Methods for producing Cas3 protein

Abstract

According to the present invention, it was discovered that a recombinant Cas3 protein can be efficiently expressed while maintaining the activity thereof by culturing, at a relatively low temperature, insect cells into which Cas3 gene is introduced, and that it is possible to recover an active form of the recombinant Cas3 protein with high purity at a high yield by purifying a solubilized fraction of the cells.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a Cas3 protein, and more specifically to a method for producing a recombinant Cas3 protein in high purity and high yield while maintaining its activity. [Background technology]

[0002] Genome editing technology specifically cuts genomic DNA sequences within animal or plant cells and uses their inherent repair mechanisms to freely rewrite them into any sequence. Its use is expanding worldwide, not only in bioscience research but also in crop and livestock breeding, regenerative medicine, and gene therapy.

[0003] CRISPR-Cas systems found in bacteria and archaea are divided into Class 1, which cleaves target sequences using a complex of multiple proteins, and Class 2, which cleaves using a single protein. Genome editing tools developed to date, such as CRISPR-Cas9, CRISPR-Cas12 (Cpf1), and CRISPR-Cas13, are all classified as Class 2. However, recently, it has been discovered that CRISPR-Cas3, a Type I CRISPR belonging to Class 1, can be used as a genome editing tool in eukaryotic cells (Patent Document 1). Among these, Type IE CRISPR-Cas3 derived from Escherichia coli K strain has been shown to recognize and bind to a 27-base target in addition to a 3-base PAM sequence, enabling highly efficient introduction of extensive deletion mutations of several hundred to several kb upstream of the target sequence in human cultured cells. Furthermore, compared to CRISPR-Cas9, it is considered safer due to the longer target recognition sequence in the guide RNA and the reduced risk of nonspecific cleavage.

[0004] CRISPR-Cas3 is generally used by introducing it into cells as an expression plasmid and expressing it there, but depending on the cell type, this can be difficult to introduce and express, and sufficient genome editing efficiency may not be achieved. For this reason, it is desirable to introduce CRISPR-Cas3 into cells in the form of guide RNA (crRNA) and proteins (Cas3 protein and Cascade protein).

[0005] However, among the proteins that make up CRISPR-Cas3, it has been particularly difficult to prepare Cas3 proteins in sufficient quality and quantity to be practically viable. For example, existing purification methods for Cas3 proteins have been reported for the thermophilic bacterium Thermobifida fusca (TfuCas3) and the Escherichia coli K strain (EcoCss3) (Non-Patent Documents 1-3). However, because TfuCas3 is derived from a thermophilic bacterium, it is not suitable for use at temperatures that many cells grow at. On the other hand, EcoCas3 is derived from Escherichia coli and exhibits activity at temperatures close to the growth temperatures of many cells, including animal cells, making it suitable for genome editing in a wide range of organisms, potentially for industrial applications. However, conventional methods have proven difficult to purify to high purity and high concentration while maintaining activity (see Comparative Example 1, described below). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 225858 [Non-patent literature]

[0007] [Non-Patent Document 1] Huo Y. et al., Nat. Struct. Mol. Biol. 2014 Sep;21(9):771-777 [Non-patent document 2] Mulepati S. & Bailey S., J. Biol. Chem. 2013 Aug 2;288(31):22184-22192 [Non-patent document 3] Robert P. Hayes et al., Nature 2016 530:499-503 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in consideration of the problems of the above-mentioned conventional technology, and its purpose is to provide a method for producing an active form of Cas3 protein that can be used for genome editing in various organisms with high purity and high yield. [Means for solving the problem]

[0009] As a result of extensive research aimed at solving the above problems, the inventors discovered that the Cas3 protein derived from Escherichia coli has low thermal stability, and even when the recombinant protein is expressed by culturing it at the normal culture temperature of Escherichia coli, it is denatured and its activity is reduced.

[0010] Based on this unexpected finding, the inventors selected insect cells, which can be cultured at relatively low temperatures, introduced the Cas3 gene into them, and cultured them under various temperature conditions. They found that culturing at 20 to 28°C efficiently expressed the Cas3 protein and maintained its activity.

[0011] Furthermore, the inventors investigated the purification conditions for recombinant Cas3 protein expressed in insect cells and found that purification in phosphate buffer enabled the recovery of active recombinant Cas3 protein in high purity and high yield.

[0012] Furthermore, the inventors discovered that the recombinant Cas3 protein thus recovered exhibits high activity even at the culture temperature of animal cells, etc., during the relatively short period required for genome editing processing, and thus completed the present invention.

[0013] That is, the present invention relates to a method for producing a recombinant Cas3 protein that can be used for genome editing in various cells at high purity and high concentration while maintaining its activity, and more specifically, provides the following inventions.

[0014] (1) A method for producing a Cas3 protein, comprising: (a) culturing insect cells into which a Cas3 gene has been introduced at 20 to 28°C to express the Cas3 protein in the insect cells; and (b) recovering the expressed Cas3 protein; A method comprising:

[0015] (2) The method according to (1), wherein the Cas3 protein is derived from Escherichia coli.

[0016] (3) The method according to (1) or (2), wherein the insect cells are Sf9 cells.

[0017] (4) The method according to any one of (1) to (3), wherein the recovery of the expressed Cas3 protein includes purification of the Cas3 protein.

[0018] (5) The method according to (4), wherein a tag is attached to the Cas3 protein, and purification of the Cas3 protein comprises affinity purification for the tag.

[0019] (6) The method according to (5), wherein the tag comprises an HN tag.

[0020] (7) The method according to any one of (4) to (6), wherein the purification of the Cas3 protein comprises purification by gel filtration chromatography.

[0021] (8) The method according to any one of (4) to (7), wherein the buffer used for purification is a phosphate buffer. [Effects of the Invention]

[0022] According to the present invention, it is possible to produce an active form of a recombinant Cas3 protein with high purity and high yield. The method of the present invention can be used to produce various recombinant Cas3 proteins, but is particularly useful for Cas3 proteins with low thermostability.

[0023] Furthermore, according to the present invention, it is possible to prepare the CRISPR-Cas3 system in a usable state before genome editing. Furthermore, it is possible to efficiently edit genomes even in cells in which it is difficult to introduce and express Cas3 as a gene, or in cells in which sufficient genome editing efficiency cannot be achieved when Cas3 is expressed as a recombinant protein. This makes it possible to use the CRISPR-Cas3 system simply and universally. [Brief explanation of the drawings]

[0024] [Figure 1] 1 is a graph showing the purification of recombinant EcoCas3 protein expressed in E. coli by gel filtration chromatography. [Figure 2] Electrophoresis photographs showing the expression of recombinant EcoCas3 protein in insect cells cultured at various temperatures. [Figure 3] The graph (top) and SDS-PAGE photograph (bottom) show the results of purification of recombinant EcoCas3 protein expressed in insect cells by gel filtration chromatography using phosphate buffer. The lanes in the SDS-PAGE are as follows: a) supernatant, b) flow-through, c) TEV digestion, d) wash, e) backtrap flow-through, 2-20 SEC fractions, and f) concentrated fractions 13-16. [Figure 4]This is a graph (top) and an SDS-PAGE photograph (bottom) showing the results of purification of recombinant EcoCas3 protein expressed in insect cells by gel filtration chromatography using Hepes buffer. [Figure 5] 1 is a graph showing the inflection point temperature due to thermal denaturation of recombinant EcoCas3 protein, measured using TychoNT6. [Figure 6] This graph shows the stability of EcoCas3, Cas9, Cas12, and TfuCas3 at 37°C. The stability of these proteins was measured by the change in fluorescence intensity caused by binding of Sypro_orange to the solvent-exposed hydrophobic regions of the proteins as they unfolded. [Figure 7] FIG. 1 shows the plasmid for the production of the Multi-NLS Eco cascade. [Figure 8] This is a capillary electrophoresis photograph showing the results of measuring genome editing activity in vitro using a complex of purified Cas3 protein, Cascade protein, and crRNA. Red arrows indicate detected DNA degradation. The left image shows the results using the EMX1-targeted cascade, and the right image shows the results using the Tyr-targeted cascade. [Figure 9] This figure shows the results of measuring genome editing activity in cultured human cells using a complex of purified Cas3 protein, Cascade protein, and crRNA. The top graph shows a representative image obtained by FACS analysis, and the bottom graph shows the analysis results of GFP knockout efficiency calculated from the number of GFP-negative cells (n = 3). DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention provides methods for producing Cas3 proteins.

[0026] In the present invention, the term "Cas3 protein" refers to a protein that constitutes the CRISPR-Cas3 system and has nuclease and helicase activities. The Cas3 protein can cleave target DNA in cooperation with the cascade and crRNA that constitute the CRISPR-Cas3 system.

[0027] Type IE CRISPR-Cas3 systems, which are the most common among type I CRISPR-Cas3 systems, cleave DNA by the cooperation of crRNA with Cas3 and Cascade (Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, and Cas7).

[0028] The type IA system contains the cascades Cas8a1, Csa5 (Cas11), Cas5, Cas6, and Cas7; the type IB system contains the cascades Cas8b1, Cas5, Cas6, and Cas7; the type IC system contains the cascades Cas8c, Cas5, and Cas7; the type ID system contains the cascades Cas10d, Csc1 (Cas5), Cas6, and Csc2 (Cas7); the type IF system contains the cascades Csy1 (Cas8f), Csy2 (Cas5), Cas6, and Csy3 (Cas7); and the type IG system contains the cascades Cst1 (Cas8a1), Cas5, Cas6, and Cst2 (Cas7).

[0029] The Cas3 protein of the present invention may be of any origin, but is preferably derived from Escherichia coli because it is suitable for genome editing in a wide range of cells, including animal cells. The amino acid sequence of a typical Cas3 protein derived from Escherichia coli is shown in SEQ ID NO: 2, and the nucleotide sequence of the DNA encoding the protein is shown in SEQ ID NO: 1. The Cas3 protein of the present invention includes naturally occurring and artificially modified mutants.

[0030] The Cas3 protein of the present invention may be a protein consisting of an amino acid sequence highly identical to the amino acid sequence of the E. coli-derived Cas3 protein set forth in SEQ ID NO: 1. High identity refers to, for example, sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more (e.g., 91% or more, 92% or more, 93% or more, 94% or more), and even more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more). Sequence identity can be determined using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., using default, i.e., initial setting parameters). Alternatively, the Cas3 protein of the present invention may be a protein consisting of the amino acid sequence of the E. coli-derived Cas3 protein set forth in SEQ ID NO: 1, in which one or more amino acids have been substituted, deleted, added, and / or inserted. Here, "multiple" generally means 50 amino acids or less, preferably 30 amino acids or less, more preferably 20 amino acids or less, and particularly preferably 10 amino acids or less (for example, 5 amino acids or less, 3 amino acids or less, 2 amino acids or less, 1 amino acid or less).

[0031] If necessary, a functional molecule may be added to the Cas3 protein. Examples of functional molecules include, but are not limited to, a nuclear localization signal for promoting the translocation into the nucleus of a eukaryotic cell, a tag for facilitating purification, and a reporter protein for facilitating detection. These functional molecules can be added, for example, to the N-terminus and / or C-terminus of the Cas3 protein.

[0032] Examples of nuclear localization signals include PKKKRKV (SEQ ID NO: 3) and KRTADGSEFESPKKKRKV (SEQ ID NO: 4). Examples of tags include HN tags, His tags, FLAG tags, and glutathione-S-transferase (GST) tags. Examples of reporter proteins include fluorescent proteins such as green fluorescent protein (GFP) and chemiluminescent proteins such as luciferase.

[0033] In the production method of the present invention, insect cells into which a Cas3 gene has been introduced are cultured at 20 to 28°C to express the Cas3 protein in the insect cells (step (a)).

[0034] A known baculovirus expression system can be used to express a recombinant Cas3 protein in insect cells. In one example of a method using the baculovirus expression system, the Cas3 gene is first cloned into a Bac-to-Bac vector such as pFastBac1, which is then introduced into Escherichia coli containing baculovirus DNA to prepare baculovirus DNA containing the Cas3 gene. In addition to this method of preparing recombinant baculovirus DNA in E. coli, it is also possible to prepare it in insect cells. When using insect cells, a vector containing the Cas3 gene and baculovirus DNA are introduced into the insect cells to induce homologous recombination between the two. The prepared recombinant baculovirus DNA is then transfected into insect cells to prepare a recombinant baculovirus containing the Cas3 gene. The prepared recombinant baculovirus is then serially infected into insect cells to obtain high-titer baculovirus, which is then used to express the recombinant Cas3 protein. As the insect cells, Sf9 cells are preferred, but are not limited thereto.

[0035] Insect cells are preferably cultured at 20 to 28°C to express recombinant Cas3 protein. Temperatures below 20°C tend to reduce the expression efficiency of the recombinant Cas3 protein, while temperatures above 28°C tend to denature the expressed recombinant Cas3 protein during the culture process. To further suppress denaturation, 20 to 24°C is more preferred, 20 to 22°C is even more preferred, and 20°C is particularly preferred. There are no particular limitations on the culture time as long as it is sufficient for the expression of the recombinant Cas3 protein, but it is usually 24 hours or more, preferably 60 to 72 hours.

[0036] In the production method of the present invention, the expressed Cas3 protein is then collected (step (b)).

[0037] Various protein isolation and purification methods can be used to recover the expressed Cas3 protein. Cell disruption and centrifugation can be used to isolate recombinant Cas3 protein from cells. For example, cells can be disrupted by sonication, then centrifuged at 100,000 g, and the supernatant can be collected to obtain a soluble fraction containing the recombinant Cas3 protein.

[0038] When a tag is attached to the Cas3 protein, the recombinant Cas3 protein can be purified by affinity purification for the tag. For example, when the tag is an HN tag or a His tag, affinity purification can be performed using a nickel column; when the tag is a FLAG tag, affinity purification can be performed using beads bound to an antibody against the FLAG tag; and when the tag is a glutathione-S-transferase (GST) tag, affinity purification can be performed using glutathione Sepharose. From the viewpoint of electrically neutralizing the Cas3 protein and suppressing aggregation, the HN tag is preferred as the tag to be attached to the Cas3 protein.

[0039] In addition, the purification of the recombinant Cas3 protein in the present invention preferably includes purification by gel filtration chromatography. Since the Cas3 protein is a globular protein with a molecular weight of approximately 100 kDa, it is preferable to select a column with a fractionation range suitable for globular proteins of this molecular weight. Commercially available columns such as Superdex 200 Increase (Cytiva) are available.

[0040] The buffer used for purification is preferably a phosphate buffer, from the viewpoint of suppressing aggregation due to denaturation of the Cas3 protein.

[0041] The recombinant Cas3 protein prepared in this manner has excellent activity and can exert its activity without denaturation even at temperatures of 37°C, within the relatively short time required for genome editing, within a few hours. In fact, in this example, excellent DNA cleavage activity and high genome editing efficiency were observed at 37°C. Therefore, the recombinant Cas3 protein obtained by the method of the present invention can be used in combination with Cascade proteins and crRNA to efficiently edit genomes in a wide range of cells. [Example]

[0042] The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples.

[0043] [Comparative Example 1] Preparation of EcoCas3 protein using E. coli The conventional method for producing EcoCas3 (Mulepati S. & Bailey S., J Biol Chem. 2013 Aug 2;288(31):22184-22192) had many problems, including: (i) the need to culture E. coli cells transfected with a plasmid encoding EcoCas3 at a low temperature of 20°C; (ii) the need to fuse EcoCas3 with maltose-binding protein (MBP) or small ubiquitin-like modifier (SUMO) to maintain EcoCas3 solubility; (iii) the need to co-express the chaperone HtpG protein; (iv) the yield was limited to a maximum of 1 mg per 1 L of culture; and (v) the difficulty in producing highly pure EcoCas3 due to the presence of bands for proteins other than EcoCas3 in the electrophoresis pattern. In fact, when we prepared EcoCas3 protein using E. coli according to the above literature, the purity and yield were low (Figure 1), and the activity was also extremely low.

[0044] [Example 1] Preparation of EcoCas3 protein using insect cells Sf9 When a prokaryotic protein is expressed as a recombinant protein in a higher eukaryote, post-translational modifications that do not occur in prokaryotes may occur. For this reason, EcoCas3 protein has not been produced in eukaryotes. However, in light of the problems described in Comparative Example 1, the present inventors attempted to prepare EcoCas3 protein using insect cells Sf9.

[0045] (1) Construction of a plasmid for expressing the recombinant EcoCas3 protein We synthesized a gene in which an 8HN tag (a tag formed by fusing a His tag and an HN tag with a GS linker between them; SEQ ID NO: 5) and an NLS (SEQ ID NO: 3) were fused to the N-terminus of EcoCas3, and an NLS (SEQ ID NO: 3) was also fused to the C-terminus (SEQ ID NO: 6, 7). The HN tag (a histidine-asparagine repeat sequence) was used to neutralize the positive charge of the His tag, which has the potential to cause aggregation of the target protein. To confirm expression, we also created a gene in which EGFP was fused to the 3' end of the 8HN tag as a reporter (SEQ ID NO: 8, 9).

[0046] The fusion gene was cloned into the pFastbac-1 plasmid (ThermoFishers). The resulting EcoCas3 / pFastbac-1 plasmid was transformed into DH10bac and integrated into the baculovirus genome within DH10bac by homologous recombination. The baculovirus genome containing the EcoCas3 gene was then extracted.

[0047] (2) Expression of recombinant EcoCas3 protein Baculovirus genomes containing the EcoCas3 gene or the EGFP-fused EcoCas3 gene were transfected into Sf9 cells, and baculoviruses containing the EcoCas3 gene were generated in the Sf9 cells. These baculoviruses were then serially infected into Sf9 cells to obtain high-titer virus for EcoCas3 expression. These high-titer virus was then used to infect Sf9 cells, where EcoCas3 was expressed as a recombinant protein.

[0048] To investigate the expression of the recombinant EcoCas3 protein, we used a baculovirus containing the EGFP-fused EcoCas3 gene. Sf9 cells were infected with the baculovirus for 24 hours at 28°C, and then cultured at various temperatures (12°C to 28°C) for 60 hours to express the recombinant EcoCas3 protein. The cells were sonicated and centrifuged at 100,000g. The supernatant (soluble fraction) was collected and subjected to electrophoresis for fluorescence detection.

[0049] As a result, we confirmed the expression of recombinant EcoCas3 protein in the soluble fraction at temperatures above 16°C, but the expression efficiency in the soluble fraction was particularly high at temperatures above 20°C (Figure 2). In the following experiments, we used recombinant EcoCas3 protein expressed in Sf9 cells at 20°C.

[0050] (3) Preparation of recombinant EcoCas3 protein The recombinant EcoCas3 protein, which had a His-like 8HN tag attached, was affinity purified on a nickel column and then finally purified by gel filtration chromatography.

[0051] Insect cells expressing the recombinant EcoCas3 protein were sonicated, centrifuged at 100,000 g, and the supernatant (soluble fraction) was mixed with nickel agarose resin (Qiagen) to bind the recombinant EcoCas3 protein to the resin. The recombinant EcoCas3 protein was then eluted from the resin with washing buffer (20 mM HEPES or 20 mM KH2PO4, 350 mM NaCl, 40 mM imidazole, 0.5 mM DTT, pH 7.0) and eluted with elution buffer (20 mM Hepes or 20 mM KH2PO4, 350 mM NaCl, 200 mM imidazole, 0.5 mM DTT, pH 7.0).

[0052] The eluted fraction was digested with TEV to remove the 8HN tag attached to the N-terminus of the recombinant EcoCas3 protein. Finally, the recombinant EcoCas3 protein was purified by gel filtration chromatography. A Superdex 200 increase (Cytiva) column was used for purification, and the mobile phase buffer was 20 mM HEPES or 20 mM KH2PO4, 200 mM NaCl, 1.0 mM DTT, pH 7.0.

[0053] When purified using HEPES buffer, a large amount of the void fraction, containing EcoCas3 that appeared to have aggregated during gel filtration chromatography, was observed (Figure 3). On the other hand, when purified using phosphate buffer, the inflection point temperature (Ti) improved by approximately 2°C, and ultimately, we succeeded in obtaining up to 2 mg of purified EcoCas3 protein per 1 L of culture (Figure 4). The use of phosphate buffer appears to have suppressed the separation of EcoCas3 into the void fraction. When the purity was confirmed by SDS-PAGE, no bands representing proteins other than the target EcoCas3 protein were detected, demonstrating that the purified EcoCas3 protein was significantly more pure than the conventional EcoCas3 protein purified using an E. coli expression system.

[0054] [Example 2] Measurement of the thermal stability of recombinant EcoCas3 protein The thermal stability of the recombinant EcoCas3 protein was evaluated by measuring the inflection point temperature (Ti) of the thermal denaturation profile using a TychoNT6 (NanoTempar). The peak shift of the autofluorescence from tryptophan residues in the molecule, which accompanies thermal unfolding, was detected at two wavelengths, 330 nm and 350 nm, and the inflection point temperature (Ti) of the thermal denaturation profile was determined by plotting the ratio of the fluorescence intensities against temperature.

[0055] We also measured the thermal stability of the recombinant EcoCas3 protein using SYPRO Orange fluorescent reagent. SYPRO Orange emits fluorescence by binding to the denatured hydrophobic region of the protein. The increase in SYPRO Orange binding and fluorescence intensity, which depended on the exposure of the hydrophobic region of the protein to the solvent due to the structural change associated with thermal denaturation, was detected using a real-time PCR system. Fluorescence detection was performed at an excitation wavelength of 473 nm and an emission wavelength of 520 nm.

[0056] The results of the above experiments using TychoNT6 revealed that the inflection point temperature of the recombinant EcoCas3 protein was similar to that of Cas9 (Figure 5). However, stability measurements using SYPRO Orange fluorescent reagent at a constant temperature of 37°C revealed that the recombinant EcoCas3 protein completely denatured after approximately 8 hours (Figure 6). These results suggest that the recombinant EcoCas3 protein is in a state of high entropy, which indicates molecular disorder, and therefore exhibits large fluctuations. Therefore, the Gibbs free energy change, a thermodynamic parameter, is small, making it less stable than Cas9, Cas12, and TfuCas3. These results on the stability of the recombinant EcoCas3 protein clearly explain the highly efficient expression of the protein in Sf9 cells at temperatures lower than the typical insect cell culture temperature (28°C) (see Example 1(2)). On the other hand, the purified recombinant Cas3 protein was found to have high activity for several hours even at 37°C, which clearly explains why it can be used without any problems to cleave target DNA in vitro or in various cells (see the results of Examples 3 and 4 below).

[0057] [Example 3] Preparation of Eco cascade complex In order to simultaneously introduce the recombinant EcoCascade into cells and the recombinant EcoCas3 protein for genome editing, it is important to efficiently translocate the EcoCascade into the nucleus. However, the molecular weight of the EcoCascade is large, approximately 0.4 MDa, raising concerns about its nuclear translocation rate. Therefore, the inventors attempted to introduce more NLS into the cascade by splitting the five-gene operon that constitutes the EcoCascade into two operons and adding NLS to the 3' end of each operon, thereby achieving highly efficient translocation into the nucleus.

[0058] The EcoCascade is a supramolecular complex consisting of Cas8, Cas11, Cas7, Cas5, and Cas6. It consists of one Cas8 molecule, two Cas11 molecules, five Cas7 molecules, two Cas5 molecules, and one Cas6 molecule. The present inventors constructed three plasmids: a His-tagged Cas11-NLS operon in the pCDFuet-1 plasmid, a Cas8-Cas11-Cas7 operon with an NLS attached to the 3' end, and a Cas5-Cas6 operon with an NLS attached to the 3' end in the pRSFDuet-1 plasmid, and a crRNA in the pACYCDuet-1 plasmid (Figure 7, SEQ ID NOs: 10-24).

[0059] These three plasmids were then transformed into E. coli JM109(DE3) (a lysogenic strain of E. coli harboring the bacteriophage λDE3, which contains a T7 RNA polymerase gene under the control of the lacUV5 promoter), and the recombinant proteins or crRNA were expressed using IPTG. The Multi-NLS-Eco cascade was affinity purified using a nickel column via the His tag introduced into Cas11, followed by gel filtration chromatography. Approximately 1 mg of the Multi-NLS-Eco cascade was successfully produced from a 2 L culture.

[0060] The target sequences of the crRNA were the sequence within the human EMX1 gene, the sequence within the mouse Tyr gene, and the sequence within the Aequorea victoria GFP gene.

[0061] [Example 4] In vitro DNA cleavage measurement The target DNA cleavage activity of purified EcoCas3 and EcoCascade proteins was examined in vitro using double-stranded DNA. Cas3 protein (20 nM), crRNA-Cascade complex (20 nM), and double-stranded DNA containing the target sequence (60 ng / μL) were mixed in a reaction buffer (5 mM HEPES-K pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 μM CoCl2, 2.5 mM ATP). The reaction solution was incubated at 37°C for 1 hour and then subjected to capillary electrophoresis using a MultiNa (Shimadzu Corporation). The target sequences were the EMX1 gene region and the Tyr gene region. For EMX1, we also prepared a double-stranded DNA sequence in which the PAM sequence (AAG), which can be recognized by E. coli type IE CRISPR, was changed to (CCA), which cannot be recognized by E. coli type IE CRISPR.

[0062] As a result, when double-stranded donor DNA containing the AAG target sequence, which can be recognized by the PAM sequence, was mixed, degradation of the donor DNA was observed (Figure 8). On the other hand, when donor DNA in which the PAM sequence had been changed to CCA, which cannot be recognized, was mixed, no DNA degradation was observed. When the Cas3 protein alone or the Cascade complex alone was used, no DNA cleavage activity was observed. On the other hand, when the Cascade complex alone was used, a band was detected above normal. This is thought to be because the Cascade complex recognized and bound to the DNA, which increased its size under non-denaturing conditions, resulting in a gel shift.

[0063] [Example 5] Activity measurement in human cultured cells HEK293T We investigated the mutagenesis efficiency of purified EcoCas3 and EcoCascade proteins in human cells using reporter HEK293T cells carrying mCherry-P2A-EGFP. Cas3 protein (30 μM or 45 μM) and the GFP-targeting crRNA-Cascade complex (30 μM or 45 μM) were introduced into reporter cells by electroporation using the Neon Transfection System (Thermo Fisher Scientific). After culturing the cells at 37°C and 5% CO2 for 5 days, all cells were harvested and the number of GFP-negative cells was counted using an SH800 (Sony) microscope. The mutagenesis efficiency was calculated.

[0064] As a result, approximately 20% of cells were GFP-negative when transfected at 30 μM, and approximately 40% when transfected at 45 μM (Figure 9). This indicates that genome editing using CRISPR-Cas3 proteins can be performed with high efficiency, up to approximately 40%. [Industrial Applicability]

[0065] The Cas3 protein produced by the method of the present invention can be used for genome editing of various cells, and therefore can be used not only in basic research but also in various applied fields of genome editing technology, such as medicine, agriculture, and industry.

Claims

1. A method for producing a Cas3 protein, comprising: (a) culturing insect cells into which a Cas3 gene has been introduced at 20 to 24°C to express the Cas3 protein in the insect cells; and (b) recovering the expressed Cas3 protein; Including, The Cas3 protein is derived from Escherichia coli; method.

2. The method of claim 1 , wherein the insect cells are Sf9 cells.

3. The method of claim 1 or 2, wherein the recovery of the expressed Cas3 protein comprises purifying the Cas3 protein.

4. The method of claim 3, wherein a tag is attached to the Cas3 protein and purification of the Cas3 protein comprises affinity purification against the tag.

5. The method of claim 4 , wherein the tag comprises an HN tag.

6. The method of any one of claims 3 to 5, wherein the purification of the Cas3 protein comprises purification by gel filtration chromatography.

7. The method according to any one of claims 3 to 6, wherein the buffer used for purification is a phosphate buffer.

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