WO2022186063A1 - Cas3タンパク質を製造する方法 - Google Patents
Cas3タンパク質を製造する方法 Download PDFInfo
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Definitions
- the present invention relates to a method for producing Cas3 protein, and more specifically to a method for producing recombinant Cas3 protein with high purity and high yield while maintaining activity.
- Genome editing technology is a technology that can be freely rewritten to any sequence by specifically cutting the genomic DNA sequence in animal or plant cells and using the endogenous repair mechanism. Its use is expanding all over the world, not only in bioscience research, but also in the breeding of crops and livestock animals, regenerative medicine, and gene therapy.
- CRISPR-Cas system possessed by bacteria and archaea is divided into class 1, which cleaves the target sequence with a complex of multiple proteins, and class 2, which cleaves with a single protein.
- CRISPR-Cas9, CRISPR-Cas12 (Cpf1), CRISPR-Cas13, etc. which have been developed as genome editing tools so far, are all classified as class 2, but recently CRISPR-Cas3, a type I CRISPR belonging to class 1 can be used as a genome editing tool for eukaryotic cells (Patent Document 1).
- type IE CRISPR-Cas3 derived from Escherichia coli K strain recognizes and binds to 27 bases in addition to the 3-base PAM sequence as a target, and is located several hundred to several kb upstream of the target sequence in cultured human cells. It has been shown to be capable of introducing wide-ranging deletion mutations with high efficiency.
- the target recognition sequence in the guide RNA is long, non-specific cleavage is less likely to occur, it is considered to be highly safe.
- CRISPR-Cas3 is generally used by introducing it into cells as an expression plasmid and expressing it, but depending on the type of cell, it is difficult to introduce and express it, and if sufficient genome editing efficiency cannot be obtained. There is Therefore, it is desirable to introduce CRISPR-Cas3 into cells in the form of guide RNA (crRNA) and proteins (Cas3 protein and Cascade protein).
- crRNA guide RNA
- proteins Cas3 protein and Cascade protein
- the present invention has been made in view of the problems of the above-described prior art, and its object is to produce an active form of Cas3 protein that can be used for genome editing in various organisms with high purity and high yield. is to provide
- the present inventors have made intensive studies to solve the above problems, the Escherichia coli-derived Cas3 protein has low thermostability, when the recombinant protein is expressed by culturing at a normal E. coli culture temperature. However, it was found that the activity was attenuated due to denaturation.
- the present inventors selected insect cells as cells that can be cultured at a relatively low temperature, introduced the Cas3 gene and cultured under various temperature conditions. By culturing, the Cas3 protein was efficiently expressed and found to maintain its activity.
- the present inventors have investigated the purification conditions of the recombinant Cas3 protein expressed in insect cells, and by performing purification in a phosphate buffer, the active form of the recombinant Cas3 protein is highly purified and We have found that it is possible to recover in high yields.
- the present inventors have found that the recombinant Cas3 protein thus recovered exhibits high activity even at culture temperatures such as animal cells in the relatively short time required for genome editing treatment, and completed the present invention. came to.
- the present invention relates to a method for producing a recombinant Cas3 protein that can be used for genome editing of various cells with high purity and high concentration while maintaining activity, and more specifically, provides the following invention. be.
- a method for producing a Cas3 protein (A) culturing the Cas3 gene-introduced insect cells at 20 to 28 ° C. to express the Cas3 protein in the insect cells, and (b) recovering the expressed Cas3 protein, method including.
- the present invention it is possible to produce an active form of recombinant Cas3 protein with high purity and high yield.
- the method of the present invention can be used to produce a variety of recombinant Cas3 proteins, it is particularly useful in application to Cas3 proteins with low thermostability.
- the present invention it is possible to prepare the CRISPR-Cas3 system in a usable state before performing genome editing. Furthermore, efficient genome editing can be performed even in cells where it is difficult to introduce and express Cas3 as a gene, or in cells where sufficient genome editing efficiency cannot be obtained when Cas3 is expressed as a recombinant protein. becomes possible. Therefore, it is possible to use the CRISPR-Cas3 system simply and universally.
- FIG. 4 is a graph showing purification by gel filtration chromatography of recombinant EcoCas3 protein expressed in E. coli. It is an electropherogram showing expression of recombinant EcoCas3 protein in insect cells cultured at each temperature. A graph (upper) and an SDS-PAGE photograph (lower) showing the results of purifying the recombinant EcoCas3 protein expressed in insect cells by gel filtration chromatography using a phosphate buffer are shown. Each lane in SDS-PAGE is as follows. a. supernatant, b. flow through, c. TEV digestion, d. washing e. Back trap flow-through, 2-20. SEC fraction f. Concentrated 13-16 fractions.
- FIG. 10 is a graph showing the inflection point temperature due to heat denaturation of recombinant EcoCas3 protein.
- the inflection point temperature was measured by TychoNT6.
- FIG. 10 is a graph showing 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 the binding of Sypro_orange to the hydrophobic region exposed to the solvent surface as the proteins were denatured.
- FIG. 10 is a graph showing the inflection point temperature due to heat denaturation of recombinant EcoCas3 protein.
- the inflection point temperature was measured by TychoNT6.
- FIG. 10 is a graph showing 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 the binding of Sypro_orange to the hydro
- the present invention provides a method for producing Cas3 protein.
- Cas3 protein in the present invention is a protein that constitutes the CRISPR-Cas3 system and has nuclease activity and helicase activity.
- the Cas3 protein can cleave target DNA by cooperating with the cascades and crRNAs that make up the CRISPR-Cas3 system.
- the Type I-E CRISPR-Cas3 system which is common among Type I CRISPR-Cas3 systems, co-operates crRNA with Cas3 and cascades (Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, and Cas7) This cuts the DNA.
- Type IA systems Cas8a1, Csa5 (Cas11), Cas5, Cas6, and Cas7 as cascades; in Type IB, Cas8b1, Cas5, Cas6, and Cas7 as cascades; in Type IC, cascades Cas8c, Cas5, and Cas7 as Cas10d, Csc1 (Cas5), Cas6, and Csc2 (Cas7) as cascades in types ID, and Csy1 (Cas8f), Csy2 (Cas5) as cascades in types I-F , Cas6, and Csy3 (Cas7), and in systems of types IG, Cst1 (Cas8a1), Cas5, Cas6, and Cst2 (Cas7) as cascades, respectively.
- the Cas3 protein derived from Escherichia coli is preferable from the viewpoint that it is suitable for genome editing in a wide range of cells including animal cells.
- the amino acid sequence of a typical Escherichia coli-derived Cas3 protein 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 proteins of the present invention include naturally occurring or artificially modified variants.
- the Cas3 protein of the present invention can be a protein consisting of an amino acid sequence having high identity with the amino acid sequence of the Cas3 protein derived from E. coli described in SEQ ID NO: 1.
- High identity is, for example, 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), more preferably 95% or more (eg, 96% or greater, 97% or greater, 98% or greater, 99% or greater) sequence identity.
- Sequence identity is determined using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) (e.g., default parameters ) can be determined.
- the Cas3 protein of the present invention is a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of the Cas3 protein derived from E. coli described in SEQ ID NO: 1.
- “plurality” is usually within 50 amino acids, preferably within 30 amino acids, more preferably within 20 amino acids, particularly preferably within 10 amino acids (e.g., within 5 amino acids, within 3 amino acids, within 2 amino acids, 1 amino acid ).
- a functional molecule may be added to the Cas3 protein.
- Functional molecules include, for example, nuclear localization signals for promoting translocation into the nucleus of eukaryotic cells, tags for facilitating purification, reporter proteins for facilitating detection, etc. but not limited to these. These functional molecules can be added, for example, to the N-terminal side and/or the C-terminal side of the Cas3 protein.
- nuclear localization signals examples include PKKKRKV (SEQ ID NO: 3) and KRTADGSEFESPKKKRKV (SEQ ID NO: 4).
- tags include HN tag, His tag, FLAG tag, glutathione-S-transferase (GST) tag and the like.
- reporter proteins include fluorescent proteins such as green fluorescent protein (GFP) and chemiluminescent proteins such as luciferase.
- the Cas3 gene-introduced insect cells are cultured at 20 to 28 ° C to express the Cas3 protein in the insect cells (step (a)).
- a known baculovirus expression system can be used as a method for expressing recombinant Cas3 protein in insect cells.
- a method using a baculovirus expression system first, the Cas3 gene is cloned into a Bac-to-Bac vector such as pFastBac1, which is introduced into E. coli having baculovirus DNA, and the Cas3 gene is introduced.
- a Bac-to-Bac vector such as pFastBac1
- the Cas3 gene is introduced.
- Prepare baculovirus DNA In addition to the method of preparing recombinant baculovirus DNA in Escherichia coli as described above, it is also possible to use a method of preparing in insect cells.
- a vector containing the Cas3 gene and baculovirus DNA may be introduced into insect cells to induce homologous recombination between them.
- insect cells are transfected with the prepared recombinant baculovirus DNA to prepare a recombinant baculovirus containing the Cas3 gene.
- the prepared recombinant baculovirus is then subcultured into insect cells to obtain high titer baculovirus, which is then infected into insect cells to express the recombinant Cas3 protein.
- Sf9 cells are suitable as insect cells, but are not limited thereto.
- Insect cells are preferably cultured at 20-28°C to express the recombinant Cas3 protein. Below 20°C, the expression efficiency of the recombinant Cas3 protein tends to decrease, while above 28°C, the recombinant Cas3 protein expressed during the culture process tends to denature. From the viewpoint of further suppressing denaturation, the temperature is more preferably 20 to 24°C, still more preferably 20 to 22°C, and particularly preferably 20°C.
- the culture time is not particularly limited 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.
- the expressed Cas3 protein is then recovered (step (b)).
- Various protein separation and purification methods can be used to recover the expressed Cas3 protein.
- Cell disruption and centrifugation can be used to separate recombinant Cas3 protein from cells.
- a soluble fraction containing recombinant Cas3 protein can be obtained by disrupting cells with ultrasound, then centrifuging at 100,000 g, and collecting the supernatant.
- affinity purification for the tag can be used in purifying the recombinant Cas3 protein.
- the tag is an HN tag or His tag
- a nickel column is used.
- the tag is a FLAG tag
- beads bound with an antibody against the FLAG tag are used.
- glutathione sepharose respectively, can be used for affinity purification.
- the tag added to the Cas3 protein is preferably an HN tag.
- 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 about 100 kDa, it is preferable to select a column having a fractionation range suitable for globular proteins of this molecular weight. As such a column, for example, a commercially available product such as Superdex 200 Increase (Cytiva) can be used.
- the buffer used for purification is preferably a phosphate buffer from the viewpoint of suppressing aggregation due to denaturation of the Cas3 protein.
- Recombinant Cas3 protein thus prepared has excellent activity, if relatively short time within several hours required for genome editing, even under temperature conditions of 37 ° C., without denaturation can exert its activity.
- 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 efficiently perform genome editing in a wide range of cells by combining with the cascade protein and crRNA.
- the above fusion gene was cloned into the pFastbac-1 plasmid (manufactured by ThermoFisher).
- the resulting EcoCas3/pFastbac-1 plasmid was transformed into DH10bac and incorporated into the baculovirus genome within DH10bac by homologous recombination, after which the baculovirus genome containing the EcoCas3 gene was extracted.
- baculovirus containing the EGFP-fused EcoCas3 gene was used, and baculovirus infection of Sf9 cells was performed at 28°C for 24 hours, after which Sf9 cells were incubated at each culture temperature (12 °C to 28 °C) for 60 hours to express the recombinant EcoCas3 protein.
- the cells were sonicated, centrifuged at 100,000 g, and the collected supernatant (soluble fraction) was subjected to electrophoresis and fluorescence detection.
- the insect cells expressing the recombinant EcoCas3 protein were crushed with ultrasonic waves, and the supernatant collected by centrifugation at 100,000 g (soluble fraction) was mixed with nickel agarose resin (Qiagen) to obtain the recombinant EcoCas3 protein. was bound to the resin, washing buffer (20 mM HEPES or 20 mM KH2PO4 , 350 mM NaCl, 40 mM imidazole, 0.5 mM DTT, pH 7.0) was used.
- the recombinant EcoCas3 protein was eluted from the resin with an elution buffer ( 20 mM Hepes or 20 mM KH2PO4 , 350 mM NaCl, 200 mM imidazole, 0.5 mM DTT, pH 7.0).
- an elution buffer 20 mM Hepes or 20 mM KH2PO4 , 350 mM NaCl, 200 mM imidazole, 0.5 mM DTT, pH 7.0.
- Example 2 Thermostability measurement of recombinant EcoCas3 protein
- TychoNT6 NanoTempar
- the thermal stability of the recombinant EcoCas3 protein was evaluated.
- rice field The peak shift of autofluorescence derived from intramolecular tryptophan residues accompanying unfolding of protein molecules by heat is detected at two wavelengths of 330 nm and 350 nm, and the ratio of fluorescence intensity is plotted against temperature to obtain a thermal denaturation profile. was determined as the inflection point temperature Ti.
- SYPRO Orange exhibits fluorescence by binding to denatured hydrophobic regions of proteins.
- the binding of SYPRO Orange and the increase in fluorescence intensity depending on the solvent exposure of the hydrophobic region of the protein due to the structural change accompanying heat denaturation were detected by a real-time PCR device. Fluorescence detection was performed at an excitation wavelength of 473 nm and an emission wavelength of 520 nm.
- the Eco cascade is a supramolecular complex consisting of Cas8-Cas11-Cas7-Cas5-Cas6.
- the number of constituents is 1 molecule of Cas8, 2 molecules of Cas11, 5 molecules of Cas7, 2 molecules of Cas5, and 1 molecule of Cas6.
- the present inventors have added Cas11-NLS fused with His tag to pCDFuet-1 plasmid, added NLS to 3' end "Cas8-Cas11-Cas7 operon", and added NLS to 3' end "Cas5- Three plasmids were constructed that incorporated the Cas6 operon into the pRSFDuet-1 plasmid and the crRNA into the pACYCDuet-1 (FIG. 7, SEQ ID NOS: 10-24).
- the target sequences of crRNA were the human EMX1 gene sequence, mouse Tyr gene sequence, and Aequorea victoria GFP gene sequence.
- the target sequences were the EMX1 gene region and the Tyr gene region.
- EMX1 a sequence was also prepared and examined by changing the PAM sequence of the double-stranded DNA from "AAG”, which can be recognized by E. coli-derived type IE CRISPR, to "CCA”, which cannot be recognized.
- Example 5 Measurement of activity in human cultured cells HEK293T Using reporter HEK293T cells with mCherry-P2A-EGFP, the mutagenesis efficiency of purified EcoCas3 and Eco cascade proteins in human cells was examined.
- Cas3 protein (30 ⁇ M or 45 ⁇ M), GFP-targeted crRNA and cascade complex (30 ⁇ M or 45 ⁇ M) were introduced into reporter cells by electroporation using Neon Transfection System (Thermo Fisher Scientific). After culturing the cells at 37° C. and 5% CO 2 for 5 days, all the cells were collected and the number of GFP-negative cells was counted using SH800 (SONY) to calculate the efficiency of mutagenesis.
- Cas3 protein produced by the method of the present invention can be used for genome editing of various cells, not only basic research but also various fields of application of genome editing technology such as medicine, agriculture, industry can be used in
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Abstract
Description
(a)Cas3遺伝子を導入した昆虫細胞を20~28℃で培養し、当該昆虫細胞内でCas3タンパク質を発現させる工程、および
(b)発現させたCas3タンパク質を回収する工程、
を含む方法。
従来のEcoCas3の製造方法(Mulepati S. & Bailey S., J Biol Chem. 2013 Aug 2;288(31):22184-22192)には、(i)EcoCas3をコードするプラスミドを導入した大腸菌を20℃という低温で培養を行う必要がある点、(ii)EcoCas3の溶解度を保つために、EcoCas3にmaltose-binding protein(MBP)やsmall ubiquitin-like modifier(SUMO)を融合させる必要がある点、(iii)シャペロン分子であるHtpGタンパク質を共発現させることも必要になる点、(iv)収量が1L培養あたり最大でも1mgである点、(v)電気泳動パターンとしてEcoCas3以外のタンパク質のバンドが確認されることから高純度なEcoCas3を製造することが困難である点、など多くの問題があった。実際、上記文献に従って、大腸菌を利用してEcoCas3タンパク質の調製を行ったところ、低純度かつ低収量であり(図1)、活性も極めて低かった。
原核生物由来のタンパク質を、より高次の真核生物において、組換えタンパク質として発現させた場合、原核生物内では起こり得ない翻訳後修飾が起こる可能性がある。このため、EcoCas3タンパク質の製造を真核生物で行うことは行われてこなかったが、本発明者は、比較例1に記載の課題に鑑み、敢えて昆虫細胞Sf9を用いたEcoCas3タンパク質の調製を試みた。
EcoCas3のN末端に8HNタグ(GSリンカーを挟んで、HisタグとHNタグを融合したタグ/配列番号:5)およびNLS(配列番号:3)を融合させ、さらにC末端にもNLS(配列番号:3)を融合させた遺伝子を合成した(配列番号:6、7)。HNタグ(ヒスチジンとアスパラギンのリピート配列)を使用したのは、Hisタグは正電荷の偏りが強く、目的タンパク質を凝集させる可能性があることから、当該正電荷を中和するためである。また、発現確認のために、レポーターとしてのEGFPを8HNタグの3’末端に融合させた遺伝子も作成した(配列番号:8、9)。
EcoCas3遺伝子またはEGFP融合EcoCas3遺伝子を含むバキュロウイルスゲノムをSf9細胞にトランスフェクションし、Sf9細胞内で、EcoCas3遺伝子を含むバキュロウイルスを作成した。このバキュロウイルスをSf9細胞へ継代感染させ、EcoCas3発現用の高力価ウイルスを獲得した。この高力価ウイルスをSf9細胞に感染させ、EcoCas3を組換えタンパク質として発現させた。
Hisタグ様の8HNタグが付加されている組換えEcoCas3タンパク質を、ニッケルカラムにてアフィニティー精製した後、ゲル濾過クロマトグラフィーにて最終精製を行った。
TychoNT6(NanoTempar社)を用いた熱変性プロファイルの変曲点温度Tiを測定することにより、組換えEcoCas3タンパク質の熱安定性の評価を行った。熱によるタンパク質分子のアンフォールディングに伴う分子内トリプトファン残基由来の自家蛍光のピークシフトを、330nmと350nmの二波長で検出し、その蛍光強度の比を温度に対してプロットすることで熱変性プロファイルの変曲点温度Tiを決定した。
組換えEcoカスケードを、組換えEcoCas3タンパク質と同時に細胞へ導入してゲノム編集を行うためには、細胞内で効率よく核内へ移行させることが重要である。しかしながら、Ecoカスケードは、分子量がおよそ0.4MDaと大きく、核内移行率が懸念される。そこで、本発明者らは、Ecoカスケードを構成する5つの遺伝子のオペロンを2つに分断し、それぞれの3’末端にNLSを付加させることで、より多くのNLSをカスケードに導入し、細胞核内への高効率移行を試みた。
二本鎖DNAを用いて、精製したEcoCas3およびEcoカスケードタンパク質による標的DNA切断活性をin vitroで検討した。反応バッファー(5mM HEPES-K pH7.5、60mM KCl、10mM MgCl2、10μM CoCl2、2.5mM ATP)中に、Cas3タンパク質(20nM)、crRNAとカスケードの複合体(20nM)、標的配列を含む二本鎖DNA(60ng/μL)を混合した。この反応溶液を37℃で1時間インキュベートし、MultiNa(島津製作所)を用いてキャピラリー電気泳動を行った。標的配列は、EMX1遺伝子領域およびTyr遺伝子領域とした。なお、EMX1については、二本鎖DNAのPAM配列を、大腸菌由来タイプI-E CRISPRが認識できる「AAG」から認識できない「CCA」に変更した配列も作製して検討した。
mCherry-P2A-EGFPを持ったレポーターHEK293T細胞を用いて、精製したEcoCas3およびEcoカスケードタンパク質のヒト細胞内での変異導入効率を検討した。Cas3タンパク質(30μMまたは45μM)、GFP標的crRNAとカスケードの複合体(30μMまたは45μM)をNeon Transfection System(Thermo Fisher Scientific社)を用いたエレクトロポレーション法によってレポーター細胞に導入した。細胞を37℃、5% CO2で5日間培養後、全細胞を回収してSH800(SONY社)を用いてGFP陰性細胞数をカウントし、変異導入効率を算出した。
Claims (8)
- Cas3タンパク質の製造方法であって、
(a)Cas3遺伝子を導入した昆虫細胞を20~28℃で培養し、当該昆虫細胞内でCas3タンパク質を発現させる工程、および
(b)発現させたCas3タンパク質を回収する工程、
を含む方法。 - Cas3タンパク質が大腸菌由来である、請求項1に記載の方法。
- 昆虫細胞がSf9細胞である、請求項1または2に記載の方法。
- 発現させたCas3タンパク質の回収が、Cas3タンパク質の精製を含む、請求項1から3のいずれかに記載の方法。
- Cas3タンパク質にタグが付加されており、Cas3タンパク質の精製が当該タグに対するアフィニティー精製を含む、請求項4に記載の方法。
- タグがHNタグを含む、請求項5に記載の方法。
- Cas3タンパク質の精製が、ゲル濾過クロマトグラフィーによる精製を含む、請求項4から6のいずれかに記載の方法。
- 精製に使用するバッファーがリン酸バッファーである、請求項4から7のいずれかに記載の方法。
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