Analysis of crispr / CAS9 system associated molecules using capillary zone electrophoresis

Abstract

The disclosed technology provides capillary electrophoresis methods and kits for the separation of multiple components in one or two runs with high resolution.

Description

ANAUYSIS OF CRISPR / CAS9 SYSTEM ASSOCIATED MOEECUEES USING CAPIEEARY ZONE EEECTROPHORESISREEATED APPEICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 511,228, filed June 30, 2023, the content of which is hereby incorporated by reference in its entirety into this disclosure.BACKGROUND

[0002] CRISPR (clustered regularly interspaced short palindromic repeats) is an immunological defense mechanism employed by bacteria against invading pathogens and is the most widely used gene editing technology due to its easy design, low cost, and high efficacy. Two molecules are essential for CRISPR: CRISPR associated protein 9 (Cas9), an RNA-guided DNA endonuclease, and single guide RNA (sgRNA). Due to its ability to perform site directed DNA cleavage and to trigger homologous recombination, Cas9 has been utilized as a gene editing tool to introduce gene inactivation and genome modification. CRISPR can improve existing therapies in the pharmaceutical industry, help find drug targets, and test drug candidates.

[0003] The Cas9 ribonucleoprotein (RNP) complex, which is formed by the sgRNA and Cas9 protein, targets the regions of genes for double-stranded breaks and DNA repair. The formation of the Cas9 RNP complex is crucial for high on-target editing efficiency and low off-target editing as it can impact the gene-editing accuracy and efficiency, and the quality of the final cellular product. A simple, efficient, and reliable analytical method is required to separate the Cas9 RNP complex from Cas9 protein and sgRNA. A method to determine the percent of Cas9, sgRNA, and / or Cas9 RNP complex in a sample and the ability to monitor the stability of the Cas9 RNP complex is also desired.SUMMARY

[0004] The claimed and described capillary electrophoresis methods offer a simple way to separate multiple components in one or two runs with high resolution for accurate quantification of those components. The claimed and described capillary electrophoresis methods also provide analytical methods for the separation of Cas9 complex components and quantitative solutions for evaluating Cas9 complex components in a sample.

[0005] One aspect of the disclosure is a first capillary electrophoresis method for analyzing a sample. The method includes loading a first sample, the first sample including a protein and / or a polynucleotide, and a complex, on a capillary electrophoresis (CE) capillary, the CE capillary comprising a buffer, wherein the buffer has a pH greater than the Isoelectric Point(pl) of the protein, initiating a separation by applying a separation voltage to the CE capillary, wherein the CE capillary is run using a reverse polarity separation mode, and, detecting the separated protein and / or the separated polynucleotide and the separated complex with a detector.

[0006] One aspect of the disclosure is a second capillary electrophoresis method for analyzing a sample. The method includes loading a first sample, the first sample including a protein and / or a polynucleotide, and a complex, on a CE capillary, the CE capillary comprising a buffer, wherein the buffer has a pH less than or equal to the pl of the polynucleotide, initiating a separation by applying a separation voltage to the CE capillary, wherein the CE capillary is run using a normal polarity separation mode, and, detecting the separated complex, the separated protein and / or the separated polynucleotide with a detector.

[0007] One aspect of the disclosure is a third capillary electrophoresis method for analyzing a sample. The method includes loading a first sample, the first sample including a protein and / or a polynucleotide, and a complex, on a CE capillary, the CE capillary comprising a first buffer, wherein the first buffer has a pH between the pl of the complex and the pl of the protein, initiating a separation by applying a separation voltage to the CE capillary, wherein the CE capillary is run using a reverse polarity separation mode, detecting the separated complex and / or the separated polynucleotide with a detector; and loading a second sample, the second sample including a protein and / or a polynucleotide, and a complex, on a CE capillary, the CE capillary comprising a second buffer, wherein the second buffer has a pH between the pl of the complex and the pl of the protein, initiating a separation by applying a separation voltage to the CE capillary, wherein the CE capillary is run using a normal polarity separation mode, and detecting the separated protein with a detector.

[0008] One aspect of the disclosure is a fourth capillary electrophoresis method for analyzing a sample. The method includes loading a first sample, the first sample including a protein and / or a polynucleotide, and a complex, on a CE capillary, the CE capillary comprising a first buffer, wherein the first buffer has a pH between the pl of the polynucleotide and the pl of complex, initiating a separation by applying a separation voltage to the CE capillary, wherein the CE capillary is run using a normal polarity separation mode, and, detecting the separated complex and / or separated protein with a detector, and loading a second sample, the second sample including a protein and / or a polynucleotide, and a complex, on a CE capillary, the CE capillary comprising a second buffer, wherein the second buffer has a pH between the pl of the polynucleotide and the pl of complex, initiating a separation by applying a separation voltage to the CE capillary, wherein the CE capillary is run using a reverse polarity separation mode, and detecting the separated polynucleotide with a detector.

[0009] In some aspects, the first sample and the second sample are the same. In another aspect, the first buffer and the second buffer are the same.

[0010] In some aspects, the method further includes producing an electropherogram comprising corrected peak area of the separated complex, the separated protein and / or the separated polynucleotide and generating a corresponding set of values for the separated complex, the separated protein and / or the separated polynucleotide.

[0011] In some aspects, the method further includes determining a percentage of the separated complex, the separated protein and / or the separated polynucleotide in the first sample or second sample, using the corresponding set of values.

[0012] In some aspects, the capillary electrophoresis is capillary zone electrophoresis (CZE).

[0013] In some aspects, the first sample and second sample are loaded onto separate capillaries or loaded sequentially on the same capillary.

[0014] In some aspects, the protein is a Cas9 protein or an engineered Cas9 protein. In another aspect, the pl of the protein is about 7.6. In some aspects, the polynucleotide is a single guide RNA (sgRNA) or a prime editing guide (pegRNA). In another aspect, the pl of the polynucleotide is between about 4 to about 4.5. In some aspects, the complex is a Cas9 ribonucleoprotein (RNP) complex. In some aspects, binding of a polynucleotide by a Cas9 protein results in the formationof the Cas9 RNP complex. In another aspect, the pl of the Cas9 RNP complex is between about 4 to about 7.6.

[0015] In some aspects, during the separation, pressure is applied to the CE capillary. In another aspect, the pressure is at least about 0.1 psi, alternatively at least about 0.2 psi, alternatively at least about 0.3 psi, alternatively at least about 0.4 psi, alternatively at least about 0.5 psi, alternatively at least about 1 psi. In some aspects, a gradient voltage is applied.

[0016] In some aspects, the first sample is diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary. In some aspects, the second sample is diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary.

[0017] In some aspects, the detector is a UV detector. In another aspect, the detector is a fluorescence detector, a laser-induced fluorescence (LIF) detector, a lamp-based fluorescence detector, or a native fluorescence detector. In some aspects, the complex, the protein and / or the polynucleotide in the first sample is fluorescently labeled with a fluorescent dye prior to CE separation. In some aspects, the complex, the protein and / or the polynucleotide in the second sample is fluorescently labeled with a fluorescent dye prior to CE separation.

[0018] Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the disclosure in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE FIGURES

[0019] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0020] FIG. 1 is an illustration of the formation of Cas9 / gRNA RNP complex.

[0021] FIG. 2 is a workflow diagram for analyzing a sample using a reverse polarity separation mode according to an aspect of this disclosure.

[0022] FIG. 3 is a workflow diagram for analyzing a sample using a normal polarity separation mode according to an aspect of this disclosure.

[0023] FIG. 4 is a workflow diagram for analyzing a first sample using a reverse polarityseparation mode and a second sample using a normal polarity separation mode according to an aspect of this disclosure.

[0024] FIG. 5 is a workflow diagram for analyzing a first sample using a normal polarity separation mode and a second sample using a reverse polarity separation mode according to an aspect of this disclosure.

[0025] FIG. 6 depicts polarity modes that may be used for the separation and quantitative analysis of the Cas9 RNP complex samples depending on the pH of the separation buffer according to an aspect of this disclosure.DETAILED DESCRIPTION

[0026] The disclosure generally relates to capillary electrophoresis methods and kits for separating multiple components in one or two high-resolution runs, allowing for accurate detection and quantification of those components. In some non-limiting aspects, the components may include those found in the CRISPR / Cas9 system (e.g., Cas9 protein, sgRNA, and Cas9 RNP complex) and as depicted in FIG. 1. It is well-known in the art that Cas9 protein, sgRNA, and Cas9 RNP complex are present in CRISPR / Cas9 systems (Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR / Cas9 genome editing. Theranostics. 2021 Jan 1;11(2):614- 648. doi: 10.7150 / thno.47007. PMID: 33391496; PMCID: PMC7738854). It is also well-known in the art that the pl of the sgRNA, the pl of the Cas9 RNP complex, and the pl of the Cas9 protein are all different. (Kalebic N, Taverna E, Tavano S, Wong FK, Suchold D, Winkler S, Huttner WB, Sarov M. CRISPR / Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo. EMBO Rep. 2016 Mar;17(3):338-48. doi: 10.15252 / embr.201541715. Epub 2016 Jan 12. PMID: 26758805; PMCID: PMC4772980).

[0027] Capillary electrophoresis (CE) is an effective technique for the separation of samples containing biological material, such as, but not limited to, proteins, peptides, amino acids, and nucleic acids. Various capillary electrophoretic techniques include capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF), capillary isotachophoresis, and micellar electrokinetic chromatography (MEKC). In a non-limiting example, aspects of this disclosure may be performed using CZE.

[0028] It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described herein and as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure or the appended claims.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong.

[0030] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, “x, y, and / or z” means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0031] The term "about" as used in connection with a numerical value throughout the specification, and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%.

[0032] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0033] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0034] CE can be carried out in a normal or reverse polarity separation mode. During a normal separation polarity separation mode, the order of migration will be cations, neutrals, and anions, with the cathode residing beyond the detector window. Zwitterions, such as amino acids, proteins, and peptides, may exhibit charge reversal at their isoelectric points (pl) and, likewise, shifts in the direction of electrophoretic mobility due to various factors including, but not limited to, environmental pH changes (e.g., sample buffer pH). If the sample consists of negatively chargedcompounds, detection requires reversing the polarity of the electrodes (reverse polarity separation mode), whereby the anode is beyond the detector window. Additionally, a mixture of both cations and anions can still be measured, although not in a single run. In these situations, the sample may be analyzed twice, first in normal polarity separation mode followed by reverse polarity separation mode (or vice versa.)

[0035] Referring now to FIG. 2, in one aspect of the disclosure 200, a first sample including a protein and / or a polynucleotide, and a complex, is loaded on a CE capillary 202. The CE capillary includes a buffer with a pH greater than the pl of the protein.

[0036] As used herein, "capillary" refers to a channel, tube, or other structure capable of supporting a volume of separation medium for performing electrophoresis. Capillary geometry can vary and includes structures having circular, rectangular, or square cross-sections, channels, grooves, plates, and the like that can be fabricated by technologies known in the art. Capillaries of the present disclosure can be made of materials such as, but not limited to, silica, fused silica, quartz, silicate-based glass such as borosilicate glass, phosphate glass, alumina-containing glass, and other silica-like materials. Capillaries of the present disclosure can be uncoated (e.g., Bare Fused Silica (BFS)) or coated. In some aspects, the methods can be adapted and used in any generally known electrophoresis platform, such as, for example, electrophoresis devices comprising single or multiple microfluidic channels, etched microfluidic capillaries, as well as slab gel and thin-plate gel electrophoresis.

[0037] In this aspect, electrophoretic separation is initiated by separation by applying a separation voltage to the CE capillary 204. The pH of the buffer affects the charge of the sample resulting in negatively charged components. Therefore the CE capillary is run using a reverse polarity separation mode 204. The separated protein and / or the separated polynucleotide and the separated complex are then detected using a detector 206. The detector can be, for example, a UV detector. For example, the detector may also be a fluorescence detector, a laser-induced fluorescence (LIF) detector, a lamp-based fluorescence detector, or a native fluorescence detector. The desired quantitation sensitivity will determine the type of detector used. LIF detection offers the benefit of an increase in sensitivity, yet it also requires additional sample manipulation.

[0038] If a fluorescence detector is used, the complex, the protein and / or the polynucleotide are fluorescently labeled with a fluorescent dye prior to CE separation. In these aspects, the fluorescentdye may be a cyanine-based dye. Cyanine-based dyes of the disclosure include, not are not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5,5, Cy7, SYBR Green I, SYBR Green II, PicoGreen, Thiazole orange, and Oxazole yellow.

[0039] Referring now to FIG. 3, in one aspect of the disclosure 300, a first sample including a protein and / or a polynucleotide, and a complex, is loaded on a CE capillary 302. The CE capillary includes a buffer with a pH less than or equal to the pl of the polynucleotide. The electrophoretic separation is initiated by separation by applying a separation voltage to the CE capillary 304. The buffer's pH affects the sample's charge, resulting in positively charged components. Therefore the CE capillary is run using a normal polarity separation mode 304. The separated protein and / or the separated polynucleotide and the separated complex are then detected using a detector 306.

[0040] Referring now to FIG. 4, in one aspect of the disclosure 400, a first sample including a protein and / or a polynucleotide, and a complex, is loaded on a CE capillary 402. The CE capillary includes a first buffer, wherein the first buffer has a pH between the complex's pl and the protein's pl. The electrophoretic separation is initiated by separation by applying a separation voltage to the CE capillary 404. The pH of the buffer affects the charge of the sample resulting in negatively charged components, and the CE capillary is run using a reverse polarity separation mode 404. The separated complex and / or the separated polynucleotide are then detected with a detector 406.

[0041] The aspect further includes loading a second sample, including a protein and / or a polynucleotide, and a complex, on a CE capillary 408. The CE capillary includes a second buffer, wherein the second buffer has a pH between the complex's pl and the protein's pl. The electrophoretic separation is initiated by separation by applying a separation voltage to the CE capillary 410. The pH of the buffer affects the charge of the sample resulting in positively charged components, and the CE capillary is run using a normal polarity separation mode 410. The separated protein is then detected with a detector 412.

[0042] Referring now to FIG. 5, in one aspect of the disclosure 500, a first sample including a protein and / or a polynucleotide, and a complex, is loaded on a CE capillary 502. The CE capillary includes a first buffer, wherein the first buffer has a pH between the pl of the polynucleotide and the pl of complex. The electrophoretic separation is initiated by separation by applying a separation voltage to the CE capillary 504. The pH of the buffer affects the charge of the sample resulting in positively charged components and the CE capillary is run using a normal polarity separation mode504. The separated complex and / or the separated protein is then detected with a detector 506.

[0043] The aspect further includes loading a second sample including a protein and / or a polynucleotide, and a complex, on a CE capillary 508. The CE capillary includes a second buffer, wherein the second buffer has a pH between the pl of the polynucleotide and the pl of complex. The electrophoretic separation is initiated by separation by applying a separation voltage to the CE capillary 510. The pH of the buffer affects the charge of the sample resulting in negatively charged components and the CE capillary is run using a reverse polarity separation mode 510. The separated polynucleotide is then detected with a detector 512.

[0044] In some non-limiting examples, the first sample and the second sample are the same and / or the first buffer and the second buffer are the same. In some non-limiting examples, the first sample and second sample are loaded onto separate capillaries or loaded sequentially on the same capillary.

[0045] In some aspects, the first sample and / or second sample are diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary.

[0046] An "electropherogram" (or "e-gram") refers to a series of peaks that can be converted to determine the size and / or quantity of a sample. Peaks are integrated for area as a measure of quantity, and can be corrected for mobility differences between different sized peaks. In some aspects, the methods further include producing an electropherogram comprising corrected peak area of the separated complex, the separated protein and / or the separated polynucleotide. The electropherograms can be used to generate a corresponding set of values for the separated complex, the separated protein and / or the separated polynucleotide.

[0047] In some non-limiting examples, the methods can be used to separate components in the direct delivery CRISPR / Cas9 system. The method allows for the separation of the Cas9 / RNA complex from RNA and protein, and, using generated values, a percentage of the separated complex, the separated protein and / or the separated polynucleotide in the first sample or second sample can be determined.

[0048] In this example, the protein may be Cas9 protein or an engineered Cas9 protein. The protein may also have a pl of about 7.6. The polynucleotide may be sgRNA or a prime editing guide (pegRNA). The polynucleotide may have a pl of about 4 to about 4.6. The complex may be a Cas9 RNP complex formed via the binding of a polynucleotide by a Cas9 protein. Cas9 RNP complexmay have a pl of between about 4 to about 7.6

[0049] As shown in FIG 6, one or two polarity modes can be used for the separation and quantitative analysis of the Cas9 RNP complex samples depending on the pH of the separation buffer. The three components can be separated and quantitatively analyzed in one run using a buffer with a pH lower than the pl of sgRNA and normal polarity or using a buffer with a pH higher than the pl of the Cas9 protein and reverse polarity.

[0050] The three components can also be separated and analyzed in two runs on the same CE system. In one example, the Cas9 RNP complex can be separated from the Cas9 protein in normal polarity separation mode using a buffer with pH between the pl of sgRNA and pl of the Cas9 RNP complex. In another example, the Cas9RNP complex can be separated from the sgRNA in reverse polarity separation mode using a buffer with pH between the pl of Cas9 protein and pl of the Cas9 RNP complex.

[0051] In some aspects, a pressure push can be applied to the CE capillary during the separation. This pressure push may optimize the run time and peak shape. In some non-limiting aspects, the pressure is at least about 0.1 psi, alternatively at least about 0.2 psi, alternatively at least about 0.3 psi, alternatively at least about 0.4 psi, alternatively at least about 0.5 psi, alternatively at least about 1 psi. A gradient polarity may also be used. The gradient polarity may also optimize the resolution, peak shape, and run time.

[0052] EXAMPLES

[0053] Exemplary separation and analysis method for Cas9 RNP complex

[0054] A sample containing a Cas9 RNP complex, Cas9 protein, and sgRNA was provided. PA 800 Plus pharmaceutical analysis CE system (SCIEX) equipped with a laser-induced fluorescence (LIF) detector with a 488 nm solid state laser and a 520 nm emission filter was used for all separations. The LIF detector was calibrated according to the user guide. Data acquisition was performed using 32 Karat software version 10 and exported to ASCII format, later imported and processed using the BioPhase Software for integration and calculations including Signal intensity, Corrected Area, and Corrected Area%.

[0055] The sample, buffer, and other reagents needed for this application were placed into the PA 800 Plus universal vials and loaded onto the instrument according to the reagent plate map in theuser guide. The separation method can be downloaded from Sciex.com or created following the user guide.

[0056] In an exemplary normal polarity CE run the pH of the buffer was in between the pl of the sgRNA and Cas9 RNP complex, causing the Cas9 RNP complex and Cas9 protein in the sample to carry positive charges and move toward the detector. The sgRNA carries a negative charge and cannot be detected.

[0057] In an exemplary normal polarity CE run with a pressure push applied during separationthe pH of the buffer was lower than the pl for all three components in the sample, resulting in positively charged Cas9 RNP complex, Cas9 protein, and sgRNA.

[0058] In an exemplary reverse polarity CE run with a pressure push applied during separationthe pH of the buffer was in between the pl of the Cas9 RNP complex and the Cas9 protein, causing the sgRNA and Cas9 RNP complex in the sample to carry negative charges and move toward the detector. The Cas9 protein carries a positive charge and cannot be detected.

[0059] While the present disclosure has been described with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the present disclosure or appended claims. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present disclosure without departing from its scope. Therefore, it is intended that the present disclosure not be limited to the particular embodiments disclosed, but that the present disclosure will include all aspects falling within the scope of the appended claims.

[0060] All patents, patent applications, publications, and descriptions mentioned above are herein incorporated by reference in their entirety.

Claims

CLAIMSWhat is Claimed is:

1. A capillary electrophoresis method for analyzing a sample, the method comprising: loading a first sample, the first sample comprising a protein and / or a polynucleotide, and a complex, on a capillary electrophoresis (CE) capillary, the CE capillary comprising a buffer, wherein the buffer has a pH greater than the Isoelectric Point(pl) of the protein, initiating a separation by applying a separation voltage to the CE capillary, wherein the CE capillary is run using a reverse polarity separation mode, and, detecting the separated protein and / or the separated polynucleotide and the separated complex with a detector.

2. A capillary electrophoresis method for analyzing a sample, the method comprising: loading a first sample, the first sample comprising a protein and / or a polynucleotide, and a complex, on a CE capillary, the CE capillary comprising a buffer, wherein the buffer has a pH less than or equal to the pl of the polynucleotide, initiating a separation by applying a separation voltage to the CE capillary, wherein the CE capillary is run using a normal polarity separation mode, and, detecting the separated complex, the separated protein and / or the separated polynucleotide with a detector.

3. A capillary electrophoresis method for analyzing a sample, the method comprising: loading a first sample, the first sample comprising a protein and / or a polynucleotide, and a complex, on a CE capillary, the CE capillary comprising a first buffer, wherein the first buffer has a pH between the pl of the complex and the pl of the protein, initiating a separation by applying a separation voltage to the CE capillary, wherein the CE capillary is run using a reverse polarity separation mode,detecting the separated complex and / or the separated polynucleotide with a detector; and loading a second sample, the second sample comprising a protein and / or a polynucleotide, and a complex, on a CE capillary, the CE capillary comprising a second buffer, wherein the second buffer has a pH between the pl of the complex and the pl of the protein, initiating a separation by applying a separation voltage to the CE capillary, wherein the CE capillary is run using a normal polarity separation mode, and detecting the separated protein with a detector.

4. A capillary electrophoresis method for analyzing a sample, the method comprising: loading a first sample, the first sample comprising a protein and / or a polynucleotide, and a complex, on a CE capillary, the CE capillary comprising a first buffer, wherein the first buffer has a pH between the pl of the polynucleotide and the pl of complex, initiating a separation by applying a separation voltage to the CE capillary, wherein the CE capillary is run using a normal polarity separation mode, and, detecting the separated complex and / or separated protein with a detector, and loading a second sample, the second sample comprising a protein and / or a polynucleotide, and a complex, on a CE capillary, the CE capillary comprising a second buffer, wherein the second buffer has a pH between the pl of the polynucleotide and the pl of complex, initiating a separation by applying a separation voltage to the CE capillary, wherein the CE capillary is run using a reverse polarity separation mode, and detecting the separated polynucleotide with a detector.

5. The method of claim 3 or claim 4, wherein the first sample and the second sample are the same.

6. The method of any one of claims 3 to 5, wherein the first buffer and the second buffer are the same.

7. The method of any one of the preceding claims, further comprising producing an electropherogram comprising corrected peak area of the separated complex, the separated protein and / or the separated polynucleotide and generating a corresponding set of values for the separated complex, the separated protein and / or the separated polynucleotide.

8. The method of claim 7, further comprising determining a percentage of the separated complex, the separated protein and / or the separated polynucleotide in the first sample or second sample, using the corresponding set of values.

9. The method of any one of the preceding claims, wherein the capillary electrophoresis is capillary zone electrophoresis (CZE).

10. The method of any one claims 3 to 8, wherein the first sample and second sample are loaded onto separate capillaries or loaded sequentially on the same capillary.

11. The method of any one of the preceding claims, wherein the protein is a Cas9 protein or an engineered Cas9 protein.

12. The method of claim 11, wherein the pl of the protein is about 7.6.

13. The method of any one of the preceding claims, wherein the polynucleotide is a single guide RNA (sgRNA) or a prime editing guide (pegRNA).

14. The method of claim 13, wherein the pl of the polynucleotide is between about 4 to about 4.5.

15. The method of any one of the preceding claims, wherein the complex is a Cas9 ribonucleoprotein (RNP) complex.

16. The method of claim 15, wherein binding of a polynucleotide by a Cas9 protein results in the formation of the Cas9 RNP complex.

17. The method of claim 15 or claim 16, wherein the pl of the Cas9 RNP complex is between about 4 to about 7.6.

18. The method of any one of the preceding claims, wherein during the separation, pressure is applied to the CE capillary.

19. The method of claim 18, wherein the pressure is at least about 0.1 psi, alternatively at least about 0.2 psi, alternatively at least about 0.3 psi, alternatively at least about 0.4 psi, alternatively at least about 0.5 psi, alternatively at least about 1 psi20. The method of any one of the preceding claims, wherein a gradient voltage is applied.

21. The method of any one of the preceding claims, wherein the first sample is diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary.

22. The method of any one claims 3 to 20, wherein the second sample is diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary.

23. The method of any one of the preceding claims, wherein the detector is a UV detector.

24. The method of any one of the preceding claims, wherein the detector is a fluorescence detector, a laser-induced fluorescence (LIF) detector, a lamp-based fluorescence detector, or a native fluorescence detector.

25. The method of claim 24, wherein the complex, the protein and / or the polynucleotide in the first sample is fluorescently labeled with a fluorescent dye prior to CE separation.

26. The method of claim 24, wherein the complex, the protein and / or the polynucleotide in the second sample is fluorescently labeled with a fluorescent dye prior to CE separation.

Images