WO2024112036A1 - Method for removing adapter dimer in high-efficiency adapter ligation reaction using cas9 protein - Google Patents
Method for removing adapter dimer in high-efficiency adapter ligation reaction using cas9 protein Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6855—Ligating adaptors
Definitions
- the present invention relates to a method for removing adapter dimers in a highly efficient adapter ligation reaction using Cas9 protein.
- NGS Next-Generation Sequencing
- NGS is carried out in the following order: sample preparation, library production, cluster production, sequencing, and result analysis.
- the library production process involves end repair of DNA fragments, followed by primers for amplification.
- It involves the process of linking adapters with various functional sequences, such as binding sequences, to DNA fragments.
- an A-tailing process is performed to connect adenine to a DNA fragment
- a sticky-end ligation process is performed to connect an adapter with thymine protruding from the end. do.
- it undergoes a purification process using magnetic beads, etc. to obtain only amplification and amplification products through Polymerase Chain Reaction (PCR).
- PCR Polymerase Chain Reaction
- this analysis through NGS can also be used to diagnose diseases by sequencing mutated DNA that exists at a very low rate.
- it is important to reduce losses as much as possible in each process, and is involved in the reaction to produce accurate results.
- the present inventors established the optimal reaction conditions for high-efficiency adapter ligation that can improve the reaction efficiency in the adapter ligation process in the NGS reaction process, and sequence-specifically cleaved the adapter dimer generated from high-efficiency adapter ligation using the Cas9 protein.
- the present invention was completed by developing a method to improve the sensitivity and accuracy of sequencing.
- the object of the present invention is to provide a gRNA (guide RNA) for cutting adapter dimers generated during NGS (Next Generation Sequencing) library manufacturing, including a spacer sequence consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3. It is done.
- a gRNA guide RNA
- NGS Next Generation Sequencing
- Another object of the present invention is to provide an RNP (ribonucleoprotein) complex for cleavage of adapter dimers generated during NGS (Next Generation Sequencing) library production, which is composed of the gRNA (guide RNA) and Cas9 protein of the present invention.
- RNP ribonucleoprotein
- Another object of the present invention is to provide a method for cutting and removing adapter dimers generated during NGS (Next Generation Sequencing) library production using the ribonucleoprotein (RNP) complex for adapter dimer cleavage of the present invention.
- NGS Next Generation Sequencing
- Another object of the present invention is to use adapter ligation reaction in the NGS (Next Generation Sequencing) library manufacturing process, target DNA and adapter at a molar ratio of 1:20 to 1:100, and PEG-6000 (Polyethylene glycol-6000).
- the aim is to provide a highly efficient adapter connection method, characterized in that it reacts by adding it at a concentration of 8 to 10%.
- the present invention provides a gRNA (guide RNA) for cutting adapter dimers generated during NGS (Next Generation Sequencing) library manufacturing, including a spacer sequence consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3. .
- gRNA (guide RNA) containing the spacer sequence of SEQ ID NO: 1 may bind complementary to an adapter dimer of sticky end ligation.
- gRNA (guide RNA) containing the spacer sequence of SEQ ID NO: 3 may bind complementary to an adapter dimer of blunt end ligation.
- the gRNA for cutting the adapter dimer may be composed of the base sequence of SEQ ID NO: 2.
- the gRNA for cutting the adapter dimer may be composed of the base sequence of SEQ ID NO: 4.
- the present invention provides an RNP (ribonucleoprotein) complex for adapter dimer cleavage generated during NGS (Next Generation Sequencing) library production, which is composed of gRNA (guide RNA) and Cas9 protein of the present invention.
- RNP ribonucleoprotein
- the present invention provides a method for cutting and removing adapter dimers generated during NGS (Next Generation Sequencing) library production using the ribonucleoprotein (RNP) complex for adapter dimer cleavage of the present invention.
- NGS Next Generation Sequencing
- the adapter dimer may be generated during a high-efficiency ligation reaction of the adapter to the target DNA during the NGS library production process.
- the high-efficiency ligation reaction uses target DNA and adapter at a molar ratio of 1:20 to 1:100, and PEG-6000 (Polyethylene glycol-6000) is added at a concentration of 8 to 10%. It may be a reaction.
- PEG-6000 Polyethylene glycol-6000
- the high-efficiency ligation reaction may be performed by using target DNA and adapter at a molar ratio of 1:50 and adding PEG-6000 (Polyethylene glycol-6000) at a concentration of 10%. .
- PEG-6000 Polyethylene glycol-6000
- the present invention uses an adapter ligation reaction in the NGS (Next Generation Sequencing) library manufacturing process, target DNA and adapter at a molar ratio of 1:20 to 1:100, and PEG-6000 (Polyethylene glycol-6000) at a molar ratio of 8 to 10.
- NGS Next Generation Sequencing
- PEG-6000 Polyethylene glycol-6000
- the step of processing the RNP (ribonucleoprotein) complex for adapter dimer cleavage consisting of the gRNA (guide RNA) of the present invention and the Cas9 protein may be further included.
- the present invention provides a technology that can effectively remove adapter dimers formed during adapter ligation reactions in the conventional NGS library production process, and provides a Cas9 gRNA spacer that can specifically bind only to adapter dimers based on the base sequence of the adapter dimers.
- the sequence is designed and the RNP complex containing gRNA and Cas9 developed in the present invention is used, only the adapter dimer can be specifically cut and removed from the sample in which the adapter has been linked to the target DNA.
- the RNP (ribonucleoprotein) complex for adapter dimer cleavage provided in the present invention has the effect of minimizing library loss and simultaneously improving the accuracy and sensitivity of sequencing.
- Figure 1 is a schematic diagram showing gRNA design for adapter dimer cleavage according to the present invention.
- Figure 2 confirms the results of the adapter ligation reaction performed by varying the mixing molar ratio of the target DNA and the adapter in order to establish high-efficiency adapter ligation reaction conditions for the target DNA in one embodiment of the present invention.
- Figure 3 shows the results of adapter ligation reaction according to the concentration of PEG-6000 used in order to establish conditions for high-efficiency adapter ligation reaction for target DNA in one embodiment of the present invention.
- Figure 4 shows the results of comparing the delivery effect of the adapter dimer in a group treated with Cas9 RNP containing the gRNA of the present invention and a group not treated with the Cas9 RNP containing the gRNA of the present invention under the high-efficiency adapter ligation reaction conditions established in the present invention.
- Figure 5 shows the results of confirming whether the adapter dimer cleavage effect occurs by applying the adapter dimer cleavage (removal) method using Cas9 RNP containing the gRNA of the present invention to NGS library production.
- the present invention is characterized by providing a method for removing adapter dimers in a highly efficient adapter ligation reaction using the Cas9 protein.
- NGS (Next-Generation Sequencing) analysis is a method of analyzing DNA strands one by one and has the advantage of being able to perform sequence analysis very quickly and inexpensively compared to existing direct sequencing.
- This NGS involves segmenting DNA into certain fragments and attaching oligonucleotides with specific base sequences that can be recognized by the equipment to create a library.
- the base sequence of each library DNA strand is read by the equipment. It consists of processing the data generated from the steps and equipment and analyzing it with an algorithm.
- the library preparation process includes nucleic acid fragmentation, end-repair, 3′-end adenine addition (A-tailing), adapter ligation, and library amplification.
- the adapter dimer formed during the adapter connection process is a problem that reduces the accuracy and sensitivity of the analysis.
- the present inventors discovered a new RNP (ribonucleoprotein) composed of gRNA (guide RNA) and Cas9 protein that can specifically bind to and cleave adapter dimers to remove them. ) The composite was prepared.
- CRISPR Clustered regularly interspaced short palindromic repeats
- Cas9 technology is a gene editing technology that originates from the adaptive immunity phenomenon of microorganisms. It precisely cuts the target DNA and then allows the DNA to be repaired naturally, making genetic manipulation simpler and more efficient. Make it possible.
- This system consists of gRNA (guide RNA) and Cas9 nuclease protein, and is also called RNA-guided engineered nucleases (RGENs) technology.
- Cas9 protein is an endonuclease induced by guide RNA (gRNA), and has a target sequence complementary to the 20 nucleotide-long spacer region of gRNA and NGG. It binds to double-stranded DNA with the PAM (Protospacer Adjacent Motif) sequence and induces DNA double-strand break at a specific site. Because of its sequence-specific cutting function, Cas9 is being applied to a wide variety of fields, including genome editing.
- gRNA guide RNA
- PAM Protospacer Adjacent Motif
- gRNA is composed of crRNA (spacer region) that binds complementary to the target sequence and tracrRNA for binding to Cas9.
- crRNA spacer region
- gRNA serves to guide Cas9 to the target site and binds complementary to the target DNA sequence.
- the 3' end of the target DNA sequence must have a PAM (Protospacer adjacent motif) sequence of the NGG sequence.
- PAM Protospacer adjacent motif
- the PAM sequence is a protospacer associated motif that helps nucleic acid hydrolases recognize target DNA in the CRISPR system.
- the PAM sequence is composed of NGG (N is A, T, C or G).
- the present inventors used this gRNA-Cas9 protein to design a guide RNA composed of a specific base sequence that can be produced by specifically cutting the adapter dimer generated during the NGS library production process.
- the gRNA (guide RNA) for adapter dimer cleavage generated during the NGS library manufacturing process designed in the present invention is characterized by including a spacer sequence consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
- the spacer base sequence of the gRNA of SEQ ID NO: 1 or SEQ ID NO: 3 of the present invention was conceived by looking at the base sequence composition of the adapter dimer. When two adapters are connected, it is a sequence that includes both adapters starting from the connection site, and is a Cas9 target.
- the sequence and gRNA spacer sequence were designed as SEQ ID NO: 1 or SEQ ID NO: 3 to be about 20 nt in length.
- the gRNA (guide RNA) containing the spacer sequence of SEQ ID NO: 1 may bind complementary to the adapter dimer of sticky end ligation, and the gRNA (guide RNA) containing the spacer sequence of SEQ ID NO: 3 guide RNA) can bind complementary to the adapter dimer of blunt end ligation.
- the gRNA containing the spacer sequence of SEQ ID NO: 1 can be used to remove adapter dimers having sticky end connections
- the gRNA containing the spacer sequence of SEQ ID NO: 3 can be used to remove adapter dimers having blunt end connections. You can.
- the entire base sequence of the gRNA for cleaving the adapter dimer according to the present invention may consist of SEQ ID NO: 2 or SEQ ID NO: 4.
- the present invention can provide an RNP (ribonucleoprotein) complex for adapter dimer cleavage generated during the NGS library manufacturing process, which is composed of gRNA (guide RNA) for adapter dimer cleavage and Cas9 protein according to the present invention.
- RNP ribonucleoprotein
- the gRNA for adapter dimer cleavage in the RNP (ribonucleoprotein) complex is a complex of a Cas9 protein and a gRNA (guide RNA) containing a spacer sequence consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3. .
- the present invention provides a method for cutting and removing adapter dimers generated during NGS (Next Generation Sequencing) library production using the ribonucleoprotein (RNP) complex for adapter dimer cleavage of the present invention.
- NGS Next Generation Sequencing
- the adapter dimer present in the reaction was analyzed for the group treated with the RNP complex for adapter dimer cleavage of the present invention and the group not treated, In the group treated with the RNP complex of the present invention, most adapter dimers were cleaved and hardly identified, whereas in the group not treated with the RNP complex of the present invention, a significant number of adapter dimers were found to be formed.
- the present inventors were able to see that when using the adapter dimer cleavage gRNA and Cas9 dimer cleavage RNP complex designed in the present invention, adapter dimers generated during the NGS library production process can be effectively removed.
- the present invention can provide a highly efficient adapter linking method that can improve the efficiency of the adapter linking reaction in the NGS library manufacturing process.
- the high-efficiency adapter ligation method provided by the present invention uses target DNA and adapter at a molar ratio of 1:20 to 1:100, and adds PEG-6000 (Polyethylene glycol-6000) at a concentration of 8 to 10% to carry out the ligation reaction.
- PEG-6000 Polyethylene glycol-6000
- an experiment was performed to identify optimal reaction conditions that can improve the efficiency of the adapter ligation reaction in NGS library production.
- the target DNA and adapter were used in the reaction at various mixing ratios, and PEG-6000 (Polyethylene glycol-6000) was used in the reaction at various concentrations.
- the target DNA and adapter were used in the reaction at a mixing ratio of 1:20 to 1:100.
- PEG-6000 Polyethylene glycol-6000
- the adapter linkage reaction efficiency was found to be excellent.
- target DNA and adapter are used at a molar ratio of 1:50 and PEG-6000 (Polyethylene glycol-6000) is added at a concentration of 10% for reaction, intermediate reaction products and reactions in which the adapter is connected to only one side of the target DNA There was almost no target DNA where this did not occur, and it was confirmed that most adapters were connected to both sides of the target DNA.
- PEG-6000 Polyethylene glycol-6000
- the present inventors have designed a method that uses Cas9 protein as a method to effectively remove adapter dimers generated during the library preparation process of NGS reaction, and the Cas9 protein acts only on adapter dimers to cleave gRNA as follows. A specific base sequence in the spacer region was designed.
- the Cas9 protein bound to gRNA must act only on the adapter dimer to perform the cleavage function. If Cas9 RNP acts on the adapter monomer, the target product of the adapter linkage, in which one adapter monomer is connected to each side, may also be cleaved by Cas9. Therefore, the target sequence of Cas9 and the gRNA spacer sequence were selected to be 20 nt in length, which are sequences created only when an adapter dimer is formed, that is, when two adapters are connected, and include both sides of the connection site. At the same time, it was designed to have the NGG sequence, which is the PAM sequence, at the appropriate position (see Figure 1). The adapter sequence and the spacer sequence of the gRNA for adapter dimer removal designed in the present invention are shown in Table 1 below.
- Adapter sequence and spacer sequence and gRNA sequence of gRNA for adapter dimer removal designed in the present invention name Sequence (5’ ⁇ 3’) Adapter /5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCUACACTCTTTCCCTACACGACGCTCTTCCGATCT gRNA spacer G*U*G UGCUCUUCCGAUCAGAU [SEQ ID NO: 1] gRNA (crRNA) G*U*G UGCUCUUCCGAUCAGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UU*U [SEQ ID NO: 2] gRNA spacer A*U*G UGGCUCUUCCGAUCGAU [SEQ ID NO: 3] gRNA (crRNA) A*U*G UGGCUCUUCCGAUCGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC
- the present inventors performed experiments to establish reaction conditions that could improve the efficiency of the adapter ligation reaction during the NGS library production process.
- the reaction product was confirmed by 7.5% Native PAGE.
- the target DNA in which the adapter ligation reaction did not occur is indicated by a green box, and the intermediate in which the adapter binds to only one side of the target DNA is indicated by a purple box.
- the final product, in which the adapter is bound to both sides of the target DNA, is indicated by a red box. Additionally, the adapter dimer is indicated by a blue box.
- the efficiency of the ligation reaction was found to increase as the adapter ratio increased, and the formation of adapter dimers was also found to decrease as the adapter ratio increased.
- the ratio of target DNA and adapter is 1:50
- the formation of adapter dimers is small and the amount of adapters linked to both sides of the target DNA is the largest. Therefore, it is recommended to react at the above addition ratio. It was found that the adapter connection reaction occurred most effectively.
- adapter ligation reaction was performed by varying the amount of PEG-6000 added.
- the use of PEG-6000 can increase the efficiency of the adapter connection by inducing a density-enhancing effect in the adapter connection reaction, but the accuracy of the T4 ligase used is limited, so the efficiency of the adapter connection reaction increases. As a result, there is a problem that adapter dimers are inevitably generated.
- Example ⁇ 2-1> Through the experiment in Example ⁇ 2-1>, it was confirmed that the optimal reaction conditions for adapter linkage were to set the molar ratio of target DNA to adapter at 1:50 and use 10% PEG-6000. Meanwhile, as it was confirmed that an adapter dimer is generated under the optimal ligation reaction conditions, it was investigated whether the formed adapter dimer can be effectively cut (removed) when using the gRNA and Cas9 designed in the present invention under the above ligation reaction conditions. Confirmed. For this purpose, an experiment was performed as follows.
- the present inventors found that when using the Cas9 RNP complex using Cas9 and a gRNA containing a gRNA spacer sequence composed of a specific sequence that can specifically bind to the adapter sequence designed in the present invention, there is a problem in NGS library production. It was found that the adapter dimer could be specifically cut and removed.
- the present inventors conducted an experiment to confirm whether the adapter dimer removal technology using gRNA and Cas9 for adapter dimer cleavage designed in the present invention can be effectively applied to NGS library production.
- RNA for this purpose, 40 ng of target DNA is diluted in 50 uL of 0.1X TE buffer and then mixed with 1X Ultra II End prep module (NEB) to perform end-repair and A-tailing reactions. did.
- NEB 1X Ultra II End prep module
- the adapter is treated at 95°C for 5 minutes and then ramped down to 25°C at a rate of 0.1°C/s to form a hairpin structure through a 15-minute reaction at 25°C. was formed, and then the adapter ligation reaction was performed under the optimal high-efficiency adapter ligation reaction conditions established in the present invention.
- the high adapter ligation reaction efficiency is maintained (a large amount of adapter-linked products are formed on both sides of the target DNA), and at the same time, the adapter dimer is completely cleaved and removed. It was confirmed that there was almost no target DNA that did not react.
- the present inventors were able to see that when performing the high-efficiency adapter ligation reaction conditions identified in the present invention, it is possible to very effectively form a product in which adapters are linked to both sides of the target DNA. It was found that when gRNA and Cas9 with a specific spacer sequence are used, adapter dimers generated in the adapter ligation reaction can be effectively removed, making it useful for obtaining more accurate and effective NGS analysis results.
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Abstract
The present invention relates to a method for removing an adapter dimer in a high-efficiency adapter ligation reaction using a Cas9 protein, and more specifically to: a guide RNA (gRNA) for cutting an adapter dimer generated in a process of manufacturing a next generation sequencing (NGS) library including a spacer sequence composed of a base sequence of SEQ ID NO: 1 or SEQ ID NO: 3; a ribonucleoprotein (RNP) complex for cutting an adapter dimer generated in the process of manufacturing the NGS library composed of the gRNA and Cas9 proteins; a method for removing an adapter dimer using the RNP complex; and a high-efficiency adapter ligation method in which an adapter ligation reaction in the process of manufacturing the NGS library is carried out by using a target DNA and an adapter in a molar ratio of 1:20 to 1:100 and adding polyethylene glycol-6000 (PEG-6000) in a concentration of 8-10%.
Description
본 발명은 Cas9 단백질을 이용한 고효율 어댑터 연결 반응에서 어댑터 이합체의 제거 방법에 관한 것이다.The present invention relates to a method for removing adapter dimers in a highly efficient adapter ligation reaction using Cas9 protein.
NGS(Next-Generation Sequencing)는 유전체 염기서열 분석법으로 분석하고자 하는 DNA를 분절화(Fragmentation)하여 병렬로 동시에 읽어내는 특징을 가지며 따라서 매우 빠른 분석이 가능한 방법이다.NGS (Next-Generation Sequencing) is a genome sequencing method that fragments the DNA to be analyzed and reads it simultaneously in parallel, making it a method that allows for very fast analysis.
NGS는 샘플 준비, 라이브러리 제작, 클러스터(Cluster) 제작, 시퀀싱, 결과 분석의 순서로 진행되는데, 그 중 라이브러리 제작 과정에는 DNA 파편에 대한 말단 수리(End repair) 과정을 거친 뒤에 증폭을 위한 프라이머(Primer) 결합 서열 등 다양한 기능적 서열을 갖는 어댑터(Adapter)를 DNA 파편에 연결하는 과정이 포함된다. 일반적으로 DNA 파편에 아데닌(Adenine)을 연결하는 A-테일링(A-tailing) 과정을 진행하고 티민(Thymine)이 말단에 돌출되어 있는 어댑터를 연결하는 점착 말단 연결(Sticky-end ligation) 과정을 진행한다. 이후, 분석 목적에 따라 중합효소연쇄반응(Polymerase Chain Reaction, 이하 PCR)을 통한 증폭 및 증폭 생성물만을 얻어내기 위해 자성 비드 (Magnetic bead) 등을 통한 정제 과정을 거치게 된다.NGS is carried out in the following order: sample preparation, library production, cluster production, sequencing, and result analysis. Among them, the library production process involves end repair of DNA fragments, followed by primers for amplification. ) It involves the process of linking adapters with various functional sequences, such as binding sequences, to DNA fragments. In general, an A-tailing process is performed to connect adenine to a DNA fragment, and a sticky-end ligation process is performed to connect an adapter with thymine protruding from the end. do. Afterwards, depending on the purpose of analysis, it undergoes a purification process using magnetic beads, etc. to obtain only amplification and amplification products through Polymerase Chain Reaction (PCR).
한편, 이러한 NGS를 통한 분석은 매우 낮은 비율로 존재하는 돌연변이가 일어난 DNA를 시퀀싱하여 질병의 진단에도 이용할 수 있는데, 이 경우 각 과정 모두에서 손실을 최대한 줄이는 것이 중요한데, 정확한 결과 도출을 위한 반응에 관여하는 반응물들의 손실 및 라이브러리 손실이 발생하는 문제가 있고, 또한 어댑터의 연결 과정에서 어댑터끼리의 연결로 인한 이합체(Adapter dimer) 형성으로 이후의 증폭 및 시퀀싱 과정에도 영향을 미쳐 정확한 분석결과를 얻지 못한다는 문제점이 있다.Meanwhile, this analysis through NGS can also be used to diagnose diseases by sequencing mutated DNA that exists at a very low rate. In this case, it is important to reduce losses as much as possible in each process, and is involved in the reaction to produce accurate results. There is a problem of loss of reactants and library loss, and in addition, the formation of adapter dimers due to the connection of adapters during the adapter connection process affects the subsequent amplification and sequencing process, making it difficult to obtain accurate analysis results. There is a problem.
따라서 NGS가 갖는 이러한 문제점을 개선할 수 있는 기술의 개발이 요구되고 있다.Therefore, there is a need to develop technologies that can improve these problems with NGS.
이에 본 발명자들은 NGS 반응 과정에서 어댑터 연결 과정에서의 반응 효율을 향상시킬 수 있는 고효율 어댑터 연결의 최적 반응조건을 확립하였고, 고효율 어댑터 연결에서 발생하는 어댑터 이합체를 Cas9 단백질을 이용하여 서열 특이적으로 절단함으로써 시퀀싱의 민감도와 정확도를 향상시킬 수 있는 방법을 개발함으로써 본 발명을 완성하였다.Accordingly, the present inventors established the optimal reaction conditions for high-efficiency adapter ligation that can improve the reaction efficiency in the adapter ligation process in the NGS reaction process, and sequence-specifically cleaved the adapter dimer generated from high-efficiency adapter ligation using the Cas9 protein. By doing so, the present invention was completed by developing a method to improve the sensitivity and accuracy of sequencing.
그러므로 본 발명의 목적은 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 스페이서(spacer) 서열을 포함하는, NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체 절단용 gRNA(guide RNA)를 제공하는 것이다.Therefore, the object of the present invention is to provide a gRNA (guide RNA) for cutting adapter dimers generated during NGS (Next Generation Sequencing) library manufacturing, including a spacer sequence consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3. It is done.
본 발명의 다른 목적은 상기 본 발명의 gRNA(guide RNA) 및 Cas9 단백질로 구성되는, NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체를 제공하는 것이다.Another object of the present invention is to provide an RNP (ribonucleoprotein) complex for cleavage of adapter dimers generated during NGS (Next Generation Sequencing) library production, which is composed of the gRNA (guide RNA) and Cas9 protein of the present invention.
본 발명의 또 다른 목적은 본 발명의 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체를 이용하여 NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체를 절단하여 제거하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for cutting and removing adapter dimers generated during NGS (Next Generation Sequencing) library production using the ribonucleoprotein (RNP) complex for adapter dimer cleavage of the present invention.
본 발명의 또 다른 목적은 NGS(Next Generation Sequencing) 라이브러리 제조과정에서 어댑터 연결반응을, 표적 DNA와 어댑터를 1:20~1:100의 몰비로 사용하고, PEG-6000(Polyethylene glycol-6000)을 8~10%의 농도로 첨가하여 반응하는 것을 특징으로 하는, 고효율 어댑터 연결방법을 제공하는 것이다.Another object of the present invention is to use adapter ligation reaction in the NGS (Next Generation Sequencing) library manufacturing process, target DNA and adapter at a molar ratio of 1:20 to 1:100, and PEG-6000 (Polyethylene glycol-6000). The aim is to provide a highly efficient adapter connection method, characterized in that it reacts by adding it at a concentration of 8 to 10%.
따라서 본 발명은, 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 스페이서(spacer) 서열을 포함하는, NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체 절단용 gRNA(guide RNA)를 제공한다.Therefore, the present invention provides a gRNA (guide RNA) for cutting adapter dimers generated during NGS (Next Generation Sequencing) library manufacturing, including a spacer sequence consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3. .
본 발명의 일실시예에 있어서, 상기 서열번호 1의 스페이서 서열을 포함하는 gRNA(guide RNA)는 점착 말단 연결(sticky end ligation)의 어댑터 이합체에 상보적으로 결합하는 것일 수 있다.In one embodiment of the present invention, gRNA (guide RNA) containing the spacer sequence of SEQ ID NO: 1 may bind complementary to an adapter dimer of sticky end ligation.
본 발명의 일실시예에 있어서, 상기 서열번호 3의 스페이서 서열을 포함하는 gRNA(guide RNA)는 평활 말단 연결(blunt end ligation)의 어댑터 이합체에 상보적으로 결합하는 것일 수 있다.In one embodiment of the present invention, gRNA (guide RNA) containing the spacer sequence of SEQ ID NO: 3 may bind complementary to an adapter dimer of blunt end ligation.
본 발명의 일실시예에 있어서, 상기 어댑터 이합체 절단용 gRNA는 서열번호 2의 염기서열로 이루어진 것일 수 있다.In one embodiment of the present invention, the gRNA for cutting the adapter dimer may be composed of the base sequence of SEQ ID NO: 2.
본 발명의 일실시예에 있어서, 상기 어댑터 이합체 절단용 gRNA는 서열번호 4의 염기서열로 이루어진 것일 수 있다.In one embodiment of the present invention, the gRNA for cutting the adapter dimer may be composed of the base sequence of SEQ ID NO: 4.
또한 본 발명은 본 발명의 gRNA(guide RNA) 및 Cas9 단백질로 구성되는, NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체를 제공한다.Additionally, the present invention provides an RNP (ribonucleoprotein) complex for adapter dimer cleavage generated during NGS (Next Generation Sequencing) library production, which is composed of gRNA (guide RNA) and Cas9 protein of the present invention.
또한 본 발명은 본 발명의 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체를 이용하여 NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체를 절단하여 제거하는 방법을 제공한다.In addition, the present invention provides a method for cutting and removing adapter dimers generated during NGS (Next Generation Sequencing) library production using the ribonucleoprotein (RNP) complex for adapter dimer cleavage of the present invention.
본 발명의 일실시예에 있어서, 상기 어댑터 이합체는 NGS 라이브러리 제조 과정 시, 표적 DNA에 대한 어댑터의 고효율 연결반응 시 생성되는 것일 수 있다.In one embodiment of the present invention, the adapter dimer may be generated during a high-efficiency ligation reaction of the adapter to the target DNA during the NGS library production process.
본 발명의 일실시예에 있어서, 상기 고효율 연결반응은 표적 DNA와 어댑터를 1:20~1:100의 몰비로 사용하고, PEG-6000(Polyethylene glycol-6000)을 8~10%의 농도로 첨가하여 반응하는 것일 수 있다.In one embodiment of the present invention, the high-efficiency ligation reaction uses target DNA and adapter at a molar ratio of 1:20 to 1:100, and PEG-6000 (Polyethylene glycol-6000) is added at a concentration of 8 to 10%. It may be a reaction.
본 발명의 일실시예에 있어서, 상기 고효율 연결반응은 표적 DNA와 어댑터를 1:50의 몰비로 사용하고, PEG-6000(Polyethylene glycol-6000)을 10%의 농도로 첨가하여 반응하는 것일 수 있다.In one embodiment of the present invention, the high-efficiency ligation reaction may be performed by using target DNA and adapter at a molar ratio of 1:50 and adding PEG-6000 (Polyethylene glycol-6000) at a concentration of 10%. .
나아가 본 발명은 NGS(Next Generation Sequencing) 라이브러리 제조과정에서 어댑터 연결반응을, 표적 DNA와 어댑터를 1:20~1:100의 몰비로 사용하고, PEG-6000(Polyethylene glycol-6000)을 8~10%의 농도로 첨가하여 반응하는 것을 특징으로 하는, 고효율 어댑터 연결방법을 제공한다.Furthermore, the present invention uses an adapter ligation reaction in the NGS (Next Generation Sequencing) library manufacturing process, target DNA and adapter at a molar ratio of 1:20 to 1:100, and PEG-6000 (Polyethylene glycol-6000) at a molar ratio of 8 to 10. A high-efficiency adapter connection method is provided, which is characterized in that it reacts by adding at a concentration of %.
본 발명의 일실시예에 있어서, 어댑터 연결반응 후, 상기 본 발명의 gRNA(guide RNA) 및 Cas9 단백질로 구성되는 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체를 처리하는 단계를 더 포함하는 것일 수 있다.In one embodiment of the present invention, after the adapter ligation reaction, the step of processing the RNP (ribonucleoprotein) complex for adapter dimer cleavage consisting of the gRNA (guide RNA) of the present invention and the Cas9 protein may be further included.
본 발명은 종래 NGS 라이브러리 제작 과정에서 어댑터 연결 반응 시 형성되는 어댑터 이합체를 효과적으로 제거할 수 있는 기술을 제공하는 것으로, 어댑터 이합체의 염기서열을 토대로 어댑터 이합체에만 특이적으로 결합할 수 있는 Cas9의 gRNA 스페이서 서열을 디자인하였고, 본 발명에서 개발한 gRNA 및 Cas9을 포함하는 RNP 복합체를 이용할 경우, 표적 DNA에 어댑터 연결을 진행한 시료로부터 어댑터 이합체만을 특이적으로 절단하여 제거할 수 있는 효과가 있어, 이후 진행되는 NGS 분석에서 정제, 증폭 및 시퀀싱에서 원하는 표적 DNA의 서열만을 민감하게 얻어낼 수 있는 효과가 있다. 따라서 본 발명에서 제공하는 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체는 라이브러리의 손실을 최소화함과 동시에 시퀀싱의 정확도와 민감도를 향상시킬 수 있는 효과가 있다.The present invention provides a technology that can effectively remove adapter dimers formed during adapter ligation reactions in the conventional NGS library production process, and provides a Cas9 gRNA spacer that can specifically bind only to adapter dimers based on the base sequence of the adapter dimers. When the sequence is designed and the RNP complex containing gRNA and Cas9 developed in the present invention is used, only the adapter dimer can be specifically cut and removed from the sample in which the adapter has been linked to the target DNA. In NGS analysis, only the desired target DNA sequence can be sensitively obtained through purification, amplification, and sequencing. Therefore, the RNP (ribonucleoprotein) complex for adapter dimer cleavage provided in the present invention has the effect of minimizing library loss and simultaneously improving the accuracy and sensitivity of sequencing.
도 1은 본 발명에 따른 어댑터 이합체 절단을 위한 gRNA 디자인을 나타낸 모식도이다.Figure 1 is a schematic diagram showing gRNA design for adapter dimer cleavage according to the present invention.
도 2는 본 발명의 일실시예에서 표적 DNA에 대한 고효율 어댑터 연결 반응 조건 확립을 위해, 표적 DNA와 어댑터의 혼합 몰비를 각기 달리하여 수행한 어댑터 연결반응 결과를 확인한 것이다.Figure 2 confirms the results of the adapter ligation reaction performed by varying the mixing molar ratio of the target DNA and the adapter in order to establish high-efficiency adapter ligation reaction conditions for the target DNA in one embodiment of the present invention.
도 3은 본 발명의 일실시예에서 표적 DNA에 대한 고효율 어댑터 연결 반응 조건 확립을 위해, PEG-6000의 사용 농도에 따른 어댑터 연결반응 결과를 확인한 것이다.Figure 3 shows the results of adapter ligation reaction according to the concentration of PEG-6000 used in order to establish conditions for high-efficiency adapter ligation reaction for target DNA in one embodiment of the present invention.
도 4는 본 발명에서 확립한 고효율 어댑터 연결반응 조건에 있어서, 본 발명의 gRNA를 포함하는 Cas9 RNP를 처리한 군과 처리하지 않은 군을 대상으로 어댑터 이합체의 전달 효과를 비교한 결과를 나타낸 것이다.Figure 4 shows the results of comparing the delivery effect of the adapter dimer in a group treated with Cas9 RNP containing the gRNA of the present invention and a group not treated with the Cas9 RNP containing the gRNA of the present invention under the high-efficiency adapter ligation reaction conditions established in the present invention.
도 5는 본 발명의 gRNA를 포함하는 Cas9 RNP를 이용한 어댑터 이합체 절단(제거) 방법을 NGS 라이브러리 제작에 적용하여 어댑터 이합체 절단 효과가 발생하는지를 확인한 결과를 나타낸 것이다.Figure 5 shows the results of confirming whether the adapter dimer cleavage effect occurs by applying the adapter dimer cleavage (removal) method using Cas9 RNP containing the gRNA of the present invention to NGS library production.
본 발명은 Cas9 단백질을 이용한 고효율 어댑터 연결 반응에서 어댑터 이합체의 제거 방법을 제공함에 특징이 있다.The present invention is characterized by providing a method for removing adapter dimers in a highly efficient adapter ligation reaction using the Cas9 protein.
차세대염기서열분석인 NGS(Next-Generation Sequencing) 분석은 DNA 가닥을 하나씩 분석하는 방법으로 기존의 직접 염기서열 분석에 비해 매우 빠르고 저렴하게 염기서열 분석이 가능한 장점이 있다.NGS (Next-Generation Sequencing) analysis, a next-generation sequencing method, is a method of analyzing DNA strands one by one and has the advantage of being able to perform sequence analysis very quickly and inexpensively compared to existing direct sequencing.
이러한 NGS는 DNA를 일정한 조각(fragment)으로 분절화시키고 장비가 인식할 수 있는 특정 염기서열을 가진 올리고뉴클레오티드(oligonucleotide)를 붙여주는 라이브러리(library) 제작단계, 각 라이브러리 DNA 가닥의 염기서열을 장비에서 읽는 단계 및 장비에서 생성된 데이터를 가공하여 알고리즘으로 분석하는 단계로 구성된다.This NGS involves segmenting DNA into certain fragments and attaching oligonucleotides with specific base sequences that can be recognized by the equipment to create a library. The base sequence of each library DNA strand is read by the equipment. It consists of processing the data generated from the steps and equipment and analyzing it with an algorithm.
상기 과정에서 라이브러리 제조과정은 핵산 분절화(fragmentation), 말단수리(end-repair), 3′-말단 아데닌 추가(A-tailing), 어댑터 연결(부착)(adapter ligation) 및 라이브러리 증폭(library amplification) 과정으로 수행되는데, 어댑터 연결 과정에서 형성되는 어댑터 이합체가 분석의 정확도 및 민감도를 떨어뜨리는 문제점이 되고 있다.In the above process, the library preparation process includes nucleic acid fragmentation, end-repair, 3′-end adenine addition (A-tailing), adapter ligation, and library amplification. However, the adapter dimer formed during the adapter connection process is a problem that reduces the accuracy and sensitivity of the analysis.
이에 본 발명자들은 이러한 어댑터 이합체를 효과적으로 감소 또는 제거할 수 있는 방법을 연구하던 중, 어댑터 이합체에만 특이적으로 결합하여 이를 절단하여 제거할 수 있는 gRNA(guide RNA) 및 Cas9 단백질로 구성된 새로운 RNP(ribonucleoprotein) 복합체를 제조하였다.Accordingly, while researching a method to effectively reduce or remove these adapter dimers, the present inventors discovered a new RNP (ribonucleoprotein) composed of gRNA (guide RNA) and Cas9 protein that can specifically bind to and cleave adapter dimers to remove them. ) The composite was prepared.
한편, CRISPR(Clustered regularly interspaced short palindromic repeats)-Cas9 기술은 미생물의 적응면역 현상에서 기인한 유전자 편집 기술로, target DNA를 정밀하게 잘라낸 다음 자연적으로 DNA가 복구되도록 함으로써 보다 간단하고 효율적으로 유전자 조작을 가능하게 한다. 이 시스템은 gRNA(guide RNA)와 Cas9 nuclease 단백질로 구성되며, RNA 유전자 가위(RNA-guided engineered nucleases, RGENs) 기술이라고도 불린다.Meanwhile, CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 technology is a gene editing technology that originates from the adaptive immunity phenomenon of microorganisms. It precisely cuts the target DNA and then allows the DNA to be repaired naturally, making genetic manipulation simpler and more efficient. Make it possible. This system consists of gRNA (guide RNA) and Cas9 nuclease protein, and is also called RNA-guided engineered nucleases (RGENs) technology.
Cas9 단백질은 가이드 RNA(guide RNA, 이하 gRNA)에 의해 유도되는 핵산중간분해효소(Endonuclease)로, gRNA의 20 뉴클레오타이드(Nucleotide) 길이를 갖는 스페이서(Spacer) 부위와 상보적인 표적 서열을 가짐과 동시에 NGG 서열의 PAM(Protospacer Adjacent Motif) 서열을 갖는 이중가닥 DNA에 결합하고 특정 부위에 DNA 이중 가닥 절단(Double-strand break)를 유도한다. 이러한 서열 특이적 절단 기능을 가짐으로 인해 Cas9는 유전체 편집 등 매우 다양한 분야에 응용되고 있다.Cas9 protein is an endonuclease induced by guide RNA (gRNA), and has a target sequence complementary to the 20 nucleotide-long spacer region of gRNA and NGG. It binds to double-stranded DNA with the PAM (Protospacer Adjacent Motif) sequence and induces DNA double-strand break at a specific site. Because of its sequence-specific cutting function, Cas9 is being applied to a wide variety of fields, including genome editing.
또한, gRNA는 타겟 서열에 상보적으로 결합하는 crRNA(spacer 영역)와 Cas9과 결합을 위한 tracrRNA로 구성된다. gRNA는 Cas9을 타겟 부위로 안내하는 역할을 하며, 타겟 DNA 서열과 상보적으로 결합한다. 타겟 DNA 서열의 3’ 말단에는 NGG 서열의 PAM(Protospacer adjacent motif) 서열이 있어야 한다. gRNA가 Cas9 nuclease와 복합체를 형성하고(RNP 형성), 타겟 서열에 특이적으로 결합하면 Cas9 nuclease는 PAM 서열을 인식하고 PAM의 3 bp 업스트림(upstream) 부분에 이중 가닥 손상(Double Strand Break, DSB)을 유도하여 DNA를 절단한다.In addition, gRNA is composed of crRNA (spacer region) that binds complementary to the target sequence and tracrRNA for binding to Cas9. gRNA serves to guide Cas9 to the target site and binds complementary to the target DNA sequence. The 3' end of the target DNA sequence must have a PAM (Protospacer adjacent motif) sequence of the NGG sequence. When the gRNA forms a complex with the Cas9 nuclease (RNP formation) and specifically binds to the target sequence, the Cas9 nuclease recognizes the PAM sequence and causes a double strand break (DSB) 3 bp upstream of the PAM. induces cleavage of DNA.
상기 PAM 서열은 protospacer associated motif로써 CRISPR 시스템에서 핵산가수분해효소가 타겟 DNA를 인지하도록 도와주는 서열로서, PAM 서열은 NGG(N은 A, T, C 또는 G임)로 구성된다.The PAM sequence is a protospacer associated motif that helps nucleic acid hydrolases recognize target DNA in the CRISPR system. The PAM sequence is composed of NGG (N is A, T, C or G).
이에 본 발명자들은 이러한 gRNA-Cas9 단백질을 이용하여 NGS 라이브러리 제조과정에서 발생하는 어댑터 이합체를 특이적으로 절단하여 제조할 수 있는 가이드 RNA을 특정 염기서열로 구성하여 디자인하였다.Accordingly, the present inventors used this gRNA-Cas9 protein to design a guide RNA composed of a specific base sequence that can be produced by specifically cutting the adapter dimer generated during the NGS library production process.
본 발명에서 고안한 NGS 라이브러리 제조과정에서 발생하는 어댑터 이합체 절단용 gRNA(guide RNA)는, 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 스페이서(spacer) 서열을 포함하는 것을 특징으로 한다.The gRNA (guide RNA) for adapter dimer cleavage generated during the NGS library manufacturing process designed in the present invention is characterized by including a spacer sequence consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
본 발명의 서열번호 1 또는 서열번호 3의 gRNA의 스페이서 염기서열은 어댑터 이합체의 염기서열 구성을 보고 착안한 것으로, 어댑터 2개가 연결되었을 때 연결 부위를 기점으로 어댑터 양쪽을 모두 포함하는 서열로 Cas9 표적서열 및 gRNA 스페이서 서열을 약 20nt의 길이가 되도록 서열번호 1 또는 서열번호 3의 서열로 디자인하였다.The spacer base sequence of the gRNA of SEQ ID NO: 1 or SEQ ID NO: 3 of the present invention was conceived by looking at the base sequence composition of the adapter dimer. When two adapters are connected, it is a sequence that includes both adapters starting from the connection site, and is a Cas9 target. The sequence and gRNA spacer sequence were designed as SEQ ID NO: 1 or SEQ ID NO: 3 to be about 20 nt in length.
이때 상기 서열번호 1의 스페이서 서열을 포함하는 gRNA(guide RNA)는 점착 말단 연결(sticky end ligation)의 어댑터 이합체에 상보적으로 결합하는 것일 수 있고, 상기 서열번호 3의 스페이서 서열을 포함하는 gRNA(guide RNA)는 평활 말단 연결(blunt end ligation)의 어댑터 이합체에 상보적으로 결합할 수 있다.At this time, the gRNA (guide RNA) containing the spacer sequence of SEQ ID NO: 1 may bind complementary to the adapter dimer of sticky end ligation, and the gRNA (guide RNA) containing the spacer sequence of SEQ ID NO: 3 guide RNA) can bind complementary to the adapter dimer of blunt end ligation.
따라서 서열번호 1의 스페이서 서열을 포함하는 gRNA는 점착 말단의 연결을 갖는 어댑터 이합체의 제거에 사용될 수 있고, 서열번호 3의 스페이서 서열을 포함하는 gRNA는 평활 말단의 연결을 갖는 어댑터 이합체의 제거에 사용될 수 있다.Therefore, the gRNA containing the spacer sequence of SEQ ID NO: 1 can be used to remove adapter dimers having sticky end connections, and the gRNA containing the spacer sequence of SEQ ID NO: 3 can be used to remove adapter dimers having blunt end connections. You can.
보다 구체적으로 본 발명에 따른 상기 어댑터 이합체 절단용 gRNA의 전체 염기서열은 서열번호 2 또는 서열번호 4로 이루어진 것일 수 있다.More specifically, the entire base sequence of the gRNA for cleaving the adapter dimer according to the present invention may consist of SEQ ID NO: 2 or SEQ ID NO: 4.
또한 본 발명은 상기 본 발명에 따른 어댑터 이합체 절단용 gRNA(guide RNA) 및 Cas9 단백질로 구성되는, NGS 라이브러리 제조과정에서 발생하는 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체를 제공할 수 있다.In addition, the present invention can provide an RNP (ribonucleoprotein) complex for adapter dimer cleavage generated during the NGS library manufacturing process, which is composed of gRNA (guide RNA) for adapter dimer cleavage and Cas9 protein according to the present invention.
상기 RNP(ribonucleoprotein) 복합체에서 어댑터 이합체 절단용 gRNA는 앞서 기술한 바와 같이, 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 스페이서(spacer) 서열을 포함하는 gRNA(guide RNA)와 Cas9 단백질의 복합체이다.As described above, the gRNA for adapter dimer cleavage in the RNP (ribonucleoprotein) complex is a complex of a Cas9 protein and a gRNA (guide RNA) containing a spacer sequence consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3. .
또한 본 발명은 본 발명의 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체를 이용하여 NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체를 절단하여 제거하는 방법을 제공한다.In addition, the present invention provides a method for cutting and removing adapter dimers generated during NGS (Next Generation Sequencing) library production using the ribonucleoprotein (RNP) complex for adapter dimer cleavage of the present invention.
본 발명의 일실시예에서는 NGS 라이브러리 제조 과정에서 어댑터 연결 반응 후, 본 발명의 어댑터 이합체 절단용 RNP 복합체를 처리한 군과 처리하지 않은 군을 대상으로, 반응물 내에 존재하는 어댑터 이합체를 분석한 결과, 본 발명의 RNP 복합체를 처리한 군은 어댑터 이합체가 대부분 절단되어 거의 확인되지 않는 것으로 나타난 반면, 본 발명의 RNP를 처리하지 않은 군은 어댑터 이합체가 상당수 형성되어 있는 것으로 나타났다.In one embodiment of the present invention, after the adapter ligation reaction in the NGS library production process, the adapter dimer present in the reaction was analyzed for the group treated with the RNP complex for adapter dimer cleavage of the present invention and the group not treated, In the group treated with the RNP complex of the present invention, most adapter dimers were cleaved and hardly identified, whereas in the group not treated with the RNP complex of the present invention, a significant number of adapter dimers were found to be formed.
따라서 이러한 결과를 통해 본 발명자들은 본 발명에서 디자인한 어댑터 이합체 절단용 gRNA 및 Cas9의 이합체 절단용 RNP 복합체를 이용할 경우, NGS 라이브러리 제조 과정에서 발생하는 어댑터 이합체를 효과적으로 제거할 수 있음을 알 수 있었다.Therefore, through these results, the present inventors were able to see that when using the adapter dimer cleavage gRNA and Cas9 dimer cleavage RNP complex designed in the present invention, adapter dimers generated during the NGS library production process can be effectively removed.
나아가 본 발명은 NGS 라이브러리 제조 과정에서 어댑터 연결반응의 효율을 향상시킬 수 있는 고효율 어댑터 연결방법을 제공할 수 있다.Furthermore, the present invention can provide a highly efficient adapter linking method that can improve the efficiency of the adapter linking reaction in the NGS library manufacturing process.
본 발명에서 제공하는 고효율 어댑터 연결방법은 표적 DNA와 어댑터를 1:20~1:100의 몰비로 사용하고, PEG-6000(Polyethylene glycol-6000)을 8~10%의 농도로 첨가하여 연결반응을 수행한다는 점에 특징이 있다.The high-efficiency adapter ligation method provided by the present invention uses target DNA and adapter at a molar ratio of 1:20 to 1:100, and adds PEG-6000 (Polyethylene glycol-6000) at a concentration of 8 to 10% to carry out the ligation reaction. The characteristic is that it is performed.
본 발명의 일실시예에서는, NGS 라이브러리 제조에 있어서 어댑터 연결반응을 효율을 향상시킬 수 있는 최적의 반응조건을 규명하기 위한 실험을 수행하였다. 그 결과, 표적 DNA와 어댑터를 다양한 혼합비로 반응에 사용하였고, PEG-6000(Polyethylene glycol-6000)을 다양한 농도로 반응에 사용하였는데, 그 결과, 표적 DNA와 어댑터를 1:20~1:100의 몰비로 사용하고, PEG-6000(Polyethylene glycol-6000)을 8~10%의 농도로 사용할 경우, 어댑터 연결반응 효율이 우수한 것으로 나타났다.In one embodiment of the present invention, an experiment was performed to identify optimal reaction conditions that can improve the efficiency of the adapter ligation reaction in NGS library production. As a result, the target DNA and adapter were used in the reaction at various mixing ratios, and PEG-6000 (Polyethylene glycol-6000) was used in the reaction at various concentrations. As a result, the target DNA and adapter were used in the reaction at a mixing ratio of 1:20 to 1:100. When used in molar ratio and PEG-6000 (Polyethylene glycol-6000) at a concentration of 8 to 10%, the adapter linkage reaction efficiency was found to be excellent.
특히 표적 DNA와 어댑터를 1:50의 몰비로 사용하고, PEG-6000(Polyethylene glycol-6000)을 10%의 농도로 첨가하여 반응한 경우, 표적 DNA의 한 쪽에만 어댑터가 연결된 중간체 반응산물 및 반응이 일어나지 않은 표적 DNA가 거의 존재하지 않고, 대부분 모두 표적 DNA의 양쪽에 어댑터가 연결된 것을 확인할 수 있었다.In particular, when target DNA and adapter are used at a molar ratio of 1:50 and PEG-6000 (Polyethylene glycol-6000) is added at a concentration of 10% for reaction, intermediate reaction products and reactions in which the adapter is connected to only one side of the target DNA There was almost no target DNA where this did not occur, and it was confirmed that most adapters were connected to both sides of the target DNA.
따라서 본 발명에서는 고효율의 어댑터 연결반응 조건을 확립하였다.Therefore, in the present invention, conditions for a highly efficient adapter linkage reaction were established.
또한, 본 발명의 다른 일실시예에서는 본 발명에서 확립한 고효율 어댑터 연결반응 시, 어댑터 이합체가 생성되는 문제점이 여전히 존재하고 있는 것으로 나타나, 어댑터 연결반응 후, 본 발명의 gRNA(guide RNA) 및 Cas9 단백질로 구성되는 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체를 처리한 결과, 어댑터 이합체가 대부분 절단되어 제거되는 것을 확인할 수 있었다.In addition, in another embodiment of the present invention, it appears that there still exists a problem of adapter dimers being generated during the high-efficiency adapter ligation reaction established in the present invention, and after the adapter ligation reaction, the gRNA (guide RNA) of the present invention and Cas9 As a result of treating the RNP (ribonucleoprotein) complex for cleaving adapter dimers composed of proteins, it was confirmed that most of the adapter dimers were cleaved and removed.
이러한 결과를 통해 본 발명에서 확립한 고효율 어댑터 연결 반응을 수행하고 이후 본 발명의 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체를 처리하는 과정을 수행할 경우, 정확도 및 민감도가 우수한 NGS 분석이 가능하다는 것을 알 수 있었다.These results show that NGS analysis with excellent accuracy and sensitivity is possible when performing the high-efficiency adapter ligation reaction established in the present invention and then processing the RNP (ribonucleoprotein) complex for adapter dimer cleavage of the present invention. I was able to.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.
<실시예 1><Example 1>
어댑터 이합체 제거용 gRNA 스페이서 디자인gRNA spacer design for adapter dimer removal
본 발명자들은 NGS 반응의 라이브러리 제조 과정에서 발생하는 어댑터 이합체를 효과적으로 제거할 수 있는 방법으로, Cas9 단백질을 이용할 수 있는 방법을 고안하였고, 다음과 같이 Cas9 단백질이 어댑터 이합체에만 작용하여 절단할 수 있는 gRNA의 스페이서(space) 영역의 특정 염기서열을 디자인하였다.The present inventors have designed a method that uses Cas9 protein as a method to effectively remove adapter dimers generated during the library preparation process of NGS reaction, and the Cas9 protein acts only on adapter dimers to cleave gRNA as follows. A specific base sequence in the spacer region was designed.
gRNA와 결합한 Cas9 단백질은 어댑터 이합체에만 작용하여 절단 기능을 수행하여야 한다. 어댑터 단량체(Monomer)에도 Cas9 RNP가 작용하게 되면 결과적으로 어댑터 단량체가 양쪽에 하나씩 연결된, 어댑터 연결의 목표 산물도 Cas9에 의해 절단되는 문제가 발생할 수 있기 때문이다. 따라서 어댑터 이합체가 형성되었을 때만 만들어지는, 즉 어댑터 두 개가 연결되었을 때 연결 부위를 기점으로 양쪽을 모두 포함하는 서열로 Cas9의 표적 서열 및 gRNA 스페이서 서열을 길이 20nt가 되도록 선정하였다. 그와 동시에 적절한 위치에 PAM 서열인 NGG 서열이 있도록 디자인하였다(도 1 참조). 어댑터 서열 및 본 발명에서 디자인한 어댑터 이합체 제거용 gRNA의 스페이서 서열은 하기 표 1에 나타내었다.The Cas9 protein bound to gRNA must act only on the adapter dimer to perform the cleavage function. If Cas9 RNP acts on the adapter monomer, the target product of the adapter linkage, in which one adapter monomer is connected to each side, may also be cleaved by Cas9. Therefore, the target sequence of Cas9 and the gRNA spacer sequence were selected to be 20 nt in length, which are sequences created only when an adapter dimer is formed, that is, when two adapters are connected, and include both sides of the connection site. At the same time, it was designed to have the NGG sequence, which is the PAM sequence, at the appropriate position (see Figure 1). The adapter sequence and the spacer sequence of the gRNA for adapter dimer removal designed in the present invention are shown in Table 1 below.
| 이름name | 서열 (5’ → 3’)Sequence (5’ → 3’) |
| AdapterAdapter | /5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCUACACTCTTTCCCTACACGACGCTCTTCCGATCT/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCUACACTCTTTCCCTACACGACGCTCTTCCGATCT |
| gRNA spacergRNA spacer | G*U*GUGCUCUUCCGAUCAGAU [서열번호 1] G*U*G UGCUCUUCCGAUCAGAU [SEQ ID NO: 1] |
|
gRNA (cr RNA) gRNA (crRNA) |
G*U*GUGCUCUUCCGAUCAGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU*U [서열번호 2] G*U*G UGCUCUUCCGAUCAGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UU*U [SEQ ID NO: 2] |
| gRNA spacergRNA spacer | A*U*GUGGCUCUUCCGAUCGAU [서열번호 3] A*U*G UGGCUCUUCCGAUCGAU [SEQ ID NO: 3] |
|
gRNA (cr RNA) gRNA (crRNA) |
A*U*GUGGCUCUUCCGAUCGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU*U [서열번호 4] A*U*G UGGCUCUUCCGAUCGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UU*U [SEQ ID NO. 4] |
상기 표 1에서 밑줄은 2‘-O-메틸 유사체(2’-O-methyl analog)를 표시한 것이고, *는 포스포로티오에이트 뉴클레오티드 간 결합(phosphorothioate internucleotide linkage)을 나타낸 것이다.In Table 1, the underline indicates a 2'-O-methyl analog, and * indicates a phosphorothioate internucleotide linkage.
<실시예 2><Example 2>
고효율 어댑터 연결반응의 조건 확립 및 본 발명의 gRNA와 Cas9을 이용한 어댑터 이합체 절단 효과 확인Establishment of conditions for high-efficiency adapter ligation reaction and confirmation of adapter dimer cleavage effect using gRNA of the present invention and Cas9
<2-1> 고효율 어댑터 연결반응의 최적 조건 확립<2-1> Establishment of optimal conditions for high-efficiency adapter coupling reaction
본 발명자들은 NGS 라이브러리 제조 과정에서, 어댑터 연결반응의 효율을 향상시킬 수 있는 반응 조건을 확립하기 위한 실험을 수행하였다.The present inventors performed experiments to establish reaction conditions that could improve the efficiency of the adapter ligation reaction during the NGS library production process.
종래 NGS 라이브러리 제조에 있어서 어댑터 연결의 효율이 낮아 어댑터 이합체가 생성되는 문제점이 있었다. 이에 본 발명자들은 어댑터 연결반응에 사용되는 어댑터들이 최대한 모두 반응에 참여하여 어댑터 이합체의 형성을 감소시키고 동시에 연결 반응의 효율을 증가시킬 수 있는 조건을 찾기 위하여, 어댑터를 연결시킬 표적 DNA로, 인간 게놈 DNA(Human genomic DNA)로부터 KRAS 유전자의 일부를 PCR로 증폭한 149 뉴클레오타이드의 길이를 갖는 DNA 파편을 이용하였고, 프라이머는 IDT로부터 구매한 것을 사용하였는데, PCR 프라이머 서열과 증폭 산물로 생성되는 표적 DNA의 서열은 하기 표 2에 나타내었다.In conventional NGS library production, there was a problem in that adapter dimers were generated due to low adapter ligation efficiency. Accordingly, in order to find conditions in which all adapters used in the adapter ligation reaction can participate in the reaction as much as possible to reduce the formation of adapter dimers and at the same time increase the efficiency of the ligation reaction, the present inventors used the target DNA to link the adapter to the human genome. A DNA fragment with a length of 149 nucleotides was used by amplifying part of the KRAS gene from DNA (human genomic DNA) by PCR, and primers purchased from IDT were used. The PCR primer sequence and the target DNA generated as an amplification product were used. The sequences are shown in Table 2 below.
연결반응은 A-테일링이 완료된 표적 DNA, 어댑터, PEG-6000, 1X Ultra II Ligation module(NEB), 증류수를 혼합하여 20℃에서 15분간 동안 수행하였으며, 최적의 연결반응을 위한 조건 확립을 위해 표적 DNA 주형과 어댑터의 첨가량을 각각 다른 몰비(표적 DNA 주형:어댑터=1:3.75, 1:20, 1:50, 1:100)를 사용하였고, PEG-6000 첨가 농도를 각기 달리 첨가하여 수행하였다.The ligation reaction was performed for 15 minutes at 20°C by mixing A-tailed target DNA, adapter, PEG-6000, 1X Ultra II Ligation module (NEB), and distilled water. To establish conditions for optimal ligation reaction, Different molar ratios of DNA template and adapter were used (target DNA template: adapter = 1:3.75, 1:20, 1:50, 1:100), and PEG-6000 was added at different concentrations.
| 이름name | 서열 (5’ → 3’)Sequence (5’ → 3’) |
Tm value (℃)TM value (℃) |
| KRAS_FPKRAS_FP | /5Phos/TTGTGGTAGTTGGAGCTGGT/5Phos/TTGTGGTAGTTGGAGCTGGT | 61.361.3 |
| KRAS_RPKRAS_RP | /5Phos/CTGTATCAAAGAATGGTCCTGCAC/5Phos/CTGTATCAAAGAATGGTCCTGCAC | 61.661.6 |
| KRAS targetKRAS target | /5Phos/TTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAG/5Phos/TTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCACAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAG | -- |
그 결과, 도 2에 나타낸 바와 같이, 반응 결과물은 7.5% Native PAGE로 확인하였는데, 어댑터 연결 반응이 일어나지 않은 표적 DNA는 초록색 박스로 표시하였고, 표적 DNA 한 쪽에만 어댑터가 결합한 중간체는 보라색 박스로 표시하였고, 표적 DNA 양쪽에 어댑터가 결합한 최종 생성물은 빨간색 박스로 표시하였다. 또한, 어댑터 이합체는 파란색 박스로 표시하였다.As a result, as shown in Figure 2, the reaction product was confirmed by 7.5% Native PAGE. The target DNA in which the adapter ligation reaction did not occur is indicated by a green box, and the intermediate in which the adapter binds to only one side of the target DNA is indicated by a purple box. The final product, in which the adapter is bound to both sides of the target DNA, is indicated by a red box. Additionally, the adapter dimer is indicated by a blue box.
표적 DNA와 어댑터의 혼합비를 달리하여 분석한 결과, 어댑터 비율이 증가할수록 연결 반응의 효율이 증가하는 것으로 나타났고, 어댑터 이합체의 형성도 어댑터 비율 증가에 따라 감소하는 것으로 나타났다. 본 발명의 실험에 의하면, 표적 DNA와 어댑터의 비율이 1:50인 경우, 어댑터 이합체의 형성이 작고 표적 DNA의 양쪽에 모두 어댑터가 연결된 양이 가장 많은 것을 확인함에 따라 상기 첨가 비율로 반응시키는 것이 가장 효과적으로 어댑터 연결반응이 이루어진다는 것을 알 수 있었다.As a result of analyzing different mixing ratios of target DNA and adapter, the efficiency of the ligation reaction was found to increase as the adapter ratio increased, and the formation of adapter dimers was also found to decrease as the adapter ratio increased. According to the experiment of the present invention, when the ratio of target DNA and adapter is 1:50, the formation of adapter dimers is small and the amount of adapters linked to both sides of the target DNA is the largest. Therefore, it is recommended to react at the above addition ratio. It was found that the adapter connection reaction occurred most effectively.
또한, PEG-6000의 첨가량을 달리하여 어댑터 연결 반응을 수행하였다. PEG-6000의 사용에 따라 어댑터 연결반응에 밀도를 증진시키는 효과를 유도하여 어댑터 연결 효율을 상승시킬 수 있으나, 사용하는 T4 라이게이즈(T4 ligase)의 정확도가 제한되므로 어댑터 연결 반응의 효율이 상승함에 따라 불가피하게 어댑터 이합체가 생성되는 문제점이 있다.Additionally, adapter ligation reaction was performed by varying the amount of PEG-6000 added. The use of PEG-6000 can increase the efficiency of the adapter connection by inducing a density-enhancing effect in the adapter connection reaction, but the accuracy of the T4 ligase used is limited, so the efficiency of the adapter connection reaction increases. As a result, there is a problem that adapter dimers are inevitably generated.
도 3의 결과에 의하면, PEG-6000 농도를 0%에서 10%로 점차적으로 증가시킴에 따라 어댑터 이합체 양도 증가되는 것으로 나타났다. 한편, 10%의 PEG-6000 첨가군이 다른 실험군에 비해 표적 DNA의 양쪽에 모두 어댑터가 연결된 형태의 반응 산물이 가장 많은 것으로 나타나, 고효율 어댑터 연결반응은 10%의 PEG-6000 조건에서 수행하는 것이 가장 효과적임을 알 수 있었다. 나아가 10%를 초과한 농도로 PEG-6000을 처리한 군들에서는 10%의 PEG-6000 첨가군과 거의 유사한 수준의 어댑터 연결 효과가 확인되어 10% 농도를 초과하여 PEG-6000을 사용하는 것이 경제적이지 못하다는 것을 확인하였다.According to the results in Figure 3, as the PEG-6000 concentration was gradually increased from 0% to 10%, the amount of adapter dimer also increased. Meanwhile, the 10% PEG-6000 addition group showed the largest number of reaction products with adapters linked to both sides of the target DNA compared to the other experimental groups, so it is recommended that the highly efficient adapter linkage reaction be performed under 10% PEG-6000 conditions. It was found to be the most effective. Furthermore, in the groups treated with PEG-6000 at a concentration exceeding 10%, an almost similar level of adapter linking effect was confirmed as in the group with 10% PEG-6000 added, making it uneconomical to use PEG-6000 at a concentration exceeding 10%. It was confirmed that it was not possible.
<2-2> 고효율 어댑터 연결반응 조건에서 본 발명의 gRNA와 Cas9을 이용한 어댑터 이합체 절단 활성 분석<2-2> Analysis of adapter dimer cleavage activity using the gRNA of the present invention and Cas9 under high-efficiency adapter ligation reaction conditions
상기 실시예 <2-1>의 실험을 통해 어댑터 연결을 위한 최적의 반응조건이 표적 DNA와 어댑터의 몰비율을 1:50으로 하고, 10%의 PEG-6000을 이용하는 것임을 확인하였다. 한편, 상기 최적의 연결반응 조건의 경우 어댑터 이합체가 생성되는 것을 확인함에 따라, 상기 연결반응 조건에서 본 발명에서 디자인한 gRNA와 Cas9을 이용할 경우, 형성된 어댑터 이합체를 효과적으로 절단(제거)할 수 있는지를 확인하였다. 이를 위해 다음과 같이 실험을 수행하였다.Through the experiment in Example <2-1>, it was confirmed that the optimal reaction conditions for adapter linkage were to set the molar ratio of target DNA to adapter at 1:50 and use 10% PEG-6000. Meanwhile, as it was confirmed that an adapter dimer is generated under the optimal ligation reaction conditions, it was investigated whether the formed adapter dimer can be effectively cut (removed) when using the gRNA and Cas9 designed in the present invention under the above ligation reaction conditions. Confirmed. For this purpose, an experiment was performed as follows.
먼저 8 μM의 SpCas9 단백질, 8 μM의 본 발명의 gRNA, 1X NEB buffer r2.1(NEB) 및 증류수를 혼합하고 상온에서 10분간 놔두어 RNP 복합체가 형성되도록 하였다. 이후 상기 실시예 <2-1>의 연결 반응을 진행했던 샘플에 40 피코몰의 Cas9 RNP, 1X NEBuffer 2.1(NEB) 및 증류수를 첨가하고 37℃에서 60분간 반응을 진행하였다. 반응 후에는 4 μL의 Proteinase K, 4 μL의 RNase A를 처리하여 Cas9 RNP가 분해되도록 하였으며, 대조군으로는 Cas9 반응을 진행하지 않은 것을 사용하였다.First, 8 μM of SpCas9 protein, 8 μM of the gRNA of the present invention, 1X NEB buffer r2.1 (NEB), and distilled water were mixed and left at room temperature for 10 minutes to form an RNP complex. Then, 40 picomole of Cas9 RNP, 1 After the reaction, the Cas9 RNP was decomposed by treatment with 4 μL of Proteinase K and 4 μL of RNase A. As a control, one without Cas9 reaction was used.
그 결과, 도 4에 나타낸 바와 같이, 어댑터 연결반응에서 발생하는 어댑터 이합체들이 본 발명의 gRNA를 포함하는 Cas9 RNP을 처리한 경우, 어댑터 이합체가 효과적으로 절단되어 제거됨을 확인할 수 있었다. 반면, 본 발명의 gRNA를 포함하는 Cas9 RNP을 처리하지 않은 군은 어댑터 이합체가 형성된 채로 반응 생성물에 존재하고 있음을 확인하였다.As a result, as shown in Figure 4, it was confirmed that the adapter dimers generated in the adapter ligation reaction were effectively cleaved and removed when the Cas9 RNP containing the gRNA of the present invention was treated. On the other hand, it was confirmed that the group that was not treated with Cas9 RNP containing the gRNA of the present invention existed in the reaction product with the adapter dimer formed.
이러한 결과를 통해, 본 발명자들은 본 발명에서 고안한 어댑터 서열에 특이적으로 결합할 수 있는 특정 서열로 구성된 gRNA 스페이서 서열을 포함하는 gRNA와 Cas9을 이용한 Cas9 RNP 복합체를 이용할 경우, NGS 라이브러리 제조에 문제가 되고 있는 어댑터 이합체를 특이적으로 절단 및 제거할 수 있음을 알 수 있었다. Through these results, the present inventors found that when using the Cas9 RNP complex using Cas9 and a gRNA containing a gRNA spacer sequence composed of a specific sequence that can specifically bind to the adapter sequence designed in the present invention, there is a problem in NGS library production. It was found that the adapter dimer could be specifically cut and removed.
<실시예 3><Example 3>
NGS 라이브러리 제조에 있어서 본 발명에 따른 gRNA 및 Cas9을 이용한 어댑터 이합체 제거방법의 적용 확인Confirmation of application of adapter dimer removal method using gRNA and Cas9 according to the present invention in NGS library production
나아가 본 발명자들은 본 발명에서 디자인한 어댑터 이합체 절단을 위한 gRNA 및 Cas9을 이용한 어댑터 이합체 제거기술이 NGS 라이브러리 제조에 실제적으로 잘 적용되어 작동되는지를 확인하기 위한 실험을 수행하였다.Furthermore, the present inventors conducted an experiment to confirm whether the adapter dimer removal technology using gRNA and Cas9 for adapter dimer cleavage designed in the present invention can be effectively applied to NGS library production.
이를 위해, 40 ng의 표적 DNA를 0.1X TE buffer 50 uL에 희석한 후 1X Ultra II End prep module(NEB)을 혼합하여 엔드 리페어(End-repair) 및 A-테일링(A-tailing) 반응을 진행하였다. 어댑터 연결 반응을 진행하기 직전, 어댑터는 95°C에서 5분간 처리한 후 0.1°C/s의 속도로 25°C까지 램핑 다운 (Ramping down)하여 25°C에서의 15분 반응을 통해 헤어핀 구조를 형성하도록 하였고, 이후 상기 본 발명에서 확립한 최적의 고효율 어댑터 연결 반응 조건으로 어댑터 연결반응을 수행하였다. 어댑터 연결 반응이 완료된 샘플에 USER 효소(NEB)를 각 3 uL씩 처리하였고 37°C에서 15분간 반응을 진행하였다. 이후 Monarch DNA Cleanup kit (NEB)를 사용하여 정제과정을 거친 이후, 상기 실시예 <2-2>의 본 발명의 gRNA와 Cas9을 이용한 어댑터 이합체 절단 반응을 진행하였다. 어댑터 이합체 절단 반응이 완료된 샘플은 0.9X SPRIselect bead (Beckman)로 정제하였으며 Ultra II Q5 master mix (NEB), NEBNext Multiplex Oligos for Illumina(96 Unique Dual Index Primer Pairs) (NEB)를 이용하여 6 사이클의 PCR 증폭 과정을 진행했다. 최종적으로 0.9X SPRIselect bead (Beckman)를 사용하여 정제한 후 dsDNA high sensitivity assay (DeNovix)를 이용해 최종 샘플 농도를 측정하였고, Native PAGE 7.5% 전기영동을 통해 확인하였다. 또한, 대조군(Ct)으로는 상업 키트(NEB Next Ultra II DNA Library Prep Kit for Illumina, NEB)를 이용한 군을 사용하였다.For this purpose, 40 ng of target DNA is diluted in 50 uL of 0.1X TE buffer and then mixed with 1X Ultra II End prep module (NEB) to perform end-repair and A-tailing reactions. did. Immediately before proceeding with the adapter ligation reaction, the adapter is treated at 95°C for 5 minutes and then ramped down to 25°C at a rate of 0.1°C/s to form a hairpin structure through a 15-minute reaction at 25°C. was formed, and then the adapter ligation reaction was performed under the optimal high-efficiency adapter ligation reaction conditions established in the present invention. Samples for which the adapter ligation reaction was completed were treated with 3 uL of USER enzyme (NEB), and the reaction was performed at 37°C for 15 minutes. After purification using the Monarch DNA Cleanup kit (NEB), an adapter dimer cleavage reaction was performed using the gRNA of the present invention and Cas9 in Example <2-2>. The sample for which the adapter dimer cleavage reaction was completed was purified using 0.9 The amplification process was carried out. Finally, after purification using 0.9X SPRIselect beads (Beckman), the final sample concentration was measured using dsDNA high sensitivity assay (DeNovix) and confirmed through Native PAGE 7.5% electrophoresis. In addition, as the control group (Ct), a group using a commercial kit (NEB Next Ultra II DNA Library Prep Kit for Illumina, NEB) was used.
그 결과, 도 5에 나타낸 바와 같이, 상업 키트를 사용한 대조군의 경우, 표적 DNA의 한 쪽에만 어댑터가 연결된 중간체 반응산물 및 반응이 일어나지 않은 표적 DNA가 그대로 존재하는 것을 확인하였다. 이러한 산물이 존재하는 경우, NGS 분석에 적합하지 않다. 또한, 본 발명의 gRNA 및 Cas9 단백질을 처리하지 않은 군에서는 최적의 연결반응 조건에서 반응을 수행하였기에 높은 어댑터 연결효율을 보였으나, 최종 NGS 라이브러리 샘플 상에서도 어댑터 이합체 밴드가 확인되었다. 어댑터 이합체는 추후 NGS 분석 과정에서 방해물로 작용할 수 있어 해당 실험군 역시 NGS 분석에 적합하지 않다는 것을 알 수 있었다.As a result, as shown in Figure 5, in the case of the control group using a commercial kit, it was confirmed that an intermediate reaction product with an adapter connected to only one side of the target DNA and a target DNA in which no reaction occurred remained as is. If these products are present, they are not suitable for NGS analysis. In addition, the group not treated with the gRNA and Cas9 protein of the present invention showed high adapter ligation efficiency because the reaction was performed under optimal ligation reaction conditions, but an adapter dimer band was also confirmed on the final NGS library sample. Since the adapter dimer can act as an obstacle in the later NGS analysis process, it was found that the experimental group was also not suitable for NGS analysis.
반면, 본 발명의 gRNA 및 Cas9 단백질을 처리한 군의 경우, 높은 어댑터 연결 반응의 효율이 유지되면서(표적 DNA의 양쪽에 모두 어댑터가 연결된 산물이 다량 형성됨), 동시에 어댑터 이합체는 완전히 절단되어 제거되고 반응이 일어나지 않은 표적 DNA도 거의 없는 것을 확인할 수 있었다.On the other hand, in the case of the group treated with the gRNA and Cas9 protein of the present invention, the high adapter ligation reaction efficiency is maintained (a large amount of adapter-linked products are formed on both sides of the target DNA), and at the same time, the adapter dimer is completely cleaved and removed. It was confirmed that there was almost no target DNA that did not react.
이상의 결과를 통해, 본 발명자들은 본 발명에서 규명한 고효율 어댑터 연결반응 조건을 수행할 경우, 매우 효과적으로 표적 DNA의 양쪽에 모두 어댑터가 연결된 산물을 형성시킬 수 있음을 알 수 있었고, 또한 본 발명에서 고안한 특정 스페이서 서열을 갖는 gRNA 및 Cas9을 이용할 경우, 어댑터 연결반응에서 발생하는 어댑터 이합체를 효과적으로 제거할 수 있어 보다 정확하고 효과적인 NGS 분석 결과를 얻는데 유용하게 사용할 수 있음을 알 수 있었다.Through the above results, the present inventors were able to see that when performing the high-efficiency adapter ligation reaction conditions identified in the present invention, it is possible to very effectively form a product in which adapters are linked to both sides of the target DNA. It was found that when gRNA and Cas9 with a specific spacer sequence are used, adapter dimers generated in the adapter ligation reaction can be effectively removed, making it useful for obtaining more accurate and effective NGS analysis results.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.
Claims (12)
- 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 스페이서(spacer) 서열을 포함하는,Containing a spacer sequence consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3,NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체 절단용 gRNA(guide RNA).gRNA (guide RNA) for cutting adapter dimers generated during the NGS (Next Generation Sequencing) library manufacturing process.
- 제1항에 있어서,According to paragraph 1,상기 서열번호 1의 스페이서 서열을 포함하는 gRNA(guide RNA)는 점착 말단 연결(sticky end ligation)의 어댑터 이합체에 상보적으로 결합하는 것을 특징으로 하는, NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체 절단용 gRNA(guide RNA).The gRNA (guide RNA) containing the spacer sequence of SEQ ID NO: 1 is characterized in that it binds complementary to the adapter dimer of sticky end ligation, which occurs during the NGS (Next Generation Sequencing) library manufacturing process. gRNA (guide RNA) for adapter dimer cleavage.
- 제1항에 있어서,According to paragraph 1,상기 서열번호 3의 스페이서 서열을 포함하는 gRNA(guide RNA)는 평활 말단 연결(blunt end ligation)의 어댑터 이합체에 상보적으로 결합하는 것을 특징으로 하는, NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체 절단용 gRNA(guide RNA).The gRNA (guide RNA) containing the spacer sequence of SEQ ID NO: 3 is characterized in that it binds complementary to an adapter dimer of blunt end ligation, which occurs during the NGS (Next Generation Sequencing) library manufacturing process. gRNA (guide RNA) for adapter dimer cleavage.
- 제2항에 있어서,According to paragraph 2,상기 어댑터 이합체 절단용 gRNA는 서열번호 2의 염기서열로 이루어진 것을 특징으로 하는, NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체 절단용 gRNA(guide RNA).The gRNA for adapter dimer cleavage is a gRNA (guide RNA) for adapter dimer cleavage generated during NGS (Next Generation Sequencing) library manufacturing, characterized in that it consists of the base sequence of SEQ ID NO: 2.
- 제3항에 있어서,According to paragraph 3,상기 어댑터 이합체 절단용 gRNA는 서열번호 4의 염기서열로 이루어진 것을 특징으로 하는, NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체 절단용 gRNA(guide RNA).The gRNA for adapter dimer cleavage is a gRNA (guide RNA) for adapter dimer cleavage generated during NGS (Next Generation Sequencing) library manufacturing, characterized in that it consists of the base sequence of SEQ ID NO: 4.
- 제1항의 gRNA(guide RNA) 및 Cas9 단백질로 구성되는, NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체.An RNP (ribonucleoprotein) complex for adapter dimer cleavage generated during NGS (Next Generation Sequencing) library manufacturing, consisting of the gRNA (guide RNA) of claim 1 and the Cas9 protein.
- 제6항의 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체를 이용하여 NGS(Next Generation Sequencing) 라이브러리 제조과정에서 발생하는 어댑터 이합체를 절단하여 제거하는 방법. A method of cutting and removing adapter dimers generated during NGS (Next Generation Sequencing) library manufacturing using the RNP (ribonucleoprotein) complex for adapter dimer cleavage of claim 6.
- 제7항에 있어서,In clause 7,상기 어댑터 이합체는 NGS 라이브러리 제조 과정 시, 표적 DNA에 대한 어댑터의 고효율 연결반응 시 생성되는 것을 특징으로 하는, 어댑터 이합체를 절단하여 제거하는 방법.A method for cutting and removing an adapter dimer, characterized in that the adapter dimer is generated during a high-efficiency ligation reaction of the adapter to the target DNA during the NGS library manufacturing process.
- 제8항에 있어서,According to clause 8,상기 고효율 연결반응은 표적 DNA와 어댑터를 1:20~1:100의 몰비로 사용하고, PEG-6000(Polyethylene glycol-6000)을 8~10%의 농도로 첨가하여 반응하는 것을 특징으로 하는, 어댑터 이합체를 절단하여 제거하는 방법.The high-efficiency ligation reaction is characterized in that the target DNA and adapter are used at a molar ratio of 1:20 to 1:100, and PEG-6000 (Polyethylene glycol-6000) is added at a concentration of 8 to 10%. Method for cutting and removing dimers.
- 제9항에 있어서,According to clause 9,상기 고효율 연결반응은 표적 DNA와 어댑터를 1:50의 몰비로 사용하고, PEG-6000(Polyethylene glycol-6000)을 10%의 농도로 첨가하여 반응하는 것을 특징으로 하는, 어댑터 이합체를 절단하여 제거하는 방법.The high-efficiency ligation reaction uses target DNA and adapter at a molar ratio of 1:50, and PEG-6000 (Polyethylene glycol-6000) is added at a concentration of 10% to cleave and remove the adapter dimer. method.
- NGS(Next Generation Sequencing) 라이브러리 제조과정에서 어댑터 연결반응을, 표적 DNA와 어댑터를 1:20~1:100의 몰비로 사용하고, PEG-6000(Polyethylene glycol-6000)을 8~10%의 농도로 첨가하여 반응하는 것을 특징으로 하는, 고효율 어댑터 연결방법.In the NGS (Next Generation Sequencing) library manufacturing process, adapter ligation reaction is performed using target DNA and adapter at a molar ratio of 1:20 to 1:100, and PEG-6000 (Polyethylene glycol-6000) at a concentration of 8 to 10%. A high-efficiency adapter connection method characterized by adding and reacting.
- 제11항에 있어서,According to clause 11,어댑터 연결반응 후, 제1항의 gRNA(guide RNA) 및 Cas9 단백질로 구성되는 어댑터 이합체 절단용 RNP(ribonucleoprotein) 복합체를 처리하는 단계를 더 포함하는 것을 특징으로 하는, 고효율 어댑터 연결방법.After the adapter ligation reaction, a highly efficient adapter ligation method further comprising the step of processing a ribonucleoprotein (RNP) complex for adapter dimer cleavage consisting of the gRNA (guide RNA) of claim 1 and the Cas9 protein.
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