A method for studying conformational changes of Cas9 protein when it recognizes target gene sites

Abstract

The present invention discloses a method for studying the conformational changes of Cas9 protein when recognizing target gene sites. By modifying the SL2 region of sgRNA, the Cas9 protein can be fixed on a glass slide while maintaining its function, and the mutant C80S / S355C / C574S / S867C of the Cas9 protein is labeled with FRET fluorescence pairs, thereby truly reflecting the conformational state of Cas9 based on single-molecule fluorescence resonance energy transfer. This provides an effective means for studying the structure-function relationship of the CRISPR / Cas9 system in vitro, and is of great significance for designing Cas9 with optimized properties and enabling it to exert new functions.

Description

Technical Field

[0001] The present invention provides an in vitro method for studying the recognition of target gene sites by Cas9 protein, and specifically relates to the application of a single-molecule fluorescence resonance energy transfer technology system in studying the conformational changes of Cas9 protein when recognizing target gene sites. Background Art

[0002] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated protein 9 (Cas9), also known as CRISPR / Cas9, are an adaptive immune system found in bacteria that defends against invading phages and plasmids. The CRISPR / Cas9 system has been successfully modified and applied to gene editing in a variety of plant and animal cells. The CRISPR / Cas9 system primarily consists of two parts: the Cas9 protein and a single guide RNA (sgRNA). Under the guidance of the sgRNA, the Cas9 protein can introduce double-stranded DNA breaks at specific gene loci, thereby achieving site-specific editing of the genome.

[0003] Although a large number of biological studies have conducted in-depth and detailed analysis of the CRISPR / Cas9 system, the CRISPR / Cas9 system still has many problems that cannot be solved by ordinary biological methods, but they are crucial for the function of the system, such as the off-target effect - Cas9 will recognize DNA targets that are not completely complementary to the sgRNA, resulting in incorrect binding and cutting. The off-target problem has become one of the main challenges in the application of the CRISPR / Cas9 system. The answer to the off-target problem depends on the interpretation of the mechanism of Cas9 binding and cutting DNA, which is closely related to the conformation of the Cas9 protein. Therefore, studying the conformation of the Cas9 protein helps to accurately understand the structure-function relationship of the CRISPR / Cas9 system, which is of great significance for designing Cas9 with optimized properties and enabling it to exert new functions.

[0004] Single-molecule Fluorescence Resonance Energy Transfer (smFRET) is an imaging method developed based on the energy transfer phenomenon that occurs when two fluorescent molecules are in close proximity. It is currently one of the most important single-molecule techniques for studying the dynamic behavior of biomolecules in aqueous solutions. It can provide spatial information within and between individual biomolecules (proteins, nucleic acids, etc.) at the nanoscale. smFRET has been used by multiple teams to monitor conformational changes in the Cas9 protein.

[0005] However, current smFRET studies on the conformational changes of Cas9 upon DNA binding have yielded conflicting results. For example, one study found that Cas9 only briefly adopts an active closed state (high FRET state) upon DNA binding, and this is sufficient for subsequent DNA cleavage. However, another study found that Cas9 or Cas9 with nuclease-deactivated activity (dCas9) maintains the closed state (high FRET state) for a prolonged period in the presence of a DNA substrate that is fully complementary to the spacer sequence of the sgRNA. Both teams fixed the Cas9 / sgRNA complex to the slide by modifying the 3' end of the sgRNA. The former attached a biotin molecule to the 3' end of the sgRNA, utilizing the interaction between biotin and streptavidin on the slide to fix the complex to the slide. The latter extended the 3' end of the sgRNA and made it complementary to an oligonucleotide tether with biotin attached to its 5' end. The interaction between the biotin-tether and streptavidin on the slide indirectly fixed the Cas9 / sgRNA complex to the slide. Previous studies have shown that there is no difference between dCas9 and Cas9 in their ability to bind to specific gene sites under the mediation of sgRNA. Both can be used to study the mechanism by which Cas9 recognizes target gene sites. Studies in living cells have shown that modifications to different positions of the sgRNA have different effects on the ability of the dCas9 / sgRNA complex to bind to specific gene loci in living cells. Modifications to the 3' end of the sgRNA significantly reduce the labeling efficiency of dCas9 / sgRNA. These results suggest that modifications to the 3' end of the sgRNA can affect the ability of Cas9 / sgRNA to bind to DNA. Therefore, the differences in the aforementioned smFRET study results may be due to the different modifications made to the 3' sequence of the sgRNA. These modifications affect the conformational behavior of the Cas9 bound to the sgRNA, resulting in different FRET signal changes. Summary of the Invention

[0006] Existing literature shows that in living cells, dCas9 can accurately mark specific gene sites after binding to sgRNA modified by stem-loop structure 2 (Stem Loop 2, SL2), while the labeling ability of specific gene sites after binding to sgRNA modified by 3' end is significantly reduced. Therefore, the sgRNA modification method (changing the 3' region of sgRNA) currently adopted in smFRET experiments cannot clarify the true conformational state of Cas9. The present invention enables Cas9 protein to be fixed on a glass slide and maintain function by modifying the SL2 region (stem loop structure 2, Stem Loop 2) of sgRNA, and develops an in vitro smFRET research system that can most realistically reflect the conformational state of Cas9.

[0007] The technical solutions provided by the present invention are as follows:

[0008] Modify the sgRNA configuration: Insert a special sequence (TS) into the SL2 region of the sgRNA to enable hybridization and complementation with a biotin-modified oligonucleotide (biotin-oligo). The modified sgRNA is obtained by in vitro transcription and named SL2-sgRNA-TS. The sequence encoding TS used in the present embodiment is 5'-CAGGAGTTGTGTTTGTGGACGAAG-3' (SEQ ID No: 1). In addition, other sequences unrelated to sgRNA can be used as the TS sequence.

[0009] Preparation of dCas9 protein (no difference from the ability of wild-type Cas9 to bind to specific gene sites under the mediation of sgRNA): Utilize the chemical reaction between maleimide and the sulfhydryl group (-SH) of cysteine ​​(Cystine, Cys) to connect the fluorophore to the target labeling site of the protein to achieve FRET fluorescence pair labeling of the dCas9 protein. First, we performed site-directed mutagenesis on the dCas9 protein, mutating the original Cys of the wild-type dCas9 to Ser, and at the same time mutating the target labeling site - the 355th and 867th amino acids of dCas9 to Cys, to obtain a dCas9 mutant (C80S / S355C / C574S / S867C), which has been confirmed by multiple research teams to not affect its own related functions. The fragment was cloned into an Escherichia coli protein expression vector, and the Escherichia coli protein expression system was used to induce expression and purify the above-mentioned mutant dCas9 protein (named dCas9 HNH Then, the fluorophores Cy3-maleimide and Cy5-maleimide were combined with dCas9 HNHThe mixture was mixed and reacted at a ratio of 5:5:1, and unreacted free fluorophores were removed using a desalting column Sephadex G-25 (PD-10, GE) to obtain dCas9. HNH -Cy3 / Cy5. In addition, the protein can also be labeled with other FRET fluorescent pairs, such as Atto550-maleimide / Atto647N-maleimide. HNH With two labelable sites (positions 355 and 867) and a FRET fluorophore pair attached to the labeling site (Cys) using the same labeling principle, when dual-color labeling is performed on a protein (labeled A (acceptor) and D (donor)), three possible dual-site labeling results are obtained: AA, AD, and DD, with probabilities of 25%, 50%, and 25%, respectively. Proteins labeled with two A fluorophores simultaneously are not excited by the laser, so their signals are not detected and do not interfere with subsequent results. Protein signals labeled with two D fluorophores simultaneously can be filtered out in subsequent analysis by screening out non-single molecule signals.

[0010] Preparation of functionalized coverslips with PEG modification: Polyethylene glycol (PEG) modification of the glass slide surface can reduce the nonspecific adsorption of proteins on the one hand, and on the other hand, biotin-oligo can be attached to the glass slide surface through the interaction of (biotin-PEG)-neutravidin (NeutrAvidin)-(biotin-oligo) ( Figure 1 ).

[0011] Prepare smFRET experimental samples: assemble the fluorophore-labeled dCas9 protein and SL2-sgRNA-TS in vitro to obtain a dCas9 / sgRNA complex. For the preparation of target DNA: the target DNA contains a target sequence complementary to the spacer sequence of the sgRNA and a PAM sequence adjacent to it. The company can be commissioned to synthesize the two single strands of the DNA, and the target DNA double strands are prepared by high-temperature denaturation and then slowly cooling and annealing. Mix and incubate dCas9, sgRNA, and target DNA in vitro to assemble into a dCas9 / sgRNA / DNA complex. Next, incubate the dCas9 / sgRNA or dCas9 / sgRNA / DNA complex on the surface of the glass slide, and use the complementarity of the TS sequence in the sgRNA and the biotin-oligo to specifically connect the complex to the surface of the glass slide ( Figure 1 ).

[0012] smFRET data acquisition and analysis: Single-molecule FRET fluorescence signals were collected using Total Internal Reflection Fluorescent Microscopy (TIRFM). Analyzing the efficiency of single-molecule FRET signals allowed us to analyze the conformational changes of dCas9 in different states.

[0013] The above technical solution can also be applied to the smFRET system for studying the conformation of Cas9 protein (i.e., using the Cas9 mutant (C80S / S355C / C574S / S867C) as the research object), which can minimize the influence of other factors on protein conformation in vitro and study its most realistic conformational fluctuations under specific experimental conditions.

[0014] The present invention provides a method for studying conformational changes of Cas9 protein when it recognizes target gene sites based on single-molecule fluorescence resonance energy transfer, comprising:

[0015] (1) A special sequence TS is inserted into the SL2 region of the sgRNA to enable hybridization and complementation with the biotin-modified oligonucleotide biotin-oligo, and the modified sgRNA is obtained by in vitro transcription and named SL2-sgRNA-TS;

[0016] (2) preparing a biotin-modified oligonucleotide, biotin-oligo, which contains a sequence complementary to TS hybridization;

[0017] (3) preparing a mutant C80S / S355C / C574S / S867C of Cas9 protein or dCas9 protein, and labeling the mutant protein with a FRET fluorescence pair;

[0018] (4) preparing a target double-stranded DNA containing a target sequence complementary to the spacer sequence of SL2-sgRNA-TS and a PAM sequence adjacent thereto;

[0019] (5) Modifying the surface of the cover glass with biotin-containing polyethylene glycol (Biotin-PEG) to obtain a functionalized cover glass, then incubating neutravidin on the surface of the functionalized cover glass to bind to the biotin-PEG on the cover glass surface; then incubating the biotin-oligo prepared in step (2) on the functionalized cover glass modified with neutravidin, and connecting the biotin-oligo to the cover glass through the reaction between biotin and neutravidin;

[0020] (6) mixing and incubating the mutant form of the Cas9 protein or dCas9 protein prepared in step (3) with the SL2-sgRNA-TS obtained in step (1) in vitro to form a Cas9 / sgRNA complex or a dCas9 / sgRNA complex; at the same time, mixing and incubating the mutant form of the Cas9 protein or dCas9 protein with the SL2-sgRNA-TS and the target double-stranded DNA in vitro to form a Cas9 / sgRNA / DNA complex or a dCas9 / sgRNA / DNA complex;

[0021] (7) incubating the complex obtained in step (6) on the coverslip obtained in step (5), respectively, and specifically connecting the complex to the surface of the coverslip through hybridization complementation between biotin-oligo and TS in SL2-sgRNA-TS;

[0022] (8) The single-molecule FRET fluorescence signal on the surface of the coverslip treated in step (7) is collected by total internal reflection fluorescence microscopy (TIRFM), and the conformational changes of Cas9 or dCas9 in different states are analyzed by analyzing the single-molecule FRET efficiency.

[0023] In step (1), the TS is a sequence unrelated to the sgRNA and has a length of 20 to 60 nt. The complementarity between a TS shorter than 20 nt and the biotin-oligo is not stable enough, and inserting a TS that is too long (>60 nt) may affect the sgRNA structure. In one embodiment of the present invention, the sequence encoding the TS is 5'-CAGGAGTTGTGTTTGTGGACGAAG-3', which is complementary to the sequence on the biotin-oligo.

[0024] In the above step (2), biotin is modified at the 5' end or 3' end of the oligonucleotide, and the oligonucleotide is a sequence complementary to the TS sequence. Depending on the actual experimental conditions, it can be determined whether it is necessary to introduce an unrelated sequence of appropriate length at both ends of the sequence complementary to TS to improve the binding stability of the oligonucleotide and TS on the glass slide surface.

[0025] In the above step (3), the FRET fluorescent pair can be Cy3 / Cy5, or Atto550 / Atto647N, ATTO488 / ATTO550, Alexa546 / Alexa647, etc. In an embodiment of the present invention, the mutant protein is mixed and reacted with a maleimide-modified fluorophore, and then the unreacted free fluorophore is removed to obtain a FRET fluorescent pair labeled mutant protein.

[0026] In step (4), the target sequence on the target double-stranded DNA can be any sequence of 11 to 25 bp. The specific target sequence length is determined by the research content and experimental design, but it needs to be complementary to the spacer sequence of the sgRNA. There is an arbitrary DNA sequence of ~20 bp extension at both ends of the target sequence and its adjacent PAM sequence to stabilize the binding of the Cas9 / sgRNA complex and DNA. In an embodiment of the present invention, the target sequence is: 5'-CTGTGATATCA-3'.

[0027] In step (5), the molecular weight of the biotin-containing polyethylene glycol Biotin-PEG is preferably about 5000 g / mol. Generally, the coverslip is first treated with APTES, and then Biotin-PEG-SVA is reacted with the APTES on the coverslip surface to modify the coverslip surface with Biotin-PEG.

[0028] In the above step (6), an excess of Cas9 or dCas9 mutant protein is required to be mixed and incubated with SL2-sgRNA-TS in vitro. It is recommended that the molar ratio be greater than 5:1 to promote the binding of protein and sgRNA, ensuring that sufficient protein binds to sgRNA and is fixed to the glass slide. When Cas9 or dCas9 mutant protein is mixed and incubated with SL2-sgRNA-TS and target double-stranded DNA in vitro, both protein and target double-stranded DNA need to be in excess relative to sgRNA. It is recommended that the molar ratio of protein to sgRNA be greater than 5:1 to ensure that sufficient protein binds to sgRNA and is fixed to the glass slide. The molar ratio of target double-stranded DNA to sgRNA is preferably greater than 2:1 to ensure that sufficient Cas9 / sgRNA / DNA ternary complex is formed. In the embodiments of the present invention, the molar ratio of the mutant protein of Cas9 or dCas9 to SL2-sgRNA-TS is 10:1; the molar ratio of the mutant protein of Cas9 or dCas9 to SL2-sgRNA-TS and target double-stranded DNA is 10:1:4.

[0029] In step (8) above, the FRET efficiency is calculated using formula (1):

[0030]

[0031] Among them, I A represents the signal intensity of the acceptor fluorophore, I D Represents the signal intensity of the donor fluorophore.

[0032] The FRET efficiency (E FRET ) to analyze the conformational changes of Cas9 or dCas9 in different states.

[0033] The method provided by the present invention for studying the conformational changes of the Cas9 protein when recognizing the target gene site based on single-molecule fluorescence resonance energy transfer enables the Cas9 protein to be fixed on a glass slide and maintain its function by modifying the SL2 region of the sgRNA. Compared with the method of modifying the 3' end of the sgRNA used in existing studies, it can more realistically and accurately reflect the conformational state of Cas9, providing an effective means for studying the structure-function relationship of the CRISPR / Cas9 system in vitro, and is of great significance for designing Cas9 with optimized properties and enabling it to exert new functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the smFRET system for studying conformational changes of Cas9 / dCas9 proteins by modifying the SL2 region of sgRNA.

[0035] Figure 2 Representative results of the present invention using smFRET to study conformational changes of dCas9 protein bound to sgRNA and DNA, where: the top row shows the original images at some time points; the middle image shows the trajectory of signal intensity changes over time; the bottom image shows the FRET efficiency (E FRET ) changes over time. DETAILED DESCRIPTION

[0036] The following examples examine the conformations of dCas9 / sgRNA when bound to and not bound to DNA, and describe the application of the present invention to explore dCas9 conformational changes in vitro. The specific experiments are as follows:

[0037] 1. Reagents and instruments

[0038] 1.1 Main reagents and materials

[0039] 1) Plasmid pET302-6×His-dCas9-Halo (used to construct expression of 6×His-dCas9 HNH The mutant plasmids (construction methods are described in Experimental Methods 2.1) and plasmid SL2-sgTelo-TS / EGFP / pdCas9-C1 (a DNA template for amplifying in vitro transcribed sgRNAs, in which the sgRNA scaffold in the plasmid has a TS inserted into the SL2 position) were obtained from Wulan Deng (Addgene catalog #72269) and from our laboratory (Addgene catalog #162759).

[0040] 2) Molecular cloning reagents: DNA polymerase, restriction endonuclease, T4 ligase and related buffers (available from New England Biolabs), and competent cells DH5α for plasmid amplification.

[0041] 3) Protein expression reagents: IPTG (isopropyl-β-d-thiogalactopyranoside), protein expression competent cells BL21 (DE3).

[0042] 4) Protein purification reagents: protease inhibitor cocktail (available from Thermo Scientific), buffer A1 (20 mM HEPES (pH 7.0), 500 mM NaCl, 1 mM TCEP), buffer B1 (20 mM HEPES (pH 7.0), 500 mM NaCl, 1 mM TCEP, 250 mM imidazole), buffer A2 (20 mM HEPES (pH 7.0), 200 mM KCl, 1 mM TCEP), buffer B2 (20 mM HEPES (pH 7.0), 1 M KCl, 1 mM TCEP), 30,000-MWCO centrifugal filters Amicon Ultra-15 and Amicon Ultra-0.5 (available from Millipore Amicon), Ni-NTA affinity chromatography column (HisTrap HP, available from GE Healthcare), cation exchange chromatography column (HiTrap SP HP, available from GE Healthcare). Healthcare), Coomassie Brilliant Blue Fast Staining Solution (available from beyotime), and Sephadex G-25 chromatographic column (PD-10, available from GE Healthcare).

[0043] 5) RNA in vitro transcription kit (RiboMAX TM Large Scale RNA Production System-T7, available from Promega).

[0044] 6) RNA product recovery kit (Monarch RNA Cleanup Kit (10 μg), available from New England Biolabs).

[0045] 7) Preparation of functionalized coverslips: coverslips (available from Warner Instruments), Hellmanex III, anhydrous ethanol, methanol, acetic acid, APTES (3-aminopropyl)triethoxysilane), mPEG-SVA (mPEG-Succinimidyl Valerate), SVA-PEG-SVA (Succinimidyl Valerate-PEG-SuccinimidylValerate), Biotin-PEG-SVA (Biotin-PEG-Succinimidyl Valerate) (PEG reagents all purchased from Laysan Bio), NaHCO₃, and NeutrAvidin. Unless otherwise noted, all other reagents were purchased from Sigma-Aldrich.

[0046] 8) Binding buffer (20 mM tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), 5% glycerol, 50 mg / mL heparin) and imaging buffer (binding buffer supplemented with glucose oxidase (1 mg / mL), catalase (0.04 mg / mL), 0.8% dextrose, and 2 mM Trolox). All reagents were purchased from Sigma-Aldrich.

[0047] 1.2 Main instruments

[0048] 1) PCR instrument, RT-PCR instrument, gel electrophoresis instrument, biochemical incubator, shaker, constant temperature incubator, ultrasonic cleaner.

[0049] 2) Cell culture incubator and biological safety cabinet.

[0050] 3) Fluorescence microscopy.

[0051] 2 Experimental methods

[0052] 2.1 Plasmid construction

[0053] Construction of inducible dCas9 in Escherichia coli HNH pET302-6×His-dCas9 for mutant protein expression HNH Plasmid (mutation sites are C80S / S355C / C574S / S867C). The construction method is divided into two steps. The first step is to obtain dCas9 HNHThe specific steps are as follows: using the wild-type dCas9 sequence on pET302-6×His-dCas9-Halo as a PCR template, primers P1 / P2, P3 / P4, P5 / P6, P7 / P8, and P9 / P10 (primer sequences are shown in Table 3) were used to amplify five dCas9 fragments. HNH The above five products were mixed and overlap extension PCR was used to amplify the complete dCas9 HNH Sequence (containing unique XbaI and XhoI sites at both ends). In the second step, the amplified product was ligated into the pET302 vector treated with XbaI and XhoI to obtain pET302-6×His-dCas9. HNH plasmid.

[0054] 2.2 Protein purification

[0055] pET302-6×His-dCas9 HNH The plasmid was transformed into Escherichia coli BL21(DE3) and cultured in LB medium containing ampicillin at 37°C until the absorbance at 600 nm (OD600) reached 0.6. Protein expression was then induced with 1 mM IPTG at 16°C. The next day, cells were harvested by centrifugation and resuspended in buffer A1 supplemented with a 1× protease inhibitor cocktail. The cells were lysed by sonication, and the lysate was centrifuged at 20,000 g. The supernatant was collected and filtered through a 0.2 μm filter. The target protein was extracted from the clarified lysate using a Ni-NTA affinity chromatography column. After loading the sample, the protein was eluted using a step gradient of buffer A1 and buffer B1. The buffer was exchanged with buffer A2 using a 30,000-MWCO centrifugal filter (Amicon Ultra-15). The protein was then purified using cation exchange chromatography. After loading the sample, the protein was eluted using a linear KCl gradient from 0.2 M to 1 M by mixing buffer A2 and buffer B2. The purity of the proteins in the different elution fractions was verified by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining, and the target protein fractions were stored at -80 °C for protein labeling.

[0056] 2.3 Fluorophore labeling

[0057] The purified dCas9 was filtered using a 30,000-MWCO centrifugal filter (Amicon Ultra-0.5 30,000-MWCO). HNHThe mutant protein's buffer was replaced with A2. The protein was mixed with Maleimide-Cy3 and Maleimide-Cy5 at a molar ratio of 1:5:5 and incubated overnight at 20°C in the dark with shaking. The excess unreacted dye was separated from the labeled dCas9 protein using a Sephadex G-25 column. The labeling efficiency and protein concentration were measured using a Nanodrop spectrophotometer with UV-Vis absorption spectroscopy. The labeled protein was stored at -20°C.

[0058] 2.4 In vitro transcription of sgRNA

[0059] Using the SL2-sgTelo-TS / EGFP / pdCas9-C1 plasmid (from Antony K. Chen, the original paper is ACRISPR / molecular beacon hybrid system for live-cell genomic imaging (Nucleic Acids Res. 2018 Jul 27; 46(13): e80.)) as a template, a linear DNA fragment containing T7 promoter-sgRNA was obtained by PCR amplification (using T7-sgRNA-R and T7-496-sgRNA-F in Table 3 as amplification primers); a kit (RiboMAX TM Large Scale RNA Production System-T7, Promega) was used for in vitro transcription of sgRNA; after the DNA template in the reaction system was treated with DNase I, the reaction product was recovered using an RNA extraction kit (Monarch RNA Cleanup Kit (10 μg), NEB), the concentration was measured, and the product was stored at -80°C.

[0060] 2.5 Preparation of double-stranded DNA

[0061] The two single strands used for double-stranded DNA synthesis (496-dsDNA (target strand) and 496-dsDNA (non-target strand) (see Table 3)) were ordered from Beijing Liuhe BGI Genomics Co., Ltd. DNA double strands were prepared by mixing equimolar amounts of DNA single strands, denaturing at 95°C, and then slowly cooling and annealing.

[0062] 2.6 Preparation of functionalized coverslips

[0063] 1) Slide Cleaning: Soak coverslips in Hellmanex III overnight and ultrasonicate them in an ultrasonic cleaner for 25 min (100% power). Next, pick up each coverslip with tweezers and rinse with running ultrapure water. Place the coverslip in ultrapure water and ultrasonicate for 15 min (100% power). Finally, ultrasonicate the coverslips in anhydrous ethanol at 100% power for 25 min.

[0064] 2) Treating slides with APTES: Soak the cleaned slides in a 2% APTES solution (Table 1) overnight, and then rinse with plenty of ultrapure water.

[0065] Table 1 Preparation of 2% APTES solution (150 mL)

[0066]

[0067] 3) Modify the slide surface with PEG: Prepare a 0.1M NaHCO3 solution (prepare as needed). Weigh three PEGs into an EP tube (Table 2). Dissolve and mix with 1 mL of 0.1M NaHCO3 until most of the solids are dissolved. Centrifuge at 14,800 rpm for 1 minute. Pour 50 μL of the supernatant onto an APTES-treated coverslip, then stack another slide on top. Incubate in a moist, clean container at room temperature for 3 hours. Rinse with ultrapure water. To ensure that the entire slide surface is PEG-modified and reduce nonspecific adsorption of proteins, repeat this step once without adding Biotin-PEG-SVA and incubate for 1 hour.

[0068] Table 2 Preparation of different types of PEG

[0069]

[0070] 2.7 Preparation of smFRET Samples

[0071] 1) Incubate NeutrAvidin on the surface of the functionalized coverslip to allow it to bind to the Biotin-PEG on the glass slide surface, and then rinse the unbound NeutrAvidin with a large amount of ultrapure water;

[0072] 2) Biotin-oligo was purchased from IDT (Integrated DNA Technologies, Inc.). Biotin-oligo was incubated on a coverslip functionalized with NeutrAvidin. The biotin-oligo was attached to the coverslip through the reaction between biotin and NeutrAvidin, and then the unbound biotin-oligo was rinsed with copious amounts of ultrapure water.

[0073] 3) Incubate dCas9 and sgRNA at a final concentration of 2 μM in binding buffer to prepare a dCas9 / sgRNA complex; then add target double-stranded DNA at a final concentration of 800 nM and incubate in binding buffer to prepare a dCas9 / sgRNA / DNA complex;

[0074] 4) Incubate the dCas9 / sgRNA and dCas9 / sgRNA / DNA complexes on a glass slide with biotin-oligo, attaching the dCas9 / sgRNA or dCas9 / sgRNA / DNA complexes to the glass slide surface through oligo complementarity with the modified sgRNA SL2 region, and rinse the unbound dCas9 / sgRNA or dCas9 / sgRNA / DNA complexes with a large amount of binding buffer;

[0075] 2.8smFRET Data Acquisition

[0076] Samples were observed using TIRFM in imaging buffer. Fluorescence imaging was performed using an Olympus inverted fluorescence microscope (OLYMPUS IX83). 561 nm laser excitation was used, and a UAPON OTIRF 100× / 1.49 objective lens was used. The dual-channel FRET signals were separated using a DV2-CUBE optical separation system (ET585 / 65M, 635LPXR, ET655LP, Photometrics). Images were acquired using an EMCCD camera (Andor) and imaging software (CellSens Dimension). Parameter settings: exposure time 30 ms, electron multiplication gain (EM gain) 300.

[0077] 2.9 Analysis of smFRET Data

[0078] ImageJ was used to extract the signal point positions, and overly bright fiducial signals and protein aggregate signals as well as randomly occurring background noise signals were screened out to obtain the positions of potential single-molecule signal points. MATLAB was used to extract Cy3 / Cy5 signal point intensity information based on the positions obtained above. The signal intensity was fitted in the form of a step function using the TJmultistepFinder.m function (slightly adapted from the code of Jacob W. J. Kerssemakers et al., the original document is Assembly dynamics of microtubules at molecular resolution. (Nature. 2006 Aug 10; 442 (7103): 709-12.)) to accurately screen single-molecule signals. Signals with single-molecule characteristics in both Cy3 and Cy5 intensity changes were used for subsequent analysis. FRET efficiency was calculated using formula (1):

[0079]

[0080] Among them, I A represents the signal intensity of the acceptor fluorophore (i.e., Cy5), I D Represents the signal intensity of the donor fluorophore (i.e., Cy3).

[0081] The above smFRET method was used to perform experiments and analyze the representative signal point information obtained. Figure 2 shown.

[0082] Table 3 Nucleic acid sequences used in this example

[0083] SEQUENCE LISTING <110> Beijing University <120> A method for studying conformational changes of Cas9 protein when it recognizes target gene sites <130> WX2021-03-127 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> twenty four <212> DNA <213> Artificial sequence <400> 1 caggagttgt gtttgtggac gaag 24 <210> 2 <211> 25 <212> DNA <213> Artificial sequence <400> 2 gactcactat aggggaattg tgagc 25 <210> 3 <211> 32 <212> DNA <213> Artificial sequence <400> 3 gaaaaaatct cctgtagata agaaatacga tt 32 <210> 4 <211> 26 <212> DNA <213> Artificial sequence <400> 4 cgtcggaaga atcgtatttc ttatct 26 <210> 5 <211> 29 <212> DNA <213> Artificial sequence <400> 5 ccgtttttgc attgatcaaa aaagatttc 29 <210> 6 <211> 28 <212> DNA <213> Artificial sequence <400> 6 gatcaatgca aaaacggata tgcaggtt 28 <210> 7 <211> 32 <212> DNA <213> Artificial sequence <400> 7 cctgaaattt caacactatc aaaagattct at 32 <210> 8 <211> 35 <212> DNA <213> Artificial sequence <400> 8 gattatttca aaaaaataga atcttttgat agtgt 35 <210> 9 <211> 27 <212> DNA <213> Artificial sequence <400> 9 cttcacttgg aacgttatca catttac 27 <210> 10 <211> twenty four <212> DNA <213> Artificial sequence <400> 10 aatcgtggta aatgtgataa cgtt 24 <210> 11 <211> 56 <212> DNA <213> Artificial sequence <400> 11 acctaggctc gaatatcatc gatctcgagt tagtcacctc ctagctgact caaatc 56 <210> 12 <211> twenty one <212> DNA <213> Artificial sequence <400> 12 aaagcaccga ctcggtgcca c 21 <210> 13 <211> 58 <212> DNA <213> Artificial sequence <400> 13 taatacgact cactataggg gtgatatcac aggtttaaga gctatgctgg aaacagca 58 <210> 14 <211> 55 <212> DNA <213> Artificial sequence <400> 14 agaggagctt cactgtatct accactgtga tatcatacag aggagcttca ctgta 55 <210> 15 <211> 55 <212> DNA <213> Artificial sequence <400> 15 tacagtgaag ctcctctgta tgatatcaca gtggtagata cagtgaagct cctct 55 <210> 16 <211> twenty four <212> RNA <213> Artificial sequence <220> <221> misc_feature <222> (1)..(1) <223> Biotin Marker <220> <221> misc_feature <222> (1)..(24) <223> 2'O-methylation <400> 16 cuucguccac aaacacaacu ccug 24

Claims

1. A method for studying conformational changes of Cas9 protein when it recognizes a target gene site, comprising: 1) Inserting a special sequence TS into the SL2 region of the sgRNA to enable hybridization and complementation with a biotin-modified oligonucleotide (biotin-oligo), and obtaining the modified sgRNA by in vitro transcription, named SL2-sgRNA-TS. The TS is a sequence unrelated to the sgRNA and is 20-60 nt in length. 2) preparing a biotin-modified oligonucleotide, biotin-oligo, which contains a sequence complementary to TS hybridization; 3) Prepare the mutant C80S / S355C / C574S / S867C of Cas9 protein or dCas9 protein and label the mutant protein with FRET fluorescence pair; 4) preparing a target double-stranded DNA containing a target sequence complementary to the spacer sequence of SL2-sgRNA-TS and an adjacent PAM sequence; 5) Modifying the surface of a coverslip with biotin-containing polyethylene glycol (Biotin-PEG) to obtain a functionalized coverslip, then incubating the functionalized coverslip with neutravidin to allow it to bind to the biotin-PEG on the coverslip surface; then incubating the biotin-oligo prepared in step 2) on the functionalized coverslip modified with neutravidin, and attaching the biotin-oligo to the coverslip through the reaction between biotin and neutravidin; 6) The Cas9 protein or mutant form of the dCas9 protein prepared in step 3) is mixed and incubated in vitro with the SL2-sgRNA-TS obtained in step 1), wherein the molar ratio of protein to sgRNA is greater than 5:1, to form a Cas9 / sgRNA complex or a dCas9 / sgRNA complex; simultaneously, the Cas9 protein or mutant form of the dCas9 protein is mixed and incubated in vitro with the SL2-sgRNA-TS and target double-stranded DNA, wherein the molar ratio of protein to sgRNA is greater than 5:1, and the molar ratio of target double-stranded DNA to sgRNA is greater than 2:1, to form a Cas9 / sgRNA / DNA complex or a dCas9 / sgRNA / DNA complex; 7) Incubate the complexes obtained in step 6) on the coverslips obtained in step 5) respectively, and specifically attach the complexes to the coverslip surface through hybridization complementation between biotin-oligo and TS in SL2-sgRNA-TS; 8) Using total internal reflection fluorescence microscopy, collect the single-molecule FRET fluorescence signal on the coverslip surface treated in step 7), and analyze the single-molecule FRET efficiency to analyze the conformational changes of Cas9 or dCas9 in different states.

2. The method according to claim 1, wherein The sequence encoding TS is 5'-CAGGAGTTGTGTTTGTGGACGAAG-3'.

3. The method according to claim 1, wherein In step 2), biotin is modified at the 5' or 3' end of the oligonucleotide.

4. The method according to claim 1, wherein The FRET fluorescent pair in step 3) is selected from Cy3 / Cy5, Atto550 / Atto647N, ATTO488 / ATTO550, and Alexa546 / Alexa647.

5. The method according to claim 1, wherein The target sequence length on the target double-stranded DNA in step 4) is 11 to 25 bp.

6. The method according to claim 1, wherein The target sequence in step 4) is: 5'-CTGTGATATCA-3'.

7. The method according to claim 1, wherein In step 5), the coverslip is first treated with APTES, and then Biotin-PEG-SVA is reacted with APTES on the surface of the coverslip to modify the surface of the coverslip with Biotin-PEG to obtain a functionalized coverslip.

8. The method according to claim 1, wherein In step 8), calculate the FRET efficiency using formula (1): in, I A represents the signal intensity of the acceptor fluorophore, I D Represents the signal intensity of the donor fluorophore; through FRET efficiency E FRET The time-dependent trajectories are used to analyze the conformational changes of Cas9 or dCas9 in different states.

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