A method for screening candidate target sites for genome editing that changes the expression level of a target gene in an individual organism

Abstract

Provided is a method for screening candidate target sites for genome editing that changes the expression level of a target gene in an individual organism. [Solution] A method for screening candidate target sites for genome editing that cause changes in the expression level of a target gene in an individual organism, wherein the candidate target site is included in the expression regulatory region of the target gene, and the screening method includes measuring the activity of a mutant expression regulatory region in which the nucleic acid sequence of the candidate target site has been modified by a reporter assay.

Description

[Technical Field]

[0001] The present disclosure relates to a method for screening candidate target sites for genome editing that cause changes in the expression level of a target gene in an individual organism. [Background technology]

[0002] Genome editing technology is a powerful tool for developing new varieties. In particular, gene knockout using genome editing is widely used to completely disrupt the function of a target gene, clarify its role, and at the same time, to bring out the characteristics of new varieties.

[0003] Gene knockdown technology differs from gene knockout in that it partially reduces gene expression. Even when complete gene deficiency through gene knockout results in lethality or severe phenotypes, gene knockdown technology allows for the moderate adjustment of gene expression levels. This enables the development of varieties with new characteristics not possible through gene knockout. Furthermore, by partially suppressing gene function, gene knockdown provides detailed information that cannot be obtained through knockout. For example, even if knocking out an essential gene results in lethality, knockdown allows for the analysis of its effects on the individual. By gradually changing the expression level of a gene, a deeper understanding of the role of that gene in biological processes can be gained, enabling the development of new varieties based on that understanding.

[0004] However, gene knockdown requires that target gene expression be reduced to the desired level, and this level varies depending on the gene, making it difficult to find the optimal editing method. For example, it is difficult to predict which region of the promoter will result in the change in expression level when a mutation is introduced. Therefore, the process of actually creating genome-edited plants and then evaluating them is time-consuming and labor-intensive.

[0005] Previously, genome editing targeting different sites within the promoter of a rice plant was used to generate multiple mutant plant lines, which were then verified using a dual-luciferase reporter assay via agroinfiltration into tobacco (Non-Patent Document 1). However, in Non-Patent Document 1, genome editing was performed targeting seven predicted cis-elements, and 115 plants were produced, but only 49 were mutated, and only one of the seven targets exhibited the desired trait. This method of evaluating expression levels after the production of genome-edited plants requires the production of a large number of genome-edited plants before the desired plant can be obtained, which is problematic in that it requires excessive time and effort. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Huang et al., Plant Biotechnol J. 2020 Nov; 18(11): 2164-2166. Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure has been made to solve the above-mentioned problems, and aims to provide a method for screening candidate target sites for genome editing that cause changes in the expression level of target genes in individual organisms. [Means for solving the problem]

[0008] One aspect of the present disclosure is a method for screening candidate target sites for genome editing that cause a change in the expression level of a target gene in an individual organism, wherein the candidate target site is included in an expression regulatory region of the target gene, and the screening method includes measuring the activity of a mutant expression regulatory region obtained by modifying the nucleic acid sequence of the candidate target site by a reporter assay. [Effects of the Invention]

[0009] According to the present disclosure, it is possible to provide a method for screening candidate target sites for genome editing that cause changes in the expression level of a target gene in an individual organism. [Brief explanation of the drawings]

[0010] [Figure 1A] Reporter assay using the SlPPD1 gene promoter (left) with a candidate target site (Target #1, Target #2, or Target #3) deleted. The open area of ​​each candidate target site indicates the deletion of that site. The relative activity value compared to the activity of the constitutive promoter (center) and the value obtained by dividing the relative activity value by the corresponding value of the wild-type promoter without the mutation (right) are shown. * indicates a significant difference (P<0.05) based on Student's t-test (n=3). [Figure 1B] Reporter assay using the SlPPD2 gene promoter (left) with a candidate target site (Target #1 or Target #2) deleted. The white outline of each candidate target site indicates the deletion of that site. The relative activity value compared to the activity of the constitutive promoter (center) and the value obtained by dividing the relative activity value by the corresponding value of the wild-type promoter without the mutation (right) are shown. * in the figure indicates a significant difference (P<0.05) by Student's t-test (n=3). [Figure 1C] Reporter assay using the SlSHR gene promoter (left) with candidate target sites (Target #1, Target #2, Target #3, or Target #4) deleted (results are shown for each candidate target site alone and the double-deleted promoters of Target #2 and Target #3). The white outline of each candidate target site indicates the deletion of that site. The relative activity value compared to the activity of the constitutive promoter (center) and the value obtained by dividing the relative activity value by the corresponding value for the wild-type promoter without mutations (right) are shown. * and ** in the figure indicate significant differences (P<0.05 and 0.01, respectively) by Student's t-test (n=3). [Figure 1D]Reporter assay using the SlLrgB gene promoter (left) with a candidate target site (Target #1, Target #2, Target #3, or Target #4) deleted. The white outline of each candidate target site indicates the deletion of that site. The relative activity value compared to the activity of the constitutive promoter (center) and the value obtained by dividing the relative activity value by the corresponding value of the wild-type promoter without the mutation (right) are shown. * indicates a significant difference (P<0.05) based on Student's t-test (n=3). [Figure 1E] Reporter assay using the SlNCED1 gene promoter (left) with a candidate target site (Target #1, Target #2, or Target #3) deleted. The white outline of each candidate target site indicates the deletion of that site. The relative activity value (center) compared to the activity of the constitutive promoter and the value obtained by dividing the relative activity value by the corresponding value of the wild-type promoter without the mutation (right) are shown. * and ** in the figure indicate significant differences (P<0.05 and 0.01, respectively) by Student's t-test (n=3). DETAILED DESCRIPTION OF THE INVENTION

[0011] Non-limiting embodiments of the present disclosure will be described below. The present disclosure is not limited to the examples in the following embodiments.

[0012] <Method for screening candidate target sites for genome editing that changes the expression level of a target gene in an individual organism>

[0013] The inventors have found that the process of obtaining a genome-edited individual with the desired expression level can be expedited by evaluating, prior to genome editing in an individual, a promoter sequence in which the sequence of a site expected to be modified by genome editing has been modified using a reporter assay.

[0014] In an embodiment, in a method for screening candidate target sites for genome editing that cause a change in the expression level of a target gene in an individual organism, the candidate target site is included in the expression regulatory region of the target gene, and the screening method includes measuring the activity of a mutant expression regulatory region obtained by modifying the nucleic acid sequence of the candidate target site by a reporter assay.

[0015] The species of the biological individual of the embodiment is not limited. The species of the biological individual of the embodiment may be a plant, an animal, a bacterium, a fungus, an algae, or an archaea, with a plant being particularly preferred. When the biological individual of the embodiment is a plant, the plant may be a terrestrial plant, an aquatic plant, an epiphyte, or a rock plant. The plant of the embodiment may include, but is not limited to, plants of the families Malvaceae, Rubiaceae, Brassicaceae, Poaceae, Cucurbitaceae, Asteraceae, Moraceae, Pesamiaceae, Araceae, Umbelliferae, Solanaceae, Papayaceae, Rosaceae, Amaryllidaceae, Fabaceae, Rutaceae, Oleaceae, Amaranthaceae, and Liliaceae. The families Brassicaceae, Asteraceae, Apiaceae, Solanaceae, Rosaceae, Amaranthaceae, and Fabaceae are particularly preferred. More specific examples of plants according to the embodiment include kiwifruit (Actinidia deliciosa), onion (Allium cepa), leek (Allium fistulosum), garlic (Allium sativum), celery (Apium graveolens var. dulce), Arabidopsis (Arabidopsis thaliana), peanut (Arachis hypogaea), beet (Beta vulgaris subsp. Vulgaris), cauliflower (Brassica oleracea var. botrytis), cabbage (Brassica oleracea var. capitata), broccoli (Brassica oleracea var. italica), rapeseed (Brassica rapa), bok choy (Brassica rapa var. chinensis), mizuna (Brassica rapa var. nipposinica), and komatsuna (Brassica rapa var. perviridis), turnip (Brassica rapa var.rapa), tea plant (Camellia sinensis), bell pepper (Capsicum annuum), paprika (Capsicum annuum), safflower (Carthamus tinctorius), lime (Citrus aurantifolia), lemon (Citrus limon), orange (Citrus sinensis), grapefruit (Citrus x paradisi), coconut palm (Cocos nucifera), coffee plant (Coffea arabica), taro (Colocasia esculenta), coriander (Coriandrum sativum), mitsuba (Cryptotaenia japonica), cucumber (Cucumis sativus), pumpkin (Cucurbita maxima), persimmon (Diospyros kaki), oil palm (Elaeis spp.), buckwheat (Fagopyrum esculentum), fig (Ficus carica), strawberry (Fragaria × ananassa), garland chrysanthemum (Glebionis coronaria), soybean (Glycine max), sunflower (Helianthus annuus), barley (Hordeum vulgare), sweet potato (Ipomoea batatas), morning glory (Ipomoea nil), lettuce (Lactuca sativa), lentil (Lens culinaris), apple (Malus domestica), peppermint (Mentha × piperita), banana (Musa spp.), watercress (Nasturtium officinale), tobacco (Nicotiana tabacum), basil (Ocimum basilicum), olive (Olea europaea), rice (Oryza sativa), shiso (Perilla frutescens var.crispa), avocado (Persea americana), parsley (Petroselinum crispum), kidney beans (Phaseolus vulgaris), pepper (Piper nigrum), peas (Pisum sativum), pears (Pyrus pyrifolia), rosemary (Rosmarinus officinalis), sugarcane (Saccharum officinarum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), potato (Solanum tuberosum), sorghum (Sorghum bicolor), spinach (Spinacia oleracea), cocoa (Theobroma cacao), thyme (Thymus vulgaris), wheat (Triticum aestivum), blueberries (Vaccinium spp.), grapes (Vitis spp.), adzuki beans (Vigna angularis), corn (Zea Examples of such herbs include, but are not limited to, Zingiber mays, Zingiber mioga, Zingiber officinale, and related species.

[0016] The target gene in the embodiment is not limited. In the embodiment, the target gene may be a gene of a plant, animal, bacterium, fungus, algae, or archaea, or may be a gene of these organisms. In the embodiment, the target gene preferably does not contain the coding sequence of another gene within 5000 bp, 4000 bp, 3000 bp, 2000 bp, 1500 bp, or 1000 bp upstream of the transcribed region.

[0017] The change in expression level in the embodiment may be a decrease or an increase in expression level, but is preferably a decrease in expression level. The change in expression level in the embodiment may be a decrease in transcription level, or a decrease in expression level that does not result in complete loss of expression.

[0018] In the present disclosure, "genome editing" may refer to modifying the genome of an individual organism and includes at least genome editing of an individual organism using a nuclease or deaminase. Genome editing using a nuclease or deaminase includes genome editing methods known to those skilled in the art, such as genome editing using the CRISPR-Cas system, genome editing using zinc finger nucleases, genome editing using transcription activator-like effector nucleases, genome editing using meganucleases, genome editing using base editors, and genome editing using prime editors. Nucleases used in genome editing using the CRISPR-Cas system include Cas9, Cas12a (Cpf1), and Cas14a. The types of mutations caused by genome editing envisioned in the screening methods of the embodiments are not limited and include deletions, insertions, and base substitutions.

[0019] In the present disclosure, a "candidate target site for genome editing" refers to a site on the genome that is targeted by genome editing, i.e., a sequence that can be modified by genome editing. This candidate target site can be used to design reagents such as guide RNAs to obtain genome-edited individuals with desired gene expression levels. If a promising candidate target site for genome editing is identified using the method disclosed herein, reagents such as guide RNAs can be designed based on this information to perform efficient genome editing.

[0020] Genome editing using the CRISPR-Cas system requires a protospacer adjacent motif (PAM) sequence on the target DNA for Cas nuclease to cleave the target DNA. Cas nuclease recognizes the PAM sequence, cleaves the DNA near the PAM sequence, and introduces mutations such as deletions or insertions. In embodiments, genome editing can be performed using the CRISPR-Cas system, and candidate target sites for genome editing can be located near the PAM sequence, within 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, or 5 bp of any base in the PAM sequence. The candidate target site can include the PAM sequence or a portion thereof. Several PAM sequences for different types of Cas nucleases are known to those skilled in the art. Representative PAM sequences include NGG in Streptococcus pyogenes-derived Cas9 (SpCas9), NNGRR or NNGRRT in Staphylococcus aureus-derived Cas9 (SaCas9), NNNNRYAC (N represents A, T, G, or C, R represents A, G, and Y represents C or T, respectively) in Campylobacter jejuni-derived Cas9 (CjCas9), TTTN and TTTV (V represents A, G, or C) in Acidaminococcus sp.-derived Cas12a (AsCas12a, also known as AsCpf1) and Lachnospiraceae bacterium-derived Cas12a (LbCas12a, also known as LbCpf1), TTN in Francisella novicida-derived Cas12a (FnCas12a, also known as FnCpf1), and TTTR in Cas14a. The guide RNA contains a sequence complementary to the target gene in a portion thereof. The complementary sequence may be a sequence adjacent to the PAM sequence. The length of the complementary sequence may be 19 to 23 nucleotides.

[0021] In embodiments, the candidate target site may be included in an expression regulatory region of the target gene. The expression regulatory region of the present disclosure may include coding or non-coding regions that function to regulate gene expression, but is preferably a non-coding region. The expression regulatory region may regulate gene expression or translation, preferably regulating transcription. The expression regulatory region of the embodiment may be a transcription regulatory region upstream of the transcription start site and may include a promoter or enhancer, but preferably includes a promoter. The expression regulatory region of the embodiment may be a promoter region upstream of the transcription start site. For example, the candidate target site may be a regulatory sequence motif predicted based on a database of sequence motifs found in known cis-elements, as described in Non-Patent Document 1.

[0022] The screening method of the embodiment involves measuring the activity of a mutant expression regulatory region in which the nucleic acid sequence of a candidate target site has been altered by an in vitro reporter assay. Mutant expression regulatory regions in which the nucleic acid sequence of a candidate target site has been altered can be constructed by methods known to those skilled in the art, including PCR and recombinant DNA techniques. Mutant expression regulatory regions can be those in which deletions, insertions, and / or base substitutions have been introduced into a wild-type expression regulatory region, preferably those in which deletions have been introduced. The size of the deletions introduced is not limited, but can be 1 bp to 50 bp, 1 bp to 25 bp, 1 bp to 15 bp, 1 bp to 10 bp, or 1 bp to 5 bp.

[0023] The reporter assay of the embodiment is not limited to an assay that measures the activity of an expression regulatory region of a target gene using a reporter gene (particularly its coding sequence). The reporter assay of the embodiment can be an in vitro or in vivo assay. In vitro reporter assays can use cells or protoplasts isolated from a plant. In vivo reporter assays can be performed by transforming plant organs such as leaves. The reporter assay of the embodiment can be performed by introducing a reporter gene linked to a mutant expression regulatory region in which the nucleic acid sequence of a candidate target site has been modified into plant cells, protoplasts, or organs. The reporter assay of the embodiment can be a luciferase assay, in which case the reporter gene can be a luciferase known to those skilled in the art, such as firefly luciferase (FLuc), Renilla luciferase, Gaussia luciferase, Cypridina luciferase, or NanoLuc luciferase. The assay may measure the luminescence intensity generated by adding or administering a substrate such as luciferin or coelenterazine to a target cell, protoplast, or tissue. The reporter assay of the embodiment may also be performed using a reporter gene other than luciferase known to those skilled in the art, such as β-galactosidase, β-glucuronidase, a fluorescent protein such as GFP, or chloramphenicol acetyltransferase.

[0024] The luminescence intensity measured in this manner reflects the activity of a mutant expression regulatory region resulting from an alteration of the nucleic acid sequence of a candidate target site, but may be corrected or normalized as appropriate. For example, a reporter assay may involve measuring the activity of a mutant expression regulatory region resulting from an alteration of the nucleic acid sequence of the candidate target site as a relative activity value relative to the activity of a constitutive expression regulatory region, and comparing this relative activity value with that of a corresponding wild-type expression regulatory region that does not contain a mutation. In this case, the reporter assay is performed using a mutant expression regulatory region linked to a reporter gene, as well as a wild-type expression regulatory region linked to the same reporter gene and a constitutive expression regulatory region linked to another reporter gene. These two reporter genes preferably use different substrates and encode reporter proteins that emit light at different (or largely non-overlapping) wavelengths. Measuring the activity of a mutant expression regulatory region resulting from an alteration of the nucleic acid sequence of a candidate target site as a relative activity value relative to the activity of a constitutive expression regulatory region may be performed by dividing the measured value for the mutant gene expression regulatory region by the measured value for the constitutive expression regulatory region. The reporter constructs for the mutant expression regulatory region and the reporter constructs for the constitutive expression regulatory region are preferably mixed and introduced into cells together, and more preferably contained in the same vector molecule and introduced into cells. This relative activity value can be compared with the value for the corresponding wild-type expression regulatory region that does not contain the mutation by further dividing the value obtained by the above division by the value for the corresponding wild-type expression regulatory region. The "value for the corresponding wild-type expression regulatory region" can also be a relative activity value divided by the measured value for the constitutive expression regulatory region measured together in the same measurement system. In the embodiments, the mutant gene expression regulatory region, the constitutive expression regulatory region, and the wild-type expression regulatory region can be a mutant gene promoter, a constitutive promoter, and a wild-type promoter, respectively. Examples of constitutive promoters are known to those skilled in the art and are typically promoters derived from genes different from the mutant gene promoter (and its corresponding wild-type promoter).

[0025] In embodiments, the candidate target site may be within 500 bp, 400 bp, 300 bp, 200 bp, or 100 bp of the transcription start site. The candidate target site is typically located upstream of the transcription start site. The transcription start site of a gene can be determined by methods known to those skilled in the art, including analyzing the sequence near the 5' end of the transcript of the gene. The mutant expression regulatory region used in the reporter assay of embodiments may include a nucleic acid sequence corresponding to a genomic sequence extending from the transcription start site to 5,000 bp or more, 4,000 bp or more, 3,000 bp or more, 2,000 bp or more, 1,500 bp or more, or 1,000 bp or more upstream. It has been found that by using an expression regulatory region of 5000 bp or more, 4000 bp or more, 3000 bp or more, 2000 bp or more, or 1800 bp or more in a reporter assay, a method for screening candidate target sites for genome editing can be performed with significantly improved accuracy or sensitivity, even if the candidate target site to be modified is located within 500 bp, 400 bp, 300 bp, 200 bp, or 100 bp of the transcription start site.

[0026] <Method for creating genome-edited organisms> A method for producing a genome-edited organism according to an embodiment includes the steps of selecting a target site for genome editing by carrying out any of the screening methods described in the section <Method for screening for candidate target sites for genome editing that cause a change in the expression level of a target gene in an individual organism>, and performing genome editing in the individual organism, targeting the selected target site. The explanations described in the section <Method for screening for candidate target sites for genome editing that cause a change in the expression level of a target gene in an individual organism> can be applied to the screening method carried out in the method for producing a genome-edited organism according to an embodiment.

[0027] The step of selecting a target site for genome editing in an embodiment may include evaluating the candidate target site using the method described in the section <Method for screening candidate target sites for genome editing that cause changes in expression levels of target genes in an individual organism>. If a mutation in the candidate target site results in a desired change in expression level measured by a reporter assay compared to the wild-type, the candidate site may be given a high rating. Such a desired change may be a decrease or increase in the transcription level of the target gene, and may be a decrease in transcription level but not a complete elimination of transcription. A candidate target site that is given a high rating in the reporter assay may be selected as a target site for genome editing in the step of performing genome editing targeting the selected target site. More specifically, sequence position information for the candidate target site may be used in designing reagents for genome editing. When genome editing using the CRISPR-Cas system is used, the information may be used to design a guide RNA complementary to at least a portion of the genomic region that includes the candidate target site or is within 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, or 5 bp of the candidate target site.

[0028] In the embodiments, the process of genome editing targeted at a selected target site can be performed by methods known to those skilled in the art. Genome editing using a nuclease can be performed by delivering the nuclease or a nucleic acid encoding it, and, if necessary, a guide nucleic acid such as a guide RNA or a nucleic acid encoding it, to a target organism, embryo, or embryonic stem cell. The specific procedure may vary depending on the species, but can be appropriately determined by those skilled in the art based on the characteristics of the nuclease used, information on the target site, and established genome editing techniques. The guide RNA in the embodiments may include CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), which may be separate molecules, or both may be present on a single RNA. In addition to the above, those skilled in the art will understand that guide RNAs used in genome editing can be used after being conferred desired functions or properties by various changes or modifications to their bases, backbone, or other polynucleotide structures. [Example]

[0029] Examples of the present disclosure are described below, but the present disclosure is not limited to the examples described below.

[0030] To generate knockdown mutants that reduce the expression of the tomato SlPPD1, SlPPD2, SlSHR, SlLrgB, and SlNCED1 genes, we focused on the promoter region 200 bp upstream of the transcription start sites of these genes and identified candidate genome editing targets containing PAM sequences within this region. Next, we constructed mutants in which the nucleic acid sequences up to 200 bp or 2000 bp upstream of the transcription start sites were linked to the luciferase coding sequence, and evaluated the changes in promoter activity using a luciferase assay.

[0031] Materials and Methods Plasmid construction We constructed plasmids expressing Firefly Luciferase (FLuc) under the 35SXL constitutive promoter and NanoLuc under the upstream promoter of the transcription start site of each target gene and its mutants. That is, the FLuc and NanoLuc constructs were contained together in the same plasmid molecule.

[0032] Promoter analysis Protoplasts isolated from Arabidopsis leaves were cultured using the polyethylene glycol / calcium (PEG / Ca) method. 2+ The constructed plasmid was introduced into the cells by the ELISA method. After standing overnight, the substrates for FLuc and NanoLuc were added, and luminescence was measured. Promoter activity was evaluated as the relative activity calculated from the ratio of the measured values ​​for NLuc and FLuc.

[0033] <Results and Discussion>

[0034] FIG. 1A shows the results of luciferase assays using sequences up to 200 bp or 2000 bp upstream of the transcription start site of SlPPD1.

[0035] The wild-type sequence of SlPPD1 up to 2000 bp upstream of the transcription start site is shown in SEQ ID NO: 1. The candidate target sites, Target#1, Target#2, and Target#3, are located within 200 bp upstream of the transcription start site, corresponding to bases 1804–1809, 1853–1856, and 1887–1890 in SEQ ID NO: 1, respectively. Deletion of Target#2 simulated a significant decrease in SlPPD1 expression in a reporter construct using the 2000-bp sequence (P<0.05, Figure 1A, right), indicating that this could be selected as a target site for genome editing in individuals. Deletion of any of the candidate target sites did not completely abolish expression. Because cis-elements within 200 bp are candidate target sites, intuitively, it would seem appropriate to perform reporter analysis within the 200-bp regulatory region. However, it was found that extending the analysis to 2000 bp can be extremely useful. Similar trends were observed for other genes described below.

[0036] FIG. 1B shows the results of luciferase assays using sequences up to 200 bp or 2000 bp upstream of the transcription start site of SlPPD2.

[0037] The wild-type sequence up to 2000 bp upstream of the transcription start site of SlPPD2 is shown in SEQ ID NO: 2. The candidate target sites, Target #1 and Target #2, are located within 200 bp upstream of the transcription start site, corresponding to base numbers 1917-1920 and 1993-2000 in SEQ ID NO: 2, respectively. Of these, it was found that deletion of Target #2 simulated a significant decrease in SlPPD2 expression in both reporter constructs using the 200 bp and 2000 bp sequences (both P<0.05, Figure 1B, right). Deletion of either candidate target site did not completely eliminate expression.

[0038] FIG. 1C shows the results of luciferase assays using sequences up to 200 bp or 2000 bp upstream of the transcription start site of SlSHR.

[0039] The wild-type sequence up to 2000 bp upstream of the SlSHR transcription start site is shown in SEQ ID NO: 3. The candidate target sites, Target #1, Target #2, Target #3, and Target #4, are located within 200 bp upstream of the transcription start site and correspond to bases 1873-1876, 1921-1926, 1942-1945, and 1990-1995 in SEQ ID NO: 3, respectively. It was found that single deletion of Target #1, Target #2, and Target #3 simulated a significant decrease in SlSHR expression in a reporter construct using the 2000-bp sequence (P<0.05, P<0.01, and P<0.05, respectively; Figure 1C, right). Deletion of any of the candidate target sites did not completely abolish expression.

[0040] FIG. 1D shows the results of luciferase assays using sequences up to 200 bp or 2000 bp upstream of the transcription start site of SlLrgB.

[0041] The wild-type sequence up to 2000 bp upstream of the SlLrgB transcription start site is shown in SEQ ID NO: 4. The candidate target sites, Target #1, Target #2, Target #3, and Target #4, are located within 200 bp upstream of the transcription start site and correspond to bases 1853-1856, 1896-1902, 1940-1943, and 1967-1973 in SEQ ID NO: 4, respectively. We found that deletion of Target #2, Target #3, and Target #4 significantly reduced SlLrgB expression in a reporter construct using the 2000-bp sequence (all P < 0.05, Figure 1D, right). Deletion of any of the candidate target sites did not completely abolish expression.

[0042] FIG. 1E shows the results of luciferase assays using sequences up to 200 bp or 2000 bp upstream of the transcription start site of SlNCED1.

[0043] The wild-type sequence up to 2000 bp upstream of the SlNCED1 transcription start site is shown in SEQ ID NO: 5. The candidate target sites, Target#1, Target#2, and Target#3, are located within 200 bp upstream of the transcription start site, corresponding to bases 1828–1834, 1843–1851, and 1955–1958 in SEQ ID NO: 5, respectively. We found that deletion of Target#1 significantly reduced SlNCED1 expression in both the 200-bp and 2000-bp reporter constructs (P<0.01 and P<0.05, respectively, Figure 1E, right). Furthermore, deletion of Target#2 significantly reduced SlNCED1 expression in the 200-bp reporter construct (P<0.05, Figure 1E, right). Deletion of any of the candidate target sites did not completely abolish expression.

[0044] The above examples demonstrate that genome editing sites that result in reduced expression can be efficiently selected by measuring the activity of mutant expression regulatory regions in which the nucleic acid sequence of a candidate target site has been modified using an in vitro reporter assay prior to genome editing in an individual. Furthermore, the above results suggest that the use of a region containing 2000 bp upstream of the transcription start point allows for more accurate evaluation of candidate target sites than when using shorter regions encompassing the candidate target site. While the above examples were performed in plants, genome editing and evaluation of promoter activity using reporter assays can be performed in many other biological species. Therefore, it is believed that similar methods can be used to efficiently select genome editing sites that result in reduced expression in other biological species.

[0045] Although the present disclosure has been described with reference to the above several embodiments, the present disclosure is not limited to the examples in the above embodiments. Various modifications can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0046] The present disclosure includes the following embodiments. (Section 1) A method for screening candidate target sites for genome editing that changes the expression level of a target gene in an individual organism, comprising: the candidate target site is contained in an expression regulatory region of the target gene; The screening method includes measuring the activity of a mutant expression regulatory region in which the nucleic acid sequence of the candidate target site is modified by a reporter assay. method. (Section 2) Item 1. The method according to Item 1, wherein the change in expression level is a decrease in transcription level. (Section 3) Item 3. The method according to Item 1 or 2, wherein the expression regulatory region is a promoter region upstream of the transcription start site. (Section 4) Item 4. The method according to Item 3, wherein the candidate target site is within 500 bp of the transcription start site, and the mutant expression regulatory region used in the reporter assay comprises a nucleic acid sequence corresponding to a genomic sequence extending from the transcription start site to 1800 bp or more upstream thereof. (Section 5) Item 5. The method according to any one of Items 1 to 4, wherein the individual organism is a plant and the target gene is a gene of the plant. (Section 6) Item 6. The method according to any one of Items 1 to 5, wherein the reporter assay is a luciferase assay. (Section 7) The reporter assay is an in vitro reporter assay, and includes measuring the activity of a mutant expression regulatory region in which the nucleic acid sequence of the candidate target site has been modified as a relative activity value to the activity of a constitutive expression regulatory region, and comparing the relative activity value with the value of a corresponding wild-type expression regulatory region that does not contain a mutation. (Section 8) Selecting a target site for genome editing by carrying out the screening method according to any one of items 1 to 7; and a step of performing genome editing targeting the selected target site in an individual organism. Methods for producing genome-edited organisms.

Claims

1. A method for screening candidate target sites for genome editing that changes the expression level of a target gene in an individual organism, comprising: the candidate target site is contained in an expression regulatory region of the target gene; The screening method includes measuring the activity of a mutant expression regulatory region in which the nucleic acid sequence of the candidate target site is modified by a reporter assay. method.

2. The method of claim 1 , wherein the change in expression level is a decrease in transcription level.

3. The method of claim 1, wherein the expression regulatory region is a promoter region upstream of the transcription start site.

4. The method of claim 3, wherein the candidate target site is within 500 bp of the transcription start site, and the mutant expression regulatory region used in the reporter assay comprises a nucleic acid sequence corresponding to a genomic sequence extending from the transcription start site to 1800 bp or more upstream thereof.

5. The method according to claim 1, wherein the individual organism is a plant and the target gene is a gene of the plant.

6. The method of claim 1, wherein the reporter assay is a luciferase assay.

7. The method of claim 1, wherein the reporter assay is an in vitro reporter assay, and comprises measuring the activity of a mutant expression regulatory region in which the nucleic acid sequence of the candidate target site has been modified as a relative activity value to the activity of a constitutive expression regulatory region, and comparing the relative activity value with the value of a corresponding wild-type expression regulatory region that does not contain a mutation.

8. Selecting a target site for genome editing by carrying out the screening method according to any one of claims 1 to 7; and and a step of performing genome editing targeting the selected target site in an individual organism. Methods for producing genome-edited organisms.

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