A fusion protein and application in base editor

Abstract

The application discloses a fusion protein xsp-SmacCas9, a new chimeric protein xsp-SmacCas9 is formed by recombination of the non-PI part of xCas9 and the PI of SmacCas9; it comprises: a) the amino acid sequence from the non-PI domain of xCas9; b) the amino acid sequence from the PI domain of SmacCas9 variant. The fusion protein has more relaxed PAM restriction in base editing than xCas9 and SmacCas9 (SpCas9 homolog). The emergence of the fusion protein provides a new editor selection for base editing; the application proves that the base editing editor constructed by the fusion protein has certain advantages in practical application than existing editors; the application confirms the recognition PAM range of the new chimeric protein by using a PAM plasmid library. The recognition PAM range of the known Cas9 editor is further expanded, so as to provide more support for gene editing technology in gene therapy, plant breeding and the like.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a fusion protein and its application in a base editor. Background Technology

[0002] The discovery of the CRISPR / Cas9 editing system, especially the emergence of base editing and lead editing technologies, has dramatically changed the development of life sciences. SpCas9 is the most widely used and characteristic Cas9 homolog. However, SpCas9 requires strict recognition of NGG PAM to function; this strict limitation has, to some extent, hindered the development of base editing and lead editing.

[0003] Existing Cas9 proteins generally strictly recognize one or more PAMs. For example, SpCas9 recognizes NGG, Cas9-NG recognizes NG, and WT SmacCas9 is known to recognize NAA. Numerous studies have demonstrated that Cas9 protein recognition of PAMs primarily relies on the PI domain, with non-PI domains having little impact. However, xCas9 mutations, except for E1219V, are concentrated in the non-PI domain and can recognize NG, GAA, and GAT PAMs. This indicates that, in addition to the PI domain, non-PI domains also significantly influence the range of PAM recognition. Summary of the Invention

[0004] The purpose of this invention is to recombine the non-PI portion of xCas9 with the PI portion of SmacCas9 to form a new chimeric protein, xsp-SmacCas9, and to confirm the recognition PAM range of this new chimeric protein using a PAM plasmid library; thereby further expanding the recognition PAM range of known Cas9 editors, and thus providing more support for gene editing technology in gene therapy, plant breeding, and other fields.

[0005] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution:

[0006] The first objective of this invention is to provide a fusion protein xsp-SmacCas9, the sequence of which is shown in SEQ ID NO: 1 or a DNA sequence having at least 90% sequence similarity to the gene sequence of SEQ ID NO: 1 and having the same function.

[0007] Furthermore, the fusion protein xsp-Smac Cas9 comprises:

[0008] a) The amino acid sequence of the non-PI domain from xCas9; b) The amino acid sequence of the PI domain from the SmacCas9 variant.

[0009] Furthermore, the amino acid sequence from the non-PI domain of xCas9 includes:

[0010] A262T / R324L / S409I / E480K / E543D / M694I.

[0011] Another objective of this invention is to provide a base editor constructed from the fusion protein xsp-Smac Cas9, which has a wide PAM recognition range.

[0012] Another object of the present invention is to provide an expression box or expression vector comprising the above-described base editor and guide editor.

[0013] The beneficial effects of this invention are:

[0014] This invention discloses a fusion protein xsp-SmacCas9, which is a new chimeric protein xsp-SmacCas9 formed by recombination of the non-PI portion of xCas9 and the PI portion of SmacCas9;

[0015] It comprises: a) an amino acid sequence from the non-PI domain of xCas9; and b) an amino acid sequence from the PI domain of a SmacCas9 variant. This fusion protein exhibits more relaxed PAM restrictions for base editing than both xCas9 and SmacCas9 (SpCas9 homologs). The emergence of this fusion protein provides a new editor option for base editing.

[0016] This invention discloses a base editor constructed from a fusion protein and demonstrates that this base editor constructed from a fusion protein has certain advantages over existing editors in practical applications.

[0017] This invention utilizes a PAM plasmid library to confirm the recognition PAM range of this novel chimeric protein. This further expands the known PAM recognition range of Cas9 editors, thereby providing greater support for gene editing technology in gene therapy, plant breeding, and other fields.

[0018] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the editing efficiency of the editor in verifying the endogenous target described in an embodiment of the present invention. Detailed Implementation

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below with reference to the accompanying drawings.

[0021] like Figure 1 As shown

[0022] Example 1

[0023] A fusion protein xsp-SmacCas9, comprising 1) an amino acid sequence from the non-PI domain of xCas9; A262T / R324L / S409I / E480K / E543D / M694I

[0024] 2) Amino acid sequence of the PI domain from the Smac Cas9 variant;

[0025] An ABE editor for this fusion protein was constructed, and the PAM library method was used to determine the PAM recognition range of this new fusion protein as NNAN and NANN.

[0026] Example 2

[0027] Base Editor Design and Construction:

[0028] Based on the existing ABE editor, we designed a method to fuse the PI fragment of Smac Cas9 with the non-PI fragment of xCas9 to construct ABE-xCas9-Smac. PCR amplification of the vector and target fragment was performed, and the amplification products were digested with DpnI to remove methylated template plasmids. Electrophoresis was used for verification after gel recovery. An editor plasmid was constructed using the ligation vector and target fragment, transformed, and then extracted. Sequencing was performed using the Sanger method for verification.

[0029] PAM confirmed

[0030] After co-transfection with ABE-xCas9-Smac using a PAM library containing spacer+NNNNNNNN and the corresponding target plasmid, puromycin was used for selection. After 72 hours, genomic DNA was extracted from the cells, the target band was amplified by PCR, and high-throughput sequencing was performed after recovery. The data were analyzed to determine the range of PAMs that ABE-xCas9-Smac can recognize.

[0031] Construction of PAM plasmid library

[0032] In this experiment, the PAM plasmid library used to identify the novel chimeric Cas9 was constructed via enzyme digestion and ligation. The vector was obtained by digesting the pUC19 vector with EcoRI and HindIII. The fragment was amplified using a cellular genome as a template via Vazyme 2×Rapid Taq Master Mix, followed by DNA gel electrophoresis, gel extraction, and enzyme digestion. The synthesized upstream primer had a protective base and an EcoRI restriction site at its 5' end, while the downstream primer had a protective base, a HindIII restriction site, and an 8-nucleotide random sequence at its 5' end. The specific primer sequences are shown in Table 9. The vector and fragment were ligated via enzyme digestion, and the ligation product was purified, electroporated, cultured, and the plasmid was extracted to obtain a PAM plasmid library containing the 8-nucleotide random sequence. The primers were synthesized by Qingke Biotechnology.

[0033] (1) The reaction system and procedure for PCR amplification are shown in Table 1-4.

[0034] Table 1 Max Super-Fidelity DNAPolymerase Reaction System

[0035]

[0036] Table 2 Max Super-Fidelity DNAPolymerase Reaction Procedure

[0037]

[0038] Table 3 Vazyme 2×Rapid TaqMaster Mix reaction system

[0039]

[0040] Table 4 Vazyme 2×Rapid Taq Master Mix Reaction Procedure

[0041]

[0042] (2) The enzyme digestion reaction system and conditions are shown in Table 5.

[0043] Table 5 Enzyme digestion reaction system

[0044]

[0045] The above reaction components were incubated in a PCR instrument at 37°C for 8 to 10 hours, and then inactivated at 85°C for 10 minutes.

[0046] (3) DNA gel recovery process

[0047] i. After the PCR reaction is complete, load all the PCR products that need to be recovered into the wells of an agarose gel and run the target band.

[0048] ii. Use a clean scalpel or blade to cut a gel section containing the DNA fragment. Cut as close to the DNA as possible to reduce gel volume. Place the gel section into a pre-weighed 1.5 mL centrifuge tube and weigh it, recording the weight of the gel section.

[0049] iii. Add a 1:1 volume of Binding Buffer to the gel slices and incubate the gel mixture at 50-60°C for 10 minutes or until the gel slices are completely dissolved. Invert the centrifuge tube every few minutes to promote the melting process. To ensure complete gel dissolution, the gel mixture can be briefly vortexed before being added to the column.

[0050] iv. Transfer the dissolved gel solution from step iii to a DNA purification column (including the collection tube), adding a maximum of 800 μL at a time. Centrifuge at 8000 rpm for 1 min, discard the liquid in the collection tube, and return the purification column to the same collection tube.

[0051] v. Repeat the previous step until all the liquid has been added to the purification column.

[0052] vi. Add 700 μL of DNA wash buffer (diluted with anhydrous ethanol) to the DNA purification column. Centrifuge at 12000 rpm for 1 min, discard the liquid in the collection tube, and return the purification column to the same collection tube.

[0053] vii. Repeat step vi once, and then centrifuge the empty DNA purification column at 12000 rpm for 2 min to completely remove any residual wash buffer.

[0054] viii. Transfer the DNA purification column to a clean 1.5 mL centrifuge tube, open the cap, and evaporate any remaining ethanol at room temperature or in a 55°C metal bath.

[0055] ix. Add an appropriate amount of ddH2O incubated at 55℃ to the center of the purification column membrane, and centrifuge at 12000 rpm for 2 min. Remove the DNA purification column to obtain the purified PCR product.

[0056] The reaction system and conditions for homologous recombination are shown in Table 6.

[0057] Table 6 ClonExpressII One Step Cloning Reaction System

[0058]

[0059] Prepare the above reaction system on ice, gently pipette and mix (do not shake to mix), and briefly centrifuge to collect the reaction solution to the bottom of the tube. React at 37°C for 30 min; cool to 4°C or immediately place on ice to cool.

[0060] (5) The reaction system and conditions for PAM plasmid library digestion and ligation are shown in Table 7.

[0061] Table 7 Enzyme digestion and ligation reaction system

[0062]

[0063] After preparing the above reaction system, mix it thoroughly and place it at 16℃ for 2-3 hours.

[0064] (6) Phenol-chloroform extraction and purification of PAM plasmid library ligation products

[0065] (7) Chemical transformation

[0066] i. Place competent cells in an ice bath (thaw for 1-2 minutes), add the target DNA, mix gently, and incubate on ice for 30 minutes. (The volume of DNA used should not exceed 1 / 10 of the volume of the competent cell suspension.)

[0067] ii. Place the centrifuge tube in a 42°C water bath for 90 seconds, then quickly transfer the centrifuge tube to an ice bath to cool the cells for 2 minutes. Do not shake the centrifuge tube during this process.

[0068] iii. Add 500 μL of sterile LB medium (antibiotic-free) to each centrifuge tube, mix well, and incubate at 37°C and 220 rpm for 60 minutes. The purpose is to express the relevant resistance marker gene on the plasmid and revive the bacteria.

[0069] iv. Take an appropriate volume of the transformed competent cells and add them to LB solid agar medium containing the appropriate antibiotic, spreading the competent cells evenly. Place the plate at room temperature until the liquid is absorbed, then invert the plate and incubate at 37°C for 12-16 hours.

[0070] (8) Electroconversion

[0071] i. Insert the electrospinning cup with an electrode spacing of 0.1 cm into crushed ice, compact the ice surface, and let it stand in the ice for 5 minutes to allow the electrospinning cup to cool down completely. (Method for reusing the electrospinning cup: After each use, rinse thoroughly with plenty of tap water to remove bacterial culture and DNA, wash three times with ultrapure water, soak it in 75% ethanol for 30 minutes, remove the cup, drain the liquid, place it in a clean bench to allow the ethanol to evaporate completely, cover it and store it in a dry place for later use.)

[0072] ii. Take competent cells stored at -80℃ and insert them into ice. After the cells have just thawed, add the ligation product (the ion concentration in the elution or plasmid dissolution solution should not be too high, or it should be diluted with double-distilled water). Gently mix the mixture by tapping the bottom of the tube with your finger, and immediately insert it into ice. In a laminar flow hood, use a sterile pipette tip to quickly transfer the cell / DNA mixture into an electroporation cup, avoiding the generation of air bubbles and ensuring that the cells sink to the bottom of the cup. Cover the cup and keep the empty tube for later use.

[0073] iii. Start the electroporation apparatus and set the electroporation parameters: 2.4 kV, 200 Ω, 25 μF (BTX ECM 630 or Bio-RadGene Pulser). Wipe off any moisture from the outside of the electroporation cuvette with a paper towel and place it in the electroporation tank for electroporation. After completion, place the cuvette in ice, add 950 μL of antibiotic-free LB medium, and transfer the liquid to the previously retained competent cell tubes. Incubate at 37°C and 220 rpm with shaking for 1 hour.

[0074] iv. Add the bacterial culture to 100 mL of LB medium containing ampicillin resistance and incubate at 37°C with shaking at 220 rpm for 14-16 hours.

[0075] 2. ABE-xcas9-smac recognition PAM verification

[0076] Cell culture process

[0077] The cells used in this experiment were HEK293T cells, routinely cultured in DMEM medium containing 10% fetal bovine serum (Excell) and penicillin-streptomycin. The incubator conditions were 37°C and 5% CO2. Cells were passaged when the confluence reached 90%.

[0078] Cell transfection process

[0079] (1) Inoculate an appropriate amount of cells one day before transfection to achieve a cell density of 80-90% for transfection the next day, such as 1-4×10⁴.

[0080] (2) Prepare the DNA-Buffer Transeasy complex by mixing as follows.

[0081] i. Dilute an appropriate amount of DNA in 25 μL of Opti-MEM serum-free medium and mix thoroughly. (For base editing experiments, the transfection amounts are 300 ng editor plasmid, 100 ng sgRNA plasmid, and 30 ng puromycin selection plasmid. For PAM determination experiments, the transfection amounts are 300 ng editor plasmid, 100 ng sgRNA plasmid, and 45 ng PAM plasmid library.)

[0082] ii. Gently mix BufferTranseasy, take 0.75 μL and dilute it in 25 μL of Opti-MEM serum-free medium, mix well and incubate at room temperature for 5 minutes.

[0083] iii. After 5 minutes, mix the diluted DNA with Buffer Transeasy, a total of 50 μL, mix thoroughly and let stand at room temperature for 20 minutes to form a complex.

[0084] iv. Add 50 μL of DNA-Buffer Transeasy complex to the cell culture dish and gently shake the dish to mix.

[0085] v. Replace the medium with fresh complete medium containing 3 μg / mL puromycin 24 hours after transfection, and continue culturing for 48 hours before harvesting the cell genome.

[0086] Cell genome extraction process

[0087] (1) After transfecting the cells, remove the culture medium, add 50 μL of 1×PBS to each well, gently shake to wash away excess culture medium and dead cells, then remove and discard the PBS, repeat the washing process three times.

[0088] (2) Add 100 μL of 1×PBS to each well, pipette the cells off, place them in a centrifuge tube, and centrifuge at 12,000 rpm for 3 minutes at room temperature.

[0089] (3) Discard the supernatant, add 30 μL of cell lysis working solution to the cell pellet, mix well with a pipette, and then incubate on a 55°C metal bath for 20 minutes, followed by inactivation at 95°C for 10 minutes. The cell genome is stored at -20°C for subsequent experiments.

[0090] High-throughput sequencing and data analysis

[0091] For experiments searching for Cas9 PAM using PAM plasmid libraries, we used high-throughput sequencing to analyze the editing process.

[0092] (1) Sequencing: Using the extracted cell genome as a template, the target fragment was amplified by PCR using the high-throughput sequencing primers in Table 17. The upstream primers all had a 7bp barcode sequence attached (this sequence was provided by the high-throughput sequencing company). The PCR amplification system and procedure are shown in Tables 5 and 6. The obtained products were recovered by DNA gel electrophoresis and sent to the sequencing company. The samples were quantified, pooled, and library constructed. Paired-end (PE) sequencing of the library was performed using the Illumina NovaSeq sequencing platform.

[0093] (2) Data Analysis: High-throughput sequencing results were analyzed using Microsoft Excel. First, all read sequences containing the spacer were identified using the 8bp upstream fragment of the target sequence. Then, the identified sequences were categorized into 256 groups based on the possible occurrence of PAM sequences within a 4bp random sequence. The editing efficiency on each PAM was calculated based on the sixth base of the spacer in each group (base C represents unedited, base T represents edited). The efficiency was then analyzed and plotted using GraphPad Prism 9 software.

[0094] Table 8 High-throughput sequencing primers

[0095]

[0096]

[0097]

[0098] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Many modifications and variations can be made based on the content of this specification. These embodiments have been selected and specifically described in this specification to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A fusion protein xsp-SmacCas9, characterized in that: Its sequence is shown in SEQ ID NO:

1.

2. The fusion protein xsp-SmacCas9 as described in claim 1, characterized in that: The fusion protein xsp-SmacCas9 comprises: a) The amino acid sequence of the non-PI domain from xCas9; b) The amino acid sequence of the PI domain from the SmacCas9 variant.

3. The fusion protein xsp-SmacCas9 as described in claim 2, characterized in that: The amino acid sequence from the non-PI domain of xCas9 includes: A262T / R324L / S409I / E480K / E543D / M694I.

4. A base editor, characterized in that: The base editor is constructed from the fusion protein xsp-SmacCas9 described in claim 1 and has a wide PAM recognition range.

5. An expression cassette or expression vector comprising the base editor of claim 4.

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