calculated.

[0070] Table 3: One-step RT-qPCR reagent enzyme system

[0071] The amplification curve is shown in Figure 2, and the screening results are shown in Table 4:

[0072] Table 4: Amplification reagent screening results

[0073] According to the Validation Guidelines for Quantitative Analysis Methods for Biological Samples 9012 of the 2020 edition of the Chinese Pharmacopoeia, the above results show that the quantification limit of the Q231 reagent can reach 2fg / ul (CV<20%), and the quantification limit of Q222 cannot reach 2fg / ul (CV>20%), so Q231 was selected as the subsequent detection system.

[0074] Example 4. Intracellular sgRNA residual detection process

[0075] 4.1 Kit

[0076] The kit provided in this application for detecting sgRNA residues mainly includes the following components:

[0077] (1) sgRNA standard prepared in Example 2 (160 ng / μL);

[0078] (2) One-step amplification buffer, including Tris, dNTP / dUTP Mix, Mg 2+ wait;

[0079] (3) One-step RNA amplification enzyme mix, including the reverse transcriptase, amplification polymerase, auxiliary enzyme, etc. of the Q231 system in Table 3;

[0080] (4) 50×ROX reference dye;

[0081] (5) sgRNA Primer & Probe Mix: Synthesize the sequence designed in Example 1 and dilute it with lowTE (0.1 mM EDTA, 10 mM Tris, pH 8.0). Accurately quantify the concentrations of the three samples F, R2, and P to 10 μM, mix them in a volume ratio of F: R2: P = 2:2:1, and prepare the sgRNA Primer & Probe Mix. Store at -20°C for later use.

[0082] All components in the kit provided in this application are stored at -20±5°C, among which ROX reference dye and sgRNA Primer & Probe Mix need to be stored away from light.

[0083] 4.2 Samples

[0084] Samples to be tested: Cells that have been gene-edited using the CRISPR / Cas system;

[0085] Positive standard: sgRNA standard (160 ng / μL);

[0086] Negative control: RNA diluent (low TE).

[0087] 4.3 Sample pretreatment

[0088] 4.3.1 RNA extraction from cells:

[0089] According to the instructions of the RNA extraction kit (RC201, purchased from Nanjing Vazyme), RNA was extracted and recovered from the samples to be tested. The specific procedures are as follows:

[0090] (1) Collect cells

[0091] a. Suspension culture cells: Count the number of cells, collect the cells by centrifugation at 300g for 5 minutes, carefully discard the culture medium, and proceed as per (2).

[0092] b. Adherent cells: Adherent cells can be directly lysed in the culture flask / dish, or they can be digested with trypsin and collected by centrifugation.

[0093] c. Add 1 mL of RNA Isolater (cell lysis buffer) to the cell sample. Vortex or pipette to dissolve the cell pellet.

[0094] (2) Place at room temperature for 2 to 3 minutes to allow the cells to fully lyse.

[0095] (3) Add 200 μL of RNA extraction auxiliary reagent (ES-8522, purchased from Guangzhou ECOTOP) to the lysate. Shake vigorously by hand for 15 seconds and let it stand at room temperature for 3 minutes.

[0096] (4) Centrifuge at 4°C, 12,000 rpm (13,400 × g) for 15 min. Pipette 500 μL of the supernatant into a new 1.5 mL centrifuge tube.

[0097] (5) It should be noted that: carefully aspirate the supernatant aqueous phase to avoid aspirating the middle and lower organic phases, which may affect the subsequent extraction results.

[0098] (6) Add 750 μL of anhydrous ethanol (1.5 times the volume of the supernatant) to the supernatant. Vortex and mix for 10 seconds.

[0099] (7) Place the RNA column (MiPure miRNA Column) in a 2 ml collection tube. Transfer half the volume of the mixture to the column and centrifuge at 12,000 rpm (13,400 × g) for 30 seconds.

[0100] (8) Discard the filtrate and return the RNA column to the collection tube. Transfer the remaining mixture to the RNA column and centrifuge at 12,000 rpm (13,400 × g) for 30 seconds.

[0101] (9) Discard the filtrate and return the RNA column to the collection tube. Add 500 μL of Buffer RW1 (wash solution 1, with ethanol added) to the column, incubate at room temperature for 1 min, and centrifuge at 12,000 rpm (13,400 × g) for 30 sec.

[0102] (10) Discard the filtrate and return the RNA column to the collection tube. Add 500 μL of Buffer RW2 (wash solution 2, with ethanol added) to the column, incubate at room temperature for 1 min, and centrifuge at 12,000 rpm (13,400 × g) for 30 sec.

[0103] (11) Discard the filtrate and return the RNA column to the collection tube. Add 500 μL of 80% ethanol (freshly prepared with RNase-free ddH2O) to the column, incubate at room temperature for 1 min, and centrifuge at 12,000 rpm (13,400 × g) for 30 sec.

[0104] (12) Discard the filtrate and return the RNA column to the collection tube. Centrifuge the empty column at 12,000 rpm (13,400 × g) for 5 minutes to dry the RNA column matrix.

[0105] (13) Transfer the RNA column to an RNase-free 1.5 mL centrifuge tube. Open the lid and let it air dry at room temperature for 2 minutes.

[0106] (14) Add 50 μL of preheated RNase-free ddH2O to the center of the RNA column membrane. Let stand at room temperature for 2 minutes. Centrifuge at 12,000 rpm (13,400 × g) for 1.5 minutes. Preheat to 45°C.

[0107] (15) Secondary elution: Add 50 μL of the centrifuged solution back to the center of the RNA column membrane. Let stand at room temperature for 2 minutes. Centrifuge at 12,000 rpm (13,400 × g) for 1 minute. If the RNA yield exceeds 30 μg, a secondary elution can achieve a higher yield.

[0108] (16) Discard the RNA column, mix and centrifuge the collected total RNA (including small molecule RNA) samples, and dispense them into 0.2 mL PCR tubes, 10 μL / tube, and store at -80°C until testing.

[0109] 4.3.2 Dilution: Dilute the extract of the sample to be tested with low TE to an appropriate concentration range to obtain the diluted sample to be tested.

[0110] 4.4 RT-qPCR detection

[0111] 4.4.1 Gradient dilution of sgRNA standard

[0112] Use diluent (low TE) to serially dilute the sgRNA quantitative reference at 160 ng / μL to the following concentrations: 40 ng / μL, 4 ng / μL, 400 pg / μL, 40 pg / μL, 4 pg / μL, 400 fg / μL, 40 fg / μL, and 4 fg / μL.

[0113] (1) Take 8 clean 1.5 ml centrifuge tubes and label them as GS, GS', GS2", GS1, GS2, GS3, GS4, and GS5 respectively;

[0114] (2) Add 30 μL of RNA diluent to each of the labeled GS tubes, and add 90 μL of RNA diluent to each of the labeled GS' to GS5 tubes;

[0115] (3) Take out the sgRNA standard from the -30°C refrigerator and thaw it in a 4°C refrigerator. After it is completely thawed, shake it slightly and centrifuge it for 10 seconds.

[0116] (4) Take 10 μL of sgRNA quantitative reference, add it to the GS tube, shake and mix, and then centrifuge quickly for a short time;

[0117] (5) Perform dilution operations according to Table 5:

[0118] Table 5: Dilution of sgRNA Quantification Reference

[0119] 4.4.2 Preparation of qRT-PCR reaction system for standards / test samples

[0120] (1) Calculate the number of test reaction wells required. Generally, 3 replicate wells / sample are made. Number of reaction wells = (5 concentration gradient standard curves + 1 template-free control NTC + 1 negative control NCS + sample to be tested) × 3

[0121] (2) Calculate the total amount of qRT-PCR MIX required to complete this test based on the number of reaction wells: qRT-PCR MIX = (number of reaction wells + 2) × 15 μL (Note: This includes the loss amount of 2 wells)

[0122] (3) Prepare the qRT-PCR reagents required for the standards and test samples according to Table 6 below:

[0123] Table 6: qRT-PCR MIX configuration table

[0124] 4.4.3 qRT-PCR sample loading and detection

[0125] 4.4.3.1 Sample addition

[0126] Add samples as shown in Table 7:

[0127] Table 7: Example of sample loading into each reaction well

[0128] The 96-well plate was sealed with an optical membrane, centrifuged briefly for 10 seconds, and then placed in a qPCR instrument.

[0129] 4.4.3.2 RT-qPCR Instrument Setup

[0130] (1) Set the target: the target name is "sgRNA", the reporter fluorescent group is "FAM", the quencher fluorescent group is "MGB", and the detection reference fluorescent group is "ROX".

[0131] (2) Set the reaction program as shown in Table 8 below:

[0132] Table 8: Reaction Procedure

[0133] 4.4.4 Result Analysis

[0134] (1) Open the saved file and click the Analyze button. After analyzing the data, export the data to an Excel table. The test results are shown in Figures 3, 4, 7, and Table 8.

[0135] (2) Calculate the residual RNA amount of the sample to be tested according to the following formula:

[0136] Residual amount of sgRNA in the sample to be tested = detection concentration (pg / μL) ÷ 5 (sample volume, μL) × sample dilution factor × 50 (RNA extraction volume, μL)

[0137] Example 5. Performance test

[0138] 5.1 Specificity (primer specificity) verification test

[0139] During the detection of sgRNA residues in gene-edited cells, RNA extraction is required. The residues of total RNA (i.e., all RNA except the target sgRNA to be detected) and genome (i.e., all DNA in the cell) may affect the detection of sgRNA, so interference needs to be eliminated. The samples used for this specificity verification mainly include: total RNA of HEK293T cells, and the effect of the human genome on sgRNA residues. The genomic DNA and total RNA of HEK293T cells were extracted using the Takara Cell Genomic DNA Extraction Kit and RNA Extraction Kit, respectively. The above samples were all subjected to fluorescent quantitative PCR detection according to the method shown in Example 4 to verify the specificity of the primers and probes. The results are shown in Figures 5 and 6.

[0140] As shown in Figure 5, no sgRNA was detected in either the HEK293T genome or total RNA samples, indicating that the primers and probes provided by the present invention do not induce nonspecific reactions in these samples. As shown in Figure 6, no sgRNA was detected in either the HEK293T genome or total RNA samples. In summary, the primers in the kit provided by the present invention have good specificity for sgRNA.

[0141] 5.2 Accuracy Test

[0142] Different amounts of sgRNA were added to the basic sample sgRNA to obtain four samples: sample 1 (sgRNA input amount 1 pg), sample 2 (sgRNA input amount 1 ng), sample 3 (sgRNA input amount 14 pg), and sample 4 (sgRNA input amount 0.5 pg). The samples were assayed using the kit and detection method in Example 4, and the recovery rate was calculated.

[0143] The results are shown in Table 9:

[0144] Table 9: Accuracy experimental results

[0145] The data shows that the recovery rates of each sample are between 70% and 120%, and the CV values ​​are all less than 20%, which meets the accuracy requirements of the method for determining the residual amount of exogenous DNA in 3407 of the Chinese Pharmacopoeia 2020 edition.

[0146] 5.3 Linear Relationship and Limit of Quantitation Test

[0147] The sgRNA standard was diluted with Low TE to prepare a series of sample loading concentrations: 400pg / μL, 40pg / μL, 4pg / μL, 400fg / μL, 40fg / μL, and 4fg / μL, respectively, denoted as GS0, GS1, GS2, GS3, GS4, and GS5. 4fg / μL, 2fg / μL, and 1fg / μL of sgRNA were also tested simultaneously. Ten replicate wells were tested, and the CV% value was calculated.

[0148] The linear relationship amplification curves are shown in Figures 2 and 3, and the results analysis is shown in Table 10:

[0149] Table 10: Linear relationship experimental results

[0150] The quantitative limit analysis results are shown in Table 11:

[0151] Table 11: Quantitation limit test results

[0152] From the results in Table 10, we can see that the linear range of the kit is: 4.00×10 2 pg / μL~4.00×10 -3 pg / μL, R 2 ≥0.999, the accuracy of each concentration standard is between 50%-150%, which meets the accuracy requirements of the method for determining the amount of residual exogenous DNA in 3407 of the Chinese Pharmacopoeia 2020 edition.

[0153] From the results in Table 11, it can be seen that the CV values ​​of 10 replicate wells at concentrations of 2 fg / μL and above are <20%, which means that the quantitative limit of the kit can reach 2 fg / μL.

[0154] 5.4 Precision

[0155] 5.4.1 Repeatability

[0156] The sgRNA concentrations of 4 pg / μL and 0.04 pg / μL were tested 10 times using the kit and detection method in Example 4, and the CV% was calculated. The results are shown in Table 12:

[0157] Table 12: Repeatability results analysis

[0158] The sgRNA concentrations of 4 pg / μL and 0.04 pg / μL were tested 10 times, and the CV values ​​were <15%, which meets the requirements for repeatability and precision in the Validation Guidelines for Quantitative Analysis Methods of Biological Samples 9012 of the 2020 edition of the Chinese Pharmacopoeia.

[0159] 5.4.2 Intermediate precision

[0160] Different researchers used the kit and detection method in Example 4 at different times to test sgRNA at concentrations of 40 pg / μL, 0.4 pg / μL, and 0.004 pg / μL. Each concentration was repeated three times, resulting in a total of 9 CV%. The results are shown in Table 13:

[0161] Table 13: Intermediate precision results analysis

[0162] The CV% of the test results of different experimenters at different times was less than 15%, which meets the requirements for intermediate precision in the 2020 edition of the Chinese Pharmacopoeia 9012 Validation Guidelines for Quantitative Analysis Methods for Biological Samples.

[0163] 5.5 Stability Test

[0164] 5.5.1 Freeze-thaw stability

[0165] The components of the kit in Example 4 were frozen and thawed four times (freeze-thaw treatment standard: the packaged primers and probes were stored in a refrigerator at -20±5°C, and the freeze-thaw treatment was taken out from the refrigerator at -20±5°C and thawed at room temperature until completely thawed, which was considered as one freeze-thaw treatment). The linear relationship and R of the kit were tested according to the method in 4.4 of Example 4. 2 , 4fg / μL sgRNA accuracy and blank limit. The test results are shown in Table 14. As shown, the kit components were frozen and thawed four times without affecting the performance of the kit.

[0166] Table 14: Freeze-thaw stability analysis

[0167] 5.5.2 Stability at 2-8°C

[0168] The components of the kit obtained in Example 4 were placed at 2-8°C for 14 days, and the linear relationship and R 2 The test results are shown in Table 15. As shown, the performance of the reference product, qPCR Reaction Buffer, and sgRNA Primer & Probe MIX was not affected when stored at 2-8°C for 14 days.

[0169] Table 15: Freeze-thaw stability analysis

[0170] Example 6. Application: sgRNA attenuation in HEK293T cells

[0171] 6.1 Comparison of different detection methods

[0172] Regarding the decay of sgRNA over time in HEK293T cells gene-edited using the CRISPR / Cas system, the absolute quantification kit of Example 4 ("Case 4" in Figure 7) and the relative RNA quantification method ("Relative quantification" in Figure 7, using GAPDH as an internal reference) were used to detect the residual amount of sgRNA in HEK293T cells at different time points. At the same time, the sgRNA decay curve (with 0H point as percentage) was plotted and compared with the sgRNA-fluorescence decay curve (Fam-sg in the figure, fluorescence collection and detection of cells at different time points using a flow cytometer):

[0173] As shown in Figure 7, the sgRNA decay rates measured under different detection methods are inconsistent, and the sgRNA decay under the relative quantitative detection method is faster than the absolute quantitative detection method in Example 4. The reason is that when the cell culture time exceeds 24 hours, the Ct value of GADPH gradually decreases significantly with the increase in cell number, causing ΔCt to increase, and the expression of residual sgRNA relative to the internal reference is further reduced, resulting in a smaller relative expression than the actual value. The use of flow cytometry to detect fluorescence to indirectly reflect the decay of sgRNA can only provide a reference and cannot directly reflect the residual sgRNA, because the fluorescent signal remains after sgRNA degradation. Therefore, the quantitative detection scheme we developed has both sensitivity and accuracy.

[0174] 6.2 Repeatability test

[0175] The kit in Example 4 was used to detect the residual amount of sgRNA at different time points in HEK293T cells subjected to gene editing using the CRISPR / Cas system. The parallel experiment was repeated three times, and a curve of the change in intracellular sgRNA concentration over time was plotted (see Figure 8).

[0176] As shown in Figure 8, the three parallel experiments showed consistent attenuation trends of sgRNA in gene-edited HEK293T cells, with good reproducibility.

[0177] Example 7. Other applications of the present invention

[0178] In addition to using TaqMan probes for qRT-PCR to detect residual sgRNA in HEK293T cells, the present invention can also be applied to cell lines commonly used in cell therapy, such as T cells. The procedure is consistent with that in Example 4. The results are shown in Figure 9, demonstrating that the present invention has a certain degree of cell universality.

[0179] ***

[0180] Although the above content refers to a particular preferred embodiment, it should be understood that the present invention is not limited thereto. Those skilled in the art will recognize that various modifications can be made to the disclosed embodiments, and these modifications are also intended to fall within the scope of the present invention.

Claims

1. A method for detecting sgRNA in cells, comprising: (1) Pretreating the cells, extracting RNA from the cells to obtain a sample to be detected; (2) Performing fluorescence quantitative PCR on the sample to be detected using a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is as shown in SEQ ID NO:1, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO:

2.

2. The method according to claim 1, wherein in step (2), it further comprises using a probe linked with a fluorophore and a quencher, and the nucleotide sequence of the probe is as shown in SEQ ID NO:

3.

3. The method according to claim 2, wherein the fluorescence quantitative PCR is RT-qPCR.

4. The method according to claim 2, wherein the fluorophore is one or more of FAM, JOE, JA270, TET, Cal Fluor Gold 540, HEX, VIC, Cal Fluor Orang 560, TAMRA, Cy3, Quasar 570, Cal Fluor Red 590, Rox, Texas Red, Cy5, Quasar 670, and Cy5.5, preferably FAM.

5. The method according to claim 2, wherein the quencher is one or more of MGB, TAMRA, DABCYL, BHQ1-3, and Eclipse, preferably MGB.

6. The method according to any one of claims 2-5, wherein the molar ratio of the forward primer, reverse primer, and probe is in the range of 1:1:1 to 2:2:

1.

7. The method according to any one of claims 2-5, wherein the molar ratio of the forward primer, reverse primer, and probe is about 2:2:

1.

8. The method according to any one of claims 2-5, wherein the concentrations of the forward primer, reverse primer, and probe are respectively in the range of 0.1-1.0 μM.

9. The method according to claim 8, wherein the concentrations of the forward primer, reverse primer, and probe are respectively in the range of 0.1-0.4 μM.

10. The method according to claim 3, wherein the RT-qPCR comprises using a reverse transcriptase, an amplification polymerase, and an auxiliary enzyme, wherein the amplification polymerase comprises Taq DNA polymerase, and the auxiliary enzyme comprises a ribonuclease inhibitor and / or a heat-labile UDG enzyme.

11. The method according to any one of claims 1-5, wherein the pretreatment in step (1) includes lysing the cells and extracting RNA through an RNA column, and extracting RNA through an RNA column includes adding preheated ribonuclease-free ddH2O to the center of the membrane of the RNA column, and the preheating temperature is 40-50 °C.

12. The method according to claim 11, wherein the preheating temperature is about 45 °C.

13. The method according to any one of claims 1-5, wherein the sgRNA comprises the nucleotide sequence shown in SEQ ID NO: 4 or a nucleotide sequence having at least about 95% sequence identity with SEQ ID NO:

4.

14. Use of the forward primer shown in SEQ ID NO: 1 and the reverse primer shown in SEQ ID NO: 2 in detecting sgRNA in cells.

15. The use according to claim 14, wherein the sgRNA comprises the nucleotide sequence shown in SEQ ID NO: 4 or a nucleotide sequence having at least about 95% sequence identity with SEQ ID NO:

4.

16. A composition comprising a forward primer, a reverse primer and a probe, wherein the nucleotide sequence of the forward primer is as shown in SEQ ID NO: 1, the nucleotide sequence of the reverse primer is as shown in SEQ ID NO: 2, the nucleotide sequence of the probe is as shown in SEQ ID NO: 3, and the probe is linked with a fluorophore and a quencher.

17. The composition according to claim 16, wherein the fluorophore is one or more of FAM, JOE, JA270, TET, Cal Fluor Gold 540, HEX, VIC, Cal Fluor Orang 560, TAMRA, Cy3, Quasar 570, Cal Fluor Red 590, Rox, Texas Red, Cy5, Quasar 670 and Cy5.5, preferably FAM.

18. The composition according to claim 16, wherein the quencher is one or more of MGB, TAMRA, DABCYL, BHQ1-3 and Eclipse, preferably MGB.

19. The composition according to any one of claims 16-18, wherein the molar ratio of the forward primer, the reverse primer and the probe is in the range of 1:1:1 to 2:2:

1.

20. The composition according to any one of claims 16-18, wherein the molar ratio of the forward primer, the reverse primer and the probe is about 2:2:

1.

21. The composition according to any one of claims 16-18, wherein the concentrations of the forward primer, the reverse primer and the probe are respectively in the range of 0.1-1.0 μM.

22. The composition according to claim 21, wherein the concentrations of the forward primer, the reverse primer and the probe are respectively in the range of 0.1-0.4 μM.

23. A kit comprising the composition according to any one of claims 16-22.

24. The kit according to claim 23, further comprising one or more of an sgRNA standard, an RNA amplification enzyme, and a ROX reference dye.

25. The kit according to claim 24, wherein the RNA amplification enzyme comprises a reverse transcriptase, an amplification polymerase and an auxiliary enzyme, wherein the amplification polymerase comprises Taq DNA polymerase, and the auxiliary enzyme comprises a ribonuclease inhibitor and / or a heat-labile UDG enzyme.

26. Use of the composition according to any one of claims 16 - 22 or the kit according to any one of claims 23 - 25 for detecting sgRNA in cells.

27. The use according to claim 26, wherein the sgRNA comprises the nucleotide sequence shown in SEQ ID NO:4 or a nucleotide sequence having at least about 95% sequence identity with SEQ ID NO:4.

Images