JP2021016374A - Identification method of single base - Google Patents

Abstract

To provide an identification method of the presence of a specific single base in nucleic acid.SOLUTION: An identification method of the presence of a specific single base in nucleic acid includes: (i) a process of amplifying detection nucleic acid which identifies the presence of a specific single base; (ii) a process of amplifying control nucleic acid by using at least one primer which is the same that was used in (i) above; and (iii) a process of identifying that a specific single base is present in the detection nucleic acid in the case where an amplified product in the detection nucleic acid is larger or smaller than an amplified product in the control nucleic acid.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for identifying the presence or absence of a specific single base in nucleic acid, a method for editing genomic DNA of a cell using the method, a method for producing a genome-edited cell, and the like.

The human genome, 10 ⁷ of a single nucleotide polymorphism (SNP) or monobasic Diversity (SNV) are expected to exist in the form of human, extender, and directly or indirectly to the phenotype of the predisposition or the like It is believed to be involved. In particular, disease-related SNVs are called pathogenic SNVs and are known to directly cause hereditary diseases such as multiple endocrine neoplasia type 2 (MEN2B) or malnourished epidermolysis bullosa (DEB). (Non-Patent Documents 1 and 2).

The development of a simple and rapid detection method for SNP / SNV is important in genomic medicine and genome repair medicine. Further, the artificial single nucleotide substitution site can be used as a marker for a genetically modified animal (Single Nucleotide (SN) marker), and the usefulness of the detection method is high. At present, PCR-RFLP and Taqman assay are known as methods for detecting SNP, SNV or SN markers (hereinafter also referred to as "SNP / SNV / SN markers"), but restriction enzymes that can be used for detection are known. Neither can be said to be a versatile method because there are no parts or a dedicated device is required.

Recently, HiDi DNA polymerase has been developed for detecting SNP / SNV / SN markers by PCR (Non-Patent Document 3). Since this enzyme can identify single nucleotide polymorphisms and amplify more nucleic acids having the target SNP / SNV / SN marker, it is expected as a new method for detecting SNP / SNV / SN markers.

Wellcome Trust Case Control Consortium, 2007, Nature, 447, 661-678. Wells, S.A., Jr. et al., 2013, J. Clin. Endocrinol. Metab., 98, 3149-3164. Drum, M. et al., 2014, PLoS One, 9, e96640

The present inventors have, when an experiment was conducted using HiDi DNA polymerase, it was found that amplification products detectable range distinguishable in sequence without the sequence to the target containing the target SN marker is about 10 ^2. This indicates that the sensitivity of SNP / SNV / SN marker detection by this enzyme is lower than expected, and it is necessary to strictly control the amount of template in order to detect SNP / SNV / SN markers.

In one embodiment, it is an object of the present invention to provide a method for identifying the presence or absence of a specific single base in a nucleic acid capable of detecting an SNP / SNV / SN marker.

The present inventor designed a primer in a region where the presence or absence of the template's parindrome sequence at the site corresponding to the 3'end site of the primer differs depending on the presence or absence of the SN marker, and used this to perform SN marker detection PCR. As a result, it was found that the detection sensitivity was remarkably improved.

The present invention includes the following embodiments.
(1) A method for identifying the presence or absence of a specific single base in nucleic acid.
(I) A step of amplifying a detected nucleic acid that identifies the presence or absence of a specific single base,
(Ii) The step of amplifying the control nucleic acid using at least one primer which is the same as that used in (i) above, and (iii) when the amplification product in the detection nucleic acid is larger than the amplification product in the control nucleic acid. , Including the step of identifying the presence of a specific base in the detected nucleic acid.
One base at the 3'end of the primer is not the same as the corresponding region of the control nucleic acid, and the control nucleic acid contains a palindromic sequence containing two or more bases in the region corresponding to the 3'end of the primer. ,
When a specific base is present in the detection nucleic acid, one base at the 3'end of the primer is the same as the corresponding region of the detection nucleic acid, and the detection nucleic acid is in the region corresponding to the 3'end side of the primer. A method that does not include the Palindrome sequence.
(2) A method for identifying the presence or absence of a specific single base in nucleic acid.
(I) A step of amplifying a detected nucleic acid that identifies the presence or absence of a specific single base,
(Ii) A step of amplifying the control nucleic acid using at least one primer which is the same as that used in (i) above, and (iii) when the amplification product in the detection nucleic acid is less than the amplification product in the control nucleic acid. , Including the step of identifying the presence of a specific base in the detected nucleic acid.
One base at the 3'end of the primer is identical to the corresponding region of the control nucleic acid, and the control nucleic acid contains a palindromic sequence containing two or more bases in the region corresponding to the 3'end of the primer. Zu,
When a specific base is present in the detection nucleic acid, the 1 base at the 3'end of the primer is not the same as the corresponding region of the detection nucleic acid, and the detection nucleic acid is in the region corresponding to the 3'end side of the primer. A method comprising the Palindrome sequence.
(3) The method according to (1) or (2), wherein the specific single base is a marker base that is artificially introduced.
(4) The method according to any one of (1) to (3), wherein the parindrome sequence contains a base of A or T.
(5) The method according to any one of (1) to (4), wherein the palindrome sequence contains 2 to 6 bases.
(6) The method according to (5), wherein the palindrome sequence contains 4 to 6 bases.
(7) The method according to any one of (1) to (6), wherein the nucleic acid amplification is performed by PCR or real-time PCR.
(8) The method according to any one of (1) to (7), wherein the nucleic acid amplification is carried out using a genetically modified or natural thermostable DNA polymerase capable of detecting a single nucleotide mismatch.
(9) Step of genome editing on cells,
Genome editing is performed based on the step of identifying the presence or absence of a specific single base in the nucleic acid of a cell and the identification result of the presence or absence of a specific single base according to the method according to any one of (1) to (8). The process of selecting cells, performed,
A method of producing genome-edited cells, including.

The present invention provides a method for identifying the presence or absence of a specific single base in a nucleic acid.

FIG. 1 is a schematic diagram showing a concept of an embodiment of the present invention. A is an example of introducing an SN marker into a region without a palindrome sequence, and B is an example of introducing an SN marker into a region with a palindrome sequence. The SN marker is framed. The underline shows the palindrome sequence or the sequence after the introduction of the marker. Figure 2 shows the case where the SN marker is placed at the site where there is no parindrome sequence (original template sequence corresponding to the 3'end site of the primer: gatt, and the template sequence corresponding to the 3'end site of the primer after introduction of the marker: gatc. ) Primer and template sequences, and electrophoresis results after nucleic acid amplification are shown. Figure 3 shows the case where the SN marker is placed at the site where the parindrome sequence is located (original template sequence corresponding to the 3'end site of the primer: aatt, template sequence corresponding to the 3'end site of the primer after introduction of the marker: aatc. ) Primer and template sequences, and electrophoresis results after nucleic acid amplification are shown. The underline shows the palindrome sequence or the sequence after the introduction of the marker. FIG. 4 shows the sequences of primers and templates when the SN marker is placed at the site where the palindrome sequence (ggcc) is located, and the electrophoresis results after nucleic acid amplification. The underline shows the palindrome sequence or the sequence after the introduction of the marker. FIG. 5 shows the sequences of primers and templates when the SN marker is placed at the site where the palindrome sequence (ttaa) is located, and the electrophoresis results after nucleic acid amplification. The underline shows the palindrome sequence or the sequence after the introduction of the marker. FIG. 6 shows the sequence of primers and templates when the SN marker is placed at the site where the palindrome sequence (at) is located, and the electrophoresis result after nucleic acid amplification. The underline shows the palindrome sequence or the sequence after the introduction of the marker. FIG. 7 shows the sequences of primers and templates when the SN marker is placed at the site where the palindrome sequence (gc) is located, and the electrophoresis results after nucleic acid amplification. The underline shows the palindrome sequence or the sequence after the introduction of the marker. FIG. 8 shows the sequences of primers and templates when the SN marker is placed at the site where the palindrome sequence (caattg) is located, and the electrophoresis results after nucleic acid amplification. The underline shows the palindrome sequence or the sequence after the introduction of the marker. FIG. 9 shows the results of electrophoresis after nucleic acid amplification using different enzymes using the same primers and templates as in FIG. 3 (A: GoTaq DNA polymerase, B: Tks Gflex DNA polymerase). FIG. 10 shows the results when this method was applied in the primary screening of target clones in the genome repair of disease-specific iPS cells using the same primers and templates as in FIG. The primary screening positive rate of (B) when introducing an SN marker into a region with parindrome is 5.7%, which is more remarkable than the primary screening positive rate of 1.8% when introducing an SN marker into a region without parindrome (A). It was expensive.

1. 1. Definition of terms In the present specification, "nucleic acid" means a high molecular weight organic compound having a nitrogen-containing base, sugar, and phosphoric acid derived from purine or pyrimidine as constituent units, and includes analogs of these nucleic acids. To do. The nucleic acid may be, for example, DNA, RNA, cDNA or the like. The nucleic acid may be, for example, the genomic DNA of the species from which the cells described herein are derived. The nucleic acid may also be labeled for detection.

As used herein, the term "detected nucleic acid" means a nucleic acid that identifies the presence or absence of a specific single base.

As used herein, the term "control nucleic acid" means a nucleic acid to be compared for identifying the presence or absence of a specific single base in a detected nucleic acid. The control nucleic acid may be, for example, a nucleic acid whose presence or absence of a specific single base is already known.

In the present specification, the type of "specific single base" is not limited, and includes A, T, G, C, and analogs of these bases. The specific single nucleotide may be a naturally occurring single nucleotide polymorphism (SNP) or single nucleotide polymorphism (SNV). In addition, a specific single nucleotide may be a base that constitutes a part of an insertion deletion (Insertion & Deletion, Indel) or a structural polymorphism (Structural Variant, SV).

As used herein, "single nucleotide polymorphism (SNP)" means a variation between genomes of an individual of the same species, and the frequency is usually found in about 1% or more in a population. SNP can be an addition, deletion, or substitution of a base, and includes not only a single base mutation but also a mutation of about 2 to 10 bases. In general, SNPs occur relatively frequently in the genome and contribute to genetic diversity.

As used herein, "single nucleotide polymorphism (SNV)" means a variation between genomes of an individual of the same species and refers to a single nucleotide polymorphism.

As used herein, "insertion deletion (Indel)" means a variation between genomes of an individual of the same species, and refers to a short insertion deletion of 1 base or more and less than 50 bases.

As used herein, "structural polymorphism (SV)" means intergenome variation of individuals of the same species, and refers to insertions, deletions, duplications, translocations, inversions, and tandem repeats of 50 or more bases.

In one embodiment, SNPs, SNVs, Indels, SVs can be mutations that cause diseases such as those described herein.

In one embodiment, the "specific single base" may be a single Nucleotide (SN) marker that is an artificially introduced base. When a specific base is a naturally occurring base, a marker can be designed by searching for a site that meets the requirements of the method of the present invention, but if an SN marker is introduced, the requirements of the method of the present invention are introduced. In principle, the present invention can be applied to any site as long as the primer is designed to satisfy the above conditions. The SN marker can be introduced, for example, by the genome editing method described herein, and by introducing it in addition to other editing sites, an artificial process, for example, detection of a nucleic acid whose genome has been edited. Can be used to simplify. As the SN marker, a base that causes a silent mutation that does not affect the amino acid sequence can be used.

FIG. 1B shows a schematic diagram showing the concept of one embodiment of the present invention when introducing an SN marker. In FIG. 1B, the SN marker is framed and the underline shows the palindrome sequence or the sequence after the marker is introduced. In FIG. 1B, the SN marker is introduced into the region (SEQ ID NO: 23) having the parindrome sequence (AATT), and the sequence after the introduction of the SN marker (SEQ ID NO: 24) does not have the parindrome sequence. For example, nucleic acid amplification is performed using a primer sequence having the same sequence as the sequence after introduction of the SN marker, and the presence or absence of a specific base (SN marker C in FIG. 1B) is determined by examining the amplification efficiency. Can be identified.

2. 2. Method for Identifying the Presence or absence of a Specific Single Base in Nucleic Acid In one embodiment, the present invention comprises (i) a step of amplifying a detection nucleic acid for identifying the presence or absence of a specific single base, (ii) a control nucleic acid, and (i) The step of amplifying using at least one primer which is the same as that used in the above, and (iii) when the amplification product in the detection nucleic acid is larger than the amplification product in the control nucleic acid, the detection nucleic acid contains a specific base. The present invention relates to a method for identifying the presence or absence of a specific single base in a nucleic acid, which comprises a step of identifying (the present method is also referred to as a “positive screening method”). The steps that can be included in the methods described herein are described below.

(Step of amplifying detected nucleic acid and step of amplifying control nucleic acid)
In the methods described herein, the presence or absence of a particular single base is identified based on whether the single base at the 3'end of the primer is identical to the corresponding region of the detection nucleic acid. Therefore, in one embodiment, if the 1 base at the 3'end of the primer is not the same as the corresponding region of the control nucleic acid and a specific base is present in the detection nucleic acid, then the 1 base at the 3'end of the primer is It is the same as the corresponding region of the detected nucleic acid (in the absence of a specific base, the 1 base at the 3'end of the primer is not the same as the corresponding region of the detected nucleic acid).

The method described in the present specification utilizes the fact that when nucleic acid amplification is performed by a specific method, the amplification efficiency decreases when one base at the 3'end of the primer does not completely match (when a mismatch exists). (In the present specification, such a method is referred to as "single nucleotide mismatch detection nucleic acid amplification" or the like). This can be done with sequence-specific primers with the corresponding base set at the 3'end, and such primers can be easily designed by those skilled in the art. Single nucleotide mismatch detection Nucleic acid amplification is also referred to as "amplification refractory mutation system (ARMS)" or "allele-specific amplification (ASA)" or "allele-specific PCR" when detecting polymorphisms and the like.

In one embodiment, if the control nucleic acid comprises a parindrome sequence containing two or more bases in the region corresponding to the 3'end of the primer, and the detection nucleic acid contains a particular base, then the detection nucleic acid , The primer is designed so that the region corresponding to the 3'end side of the primer does not contain the parindrome sequence (if the detection nucleic acid does not have a specific base, the detection nucleic acid is the 3'end of the primer. Including the parindrome sequence in the region corresponding to the side). The present inventor has shown in Examples that the detection sensitivity is remarkably increased when single nucleotide mismatch detection nucleic acid amplification is performed at the site. Therefore, in one embodiment, the present invention has the effect of being able to discriminate the presence or absence of a specific single base with high sensitivity.

In the present specification, the primer and the "corresponding region" in the nucleic acid strand serving as a template are intended to be a complementary region in the nucleic acid strand with respect to the region in the complementary strand that hybridizes with the primer if it is a forward primer. To do. Further, in the case of a reverse primer, a complementary region in the complementary strand is intended with respect to a region in the nucleic acid strand that hybridizes with the primer.

In the case of a forward primer, the region corresponding to one base at the 3'end of the primer may be a region capable of hybridizing with one base at the 5'end in the region in the complementary strand that hybridizes with the primer. Further, in the case of a reverse primer, the region corresponding to one base at the 3'end of the primer may be a region capable of hybridizing with one base at the 5'end in the region in the nucleic acid chain that hybridizes with the primer. ..

From the viewpoint of nucleic acid amplification efficiency, it is preferable that the sequence is completely the same as that of the template or its complementary strand, except for the difference in the corresponding region of the detected nucleic acid strand or the control nucleic acid strand at the 3'end of the primer. However, one base at the 3'end of the primer needs to be exactly the same as the sequence of the template as long as it can hybridize with the nucleic acid chain under high stringent conditions when it is the same as the corresponding region of the nucleic acid. It may contain some mutations. For example, the primer is a sequence having 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more base identity with respect to the template sequence or its complementary sequence. Well, or the template sequence or its complementary sequence may be added, deleted, and / or substituted with one or several bases.

In the present specification, "base identity" means two when two base sequences are aligned and a gap is introduced as necessary so that the degree of base matching between the two is the highest. The ratio (%) of the same base between base sequences. As used herein, the term "several pieces" means, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4, 2 to 3 or 2.

The length of the primer is not limited, but may be 10 to 40 bases, 15 to 30 bases, or 20 to 30 bases.

As used herein, "hybridizing under high stringent conditions" means performing hybridization and washing under low salt concentration and / or high temperature conditions. For example, incubate with probe in 6 × SSC, 5 × Denhardt reagent, 0.5% SDS, 100 μg / mL denatured fragmented salmon sperm DNA at 65 ° C to 68 ° C, then in a wash solution of 2 × SSC, 0.1% SDS. It is exemplified by starting from room temperature at room temperature, lowering the salt concentration in the cleaning solution to 0.1 × SSC, and raising the temperature to 68 ° C. until no background signal is detected. The conditions for highly stringent hybridization are described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. It can be used as a reference.

In one embodiment, the step of amplifying the detection nucleic acid and the step of amplifying the control nucleic acid are carried out using at least one identical primer. Other primers may or may not be the same, but from the viewpoint of making the amplification conditions the same, two or more identical primers (for example, forward and reverse primers), for example, the same primer set should be used. Is preferable. The total number of primers used in nucleic acid amplification is not limited, but may be, for example, 2 or more or 4 or more, for example 2.

As used herein, the term "palindromic sequence" or "palindromic structure" refers to the sequence of one strand and the sequence complementary to the sequence in the complementary strand of that strand, assuming the presence of a complementary strand. An array or structure that is the same. The length of the palindrome sequence is not limited as long as it is 2 or more, but may be, for example, 4 or more, 6 or more, or 8 or more, and is 20 or less, 18 or less, 16 or less, 10 or less, 8 or less, or 6 or less. It may be there. In one embodiment, the palindromic sequence comprises or consists of, for example, 2-20, 2-10, 2-6, 4-20, 4-10, or 4-6 bases.

In one embodiment, the parindrome sequence comprises a base of A or T. This embodiment may have the effect of improving the detection sensitivity as compared with a palindrome sequence consisting of only a G or C sequence. Examples of palindromic sequences include, but are not limited to, AT, GC, AATT, TTAA, GGCC, CCGG, and CAATTG.

The step of amplifying the nucleic acid is not particularly limited. Nucleic acid selected from the group consisting of PCR (Polymerase Chain Reaction) method, LAMP (Loop-Mediated Isothermal Amplification) method, RCA (Rolling Circle Amplification) method, SMAP (Smart Amplification Process) method, and RPA (Recombinase Polymerase Amplification) method. Amplification method can be mentioned. Details of these methods are known in the art and can follow their usual methods. These may be used alone or in combination of two or more. In one embodiment, the detection step is performed using a PCR method (eg, a PCR method such as quantitative PCR or a real-time PCR method).

In the PCR method, a nucleic acid having a target base sequence is amplified by using a primer pair capable of specifically amplifying the target base sequence.

The LAMP (Loop-Mediated Isothermal Amplification) method is a nucleic acid amplification method that preferably uses at least four types of oligonucleotides as primers, and double-stranded DNA, primers, strand-substituted DNA polymerase, substrate, etc. are placed in the same container. By keeping the temperature at a constant temperature (for example, around 65 ° C.), detection is performed in a one-step process (for details, refer to, for example, WO 00/28082).

The SMAP (Smart Amplification Process) method is similar to the LAMP method, but has a self-complementary sequence on the 5'side, that is, two nucleic acid sequences that hybridize with each other, called folding primers, on the same strand. It differs mainly from the LAMP method in that it uses a primer that is a folded sequence (for details, for example, Mitani Y., et al., Nature Methods, vol.4, No.3, p.257-262 (2007)). Please refer to). The RCA (Rolling Circle Amplification) method is an isothermal enzyme reaction that amplifies long single-stranded DNA or RNA with a specific DNA or RNA polymerase and cyclic DNA as a template with a short DNA or RNA primer (for details, see , For example, Ali MM. Et al., Chem. Soc. Rev., 2014, 21; 43 (10): 3324-41).

The RPA (Recombinase Polymerase Amplification) method uses two key proteins that replace the usual heat denaturation steps of PCR: E. coli RecA recombinase and single-stranded DNA-binding protein (SSB) (for more information, see, eg, Daher RK). . et al., Clin. Chem., 2016, 62 (7): 947-58). The recombinase forms a nucleoprotein filament with a single-stranded oligonucleotide primer and probe. The filament scans the target double-helix DNA (dsDNA) for homology, and once homology is found, the filament invades the dsDNA to form a D-loop structure with locally split DNA strands. Then the complementary strand is stabilized by SSB and the target strand hybridizes with the primer. Degradation of recombinases from nucleoprotein filaments is triggered by ATP, which hydrolyzes the RecA protein, allowing primer extension by strand-substituted DNA polymerase. The newly generated DNA strand will be used for the next RPA round.

The type of polymerase used to perform nucleic acid amplification is not specified. The present inventor has shown that by applying the methods described herein, single nucleotide mismatches can be detected regardless of the type of enzyme.

In one embodiment, nucleic acid amplification is performed using a genetically modified or natural thermostable DNA polymerase capable of detecting a single nucleotide mismatch. By using such an enzyme, the detection sensitivity of a specific single base can be further improved. DNA polymerases capable of detecting single nucleotide mismatches are known to those skilled in the art, such as the HiDi DNA polymerase described in Drum, M. et al., 2014, PLoS One, 9, e96640, or the mutations described in the literature. It can be made by introducing the corresponding mutation into another DNA polymerase. Specifically, in Drum, M. et al., 2014 (above), basic amino acids (arginine and lysine) that are in direct contact with the phosphate skeleton of the primer chain in the three-dimensional structure of the Clenault fragment of Taq DNA polymerase. Has been shown to be associated with nucleotide sequence identification, and analysis has shown that the one-amino acid substituent (R660V) has the highest enzymatic activity and can detect single nucleotide mismatches. Therefore, by introducing these mutations, such as R660V, or the corresponding mutations into a heat-resistant DNA polymerase (for example, a known heat-resistant DNA polymerase), a polymerase capable of detecting a single-base mismatch can be prepared. Here, the base corresponding to the above mutation is, for example, by aligning the base sequences of a plurality of DNA polymerases so as to have the highest degree of base matching between the two by introducing a gap as necessary. It can be specified by determining the position corresponding to the above 660th place.

(Step to identify the presence of a specific base in the detected nucleic acid)
In one embodiment, the method of the invention comprises (iii) identifying the presence of a particular base in the detection nucleic acid when there are more amplification products in the detection nucleic acid than in the control nucleic acid. In this step, the nucleic acids amplified in steps (i) and (ii) are quantified, and based on the comparison of the amounts of the detected nucleic acid and the control nucleic acid, it is determined whether or not a specific base is present in the detected nucleic acid. Can be done.

As used herein, "quantitative" also includes absolute quantification, semi-quantitative, quantification of relative amounts relative to other samples, and determination of the presence or absence of qualitative products.

Methods for quantifying amplification products are known to those skilled in the art and, for example, quantify nucleic acids based on fluorescence amplification, electrophoresis, phosphoric acid quantification, atomic absorption spectroscopy, absorbance measurement, etc., for example, electrophoresis or fluorescence amplification. be able to. For example, in the electrophoresis method, the nucleic acid is electrophoresed on a medium such as agarose gel, and the medium after electrophoresis is stained with a staining agent (for example, an intercalating agent such as ethidium bromide or SYBR Green). The amount of amplification product can be quantified based on the staining intensity. Further, in the fluorescence amplification method, for example, the concentration of nucleic acid can be quantified by performing PCR by adding a double-stranded DNA-binding fluorescent dye and measuring the fluorescence intensity at the same time as performing PCR. Alternatively, a probe modified with a fluorescent dye and a quencher and capable of binding to the target base sequence can be used. In this case, when the probe is decomposed in the extension reaction step, the fluorescent dye is separated from the quencher and emits fluorescence, so that the fluorescence is amplified as the nucleic acid amplification reaction progresses. The amount of nucleic acid amplification can be quantified based on the intensity of this fluorescence.

In the present specification, the order of (i) the step of amplifying the detected nucleic acid and (ii) the step of amplifying the control nucleic acid may be arbitrary and may be simultaneous or separate. By performing the same at the same time, the detection conditions can be made the same, and the difference between the amplification products of the detected nucleic acid and the quantitative nucleic acid can be more accurately specified. When performed separately, the step (i) of amplifying the detection nucleic acid can be performed before or after the step (ii) of amplifying the control nucleic acid, and the detection accuracy can be further improved by performing each step multiple times. Can be done. It is also possible to use the result of the amplification product of the control nucleic acid, for example, a plurality of amplification products (for example, the average value thereof) as a threshold value, and compare this threshold value with the amplification product of the detected nucleic acid.

The method for identifying the presence or absence of a specific single base described in the present specification may include methods other than the above steps (i) to (iii). For example, a step of preparing a dilution series of the detection nucleic acid and / or the control nucleic acid may be included before the step (i). By performing this step and subjecting the plurality of dilutions to a later step, the difference in the amount of amplification products of the detection nucleic acid and the control nucleic acid can be detected more clearly even when the detection sensitivity is not sufficient. ..

In one aspect, the present invention is a method for identifying the presence or absence of a specific single base in nucleic acid.
(I) A step of amplifying a detected nucleic acid that identifies the presence or absence of a specific single base,
(Ii) A step of amplifying the control nucleic acid using at least one primer which is the same as that used in (i) above, and (iii) when the amplification product in the detection nucleic acid is less than the amplification product in the control nucleic acid. , A method comprising a step of identifying the presence of a specific base in the detected nucleic acid (also referred to herein as a "negative screening method").

The steps (i) and (ii) in the present negative screening method are the same as the steps (i) and (ii) described for the positive screening method described in the present specification. This negative screening method is described herein in that step (iii) identifies that a particular base is present in the detected nucleic acid when there are fewer amplification products in the detection nucleic acid than in the control nucleic acid. It is different from the positive screening method of.

In this negative screening method, the correspondence between the primer and the control nucleic acid and the detection nucleic acid is opposite to the correspondence in the positive screening method described herein. That is, in this negative screening method, when one base at the 3'end of the primer is the same as the corresponding region of the control nucleic acid and a specific base is present in the detection nucleic acid, one base at the 3'end of the primer. Is not the same as the corresponding region of the detected nucleic acid (if the detected nucleic acid does not have a specific base, the 1 base at the 3'end of the primer is the same as the corresponding region of the detected nucleic acid). Further, in this negative screening method, the control nucleic acid does not contain a parindrome sequence containing two or more bases in the region corresponding to the 3'end side of the primer, and is detected when a specific base is present in the detection nucleic acid. The nucleic acid contains the parindrome sequence in the region corresponding to the 3'end side of the primer (if the detection nucleic acid does not have a specific base, the outgoing nucleic acid is in the region corresponding to the 3'end side of the primer. Does not include the primer sequence). The details of each configuration of this negative screening method are the same as those described in the positive screening method.

3. 3. Genome Editing Method In one aspect, the present invention comprises a step of performing genome editing on a cell, a step of identifying the presence or absence of a specific base in a cell's nucleic acid according to the method described herein, and a specific step. The present invention relates to a method for producing a genome-edited cell, or a method for genome editing, which comprises a step of selecting a cell for which genome editing has been performed based on an identification result of the presence or absence of one base.

The cell is not limited as long as it is an animal cell or a plant cell. Species from which animal cells are derived include, for example, mammals (eg, primates such as humans and Xenopus monkeys, experimental animals such as rats, mice, and dovetails, domestic animals such as pigs, cows, horses, sheep, and goats. In addition, pet animals such as dogs and cats, as well as bags such as kangaroos, koalas and wonbats, and single-hole animals such as kamonohashi and harimogura), birds (chicken, duck, pigeon, ostrich, emu, parrot, and Sekishokuyakei etc.), reptiles (lizards, crocodile, snakes, turtles, etc.), amphibians (Xenopus laevis and nettai tsumegael, sanshou, axolotl, etc.), fish (medaka, zebrafish, goldfish, funa, carp, salmon, trout, eel, etc. Catfish, Suzuki, Thailand, flatfish, tuna, bristle, bonito, shark, etc.), soft animals (octopus, squid, abalone, sazae, red mussel, hamaguri, asari, cattle, etc.), spiny skin animals (uni, catfish, starfish, etc.) ), Shells (crab, shrimp, shako, clawed frog, yadkari, etc.), insects (caiko, quail, oyster, chaminoga, ginger, bee, bear bee, ant, tentumushi, cricket, bat, beetle, stag beetle White ants, etc.), spore animals (hydra, jellyfish, coral, etc.) are mentioned, and mammals such as humans are preferable. The species from which the plant cells are derived is not limited, and may be any plant cell such as angiosperms including monocotyledonous plants and dicotyledonous plants, nude plants, moss plants, fern plants, herbaceous plants and woody plants. May be good. Specific examples of the origin of plant cells include rice family (rice, wheat, barley, corn, sorghum, awa, hie, shikokubie, toujinbie, honeybee, millet, sugar cane, yoshi and madake, etc.), and abrana family (white inunazuna, etc.). Abrana, broccoli, wasabi, cabbage, etc.), eggplant family (egg, tomato, tobacco, capsicum, potato, etc.), Uri family (cucumber, melon, watermelon, gourd, etc.), Higanbana family (negi, onion, garlic, etc.) , Bean family (soybean, azuki, lotus, kanzo, kudzu, senna, kibanaougi, arabic rubber tree, shitan, etc.), kinpouge family (ouren, etc.), madder family (cutinashi, coffee tree, etc.), satimo family (satoimo, konjak, hange, etc.) Karasubishaku, etc.), Yamanoimo family (Yamanoimo, Nagaimo, etc.), Hirugao family (Satsumaimo, Asagao, etc.), Todaigusa family (Cassaba, Paragomuki, etc.) (Inutade, buckwheat, Daiou, etc.), Kuwa family (Kuwa, Casinoki, etc.), Hiyu family (Amaranthus, Keito, etc.), Suberihiyu family (Suberihiyu, Matsuba button, etc.), Aoi family (Okura, Hibiscus, etc.), Kiku family (Sunflower) , Kikuimo, Artichoke, Hosoba okera, Oobana okera, etc.), Mizuki family (Sanshuyu, etc.), Rose family (rose, peach, pear, apple, strawberry, etc.), Mikan family (Mikan, Yuzu, Sansho, Kihada, etc.), Grape family (Grape, Yama grape, etc.), Button family (Button, Shakuyaku, etc.), Gomanohagusa family (Akayaziou, etc.), Shiso family (Shiso, Hakka, Rosemary, Koganebana, etc.), Mokusei family (Rengyo, etc.), Kikyo family (Kikyo, etc.) , Matatabi family (Matatabi, Sarpashi, Kiwi fruit, etc.), Omodaka family (Sagiomodaka, etc.), Janohige (Janohige, etc.), Kakinoki family (Kakinoki, Kokutan, etc.), Orchid family (Shunran, Vanilla, etc.), Basho family (Basho, Banana, etc.) Etc.), Ukogi family (Taranoki, Otaneninjin, etc.), Kusunoki family (Shinnikkei, Avocado, etc.), Crow Memodoki family (Natsume, etc.), Beech family (Beech, Nara, Kuri, etc.), Mukuroji family (Mukuroji, Tochinoki, etc.), Asa family (Hemp, etc.), Irakusa family (Rami, etc.), Kijikakushi family (Saizaruasa, Agave, etc.), Urushi family (Yamaurushi, Hazenoki, Mango, etc.), Kabanoki family (Shirakamba, Dakekamba, etc.) Etc.), Salixaceae (Nekoyanagi, Shidareyanagi, etc.), Piperaceae (Piperaceae, etc.), Pinaceae (Pineapple, etc.), Papiaceae (Papaya, etc.), Myristicaceae (Myristicaceae, etc.), Ginkgoaceae (Papaceae, etc.) Coco palm, Abra palm, etc.), Pinaceae (Red pine, Ezo pine, etc.), Maou family (Maou), Ginkgoaceae (Ginkgoaceae, etc.), Shida plants (Warabi, Sugina, Hego, etc.), Moss plants (Myristicaceae, Tsunogoke, Magoke, Sugigoke, etc.) ) Plants. These cells may be either primary cultured cells, subcultured cells, or frozen cells.

The target cell of the method may be, for example, a mammalian cell, for example, a stem cell. As used herein, the term "stem cell" refers to a cell having both the ability to differentiate into another type of cell or various types of cells and the ability to self-renew. The stem cell may be a cell population consisting only of stem cells or a cell population rich in stem cells. For example, in the case of mammals as an example of stem cells, undifferentiated cells existing in living tissues such as bone marrow, blood, skin, intestines, nerves, and fat (collectively referred to as somatic stem cells, and muse as an example). Includes cells), embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and the like. Such stem cells can be produced by a method known per se, but can be obtained from a predetermined institution or a commercially available product can be purchased.

In the step of performing genome editing (hereinafter, also referred to as "genome editing step"), for example, a mutant base sequence such as SNP / SNV in genomic DNA is replaced with a wild type base sequence, or the wild type base sequence is replaced with SNP. It can be replaced with a mutant base sequence such as / SNV. Alternatively, for example, a specific gene, a group of genes, or a group of natural or artificial base sequences associated thereto may be knocked in or knocked out at a specific site on the genome. Further, in the genome editing step, a plurality of loci may be deleted on a large scale, or all or a part of a specific gene or a group of genes may be translocated on another chromosome, and the specific gene may be translocated on another chromosome. In addition, they may be duplicated or vertically duplicated at adjacent positions on the same chromosome.

In the present specification, the "wild type" is the most abundant in nature in an allelic population having the same nucleotide sequence, and if the protein or non-coding RNA encoded by the allele has a function, it has the original function. Refers to an allele. In the present specification, the types of mutations include substitutions, insertions, deletions, and structural polymorphisms (duplication, translocation, inversion, tandem repeat, copy number polymorphism).

In one embodiment, mutant nucleotide sequences such as SNP, SNV, Indel, SV, etc. can cause disease. The type of disease is not limited as long as it can be caused by a mutant base sequence, but for example, multiple endocrine neoplasia type2A (MEN2B), multiple endocrine neoplasia type2A (MEN2A), Multiple endocrine neoplasia type 1 (MEN1), dystrophic epidermal vesicular disease (DEB), hereditary Breast and / or Ovarian Cancer Syndrome (HBOC), Li-Fraumeni syndrome (LFS) , Cowden Syndrome, Lynch Syndrome, Familial adenomatous polyposis (FAP), Hyperthyroidism Jaw Tumor Syndrome (HPT-JT), Myotonic lateral sclerosis (ALS) , Myotonic dystrophy1 (DM1), familial Parkinson's disease, hereditary Alzheimer's disease, Malfan syndrome, metatropic dysplasia, progressive ossifying fibrodysplasia, neonatal period Onset multi-organ inflammatory disease (neonatal-onset-multi-system inflammatory disease (NOMID)), FGFR3 chondropathy, type II collagen dysfunction, von-Hippel-Lindau disease (VHLD), citrin deficiency ), Transthyretin-type Familial Amyloid Polyneuropathy, Niemann-Pick type C (NPC), Charcot-Marie Tooth disease , Tay-Sachs disease, Williams syndrome, Duchenne myotonic dystrophy, Smith-Magenis syndrome, Carney Complex, Alzheimer's disease caused by APP mutation, Potocki-Lupski syndrome, Prader-Willi syndrome, Angelman syndrome, Down Syndrome, XX Male Syndrome (SRY), Myotonic Dysfunction (chr 11), Barkit It may be lymphoma, Hemophilia A, Hunter syndrome, Emery-Dreifuss muscular dystrophy, fragile X syndrome due to FMR1 mutation, Huntington's disease, spinocerebellar ataxia, etc.

As used herein, "genome editing" refers to a technique for specifically cleaving and editing a target site on the genome. In genome editing, site specific Nuclease (SSN) (also referred to as "genome editing protein" in the present specification) capable of sequence-specific cleavage, for example, TALEN (Transcription activator-like effector CRISPR). ), CRISPR / Cas9 (Clustered regularly interspaced short palindromic repeats CRISPR) / CRISPR-associated protein 9), CRISPR / Cpf1 (CRISPR from Prevotella and Francisella 1) or ZFN (zinc finger nuclease) and variants thereof. SSN also includes analogs derived from other CRISPR / Cas strains (SaCas9, ScCas9, FnCpf1, etc.), or other Cas protein groups (Cas12a, Cas12b, C2c1, C2c2, C2c3, etc.) and their variants. .. It is preferable to use a site-specific nuclease (SSN) that has accurate target recognition ability and can identify a single base substitution, and examples thereof include TALEN and its variants (for example, Platinum TALEN), Streptococcus pyogenes Cas9 (SpCas9) and the like. Variants (eg, high fidelity variants include eSpCas9-1.0 / -1.1, SpCas9-HF1 / HF2 / HF3 / HF4, HypaCas9, xCas9) and PAM variants of SpCas9 (SpCas9 (VQR), SpCas9 (eg) EQR), SpCas9 (VRER), SpCas9 (D1135E), and SpCas9 (QQR1), etc.), Staphylococcus aureus Cas9 (SaCas9) and its variants (eg, SaCas9HF and SaCas9 (KKH)), Streptococcus canis Cas9 (ScCas9) and its variants. Variants (eg ScCas9HF), Acidaminococcus sp. Cpf1 (AsCpf1) and variants thereof (eg AsCpf1 (RR), AsCpf1 (RVR)), Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) and variants thereof (eg LbCpf1 (RR)) ) And LbCpf1 (RVR)), Francisella novicida Cpf1 (FnCpf1) and its variants (eg, FnCpf1 (RR), FnCpf1 (RVR)), and Moraxella bovoculi 237 (Mb) Cpf1 and its variants (eg, MbCpf1 (eg, MbCpf1) RR) and MbCpf1 (RVR)) and the like.

In genome editing, DNA cleaved by the above nuclease or the like is repaired by homologous recombination or non-homologous end ligation, and at this time, the gene of interest can be modified. By using a nuclease with accurate target recognition ability in genome editing, for example, AsCpf1, the accuracy with which the target cell can be obtained can be improved.

When the genome editing protein is CRISPR / Cas9, CRISPR / Cpf1 or another Cas protein group, in order to perform genome editing (cutting), crRNA (or guide RNA) is introduced at the same time in addition to the genome editing protein. There is a need.

If the genome editing protein is CRISPR / Cas9, CRISPR / Cpf1 or another Cas protein group, etc., the genome editing protein, crRNA (or guide) can be used for genome editing (intended substitution, insertion or deletion, etc.). In addition to RNA), template DNA (ssODN, dsDNA, etc.) must be introduced at the same time.

In addition to the general genome editing method using SSN and template DNA, the genome editing step also includes base substitution using Cytosine Base Editor (CBE) or Adenine Base Editor (ABE). CBE is a CRISPR / Cas protein-based artificial enzyme that enables single-base translocation from G-C base pairs to T-A base pairs. Possible proteins include CBE and its variants and analogs (eg BE1, BE2, BE3, HF-BE3, BE4, BE4max, BE4-gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-BE3, VQR. -BE3, VRER-BE3, SaBE3, SaBE4, SaBE4-Gam, Sa (KKH) -BE3, Cas12a-BE, Target-AID, Target-AID-NG, xBE3, eA3A-BE3, A3A-BE3, BE-PLUS, TAM, CRISPR-X) and the like. On the other hand, ABE is a CRISPR / Cas protein-based artificial enzyme that enables single-base translocation from A-T base pairs to G-C base pairs. Possible proteins include ABE and its variants and analogs (eg, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE.7.10 *, xABE, ABESa, VQR-ABE, VRER-ABE, Sa (KKH). )-ABE) and the like.

Further, in addition to the genome editing protein (and optionally the above crRNA), a selectable marker such as antibiotic resistance for selecting the cell in which the genome editing has been performed may be introduced into the cell.

Genome-editing proteins (and optionally selectable markers) may be introduced into cells in the form of vectors containing nucleic acids encoding them. In addition to the nucleic acid encoding the genome editing protein, the nucleic acid and / or crRNA encoding the optional marker may be contained on the same vector or may be contained on a plurality of different vectors. .. The vector may also be introduced into the cell together with, for example, single-stranded DNA or double-stranded DNA as a modification template that can be incorporated into the cleaved DNA, for example, single-stranded oligodeoxynucleotides. Vectors can be introduced into cells by known methods, such as electroporation, lipofection, calcium PEG-phosphate, polyethyleneimine (PEI) mediated transfection, and microinjection, viral, as examples of introduction methods. The vector method and the like can be mentioned.

In the method of this aspect, genome editing is performed based on the step of identifying the presence or absence of a specific single base in the nucleic acid of a cell and the identification result of the presence or absence of a specific single base according to the method described in the present specification. Includes the step of selecting the damaged cells.

In one embodiment, the method of the present invention may include a selection step after or before the genome editing step and the selection step. The selection step can be performed, for example, by introducing a selectable marker such as antibiotic resistance into cells at the same time as the genome editing step and culturing the cells in the presence of the antibiotic resistance or the like. Examples of the antibiotic include puromycin, neomycin, blastsidedin, hygromyosin, and zeocin, and the type and concentration thereof can be appropriately set by those skilled in the art. Further, the culture conditions in the selection step can be appropriately set by those skilled in the art, and for example, the culture period can be 1 to 7 days, 2 to 5 days, or 2 to 3 days.

In one aspect, the invention relates to cells obtained by the method of producing genome-edited cells described herein, such as genome-edited cells. In one aspect, the present invention comprises a pharmaceutical composition comprising the genome-edited cells, eg, a pharmaceutical composition for the treatment and / or prevention of a disease, and the cells for the treatment and / or prevention of a disease. Regarding use.

Further, in one aspect, the present invention includes a step of obtaining cells whose genome has been edited by the genome editing method described in the present specification, and a step of treating and / or preventing a disease using the obtained cells. Regarding the treatment method of the disease.

Treatment and / or prevention of the disease in these embodiments can be performed, for example, by proliferating and, if necessary, differentiating the genome-edited cells and administering or transplanting them to the subject. In this embodiment, the cells for genome editing can be stem cells obtained from the subject in order to reduce rejection in the subject.

In these embodiments, the subject to which treatment and / or prevention can be made may be human. Further, the disease is not limited as long as it is a disease that can be caused by mutations such as SNP, SNV, Indel, SV, etc., and may be the diseases described in the present specification.

<Materials and methods>
1. Primer design Primer design was in accordance with the HiDi DNA polymerase (myPOLS, Germany) data sheet.

Specifically, the primer pair (single nucleotide detection primer (SN primer) and common primer) was designed to be able to amplify an amplicon of 60 to 200 bp. Primer design follows general rules, but the most important point is to keep the GC content in the 40-60% range. SN primers (using either forward or reverse primers) were designed so that one base at the 3'end of the primer corresponds to the SN marker. The base sequence of the primers used is shown in Table 1.

2. Template preparation (building ssODN)
99 mer Single-stranded oligodeoxynucleotides (ssODN) (PAGE purified grade) or 75 mer ssODN (HPLC purified grade) corresponding to genomic DNA near Human RET exon 16 were used. The base sequence of the ssODN template used is shown in Table 1. These ssODN a (100 nM) as a stock solution was prepared stepwise dilution series by 10-fold dilution from ^10-1 to ^10-10.

3. Composition of PCR reaction solution
3.1 HiDi DNA Polymerase
The composition was based on the HiDi DNA polymerase (myPOLS, Germany) data sheet. Specifically, the volume of the reaction solution per reaction was 10 μL. The final concentrations of the forward and reverse primers were 0.2 μM each. The final concentration of dNTPs was 200 μM. The final concentration of HiDi buffer was 1x. The final concentration of HiDi DNA polymerase was 2.5 Unit / reaction.

3.2 GoTaq DNA polymerase
The composition was based on the GoTaq Green Master Mix (Promega, USA) data sheet. Specifically, the volume of the reaction solution per reaction was 10 μL. The final concentrations of the forward and reverse primers were 0.2 μM each. The final concentration of Master Mix was 1x.

3.3 Tks Gflex DNA polymerase
The composition was based on the data sheet of Tks Gflex DNA polymerase (TAKARA-BIO, Japan). Specifically, the volume of the reaction solution per reaction was set to 10 μL. The final concentrations of the forward and reverse primers were 0.2 μM each. The final concentration of Gflex PCR Buffer was 1x. The final concentration of DNA polymerase was 1.25 Unit / 50 μL.

4. PCR program
4.1 HiDi DNA polymerase
The amplification program was partially modified according to the data sheet of HiDi DNA polymerase (myPOLS, Germany). The program is as follows: 1. First denaturation, 95 ° C / 2 min; 2. Degeneration, 95 ° C / 15 sec; 3. Annealing, 63-69 ° C / 10 sec; 4. Elongation, 70 ° C / 30 sec; 5. Repeating 35-37 cycles of 2-4 above; 6. Hold <10 ° C.

4.2 GoTaq DNA polymerase
The amplification program was partially modified according to the GoTaq Master Mix (Promega, USA) data sheet. The program is as follows: 1. First denaturation, 95 ° C / 2 min; 2. Degeneration, 95 ° C / 1 min; 3. Annealing, 65-68 ° C / 1 min; 4. Elongation, 70 ° C / 30 sec; 5. Repeat 2-4 above, 39 cycles; 6. Hold <10 ° C.

4.3 Tks Gflex DNA polymerase
The amplification program followed the data sheet of Tks Gflex DNA polymerase (TAKARA-BIO, Japan). The program is as follows: 1. Degeneration, 98 ° C / 10 sec; 2. Annealing, 65-68 ° C / 15 sec; 3. Elongation, 68 ° C / 30 sec; 4. Repeat 1-3 above, 37 Cycle; 5. Hold <10 ° C.

5 Genome editing
5.1 Design and construction of AsCpf1_RR and CRISPR RNA (crRNA) A pY211 mammalian expression vector containing the AsCpf1-RR and crRNA backbones to generate an all-in-one vector of AsCpf1-RR mutants carrying the puromycin resistance gene ( Gao, L. et al., 2017, Nat. Biotechnol., 35, 789-792) (Addgene plasmid number 89352 was inherited from Dr. Feng Zhang) was used. Here, the 3 × HA tag was replaced with the 3 × HA tag, T2A peptide cDNA, and SGFP2 cDNA. Briefly, 3 × HA and T2A fragments were constructed by ssODN annealing and SGFP2 cDNA was amplified from pSGFP2-C1 (Addgene plasmid number 22881 was inherited from Dr. Dorus Gadella). The pY211 vector was cleaved by BamHI / EcoRI. Finally, all the fragments were fused using the In-Fusion HD Cloning kit (Clontech / TAKARA-BIO). The resulting plasmid was named pY211-T2G. The cDNA corresponding to the puromycin resistance gene was then amplified from the pSIH-H1-Puro vector (Addgene plasmid number 26597 was inherited from Dr. Frank Sinicrope) and fused to the pY211-T2G vector at the SpeI / EcoRI site. The final all-in-one vector was named pY211-puro.

The crRNA was manually designed as previously described (Gao, L. et al., Supra). A crRNA-guided sequence template targeting RET exon 16 or COL7A1 exon 78 was cloned into a BbsI-digested pY211-puro vector as previously described (Gao, L. et al., Supra).

5.2 Construction of iPS cells
iPS cells were generated from human T cells using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific, USA) according to the manufacturer's protocol. Briefly, human T cells were isolated from peripheral blood samples and seeded at a density of 1.5 × 10 ⁶ cells / well on 6-well plates coated with anti-CD3 antibody (eBioscience). One day later, 3 × 10 ⁵ cells were infected with recombinant Sendai virus (SeV), which has a reprogramming factor with a multiplicity of infection (MOI) of 10. After culturing for 2 days, infected cells were collected and reseeded on mitomycin C (MMC) treated mouse embryo fibroblasts (MEF, acting as feeder cells) at 2 × 10 ⁴ cells per 100 mm dish. Twenty-three days after infection, colonies were harvested and recultured in human iPS cell medium (details below). To remove SeV, iPS cells were cultured at 38 ° C for 3 days and passaged once.

Five pluripotent genes (Nanog, Gdf3, Rex1, Sall4, Dnmt3B) and four Yamanaka factors (Oct-3 /) to confirm the quality of newly established iPS cells (FB4-14 and B117-3 cells) 4, Sox2, Klf4, c-Myc) were confirmed by RT-PCR using a pre-designed primer set.

5.3 Cell Disease FB4-14 cells (MEN2B-specific iPS cells) with autosomal dominant mutations at the disease locus were used. This locus carries a single allergen point mutation (T to C) in the RET gene at codon 918 of exon 16, which results in the Met918 Thr substitution.

5.4 Cultured iPS cells Cells were maintained on cell culture plates coated with 0.1% gelatin seeded with MMC-treated MEF and grown in iPS cell medium at 37 ° C. under 5% CO ₂ . This medium contains 20% KNOCKOUT ^TM serum substitute (KSR, Invitrogen), 2 mM L-glutamine (Life technologies), 0.1 mM non-essential amino acids (NEAA, SIGMA), 0.1 mM 2-mercaptoethanol (SIGMA), 0.5%. It consists of DMEM / F12 (SIGMA) supplemented with penicillin and streptomycin (Nacalai Tesque) and 5 ng / mL basic fibroblast growth factor (bFGF, Wako). Prior to transfection, iPS cells were transferred to a cell culture plate coated with Matrigel-GFR (Corning) and medium for iPS cells containing 10 μM ROCK inhibitor was conditioned with MEF (MEF-CM) for 2 days. It was cultured. Finally, cells were grown on Matrigel-GFR-coated cell culture plates in mTeSR1 medium (STEMCELL Technologies) at 37 ° C. under 5% CO ₂ .

5.5 Transfection
iPS cells were electroporated with pY211-puro vector (all-in-one mammalian expression vector containing AsCpf1-RR cDNA, CRISPR RNA, and puromycin resistance gene) and ssODN. Briefly, 1 × 10 ⁶ cells were resuspended in 100 μL OptiMEM containing 10 μg pY211-puro and 15 μg ssODN (99 nt, PAGE-purified; Sigma-Aldrich). Subsequently, cells were electroporated in a 2 mm gap cuvette using Super Electroporator NEPA21 Type 2 (NEPA GENE, Japan) (transfer pulse 20 V, pulse length 50 ms, pulse count 5). After electroporation, cells are transferred to a Matrigel-GFR coated 24-well plate, grown in mTeSR1 medium containing CloneR (STEMCELL Technologies) for 16 hours and treated with mTeSR1 medium containing CloneR and puromycin (0.5 μg / mL) for 48 hours. Then, it was grown in mTeSR1 medium containing CloneR for 1 to 2 days. The cells were then united with TrypLE (ThermoFisher Scientific, USA), seeded in mTeSR1 medium containing CloneR at low density (500-1000 cells per 100 mm plate) and cloned.

6. Extraction of genomic DNA One-third of the colonies of genome-edited iPS cells were sampled, and a crude fraction of genomic DNA was extracted by the Proteinase K extraction method. Specifically, the cell sample was resuspended in a lysis buffer containing 10 μL of Proteinase K (TAKARA-BIO, Japan), incubated at 55 ° C. for 12 hours, and then Proteinase K was heat-treated at 85 ° C. for 1 hour. Inactivated. The details of the subsequent PCR conditions are as described above.

<Result>
1. Single nucleotide detection PCR in palindrome sequence
SN marker-specific primers were designed in regions where the presence or absence of the template palindrome sequence at the 3'end site of the primer and the corresponding site differed depending on the presence or absence of the SN marker. A schematic diagram showing the concept of one embodiment of the present invention is shown in FIG.

When the SN marker is placed at the site where there is no parindrome sequence (original template sequence corresponding to the 3'end site of the primer: GATT, template sequence corresponding to the 3'end site of the primer after introduction of the marker: GATC), the control detectable range of SN markers when compared was approximately 10 ² (FIG. 2). On the other hand, when a marker is introduced at a site having a parindrome sequence so that the parindrome sequence is eliminated (original template sequence corresponding to the 3'end site of the primer: AATT, the 3'end site of the primer after introduction of the marker). corresponding template sequence to: AATC), the detectable range of SN markers when compared to a control is about 10 ^5, the detection sensitivity is significantly improved (Fig. 3, example 1).

2. Single nucleotide detection PCR in different palindrome sequences
We examined whether this method is effective even when the 4-base length palindrome sequence (AATT) is changed to another palindrome sequence of the same length. As a result, 4 when the base length palindromic sequence is GGCC, detectable range detection sensitivity is 10 ³ has been greatly improved (Example 2, FIG. 4). For the case where 4 is a base length palindromic sequence TTAA is in another position also, the detectable range is 10 ⁴ Detection sensitivity was improved significantly (Example 3, Figure 5). These results indicate that this method is effective even in a different sequence having a length of 4 bases and a different position.

3. Examination of palindrome sequence length In order to examine whether this method is effective even when the palindrome sequence length is reduced, first, the palindrome sequence of the original template sequence corresponding to the 3'end site of the primer is selected. The case of 2 base length was analyzed. If 2 bases long palindromic sequence was AT, the detectable range is 10 ⁴ Detection sensitivity was improved significantly (Example 4, FIG. 6). If 2 bases long palindromic sequence was GC, the detectable range has improved the detection sensitivity is 10 ³ (Example 5, Figure 7). Next, in order to examine whether this method is effective even when the palindrome sequence length is increased, the palindrome sequence at the 3'end of the original template sequence corresponding to the 3'end site of the primer is increased to 6 bases in length. The case was analyzed. If a 6-base long palindromic sequence was CAATTG, the detectable range is 10 ⁴ Detection sensitivity was improved significantly (Example 6, Figure 8). As described above, it was shown that this method is effective when the palindrome sequence length is in the range of 2 to 6 bases.

4. Examination of enzyme species In order to examine whether this method can be applied to general PCR DNA polymerases other than HiDi DNA polymerase for single-base mismatch detection, two enzymes (GoTaq DNA polymerase and Tks Gflex DNA) Polymerase) was analyzed using the same template and primer set as in Example 1. As a result, SN marker detectable range in the GoTaq DNA polymerase used is 10 ^4, the SN marker located at a site not palindromic sequence, as compared with the case of using the HiDi DNA polymerase, the detection sensitivity is greatly improved (Example 7, FIG. 9A). Tks Gflex DNA polymerase in use SN marker detection range is 10 ² (Example 8, FIG. 9B), the detection range, the SN marker placed at the site without palindromic sequences were used HiDi DNA polymerase It was the same as the case. GoTaq DNA polymerase and Tks Gflex DNA polymerase are not originally designed to distinguish single base differences at the 3'end. Therefore, it is considered that by applying this method, even a normal PCR enzyme can be improved so that a difference of one base can be discriminated.

These results are summarized in the table below.

5. Application to genome editing This method was applied in the primary screening of target clones in genome repair of disease-specific iPS cells using the same template and primer set as in Example 1. Specifically, ODN_Ori99_I920sC (SEQ ID NO: 5) was introduced into cells by genome editing, and then PCR was performed using a forward primer (SEQ ID NO: 6) to perform screening. As a result, SN was found at a site without a parindrome sequence. The positive rate of the target clone was significantly improved compared to the case where the marker was placed and HiDi DNA polymerase was used (ODN_Ori99_I913sC (SEQ ID NO: 2) was introduced and the forward primer (SEQ ID NO: 3) was used). Figure 10, 1.8%-> 5.7%). This result indicates that this method is effective in improving the screening efficiency of actual genome editors.

Claims (9)

A method for identifying the presence or absence of a specific single base in nucleic acid.
(I) A step of amplifying a detected nucleic acid that identifies the presence or absence of a specific single base,
(Ii) The step of amplifying the control nucleic acid using at least one primer which is the same as that used in (i) above, and (iii) when the amplification product in the detection nucleic acid is larger than the amplification product in the control nucleic acid. , Including the step of identifying the presence of a specific base in the detected nucleic acid.
One base at the 3'end of the primer is not the same as the corresponding region of the control nucleic acid, and the control nucleic acid contains a palindromic sequence containing two or more bases in the region corresponding to the 3'end of the primer. ,
When a specific base is present in the detection nucleic acid, one base at the 3'end of the primer is the same as the corresponding region of the detection nucleic acid, and the detection nucleic acid is in the region corresponding to the 3'end side of the primer. A method that does not include the Palindrome sequence. A method for identifying the presence or absence of a specific single base in nucleic acid.
(I) A step of amplifying a detected nucleic acid that identifies the presence or absence of a specific single base,
(Ii) A step of amplifying the control nucleic acid using at least one primer which is the same as that used in (i) above, and (iii) when the amplification product in the detection nucleic acid is less than the amplification product in the control nucleic acid. , Including the step of identifying the presence of a specific base in the detected nucleic acid.
One base at the 3'end of the primer is the same as the corresponding region of the control nucleic acid, and the control nucleic acid contains a palindrome sequence containing two or more bases in the region corresponding to the 3'end of the primer. Zu,
When a specific base is present in the detection nucleic acid, the 1 base at the 3'end of the primer is not the same as the corresponding region of the detection nucleic acid, and the detection nucleic acid is in the region corresponding to the 3'end side of the primer. A method comprising the Palindrome sequence. The method according to claim 1 or 2, wherein the specific single base is a marker base that is artificially introduced. The method according to any one of claims 1 to 3, wherein the palindrome sequence contains a base of A or T. The method according to any one of claims 1 to 4, wherein the palindrome sequence contains 2 to 6 bases. The method of claim 5, wherein the palindromic sequence comprises 4-6 bases. The method according to any one of claims 1 to 6, wherein the nucleic acid amplification is performed by PCR or real-time PCR. The method according to any one of claims 1 to 7, wherein the nucleic acid amplification is carried out using a genetically modified or natural thermostable DNA polymerase capable of detecting a single nucleotide mismatch. The process of genome editing on cells,
Genome editing is performed based on the step of identifying the presence or absence of a specific single base in the nucleic acid of a cell and the identification result of the presence or absence of a specific single base according to the method according to any one of claims 1 to 8. The process of selecting cells, performed,
A method of producing genome-edited cells, including.

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