JP2022116588A - Genome editing method in photosynthetic eukaryotes - Google Patents

Abstract

To establish a genome editing system using a single-stranded oligonucleotide in a photosynthetic eukaryote, which is different from conventional genome editing systems using site-specific artificial nucleases.SOLUTION: We have found that the genome of a photosynthetic eukaryote can be edited by treating it with a single-stranded oligonucleotide consisting of a base sequence modified from the sense strand base sequence of the target genome region.SELECTED DRAWING: None

Description

The present invention relates to genome editing methods in photosynthetic eukaryotes using single-stranded oligonucleotides.

The PODiR (Partly Overlapped Direct Repeat) system is a structure-dependent (sequence-independent) system discovered from a phenomenon in which genes are generated in non-coding regions of the genome when bacteria become resistant to antibiotics. It is a self-genome editing mechanism. In this system, the non-overlapping sequence (X-Y) in the nucleotide sequence (PODiR sequence) indicated by X-Y-X-Y-X, in which the sequence X-Y-X and the sequence X-Y-X overlap at X, is deleted (Patent Document 1). This phenomenon is caused by the interaction between the mRNA containing the PODiR sequence and lacking the nucleotide sequence X-Y in the genome and the DNA containing the PODiR sequence in the genome. In addition, it is speculated that extra sites (X-Y of genomic DNA) that cannot interact are cleaved by some kind of enzyme.

On the other hand, it has also been reported that a PODiR sequence is created by inserting a nucleotide sequence represented by X-Y adjacent to the 5' side of the sequence X-Y-X in the genome (Patent Document 1). interacts with DNA containing the sequence X-Y-X (non-PODiR sequence) in the genome, and the base sequence of the resulting extra site (X-Y of mRNA) that cannot interact is inserted into the genomic DNA during DNA replication, etc. It is speculated that it occurs because it is inserted.

In addition, it is essentially a system that uses site-specific artificial nucleases, which is characterized by artificially introducing RNA that serves as a template for deletion or insertion of the genome or the corresponding DNA into cells. A new and different genome editing system has been proposed (Patent Document 2).

However, the functions of this genome editing system have been verified only in the cells of some organisms such as Pseudomonas aeruginosa and human cells, and in photosynthetic eukaryotes such as eukaryotic algae and plants. not

WO2015/115610 WO2018/030536

The present invention has been made in view of such circumstances, and its purpose is to use single-stranded oligonucleotides in photosynthetic eukaryotes, which is different from genome editing systems based on conventional site-specific artificial nucleases. The purpose is to provide a genome editing system that has

In order to solve the above problems, the present inventor conducted extensive studies using eukaryotic algae and plants as examples of photosynthetic eukaryotes. As a result, the PODiR system functions in these photosynthetic eukaryotes, It was found that the base sequence of the genomic region can be modified by acting in cells a single-stranded oligonucleotide consisting of a base sequence obtained by modifying the base sequence of the sense strand of the genomic region. Furthermore, the inventors have found that the genome can be edited in the same way when using a single-stranded oligonucleotide targeting a genomic region where the PODiR sequence does not exist, and have completed the present invention.

The present invention relates to a genome editing system using single-stranded oligonucleotides in photosynthetic eukaryotes, and more specifically provides the following inventions.

(1) A method for producing a genome-edited photosynthetic eukaryote, comprising:
introducing a single-stranded polynucleotide or a vector expressing the polynucleotide into a photosynthetic eukaryotic cell;
The single-stranded polynucleotide consists of a base sequence obtained by modifying the base sequence of the target strand of the target genomic DNA region of the photosynthetic eukaryote,
From the group consisting of (a) deletion of any internal base sequence, (b) insertion of any internal base sequence, or (c) replacement of any internal base sequence with another base sequence is a selected modification, and
A method, wherein genome editing by said single-stranded polynucleotide does not involve cleavage in said target genomic DNA region by a site-specific artificial nuclease.

(2) The method according to (1), wherein the target strand of the target genomic DNA region contains a PODiR sequence.

(3) The method according to (1), wherein the target strand of the target genomic DNA region does not contain a PODiR sequence.

(4) The method according to any one of (1) to (3), wherein the photosynthetic eukaryote is eukaryotic algae or plants.

(5) A composition or kit for producing a genome-edited photosynthetic eukaryote,
A single-stranded polynucleotide consisting of a base sequence modified from the base sequence of the target strand of the target genomic DNA region of a photosynthetic eukaryote or a vector expressing the polynucleotide,
From the group consisting of (a) deletion of any internal base sequence, (b) insertion of any internal base sequence, or (c) replacement of any internal base sequence with another base sequence is a selected modification, and
Compositions or kits not used in combination with site-specific engineered nucleases.

(6) The composition or kit of (5), wherein the target genomic DNA region comprises a PODiR sequence.

(7) The composition or kit of (5), wherein the target genomic DNA region does not contain a PODiR sequence.

(8) The composition or kit according to any one of (5) to (7), wherein the photosynthetic eukaryote is eukaryotic algae or plants.

According to the present invention, only by allowing a single-stranded polynucleotide in which the base sequence of the target strand of the target genomic DNA region is modified to act in the cells of photosynthetic eukaryotes, it is possible to obtain a simple method without using a site-specific artificial nuclease system. It became possible to perform genome editing in This genome editing can target not only the PODiR sequence on the genome of photosynthetic eukaryotes, but also other nucleotide sequences, and is highly versatile.

Targeted lipase gene regions (PODiR sequences and non-PODiR sequences) in single-stranded DNA-based genome editing in eukaryotic algae (haptophytes) are shown. In the figure, the underlined nucleotide sequence indicates the nucleotide sequence used for designing the single-stranded DNA, and the squared nucleotide sequence indicates the nucleotide sequence to be deleted in the genome editing. Two arrows in the figure indicate the PODiR sequence. The target genomic DNA region containing the PODiR sequence of the lipase gene in the figure is shown in SEQ ID NO:2, and the single-stranded DNA targeting this region is shown in SEQ ID NO:1. In addition, SEQ ID NO: 4 shows the target genomic DNA region that does not contain the PODiR sequence of the lipase gene in the figure, and SEQ ID NO: 3 shows the single-stranded DNA that targets this region. FIG. 3 shows the results of genome editing using single-stranded DNA in a plant (Arabidopsis thaliana). In the figure, "a" shows the structure of the plasmid "pCAMBIA" used to confirm the functionality of the PODiR system, and "b" shows the structure of the experimental plant for confirming the functionality of the PODiR system. The culture process is shown, and "c" shows the result of detection of the functionality of the PODiR system in the experimental plant by the expression (fluorescence) of the functional GPF gene caused by the deletion of the introduced PODiR sequence.

The present invention provides a method for producing genome-edited photosynthetic eukaryotes.

"Photosynthetic eukaryotes" to be genome-edited in the method of the present invention are organisms that grow by converting light energy into energy in a biologically usable form and that have nuclei within their cells. be. Photosynthetic eukaryotes are typically eukaryotic algae and plants. Examples of eukaryotic algae include haptophytes, cryptophytes, brown algae, red algae, green algae, diatoms, golden algae, gray algae, euglenoid algae, charadium algae, and dinoflagellates. Plants include seed plants, Ferns and bryophytes can be exemplified. Experimentally or industrially useful plants include Arabidopsis thaliana, tomato, soybean, rice, wheat, barley, corn, rapeseed, tobacco, banana, peanut, sunflower, potato, cotton, and carnation.

In the method of the present invention, the "single-stranded polynucleotide" to be introduced into the cells of the photosynthetic eukaryote consists of a nucleotide sequence obtained by modifying the nucleotide sequence of the target strand of the target genomic DNA region of the photosynthetic eukaryote. A single-stranded polynucleotide can be either DNA or RNA, as long as it interacts with the target genomic DNA region and serves as a template for DNA editing of the target genomic DNA region. These single-stranded polynucleotides may be partially or wholly chemically modified.

As used herein, the term "target genomic DNA region" means a genomic DNA region to be targeted for genome editing using single-stranded polynucleotides. According to the present invention, any genomic DNA region can be targeted for genome editing. In addition, the “target strand” refers to the 5′ region (5′ homology sequence described later) and the 3′ region (3′ homology sequence described later) of the single-stranded polynucleotide of the present invention, among the double strands of genomic DNA. ) and serves as a base for designing the single-stranded polynucleotide of the present invention. For example, when desired genome editing is performed in a gene region using the single-stranded polynucleotide of the present invention, the target strand is the sense strand. The “sense strand” as used herein means the non-transcription template strand of the genomic DNA double strand.

The "nucleotide sequence of the target strand" in the method of the present invention may be a nucleotide sequence containing the PODiR sequence or a nucleotide sequence not containing the PODiR sequence (hereinafter referred to as "non-PODiR sequence"). The "PODiR sequence" is a nucleotide sequence represented by X-Y-X-Y-X, which is formed by overlapping the sequence X-Y-X and the sequence X-Y-X on the genome (see Patent Documents 1 and 2).

The number of bases of the "base sequence of the target strand of the target genomic DNA region" is not particularly limited. It is more preferably about 80-600, even more preferably about 90-300, particularly preferably about 95-200.

Nucleotide sequences obtained by modifying the base sequence of the target strand typically have the following three modes: (a) deletion of an arbitrary base sequence inside the target strand, (b) insertion of an arbitrary base sequence inside , or (c) replacement of any internal base sequence with another base sequence. Hereafter, when the base sequences of the target strand of the target genomic DNA region and the single-stranded polynucleotide are aligned, the base sequence of the single-stranded polynucleotide corresponding to the 5' region of the target genomic DNA region is referred to as "5' homology When the base sequences of the target genomic DNA region and the single-stranded polynucleotide are aligned, the base sequence of the single-stranded polynucleotide corresponding to the 3' region of the target genomic DNA region is referred to as "3' homology array”.

When the modification is deletion of an arbitrary internal base sequence, the single-stranded polynucleotide is 5' of the base sequence to be deleted in the target strand of the target genomic DNA region (hereinafter referred to as "deletion target base sequence"). It consists of a base sequence corresponding to the side region (5' homology sequence) and a base sequence corresponding to the 3' side region (3' homology sequence). On the other hand, when the modification is the insertion of an arbitrary nucleotide sequence into the interior or the substitution of an arbitrary internal nucleotide sequence with another nucleotide sequence, the single-stranded polynucleotide is placed in the 5' region of the target strand of the target genomic DNA region. Nucleotide sequence to be inserted between the corresponding nucleotide sequence (5' homology sequence) and the nucleotide sequence corresponding to the 3' region (3' homology sequence) (hereinafter referred to as "insertion nucleotide sequence") or replacement intervening nucleotide sequence (hereinafter referred to as "substituting nucleotide sequence").

The number of bases of the 5' homology sequence and 3' homology sequence is, for example, 10 to 1000, preferably 20 to 1000, more preferably 30 to 500, still more preferably 40 to 300, still more preferably 40 to 100, particularly preferably is about 40-70. The base number ratio between the 5' homology sequence and the 3' homology sequence is not particularly limited, and is, for example, 0.01-100, preferably 0.1-10, more preferably 0.2-5, further preferably 0.5-2.

The homology of the base sequence of the target strand of the target genomic DNA region corresponding to the 5' homology sequence and 3' homology sequence in the single-stranded polynucleotide does not necessarily have to be 100%. For example, 90% or more identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identity), as long as it causes genome editing by interacting with the target genomic DNA region gender). Sequence identity can be calculated using BLAST or the like (eg, default parameters).

The number of nucleotides to be deleted in the target strand of the target genomic DNA region, and the number of nucleotides in the insertion nucleotide sequence and replacement nucleotide sequence in the single-stranded polynucleotide is not particularly limited. 1000, more preferably 1 to 920, more preferably 2 to 500, even more preferably 4 to 200, still more preferably 4 to 100, still more preferably 5 to 50, still more preferably 6 to 20, more More preferably 7-12, still more preferably 8-9.

The number of bases of the nucleotide sequence to be deleted in the target strand of the target genomic DNA region, or the base sequence for insertion or replacement in the single-stranded polynucleotide, relative to the total number of bases of the 5' homology sequence and the 3' homology sequence The ratio is not particularly limited, and is, for example, 500 or less, preferably 100 or less, more preferably 10 or less, even more preferably 5 or less, even more preferably 2 or less, and even more preferably 1 or less. The lower limit of the ratio is not particularly limited, and is, for example, 0.01, 0.02, 0.04.

In the present invention, a vector that expresses a single-stranded polynucleotide can be used instead of the single-stranded polynucleotide. The vector contains one or more regulatory elements operably linked to the DNA encoding the single-stranded polynucleotide to be expressed.

Here, "operably linked" means that the DNA is expressably linked to the regulatory element. "Regulatory elements" include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, transcription termination signals, polyadenylation signals, etc.). Regulatory elements may be those that direct DNA expression only in specific cells, tissues, or organs, depending on the purpose, for example, those that direct constitutive expression of DNA in a variety of host cells may be Moreover, it may be one that directs the expression of DNA only at a specific time, or one that directs the expression of an artificially inducible DNA. Examples of promoters include FCP promoter and GAPDH promoter for expression in algae, and 35SCaMV promoter for expression in plants. A person skilled in the art can select an appropriate expression vector depending on the type of cell to be introduced. The vector may contain other elements (eg, multiple cloning site, drug resistance gene, origin of replication, etc.) as necessary.

"Cells" of photosynthetic eukaryotes into which single-stranded polynucleotides or vectors expressing them are introduced include cultured cells as well as cells in an individual. Also included are cells, such as protoplasts, that have undergone specific treatments for the introduction of single-stranded polynucleotides.

Introduction of single-stranded polynucleotides or vectors expressing them into cells can be appropriately selected according to the type of photosynthetic eukaryote, cell morphology, and the like. Examples include, but are not limited to, electroporation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, lipofection, nanoparticle-mediated transfection, virus-mediated nucleic acid delivery, and the like.

The concentration of the single-stranded polynucleotide to be introduced into the cell is not particularly limited as long as the genome editing in the present invention is possible, depending on the chain length of the single-stranded polynucleotide and the type of cells to be introduced, those skilled in the art can It can be set, for example, from 1 μg to 50 μg per 10 ⁵ cells, preferably from 2.5 μg to 15 μg per 10 ⁵ cells.

It has long been known that the somatic cells of plants have totipotency, and methods for regenerating plant bodies from plant cells have been established for various plants. Therefore, for example, a genome-edited plant can be obtained by regenerating a plant from a plant cell whose genome has been edited by introduction of the single-stranded polynucleotide of the present invention. Progeny, clones, or propagation material (eg, tubers, tubers, seeds, etc.) can also be obtained from the resulting plants. As a method of redifferentiating plant tissues by tissue culture to obtain individuals, a method established in this technical field can be used (transformation protocol [Plants], Yutaka Tabei, edited by Kagaku Dojin, pp.340- 347 (2012)).

Editing of a photosynthetic eukaryotic genome by the methods of the present invention occurs only by introducing single-stranded oligonucleotides into cells. The editing of the genome does not require cleavage at the target genomic DNA region by site-specific artificial nucleases such as ZFNs, TALENs, CRISPR-Cas.

The present invention also provides a composition or kit for producing a genome-edited photosynthetic eukaryote, comprising the single-stranded polynucleotide or a vector expressing the polynucleotide. In the composition or kit, since the single-stranded oligonucleotide itself has the ability to edit the genome, it does not need to be used in combination with a site-specific artificial nuclease.

The preparations constituting the kit of the present invention and the composition of the present invention may further contain other components, if necessary. Other components include, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, Examples include, but are not limited to, fragrances, chelating agents, and the like.

Kits of the invention can further comprise additional components. Additional components include, but are not limited to, for example, dilution buffers, wash buffers, transfection reagents, control reagents (eg, control single-stranded polynucleotides, etc.). The kit may contain instructions for carrying out the methods of the invention.

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

(1) Genome editing in eukaryotic algae using single-stranded polynucleotides (a) Analysis using single-stranded DNA targeting the PODiR sequence of the lipase gene Diploid eukaryotic algae as an example of eukaryotic algae was used.

350 μL of haptoalgal protoplasts containing 4×10 ⁵ cells containing 30 μg (30 μL of 1 μg/μL solution) of a single-stranded DNA (SEQ ID NO: 1, FIG. 1) that modifies part of the PODiR sequence of the lipase gene of the haptoalgae. It was mixed with the solution, further mixed with an equal volume (350 μL) of 40% PEG (MW=6000) solution, and allowed to stand at room temperature for 15 minutes. After that, it was transplanted to Marine Art-ESM medium and cultured for 14 days. After culturing, genomic DNA was extracted from the cells and amplified by PCR using PrimeStar (TaKaRa). In PCR, template genomic DNA was added to the reaction solution at a concentration of 30 ng/20 μl reaction solution, and the reaction was carried out under the conditions of 98° C. for 5 seconds, 59° C. for 5 seconds, and 72° C. for 15 seconds. About 10,000,000 sequences of the obtained amplified products were analyzed with a next-generation sequencer (Macrogen Co. Ltd.). As a result, the frequency of deletion of the PODiR sequence of the lipase gene (i.e., deletion due to spontaneous PODiR) was about 0.03% in the control experimental group, whereas it was about 0.27 in the single-stranded DNA introduction experimental group. %, and the deletion was more than 8 times that of the control group.

Next, the influence of introduction of the single-stranded DNA targeting the PODiR sequence of the lipase gene on mutagenesis in a gene containing a PODiR sequence other than the lipase gene (for example, the citrate synthase gene) was examined. As a result, the citrate synthase gene showed a high deletion rate of about 21% due to spontaneous PODiR in the control experimental group, while the single-stranded DNA introduction experimental group showed a high deletion rate of about 24%. There was little increase from From the above, it was shown that the introduction of single-stranded DNA targeting the PODiR sequence of the lipase gene had a minor effect on the mutagenesis of the citrate synthase gene containing the PODiR sequence.

(b) Analysis using single-stranded DNA targeting the non-PODiR sequence of the lipase gene A single-stranded DNA (SEQ ID NO: 3, Fig. 1) that modifies part of the non-PODiR sequence of the lipase gene of haptophyceae was analyzed. was used to analyze approximately 16 million sequences in the same manner as in (a) above. As a result, while no deletion of non-PODiR sequences in the lipase gene was observed in the control experimental group, deletion was confirmed at a frequency of about 0.00006% (102 sequences) in the single-stranded DNA introduction experimental group. rice field. This result indicates that the target of genome editing by single-stranded DNA with a partial gene deletion is not limited to the PODiR sequence.

(2) Functional analysis of PODiR system in plants Arabidopsis thaliana was used as an example of plants.

First, we created Arabidopsis thaliana strains to analyze the functionality of the PODiR system. Specifically, seeds of Arabidopsis Columbia strain (Col-0) were purchased from INPLANTA INNOVATIONS, INC (Yokohama, Japan) and cultured in a growth chamber at 23°C with a cycle of 16 hours light/8 hours dark. did. Next, the plasmid "pCAMBIA" in which a cassette containing EGFP (EGFP-STNT-POD, Fig. 2a) was inserted under the 35SCaMV promoter was introduced into the Columbia strain of Arabidopsis thaliana by the agroinfiltration method, and the functionality of PODiR in the plant was investigated. We created an experimental strain for analysis and obtained a seed library. In addition, the PODiR sequence was artificially introduced into the EGFP gene of the plasmid "pCAMBIA", and when a base deletion occurred by the PODiR system, a functional EGFP gene was formed, and its expression caused fluorescence. be able to emit

Next, a rectangular medium plate was prepared, and seedlings after germination were planted at sufficient intervals from the bottom of the plate. For the first 3 days, the plates were wrapped in aluminum foil and the plants were grown under limited light conditions (plants sprouted by this dark treatment grow as bleached). After that, the cells were cultured at 23° C. for 2 weeks with a cycle of 16 hours light period/8 hours dark period. The culture process described above is shown in Fig. 2b.

As a result of observation, GFP expression was confirmed in various parts of the root (Fig. 2c). From the above, it was found that PODiR functions in plants.

INDUSTRIAL APPLICABILITY As described above, according to the present invention, a site-specific artificial nuclease can be produced by simply allowing a single-stranded polynucleotide in which the base sequence of the target strand of the target genomic DNA region is modified to act in the cells of a photosynthetic eukaryote. Genome editing can be easily performed without using a system. INDUSTRIAL APPLICABILITY The present invention can be used in a wide range of industrial fields, such as the production of useful substances using genome-edited algae and plants, and the production of genome-edited useful crops.

SEQ ID NO: 1
<223> Single-stranded DNA targeting the PODiR sequence of the lipase gene
SEQ ID NO:3
<223> Single-stranded DNA targeting non-PODiR sequences of the lipase gene

Claims (8)

A method for producing a genome-edited photosynthetic eukaryote, comprising:
introducing a single-stranded polynucleotide or a vector expressing the polynucleotide into a photosynthetic eukaryotic cell;
The single-stranded polynucleotide consists of a base sequence obtained by modifying the base sequence of the target strand of the target genomic DNA region of the photosynthetic eukaryote,
From the group consisting of (a) deletion of any internal base sequence, (b) insertion of any internal base sequence, or (c) replacement of any internal base sequence with another base sequence is a selected modification, and
A method, wherein genome editing by said single-stranded polynucleotide does not involve cleavage in said target genomic DNA region by a site-specific artificial nuclease. 2. The method of claim 1, wherein the target strand of the target genomic DNA region comprises PODiR sequences. 2. The method of claim 1, wherein the target strand of the target genomic DNA region does not contain PODiR sequences. 4. The method according to any one of claims 1 to 3, wherein the photosynthetic eukaryote is a eukaryotic algae or a plant. A composition or kit for producing genome-edited photosynthetic eukaryotes, comprising:
A single-stranded polynucleotide consisting of a nucleotide sequence modified from the target strand nucleotide sequence of the target genomic DNA region of a photosynthetic eukaryote or a vector expressing the polynucleotide,
From the group consisting of (a) deletion of any internal base sequence, (b) insertion of any internal base sequence, or (c) replacement of any internal base sequence with another base sequence is a selected modification, and
Compositions or kits not used in combination with site-specific engineered nucleases. 6. The composition or kit of claim 5, wherein the target genomic DNA region comprises PODiR sequences. 6. The composition or kit of claim 5, wherein the target genomic DNA region does not contain PODiR sequences. 8. The composition or kit of any of claims 5-7, wherein the photosynthetic eukaryote is a eukaryotic algae or a plant.

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