KR20230149445A - Recombinant vector for activity test of guide RNA for CRISPR-based base editing and uses thereof - Google Patents
Recombinant vector for activity test of guide RNA for CRISPR-based base editing and uses thereof Download PDFInfo
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Abstract
본 발명은 표적 서열 발현 카세트, 디아미나아제(deaminase) 및 엔도뉴클레아제(endonuclease) 발현 카세트 및 가이드 RNA (guide RNA) 발현 카세트를 포함하는, 크리스퍼(CRISPR) 기반 염기 교정(base editing)용 가이드 RNA의 활성 검정용 재조합 벡터 및 이의 용도에 관한 것으로, 본 발명에 따른 재조합 벡터를 이용하면 대장균에서 CRISPR 기반 염기 교정을 위한 후보 gRNA의 활성을 신속하게 스크리닝하여, 시간, 비용 및 노동 소모 없이 바로 식물체에 적용 가능한 염기교정 시스템을 개발할 수 있으므로, 유전자 교정 분야에 유용하게 사용될 수 있을 것이다.The present invention is for CRISPR-based base editing, including a target sequence expression cassette, a deaminase and endonuclease expression cassette, and a guide RNA expression cassette. This relates to a recombinant vector for testing the activity of guide RNA and its use. Using the recombinant vector according to the present invention, the activity of a candidate gRNA for CRISPR-based base correction can be quickly screened in E. coli without consuming time, money, or labor. Since it is possible to develop a base correction system applicable to plants, it will be useful in the field of gene editing.
Description
본 발명은 크리스퍼 기반 염기 교정용 가이드 RNA의 활성 검정용 재조합 벡터 및 이의 용도에 관한 것이다.The present invention relates to a recombinant vector for testing the activity of a guide RNA for CRISPR-based base correction and its use.
현대 농업에서 작물의 생산성 증대와 스트레스 저항성 형질 획득은 매우 중요한 과제이다. 최근, 목적하는 유전자만을 특이적으로 교정하는 CRISPR/Cas9 시스템을 사용한 유전체 변형 작물들이 개발되었다. 이러한 CRISPR/Cas9 시스템은 간편성과 효율성 덕에 유전자 편집 기술 분야에 폭넓게 활용되고 있으며, 해당 분야의 획기적인 발전을 이루었다. 하지만, CRISPR/Cas9 시스템은 DNA 절단 후, 생물의 무작위적인 복구 시스템을 이용하기 때문에 정확히 원하는 DNA 서열을 얻기 힘들다는 점과, 유전자의 기능을 없애는 방향으로만 사용 가능하다는 단점이 있다. 이러한 CRISPR/Cas9 시스템의 단점을 극복하기 위한 대안으로 염기교정(base editor, BE)이 개발되고 있다. In modern agriculture, increasing crop productivity and acquiring stress resistance traits are very important tasks. Recently, genome-modified crops using the CRISPR/Cas9 system, which specifically corrects only the target gene, have been developed. Thanks to its simplicity and efficiency, the CRISPR/Cas9 system is widely used in the field of gene editing technology and has achieved groundbreaking developments in the field. However, the CRISPR/Cas9 system has the disadvantage of making it difficult to obtain exactly the desired DNA sequence because it uses the organism's random repair system after DNA cutting, and that it can only be used to eliminate the function of a gene. Base editor (BE) is being developed as an alternative to overcome these shortcomings of the CRISPR/Cas9 system.
염기교정은 CRISPR/Cas9 시스템의 목적 특이성과 디아미나아제(deaminase)의 특성을 활용한 기술로, 이중가닥절단(double strand break, DSB)과 외래 DNA의 도입 없이도 원하는 서열의 돌연변이를 일으킬 수 있으며, 식물체에 적용가능한 염기교정 도구로 ABE(A to G base editor) 및 CBE(C to T base editor)가 개발되었다. 염기교정 도구 및 구성 요소(sgRNA 등)의 평가는 성공적인 유전자 교정을 위한 중요한 과정인데, 원형질체 또는 아그로박테리움 매개 형질전환법을 이용해야하는 식물 시스템은 시간, 비용 및 노동 소모가 많으므로 박테리아 시스템을 이용하는 것이 유리하다. 그러나, 박테리아에서는 CRISPR/Cas9에 의해 생성된 DNA 이중가닥절단(DSB)을 수선할 수 있는 비상동 말단 연결 경로(Non-homologous end joining, NHEJ)가 부족하여 세포 사멸이 유도되는 문제가 있어, DSB를 만들지 않는 DSB-free 염기 교정 시스템이 주로 사용되었는데 이는 강력한 프로모터를 사용하여 발현되는 BE 시약에 의해 세포 독성이 유발된다는 문제가 있다. 이에, 본 발명자는 대장균에서 세포 독성을 유발하지 않으면서, 쉽고 빠르게 유전자 교정 도구 및 구성 요소의 기능성을 평가할 수 있는 벡터 시스템을 개발하고자 하였다.Base correction is a technology that utilizes the target specificity of the CRISPR/Cas9 system and the characteristics of deaminase, and can cause mutations in the desired sequence without a double strand break (DSB) or introduction of foreign DNA. ABE (A to G base editor) and CBE (C to T base editor) were developed as base correction tools applicable to plants. Evaluation of base editing tools and components (sgRNA, etc.) is an important process for successful gene editing. Plant systems that require protoplast or Agrobacterium-mediated transformation require a lot of time, cost, and labor, so bacterial systems are used. It is advantageous. However, in bacteria, there is a problem that cell death is induced due to the lack of a non-homologous end joining (NHEJ) pathway that can repair DNA double-strand breaks (DSBs) generated by CRISPR/Cas9. A DSB-free base correction system that does not produce has been mainly used, but this has the problem of causing cytotoxicity due to BE reagents expressed using a strong promoter. Accordingly, the present inventor sought to develop a vector system that can easily and quickly evaluate the functionality of gene editing tools and components without causing cytotoxicity in E. coli.
한편, 한국공개특허 제2017-0087959호에는 '원형 폴리뉴클레오티드 변형 주형과 함께 가이드 RNA/Cas 엔도뉴클레아제 시스템을 이용하여 대장균에서 효율적으로 유전자 편집을 하기 위한 조성물 및 방법'이 개시되어 있고, 한국등록특허 제1804145호에는 '대장균의 지놈 엔지니어링용 플라스미드 및 이를 이용한 대장균의 지놈 엔지니어링 방법'이 개시되어 있으나, 본 발명의 크리스퍼 기반 염기 교정용 가이드 RNA의 활성 검정용 재조합 벡터 및 이의 용도에 대해서는 기재된 바가 없다.Meanwhile, Korean Patent Publication No. 2017-0087959 discloses 'A composition and method for efficient gene editing in E. coli using a guide RNA/Cas endonuclease system with a circular polynucleotide modification template,' and is disclosed in Korea. Registration Patent No. 1804145 discloses 'E. coli genome engineering plasmid and E. coli genome engineering method using the same', but the recombinant vector for testing the activity of the guide RNA for CRISPR-based base correction of the present invention and its use are not described. There is no bar.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명에서는 대장균에서 세포 독성을 유발하지 않는 GlpT 프로모터-L3S2P21 터미네이터 또는 35S 프로모터-35S 터미네이터 조합을 선발하여 대장균에서 작동 가능한 재조합 벡터 시스템을 구축하였고, 이미 알려져 있는 gRNA를 상기 재조합 벡터 시스템에 적용하여 염기전환율을 분석한 결과, gRNA의 활성을 효과적으로 검정할 수 있는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above needs. In the present invention, a combination of GlpT promoter-L3S2P21 terminator or 35S promoter-35S terminator that does not cause cytotoxicity in E. coli was selected to construct a recombinant vector system operable in E. coli, As a result of analyzing the base conversion rate by applying a known gRNA to the above recombinant vector system, it was confirmed that the activity of gRNA could be effectively tested, thereby completing the present invention.
상기 과제를 해결하기 위해, 본 발명은 표적 서열 발현 카세트, 디아미나아제(deaminase) 및 엔도뉴클레아제(endonuclease) 발현 카세트 및 가이드 RNA (guide RNA) 발현 카세트를 포함하는, 크리스퍼(CRISPR) 기반 염기 교정(base editing)용 가이드 RNA의 활성 검정용 재조합 벡터를 제공한다.In order to solve the above problems, the present invention is a CRISPR-based method comprising a target sequence expression cassette, a deaminase and endonuclease expression cassette, and a guide RNA expression cassette. Provides a recombinant vector for testing the activity of guide RNA for base editing.
또한, 본 발명은 상기 재조합 벡터를 유효성분으로 포함하는 크리스퍼(CRISPR) 기반 염기 교정(base editing)용 가이드 RNA (guide RNA) 활성 검정용 조성물을 제공한다.In addition, the present invention provides a composition for testing the activity of guide RNA for CRISPR-based base editing containing the recombinant vector as an active ingredient.
또한, 본 발명은 상기 재조합 벡터로 숙주세포를 형질전환하는 단계; 및 상기 형질전환된 숙주세포로부터 핵산을 추출하고 가이드 RNA의 표적 서열의 염기서열을 분석하는 단계;를 포함하는, 크리스퍼(CRISPR) 기반 염기 교정(base editing)용 가이드 RNA (guide RNA)의 활성을 검정하는 방법을 제공한다.Additionally, the present invention includes the steps of transforming a host cell with the recombinant vector; And extracting nucleic acid from the transformed host cell and analyzing the base sequence of the target sequence of the guide RNA. Activity of guide RNA for CRISPR-based base editing, including Provides a method for testing.
본 발명의 크리스퍼(CRISPR) 기반 염기 교정용 가이드 RNA의 활성 검정용 재조합 벡터를 이용하면 대장균에서 CRISPR 기반 염기 교정을 위한 후보 gRNA의 활성을 신속하게 스크리닝하여, 시간, 비용 및 노동 소모 없이 바로 식물체에 적용 가능한 염기교정 시스템을 개발할 수 있으므로, 유전자 교정 분야에 유용하게 사용될 수 있을 것이다.By using the recombinant vector for testing the activity of the guide RNA for CRISPR-based base correction of the present invention, the activity of candidate gRNA for CRISPR-based base correction can be quickly screened in E. coli, and directly transferred to the plant without time, cost, or labor. Since a base correction system applicable to can be developed, it can be usefully used in the field of gene editing.
도 1A는 표적 서열 발현 카세트, 디아미나아제(deaminase) 및 엔도뉴클레아제(endonuclease) 발현 카세트 및 가이드 RNA (guide RNA) 발현 카세트를 포함하는 본 발명의 재조합 벡터의 모식도이고, 도 1B는 도 1A의 표적 서열 발현 카세트의 제조 방법을 나타낸 것이며, 도 1C는 도 1A의 표적 서열 발현 카세트에서 표적 서열 디자인 전략을 나타낸 것이다. sfGFP(superfolder green fluorescent protein) 유전자는 형광의 유무에 따라 클로닝된 플라스미드를 효과적으로 스크리닝하기 위해 사용된 리포터 유전자이다.
도 2는 본 발명의 재조합 벡터 시스템 확립을 위해 고안한 두 가지 방법에 관한 것으로, A는 두 개의 독립된 플라스미드로 구성된 방법(two-plasmid base assay)의 모식도이고, B는 한 개의 플라스미드로 구성된 방법(single-plasmid base assay)의 모식도이다.
도 3은 본 발명의 재조합 벡터 시스템 확립을 위해 고안한 두 가지 방법에 따른 염기전환율을 분석한 결과로, A는 두 개의 독립된 플라스미드로 구성된 방법을 이용하였을 때의 염기전환율 분석 결과이고, B는 한 개의 플라스미드로 구성된 방법을 이용하였을 때의 염기전환율 분석 결과이다.
도 4는 디아미나아제(deaminase) 및 엔도뉴클레아제(endonuclease) 발현 카세트에서 GlpT 프로모터-L3S2P21 터미네이터 조합 또는 35S 프로모터-35S 터미네이터 조합에 의한 C to T 염기전환율을 분석한 결과로, A는 테스트 sgRNA1을 이용한 결과이고, B는 테스트 sgRNA2를 이용한 결과이다. 디아미나아제로는 PmCDA1을 이용하였고, gRNA 발현 카세트에는 AtU6 프로모터를 이용하였다.
도 5는 디아미나아제(deaminase) 및 엔도뉴클레아제(endonuclease) 발현 카세트에서 GlpT 프로모터-L3S2P21 터미네이터 조합 또는 35S 프로모터-35S 터미네이터 조합에 의한 A to G 염기전환율을 분석한 결과로, A는 테스트 sgRNA1을 이용한 결과이고, B는 테스트 sgRNA2를 이용한 결과이다. 디아미나아제로는 ABE8e 또는 ABE9e를 이용하였고, sgRNA 발현 카세트에는 AtU6 프로모터를 이용하였다.
도 6은 디아미나아제(deaminase) 및 엔도뉴클레아제(endonuclease) 발현 카세트에서 GlpT 프로모터-L3S2P21 터미네이터 조합 또는 35S 프로모터-35S 터미네이터 조합에 의한 염기전환율을 분석한 결과로, A는 이미 알려진 활성 sgRNA를 이용한 결과이고, B는 이미 알려진 비활성 sgRNA를 이용한 결과이다. 디아미나아제로는 ABE8e 또는 PmCDA1을 이용하였고, sgRNA 발현 카세트에는 AtU6 프로모터를 이용하였다.Figure 1A is a schematic diagram of the recombinant vector of the present invention including a target sequence expression cassette, a deaminase and endonuclease expression cassette, and a guide RNA expression cassette, and Figure 1B is a schematic diagram of Figure 1A. A method for producing a target sequence expression cassette is shown, and Figure 1C shows a target sequence design strategy in the target sequence expression cassette of Figure 1A. The sfGFP (superfolder green fluorescent protein) gene is a reporter gene used to effectively screen cloned plasmids based on the presence or absence of fluorescence.
Figure 2 relates to two methods designed to establish the recombinant vector system of the present invention. A is a schematic diagram of a method consisting of two independent plasmids (two-plasmid base assay), and B is a schematic diagram of a method consisting of one plasmid (two-plasmid base assay). This is a schematic diagram of a single-plasmid base assay.
Figure 3 shows the results of analyzing the base conversion rate according to two methods designed to establish the recombinant vector system of the present invention. A is the result of analyzing the base conversion rate when using a method consisting of two independent plasmids, and B is the result of analyzing the base conversion rate using a method consisting of two independent plasmids. This is the result of base conversion rate analysis using a method consisting of plasmids.
Figure 4 shows the results of analyzing the C to T base conversion rate by the GlpT promoter-L3S2P21 terminator combination or the 35S promoter-35S terminator combination in the deaminase and endonuclease expression cassette, where A is test sgRNA1. This is the result using , and B is the result using test sgRNA2. PmCDA1 was used as deaminase, and AtU6 promoter was used as the gRNA expression cassette.
Figure 5 shows the results of analyzing the A to G base conversion rate by the GlpT promoter-L3S2P21 terminator combination or the 35S promoter-35S terminator combination in the deaminase and endonuclease expression cassette, where A is test sgRNA1. This is the result using , and B is the result using test sgRNA2. ABE8e or ABE9e was used as the deaminase, and the AtU6 promoter was used as the sgRNA expression cassette.
Figure 6 shows the results of analyzing the base conversion rate by the GlpT promoter-L3S2P21 terminator combination or the 35S promoter-35S terminator combination in the deaminase and endonuclease expression cassette, where A represents a known active sgRNA. This is the result using an already known inactive sgRNA. ABE8e or PmCDA1 was used as the deaminase, and the AtU6 promoter was used as the sgRNA expression cassette.
본 발명의 목적을 달성하기 위하여, 본 발명은 표적 서열 발현 카세트, 디아미나아제(deaminase) 및 엔도뉴클레아제(endonuclease) 발현 카세트 및 가이드 RNA (guide RNA) 발현 카세트를 포함하는, 크리스퍼(CRISPR) 기반 염기 교정(base editing)용 가이드 RNA의 활성 검정용 재조합 벡터를 제공한다.In order to achieve the object of the present invention, the present invention is a CRISPR method comprising a target sequence expression cassette, a deaminase and endonuclease expression cassette, and a guide RNA expression cassette. ) provides a recombinant vector for testing the activity of guide RNA for base editing.
본 발명에서 용어 "재조합"은 세포가 이종의 핵산을 복제하거나, 상기 핵산을 발현하거나 또는 펩티드, 이종의 펩티드 또는 이종의 핵산에 의해 암호화된 단백질을 발현하는 세포를 지칭하는 것이다. 재조합 세포는 상기 세포의 천연 형태에서는 발견되지 않는 유전자 또는 유전자 절편을, 센스 또는 안티센스 형태 중 하나로 발현할 수 있다. 또한 재조합 세포는 천연 상태의 세포에서 발견되는 유전자를 발현할 수 있으며, 그러나 상기 유전자는 변형된 것으로써 인위적인 수단에 의해 세포 내 재도입된 것이다.As used herein, the term “recombinant” refers to a cell that replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells can express genes or gene segments that are not found in the natural form of the cell, either in sense or antisense form. Additionally, recombinant cells can express genes found in cells in their natural state, but the genes have been modified and reintroduced into the cells by artificial means.
용어 "벡터"는 세포 내로 전달하는 DNA 단편(들), 핵산 분자를 지칭할 때 사용된다. 벡터는 DNA를 복제시키고, 숙주세포에서 독립적으로 재생산될 수 있다. 용어 "전달체"는 흔히 "벡터"와 호환하여 사용된다.The term “vector” is used to refer to a DNA fragment(s) or nucleic acid molecule that is delivered into a cell. Vectors replicate DNA and can reproduce independently in host cells. The term “vector” is often used interchangeably with “vector”.
용어 "발현 카세트(expression cassette)"는 하기의 세 가지 주요한 요소를 포함한다: i) 프로모터; ii) 상기 프로모터에 작동가능하게 연결되고 상기 발현 카세트가 세포 내로 도입되는 경우 상기 프로모터에 의해 그 전사가 지시되는 것인 "코딩 폴리뉴클레오티드(coding polynucleotide)", 또는 "코딩 서열(coding sequence)(코딩 유전자라고도 함)"로 지칭될 수 있는 제2 폴리뉴클레오티드; 및 iii) 전사의 종료를 지시하고 상기 제2 폴리뉴클레오티드의 바로 하류에 위치하는 종결자(terminator) 폴리뉴클레오티드(전사종결인자라고도 함).The term “expression cassette” includes three main elements: i) a promoter; ii) a “coding polynucleotide”, or “coding sequence” (coding sequence) operably linked to the promoter and whose transcription is directed by the promoter when the expression cassette is introduced into a cell a second polynucleotide, which may be referred to as “a gene”; and iii) a terminator polynucleotide (also called a transcription terminator) that directs termination of transcription and is located immediately downstream of the second polynucleotide.
본 발명의 크리스퍼 기반 염기 교정용 가이드 RNA의 활성 검정용 재조합 벡터에 있어서, 상기 표적 서열 발현 카세트는 활성 검정용 가이드 RNA의 표적 서열 및 PAM (protospacer adjacent motif) 서열을 암호화하는 DNA를 포함할 수 있으나, 이에 제한되지 않는다. In the recombinant vector for testing the activity of the guide RNA for CRISPR-based base correction of the present invention, the target sequence expression cassette may include DNA encoding the target sequence of the guide RNA for activity testing and a PAM (protospacer adjacent motif) sequence. However, it is not limited to this.
상기 표적 서열은 BsmB Ⅰ 제한효소에 의해 절단된 부위에 목적하는 서열을 제한없이 삽입하여 사용할 수 있다(도 1B 참고).The target sequence can be used by inserting the desired sequence without limitation into the site cut by the BsmB I restriction enzyme (see Figure 1B).
용어 "가이드 RNA(guide RNA)"는 짧은 단일 가닥의 RNA로, 표적 유전자를 암호화하는 염기서열 중 표적 DNA에 특이적인 RNA를 의미하며, 표적 DNA 염기서열과 전부 또는 일부가 상보적으로 결합하여 해당 표적 DNA 염기서열로 엔도뉴클레아제 단백질을 이끄는 역할을 하는 리보핵산을 의미한다. 상기 가이드 RNA는 두 개의 RNA, 즉, crRNA (CRISPR RNA) 및 tracrRNA (trans-activating crRNA)를 구성 요소로 포함하는 이중 RNA (dual RNA); 또는 표적 유전자 내 염기서열과 전부 또는 일부 상보적인 서열을 포함하는 제1 부위 및 엔도뉴클레아제(특히, RNA-가이드 뉴클레아제)와 상호작용하는 서열을 포함하는 제2 부위를 포함하는 단일 사슬 가이드 RNA(single guide RNA, sgRNA) 형태를 말하나, 제한없이 본 발명의 범위에 포함될 수 있으며, 함께 사용된 엔도뉴클레아제의 종류 또는 엔도뉴클레아제의 유래 미생물 등을 고려하여 당업계의 공지된 기술에 따라 제조하여 사용할 수 있다. 상기 가이드 RNA는 PAM(protospacer adjacent motif) 자리와 인접하며, 편집하려는 DNA의 10~20bp의 염기 서열과 상보적인 서열을 포함하는 것일 수 있으나, 이에 제한되지 않는다.The term “guide RNA” refers to a short, single-stranded RNA that is specific to the target DNA among the base sequences encoding the target gene, and all or part of it is complementary to the target DNA base sequence to produce the corresponding target DNA. It refers to a ribonucleic acid that guides the endonuclease protein to the target DNA base sequence. The guide RNA is a dual RNA containing two RNAs, namely crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as components; or a single chain comprising a first portion comprising a sequence that is fully or partially complementary to a base sequence in the target gene and a second portion comprising a sequence that interacts with an endonuclease (particularly an RNA-guided nuclease). It refers to the form of guide RNA (single guide RNA, sgRNA), but can be included within the scope of the present invention without limitation, and is known in the art considering the type of endonuclease used together or the microorganism from which the endonuclease is derived. It can be manufactured and used according to technology. The guide RNA is adjacent to the PAM (protospacer adjacent motif) site and may include, but is not limited to, a sequence complementary to a 10 to 20 bp nucleotide sequence of the DNA to be edited.
또한, 본 발명의 크리스퍼 기반 염기 교정용 가이드 RNA의 활성 검정용 재조합 벡터에 있어서, 상기 디아미나아제 및 엔도뉴클레아제 발현 카세트는 5'에서 3' 방향으로 GlpT 프로모터 또는 35S 프로모터; 디아미나아제; 엔도뉴클레아제; 및 L3S2P21 터미네이터 또는 35S 터미네이터; 코딩 서열이 작동가능하게 연결된 것일 수 있으나, 이에 제한되지 않는다. In addition, in the recombinant vector for testing the activity of the guide RNA for CRISPR-based base correction of the present invention, the deaminase and endonuclease expression cassette includes a GlpT promoter or a 35S promoter in the 5' to 3' direction; deaminase; endonuclease; and L3S2P21 terminator or 35S terminator; The coding sequences may be operably linked, but are not limited thereto.
본 발명에서 "작동가능하게 연결된(operably linked)"이란 하나의 핵산 단편이 다른 핵산 단편과 결합되어 그의 기능 또는 발현이 다른 핵산 단편에 의해 영향을 받는 것을 말한다. 즉, 상기 디아미나아제 및 엔도뉴클레아제를 코딩하는 유전자는 벡터 내에 있는 프로모터에 의해 그 발현이 조절될 수 있도록 연결될 수 있다.In the present invention, “operably linked” refers to one nucleic acid fragment being linked to another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment. That is, the genes encoding the deaminase and endonuclease can be linked so that their expression can be controlled by a promoter in the vector.
상기 디아미나아제는 엔도뉴클레아제의 N 말단에 링커(linker)로 융합된 형태일 수 있으며, 상기 링커는 바람직하게는 XTEN 링커일 수 있으나, 이에 제한되지 않는다.The deaminase may be fused to the N terminus of the endonuclease with a linker, and the linker may preferably be an XTEN linker, but is not limited thereto.
본 발명의 일 구현 예에 따른 디아미나아제 및 엔도뉴클레아제 발현 카세트는 GlpT 프로모터-L3S2P21 터미네이터 조합 또는 35S 프로모터-35S 터미네이터 조합으로 이루어질 수 있다.The deaminase and endonuclease expression cassette according to one embodiment of the present invention may be composed of a GlpT promoter-L3S2P21 terminator combination or a 35S promoter-35S terminator combination.
용어 "프로모터"는 구조 유전자로부터의 DNA 상류(upstream)의 영역을 의미하며 전사를 개시하기 위하여 RNA 중합효소가 결합하는 DNA 분자를 말한다.The term “promoter” refers to a region of DNA upstream from a structural gene and refers to the DNA molecule to which RNA polymerase binds to initiate transcription.
상기 디아미나아제는 시티딘 디아미나아제(cytidine deaminases) 또는 아데닌 디아미나아제(adenine deaminases)일 수 있으나 이에 제한되지 않는다. 또한, 상기 GlpT(Glycerol-3-phosphate transporter) 프로모터는 서열번호 1의 염기서열로 이루어진 것일 수 있고, 상기 35S(cauliflower mosaic virus 35S, CaMV 35S) 프로모터는 서열번호 2의 염기서열로 이루어진 것일 수 있고, 상기 L3S2P21 터미네이터는 서열번호 3의 염기서열로 이루어진 것일 수 있으며, 상기 35S(cauliflower mosaic virus 35S, CaMV 35S) 터미네이터는 서열번호 4의 염기서열로 이루어진 것일 수 있으나, 이에 제한되지 않는다. 또한, 상기 엔도뉴클레아제는 DNA 이중 가닥을 절단하는 엔도뉴클레아제 활성을 상실한 Cas9 단백질 또는 Cpf1 단백질일 수 있으며, 바람직하게는 nCas9(nickase Cas9) 또는 dCas9(deactivated Cas9)일 수 있으나, 이에 제한되지 않는다.The deaminases may be cytidine deaminases or adenine deaminases, but are not limited thereto. In addition, the GlpT (Glycerol-3-phosphate transporter) promoter may consist of the base sequence of SEQ ID NO: 1, and the 35S (cauliflower mosaic virus 35S, CaMV 35S) promoter may consist of the base sequence of SEQ ID NO: 2. , the L3S2P21 terminator may be composed of the nucleotide sequence of SEQ ID NO: 3, and the 35S (cauliflower mosaic virus 35S, CaMV 35S) terminator may be composed of the nucleotide sequence of SEQ ID NO: 4, but is not limited thereto. Additionally, the endonuclease may be a Cas9 protein or a Cpf1 protein that has lost its endonuclease activity to cleave DNA double strands, preferably nCas9 (nickase Cas9) or dCas9 (deactivated Cas9), but is limited thereto. It doesn't work.
본 발명에 따른 가이드 RNA 활성 검정용 재조합 벡터에 있어서, 디아미나아제 또는 엔도뉴클레아제 코딩 서열은 숙주세포의 코돈에 최적화된 서열일 수 있으나, 이에 제한되지 않는다.In the recombinant vector for guide RNA activity assay according to the present invention, the deaminase or endonuclease coding sequence may be a sequence optimized for the codon of the host cell, but is not limited thereto.
용어 "코돈 최적화"는 코딩된 단백질이 유기체에서 보다 효율적으로 발현되도록 특정 유기체에서 우선적으로 사용되는 것으로 단백질을 코딩하는 폴리뉴클레오티드의 코돈을 변화시키는 것을 의미한다. 대부분의 아미노산이 "동의어" 또는 "동의" 코돈이라고 하는, 몇몇 코돈에 의해 표시된다는 점에서, 유전자 코드가 축퇴적이지만, 특정 유기체에 의한 코돈 용법은 임의적이지 않고 특정 코돈 트리플렛에 편향적이다. 이러한 코돈 용법 편향성은 소정 유전자, 공통 기능 또는 조상 기원의 유전자, 고도로 발현되는 단백질 대 낮은 복제수 단백질, 및 유기체 게놈의 집합적 단백질 코딩 영역과 관련하여 더 높을 수 있다.The term “codon optimization” refers to changing the codons of a polynucleotide encoding a protein to those preferentially used in a particular organism so that the encoded protein is expressed more efficiently in that organism. Although the genetic code is degenerate in the sense that most amino acids are represented by several codons, called "synonymous" or "synonymous" codons, codon usage by a particular organism is not random and is biased toward particular codon triplets. This codon usage bias may be higher with respect to certain genes, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and collective protein coding regions of the organism's genome.
또한, 본 발명의 크리스퍼 기반 염기 교정용 가이드 RNA의 활성 검정용 재조합 벡터에 있어서, 상기 가이드 RNA 발현 카세트는 5'에서 3' 방향으로 AtU6 프로모터 코딩 서열 및 활성 검정용 가이드 RNA를 암호화하는 DNA가 작동가능하게 연결된 것일 수 있으나, 이에 제한되지 않는다. In addition, in the recombinant vector for testing the activity of the guide RNA for CRISPR-based base correction of the present invention, the guide RNA expression cassette has DNA encoding an AtU6 promoter coding sequence and a guide RNA for activity testing in the 5' to 3' direction. It may be, but is not limited to, operably connected.
상기 AtU6 프로모터는 바람직하게는 서열번호 7의 염기서열로 이루어진 것일 수 있으나, 이에 제한되지 않는다.The AtU6 promoter may preferably consist of the base sequence of SEQ ID NO: 7, but is not limited thereto.
본 발명의 크리스퍼 기반 염기 교정용 가이드 RNA의 활성 검정용 재조합 벡터는 '표적 서열 발현 카세트'와 '디아미나아제(deaminase) 및 엔도뉴클레아제(endonuclease) 발현 카세트-가이드 RNA (guide RNA) 발현 카세트'가 별개의 플라스미로 구성된 two-plasmid base assay로 이용할 수도 있고, '표적 서열 발현 카세트-디아미나아제(deaminase) 및 엔도뉴클레아제(endonuclease) 발현 카세트-가이드 RNA (guide RNA) 발현 카세트'가 한개의 플라스미드로 구성된 single-plasmid base assay로 이용할 수 있는 것이 특징이다(도 2 참고).The recombinant vector for testing the activity of the guide RNA for CRISPR-based base correction of the present invention includes a 'target sequence expression cassette' and a 'deaminase and endonuclease expression cassette - guide RNA expression. It can be used as a two-plasmid base assay where the 'cassette' is composed of separate plasmids, and the 'target sequence expression cassette - deaminase and endonuclease expression cassette - guide RNA expression cassette' It is characterized by being able to be used as a single-plasmid base assay consisting of one plasmid (see Figure 2).
본 발명의 재조합 벡터는 하나 이상의 선택성 마커를 포함한다. 상기 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질전환된 세포를 비형질전환 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 그 예로는 제오신(블레오마이신), 글리포세이트(glyphosate) 또는 포스피노트리신과 같은 제초제 저항성 유전자, 카베니실린(carbenicillin), 카나마이신(kanamycin), 하이그로마이신(hygromycin), 클로람페니콜(chloramphenicol), G418과 같은 항생제 내성 유전자가 있으나, 이에 한정되는 것은 아니다.The recombinant vector of the present invention contains one or more selectable markers. The marker is a nucleic acid sequence that has characteristics that can be generally selected by chemical methods, and includes all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as zeocin, glyphosate or phosphinothricin, carbenicillin, kanamycin, hygromycin, chloramphenicol, There are antibiotic resistance genes such as G418, but they are not limited to these.
본 발명은 또한, 상기 재조합 벡터를 유효성분으로 포함하는 크리스퍼(CRISPR) 기반 염기 교정(base editing)용 가이드 RNA (guide RNA) 활성 검정용 조성물을 제공한다. The present invention also provides a composition for testing guide RNA activity for CRISPR-based base editing containing the recombinant vector as an active ingredient.
본 발명의 조성물에 있어서, 상기 재조합 벡터는 전술한 것과 같다. In the composition of the present invention, the recombinant vector is the same as described above.
본 발명에 따른 크리스퍼 기반 염기 교정용 가이드 RNA 활성 검정용 조성물은 숙주세포에 독성을 유발하지 않는 것이 특징이며, 상기 숙주세포는 바람직하게는 대장균(Escherichia coli)일 수 있으나, 이에 제한되지 않는다.The composition for testing the activity of a guide RNA for CRISPR-based base correction according to the present invention is characterized by not causing toxicity to host cells, and the host cell may preferably be Escherichia coli , but is not limited thereto.
본 발명은 또한, 상기 재조합 벡터로 숙주세포를 형질전환하는 단계; 및 상기 형질전환된 숙주세포로부터 핵산을 추출하고 가이드 RNA의 표적 서열의 염기서열을 분석하는 단계;를 포함하는, 크리스퍼(CRISPR) 기반 염기 교정(base editing)용 가이드 RNA (guide RNA)의 활성을 검정하는 방법을 제공한다.The present invention also includes the steps of transforming a host cell with the recombinant vector; And extracting nucleic acid from the transformed host cell and analyzing the base sequence of the target sequence of the guide RNA. Activity of guide RNA for CRISPR-based base editing, including Provides a method for testing.
본 발명의 방법에 있어서, 상기 재조합 벡터 및 숙주세포는 전술한 것과 같으며, 상기 형질전환, 핵산 추출 및 염기서열 분석은 본 발명이 속하는 기술 분야에서 공지된 통상적인 방법에 따라 실시될 수 있다.In the method of the present invention, the recombinant vector and host cell are the same as described above, and the transformation, nucleic acid extraction, and base sequence analysis can be performed according to conventional methods known in the technical field to which the present invention pertains.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.
재료 및 방법Materials and Methods
1. 재조합 벡터 구축에 사용한 재료1. Materials used to construct recombinant vectors
본 발명에서 사용한 대장균 균주는 10-beta, DH5α, BL21(DE3) 또는 DB3.1이며 LB(Luria-Bertani) 배지에서 37℃, 18~24시간 동안 배양하여 실험에 사용하였다. 재조합 벡터 구축을 위한 모든 클로닝 과정에서는 열 충격(heat shock) 방법을 통해 대장균을 형질전환하였다.The E. coli strain used in the present invention was 10-beta, DH5α, BL21(DE3), or DB3.1, and was cultured in LB (Luria-Bertani) medium at 37°C for 18 to 24 hours and used in the experiment. In all cloning processes for constructing recombinant vectors, E. coli was transformed using a heat shock method.
GlpT 프로모터(서열번호 1) 및 35S 프로모터(서열번호 2) 서열은 BBa_J72163(SynBioHub) 및 pICH51266(Addgene #50267) 플라스미드로부터 각각 얻었고, 35S 터미네이터(서열번호 4) 서열은 pICH41414(Addgene #50337) 플라스미드로부터 얻었다. Ec1 프로모터(서열번호 5) 서열은 pBP-SJM901(Addgene #72966) 및 pBP-RBSTL2(Addgene #72986) 플라스미드로부터 얻었고, L3S2P21 터미네이터(서열번호 3) 서열은 pBP-L3S2P21(Addgene #72999) 플라스미드로부터 얻었다. BBa_B0015 터미네이터(서열번호 6)는 pBP-BBa_B0015(Addgene #72998) 플라스미드로부터 얻었다. 시티딘 디아미나아제(cytidine deaminases)인 PmCDA1(Target-AID), evoCDA1 및 APOBEC3A 서열은 각각 PmCDA1-1×UGI(Addgene #79620), evoCDA1 pBT277(Addgene #122608) 및 A3A-PBE-ΔUGI(Addgene #119770) 플라스미드로부터 얻었다. sgRNA 발현을 위한 프로모터로 사용한 식물용 AtU6 프로모터(서열번호 7)는 pICSL01009(Addgene #46968)로부터 얻었고, 박테리아용 J23119 프로모터(서열번호 8)는 합성하여 사용하였다. nCas9(D10A) 서열(서열번호 9)은 hCas9(Addgene #49771)을 주형으로 한 PCR을 통해 합성하여 사용하였고, dCas9(서열번호 10) 서열은 합성하여 사용하였다.The GlpT promoter (SEQ ID NO: 1) and 35S promoter (SEQ ID NO: 2) sequences were obtained from BBa_J72163 (SynBioHub) and pICH51266 (Addgene #50267) plasmids, respectively, and the 35S terminator (SEQ ID NO: 4) sequence was obtained from pICH41414 (Addgene #50337) plasmid. got it The Ec1 promoter (SEQ ID NO: 5) sequence was obtained from pBP-SJM901 (Addgene #72966) and pBP-RBSTL2 (Addgene #72986) plasmids, and the L3S2P21 terminator (SEQ ID NO: 3) sequence was obtained from pBP-L3S2P21 (Addgene #72999) plasmid. . BBa_B0015 terminator (SEQ ID NO: 6) was obtained from pBP-BBa_B0015 (Addgene #72998) plasmid. The cytidine deaminases PmCDA1 (Target-AID), evoCDA1 and APOBEC3A sequences were PmCDA1-1×UGI (Addgene #79620), evoCDA1 pBT277 (Addgene #122608) and A3A-PBE-ΔUGI (Addgene #122608), respectively. 119770) obtained from the plasmid. The AtU6 promoter for plants (SEQ ID NO: 7), used as a promoter for sgRNA expression, was obtained from pICSL01009 (Addgene #46968), and the J23119 promoter for bacteria (SEQ ID NO: 8) was synthesized and used. The nCas9 (D10A) sequence (SEQ ID NO: 9) was synthesized and used through PCR using hCas9 (Addgene #49771) as a template, and the dCas9 (SEQ ID NO: 10) sequence was synthesized and used.
아데닌 디아미나아제(adenine deaminases)인 ABE8e 서열(Addgene #138489)은 바이오니아(Bioneer, 대한민국)에서 합성하였고, ABE9e 변이체(v82s/Q154R)는 ABE8e를 주형으로 한 부위 지정 돌연변이유발 PCR(site directed mutagenesis PCR)을 통해 얻었다. PmCDA1-1×UGI(Uracil Glycosylase Inhibitor)는 nCas9의 C 말단에 융합시켰고, evoCDA1, APOBEC3A(이하, A3A) 및 ABE8e는 nCas9의 N-말단에 XTEN 링커를 이용하여 융합시켰다. 2×UGI 모듈은(Addgene #122608) evoCDA1-nCas9 또는 A3A-nCas9의 C-말단에 융합시켰다. 리포터 유전자로 사용한 sfGFP(superfolder green fluorescent protein) 유전자 서열은 Lee 등(2015, ACS Synth. Biol. 4:975-986)에 개시된 정보를 이용하였다.The ABE8e sequence (Addgene #138489), an adenine deaminases, was synthesized by Bioneer (Korea), and the ABE9e variant (v82s/Q154R) was synthesized by site directed mutagenesis PCR using ABE8e as a template. ) was obtained through PmCDA1-1×UGI (Uracil Glycosylase Inhibitor) was fused to the C-terminus of nCas9, and evoCDA1, APOBEC3A (hereinafter referred to as A3A), and ABE8e were fused to the N-terminus of nCas9 using an XTEN linker. The 2×UGI module (Addgene #122608) was fused to the C-terminus of evoCDA1-nCas9 or A3A-nCas9. The sfGFP (superfolder green fluorescent protein) gene sequence used as a reporter gene used information disclosed in Lee et al. (2015, ACS Synth. Biol. 4:975-986).
2. 염기전환율(base conversion rate) 분석2. Base conversion rate analysis
형질전환된 대장균으로부터 DNA를 추출하여 SnapGene 소프트웨어(GSL Biotech)를 이용한 생거 시퀀싱(Sanger sequencing)을 통해 염기서열을 분석한 후, 온라인 분석도구인 EditR(baseeditr.com)을 이용하여 염기전환율을 계산하였고, GraphPad Prism 소프트웨어(version 9.0.0, www.graphpad.com)를 이용하여 통계 분석을 수행하였다. DNA was extracted from transformed E. coli, the base sequence was analyzed through Sanger sequencing using SnapGene software (GSL Biotech), and the base conversion rate was calculated using EditR (baseeditr.com), an online analysis tool. , Statistical analysis was performed using GraphPad Prism software (version 9.0.0, www.graphpad.com).
실시예 1. 재조합 벡터 구축Example 1. Construction of recombinant vector
재조합 벡터는 도 1A에 개시한 바와 같이, 표적 서열 발현 카세트, 디아미나아제(deaminase) 및 엔도뉴클레아제(endonuclease) 발현 카세트, 가이드 RNA (guide RNA) 발현 카세트로 구성하였다. As shown in Figure 1A, the recombinant vector consisted of a target sequence expression cassette, deaminase and endonuclease expression cassettes, and guide RNA expression cassette.
표적 서열 발현 카세트는 "GlpT 프로모터-Tag-dummy-sfGFP-BBa_B0015 터미네이터"로 이루어진 모듈에서 BsmBⅠ 제한효소 처리에 의해 dummy를 제거하고 활성을 확인하고자 하는 gRNA의 표적 부위를 삽입하여 제조하였다(도 1B). 디아미나아제 및 엔도뉴클레아제 발현 카세트와 gRNA 발현 카세트는 골든 게이트 어셈블리 프로토콜(Golden Gate assembly protocol)에 따라 원하는 프로모터를 삽입하여 다양한 조합으로 제조하였다. 이후, Rahul 등(Int. J. Mol. Sci. 2022.23;1145)에 개시된 방법에 따라 재조합 벡터를 제조하였다.The target sequence expression cassette was prepared by removing the dummy from a module consisting of “GlpT promoter-Tag-dummy- sfGFP -BBa_B0015 terminator” by BsmBⅠ restriction enzyme treatment and inserting the target site of the gRNA whose activity is to be confirmed (Figure 1B) ). Deaminase and endonuclease expression cassettes and gRNA expression cassettes were prepared in various combinations by inserting the desired promoter according to the Golden Gate assembly protocol. Afterwards, a recombinant vector was prepared according to the method disclosed in Rahul et al. (Int. J. Mol. Sci. 2022.23;1145).
실시예 2. 재조합 벡터의 이용 방법 고안Example 2. Devising a method of using recombinant vectors
본 발명의 벡터 시스템 확립을 위한 두 가지 이용 방법을 고안하였다. 첫 번째는 두 개의 독립된 플라스미드로 구성된 방법(two-plasmid based assay)으로, '표적 서열 발현 카세트'와 '디아미나아제 및 엔도뉴클레아제 발현 카세트-가이드 RNA 발현 카세트'를 사용하는 방법이다(도 2A). 두 번째는 한 개의 플라스미드로 구성된 방법(single-plasmid based assay)으로, '표적 서열 발현 카세트-디아미나아제 및 엔도뉴클레아제 발현 카세트-가이드 RNA 발현 카세트'를 사용하는 방법이다(도 2B). 상기 두 가지 방법을 이용하여 염기전환율을 각각 분석하였다. 실험에 사용한 gRNA는 sgRNA1이고, OD600=5~6인 대장균(10-beta strain, 50 ㎕)에 50 ng의 플라스미드를 혼합한 후 열 충격 방법으로 형질전환하였고, 24시간 동안 배양하였고, 디아미나아제-엔도뉴클레아제 발현용으로는 GlpT 프로모터-L3S2P21 터미네이터 조합 또는 35S 프로모터-35S 터미네이터 조합을 이용하였고, 디아미나아제로는 C to T 염기전환을 유도하는 PmCDA1을 이용하였으며, gRNA 발현용 프로모터로는 AtU6를 이용하여 C to T 염기전환율을 분석하였다.Two methods were designed to establish the vector system of the present invention. The first is a method consisting of two independent plasmids (two-plasmid based assay), which uses a 'target sequence expression cassette' and a 'deaminase and endonuclease expression cassette-guide RNA expression cassette' (Figure 2A). The second is a method consisting of a single plasmid (single-plasmid based assay), which uses 'target sequence expression cassette - deaminase and endonuclease expression cassette - guide RNA expression cassette' (Figure 2B). Base conversion rates were analyzed using the above two methods, respectively. The gRNA used in the experiment was sgRNA1, and 50 ng of plasmid was mixed with E. coli (10-beta strain, 50 ㎕) with OD 600 = 5~6, transformed by heat shock method, cultured for 24 hours, and diamina For enzyme-endonuclease expression, the GlpT promoter-L3S2P21 terminator combination or the 35S promoter-35S terminator combination was used, as the deaminase, PmCDA1, which induces C to T base conversion, was used, and as the promoter for gRNA expression. analyzed the C to T base conversion rate using AtU6.
그 결과, 두 개의 플라스미드를 이용하였을 때나 한 개의 플라스미드를 이용하였을 때나 염기전환율에는 차이가 없는 것을 확인하였고, 디아미나아제-엔도뉴클레아제 발현용으로 35S 프로모터-35S 터미네이터 조합을 이용하였을 때나 GlpT 프로모터-L3S2P21 터미네이터 조합을 이용하였을 때나 두 가지 이용 방법에 따른 차이는 없는 것을 확인하였다(도 3). As a result, it was confirmed that there was no difference in base conversion rate when using two plasmids or one plasmid, and when using the 35S promoter-35S terminator combination for deaminase-endonuclease expression, the GlpT promoter It was confirmed that there was no difference when using the -L3S2P21 terminator combination or between the two methods (Figure 3).
따라서, 이후 모든 실험은 한 개의 플라스미드를 이용한 방법으로 수행하였다. Therefore, all subsequent experiments were performed using a single plasmid.
실시예 3. 프로모터 선발을 위한 세포 독성 분석Example 3. Cytotoxicity assay for promoter selection
프로모터 선발을 위한 세포 독성 실험은 단일플라스미드 시스템을 사용하여 각각 별도의 프로모터에 의해 디아미나아제-엔도뉴클레아제 발현 카세트와 gRNA 발현 카세트 들이 발현되도록 구성되어 있다. 이에, 디아미나아제-엔도뉴클레아제 발현용으로는 Ec1 또는 GlpT 프로모터를, gRNA 발현용으로는 J23119 또는 AtU6 프로모터를 이용하였고, Cas9의 경우 nCas9(D10A) 및 dCas9(D10A+H840A)가 PmCDA1과 결합한 Target AID 시스템을 가지고 다양한 조합 따른 세포 독성을 분석하였다. Ec1 프로모터를 이용하였을 때는 L3S2P21 터미네이터를 이용하였고, GlpT 프로모터를 이용하였을 때는 L3S2P21 터미네이터를 이용하였다. 동일양의 플라스미드 DNA를 대장균(10-beta)에 형질전환시켰고, 콜로니 형성 단위(colony-forming unit, CFU)측정을 통한 대장균 생존율을 분석하였다.The cytotoxicity test for promoter selection consists of using a single plasmid system so that the deaminase-endonuclease expression cassette and the gRNA expression cassette are expressed by separate promoters. Accordingly, the Ec1 or GlpT promoter was used for deaminase-endonuclease expression, and the J23119 or AtU6 promoter was used for gRNA expression. In the case of Cas9, nCas9(D10A) and dCas9(D10A+H840A) were used in conjunction with PmCDA1. Cytotoxicity according to various combinations was analyzed using the combined Target AID system. When using the Ec1 promoter, the L3S2P21 terminator was used, and when using the GlpT promoter, the L3S2P21 terminator was used. The same amount of plasmid DNA was transformed into E. coli (10-beta), and the E. coli survival rate was analyzed by measuring colony-forming units (CFU).
그 결과, 디아미나아제-엔도뉴클레아제 발현용으로 Ec1 프로모터를 이용하였을 때는 gRNA 발현용으로 어떠한 프로모터를 이용하더라도 세포 독성이 유발되는 것을 확인하였다. 반면, 디아미나아제-엔도뉴클레아제 발현용으로 GlpT 프로모터를 이용하였을 때는 gRNA 발현용으로 J23119 프로모터를 이용하면 세포 독성이 유발되나, AtU6 프로모터를 이용하면 세포 독성이 유발되지 않는 것을 확인하였다(표 1). As a result, it was confirmed that when the Ec1 promoter was used for deaminase-endonuclease expression, cytotoxicity was induced regardless of which promoter was used for gRNA expression. On the other hand, when the GlpT promoter was used for deaminase-endonuclease expression, it was confirmed that cytotoxicity was induced when the J23119 promoter was used for gRNA expression, but when the AtU6 promoter was used, cytotoxicity was not induced (Table One).
상기 결과를 바탕으로, 디아미나아제-엔도뉴클레아제 발현용 프로모터와 gRNA 발현용 프로모터의 조합에 따라 세포 독성 결과가 상이함을 알 수 있었다. 따라서, 대장균에서 gRNA 발현용 프로모터가 같이 사용되면 세포 독성을 유발하는 Ec1 프로모터는 제외하였고, 대장균에서 세포 독성을 유발하지 않는 GlpT 및 AtU6 프로모터를 최종 선발하였다. Based on the above results, it was found that the cytotoxicity results were different depending on the combination of the promoter for deaminase-endonuclease expression and the promoter for gRNA expression. Therefore, the Ec1 promoter, which causes cytotoxicity when used together with a promoter for gRNA expression in E. coli, was excluded, and the GlpT and AtU6 promoters, which do not cause cytotoxicity in E. coli, were finally selected.
실시예 4. C to T 염기전환율 분석Example 4. C to T base conversion rate analysis
본 발명에 따른 프로모터 조합에 의한 C to T 염기전환율을 확인하기 위하여 두 가지 테스트 sgRNA를 디자인하였다(표 2). 디아미나아제-엔도뉴클레아제 발현용으로는 GlpT 프로모터-L3S2P21 터미네이터 조합 또는 35S 프로모터-35S 터미네이터 조합을 이용하였고, 디아미나아제로는 C to T 염기전환을 유도하는 PmCDA1을 이용하였으며, gRNA 발현용 프로모터로는 AtU6를 이용하여 염기전환율을 분석하였다.To confirm the C to T base conversion rate by the promoter combination according to the present invention, two test sgRNAs were designed (Table 2). For deaminase-endonuclease expression, the GlpT promoter-L3S2P21 terminator combination or the 35S promoter-35S terminator combination was used, and as the deaminase, PmCDA1, which induces C to T base conversion, was used, and for gRNA expression The base conversion rate was analyzed using AtU6 as a promoter.
그 결과, 본 발명에 따른 프로모터-터미네이터 조합에 의해 대장균에서 세포 독성을 유발하지 않고, 표적 부위에서 효과적으로 C to T 염기전환이 일어나는 것을 확인하였다. 특히, 디아미나아제-엔도뉴클레아제 발현용으로 35S 프로모터-35S 터미네이터 조합을 이용하였을 때보다 GlpT 프로모터-L3S2P21 터미네이터 조합을 이용하였을 때의 C to T 염기전환율이 우수한 것을 확인하였다(도 4).As a result, it was confirmed that the promoter-terminator combination according to the present invention does not cause cytotoxicity in E. coli and effectively causes C to T base conversion at the target site. In particular, it was confirmed that the C to T base conversion rate when using the GlpT promoter-L3S2P21 terminator combination was superior to when using the 35S promoter-35S terminator combination for deaminase-endonuclease expression (Figure 4).
실시예 5. A to G 염기전환율 분석Example 5. A to G base conversion rate analysis
본 발명에 따른 프로모터 조합에 의한 A to G 염기전환율을 확인하기 위하여 두 가지 테스트 sgRNA(표 3)를 이용하였다. 디아미나아제-엔도뉴클레아제 발현용으로는 GlpT 프로모터-L3S2P21 터미네이터 조합 또는 35S 프로모터-35S 터미네이터 조합을 이용하였고, 디아미나아제로는 A to G 염기전환을 유도하는 ABE8e 또는 ABE9e를 이용하였으며, gRNA 발현용 프로모터로는 AtU6를 이용하여 염기전환율을 각각 분석하였다.To confirm the A to G base conversion rate by the promoter combination according to the present invention, two test sgRNAs (Table 3) were used. For deaminase-endonuclease expression, the GlpT promoter-L3S2P21 terminator combination or the 35S promoter-35S terminator combination was used, and as the deaminase, ABE8e or ABE9e, which induces A to G base conversion, was used, and gRNA The base conversion rate was analyzed using AtU6 as the expression promoter.
그 결과, 본 발명에 따른 프로모터-터미네이터 조합에 의해 대장균에서 세포 독성을 유발하지 않고, 표적 부위에서 효과적으로 A to G 염기전환이 일어나는 것을 확인하였다. 특히, 디아미나아제-엔도뉴클레아제 발현용으로 35S 프로모터-35S 터미네이터 조합을 이용하였을 때보다 GlpT 프로모터-L3S2P21 터미네이터 조합을 이용하였을 때의 A to G 염기전환율이 우수한 것을 확인하였다(도 5).As a result, it was confirmed that the promoter-terminator combination according to the present invention does not cause cytotoxicity in E. coli and effectively causes A to G base conversion at the target site. In particular, it was confirmed that the A to G base conversion rate when using the GlpT promoter-L3S2P21 terminator combination was superior to when using the 35S promoter-35S terminator combination for deaminase-endonuclease expression (FIG. 5).
실시예 6. sgRNA 활성 검정Example 6. sgRNA activity assay
본 발명에 따른 재조합 벡터를 이용하여 대장균에서 sgRNA 활성을 확인할 수 있는지 검정하기 위하여 다른 연구에서 활성 sgRNA 또는 비활성 sgRNA로 이미 보고된 바 있는 sgRNA(표 3)를 이용하여 염기전환율을 각각 분석하였다. AtGL1, SIMlo1, inactive 1 또는 inactive 2는 디아미나아제로 ABE8e를 이용하여 A to G 염기전환율을 분석하였고, SIPelo는 디아미나아제로 PmCDA1을 이용하여 C to T 염기전환율을 분석하였다.In order to test whether sgRNA activity can be confirmed in E. coli using the recombinant vector according to the present invention, the base conversion rate was analyzed using sgRNA (Table 3) that has already been reported as active sgRNA or inactive sgRNA in other studies. For AtGL1, SIMlo1, inactive 1 or inactive 2, the A to G base conversion rate was analyzed using ABE8e as a deaminase, and for SIPelo, the C to T base conversion rate was analyzed using PmCDA1 as a deaminase.
그 결과, AtGL1, SIMlo1 또는 SIPelo는 각 표적 염기에서 염기전환이 효과적으로 일어나는 것을 확인한 반면, inactive 1 또는 inactive 2는 각 표적 염기에서의 염기전환율이 낮은 것을 확인하였다(도 6).As a result, it was confirmed that AtGL1, SIMlo1 or SIPelo effectively perform base conversion at each target base, while inactive 1 or inactive 2 had a low base conversion rate at each target base (FIG. 6).
상기 결과를 바탕으로, 본 발명에 따른 재조합 벡터를 이용하면 대장균에서 sgRNA의 활성을 효과적으로 검정할 수 있음을 알 수 있었다. Based on the above results, it was found that the activity of sgRNA in E. coli can be effectively tested using the recombinant vector according to the present invention.
<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Recombinant vector for activity test of guide RNA for CRISPR-based base editing and uses thereof <130> PN22020 <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 166 <212> DNA <213> Artificial Sequence <220> <223> GlpT promoter <400> 1 gaaagtgaaa cgtgatttca tgcgtcattt tgaacatttt gtaaatctta tttaataatg 60 tgtgcggcaa ttcacattta atttatgaat gttttcttaa catcgcggca actcaagaaa 120 cggcaggttc ggatcttagc tactagagaa agaggagaaa tactag 166 <210> 2 <211> 1729 <212> DNA <213> Artificial Sequence <220> <223> 35S promoter <400> 2 gaattccaat cccacaaaaa tctgagctta acagcacagt tgctcctctc agagcagaat 60 cgggtattca acaccctcat atcaactact acgttgtgta taacggtcca catgccggta 120 tatacgatga ctggggttgt acaaaggcgg caacaaacgg cgttcccgga gttgcacaca 180 agaaatttgc cactattaca gaggcaagag cagcagctga cgcgtacaca acaagtcagc 240 aaacagacag gttgaacttc atccccaaag gagaagctca actcaagccc aagagctttg 300 ctaaggccct aacaagccca ccaaagcaaa aagcccactg gctcacgcta ggaaccaaaa 360 ggcccagcag tgatccagcc ccaaaagaga tctcctttgc cccggagatt acaatggacg 420 atttcctcta tctttacgat ctaggaagga agttcgaagg tgaaggtgac gacactatgt 480 tcaccactga taatgagaag gttagcctct tcaatttcag aaagaatgct gacccacaga 540 tggttagaga ggcctacgca gcaagtctca tcaagacgat ctacccgagt aacaatctcc 600 aggagatcaa ataccttccc aagaaggtta aagatgcagt caaaagattc aggactaatt 660 gcatcaagaa cacagagaaa gacatatttc tcaagatcag aagtactatt ccagtatgga 720 cgattcaagg cttgcttcat aaaccaaggc aagtaataga gattggagtc tctaaaaagg 780 tagttcctac tgaatctaag gccatgcatg gagtctaaga ttcaaatcga ggatctaaca 840 gaactcgccg tcaagactgg cgaacagttc atacagagtc ttttacgact caatgacaag 900 aagaaaatct tcgtcaacat ggtggagcac gacactctgg tctactccaa aaatgtcaaa 960 gatacagtct cagaagatca aagggctatt gagacttttc aacaaaggat aatttcggga 1020 aacctcctcg gattccattg cccagctatc tgtcacttca tcgaaaggac agtagaaaag 1080 gaaggtggct cctacaaatg ccatcattgc gataaaggaa aggctatcat tcaagatctc 1140 tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa 1200 gaggttccaa ccacgtctac aaagcaagtg gattgatgtg acatctccac tgacgtaagg 1260 gatgacgcac aatcccacta tccttcgcaa gacccttcct ctatataagg aagttcattt 1320 catttggaga ggacacgctc gagtataagg taaatttctg tgttccttat tctctcaaaa 1380 tcttcgattt tgttttcgtt cgatcccaat ttcgtatatg ttctttggtt tagattctgt 1440 taatcttaga tcgaagatga ttttctgggt ttgatcgtta gatatcatct taattctcga 1500 ttagggtttc atagatatca tccgatttgt tcaaataatt tgagttttgt cgaataatta 1560 ctcttcgatt tgtgatttct atctagatct ggtgttagtt tctagtttgt gcgatcgaat 1620 ttgtcgatta atctgagttt ttctgattaa caggagctca tttttacaac aattaccaac 1680 aacaacaaac aacaaacaac attacaatta catttacaat tatcgatac 1729 <210> 3 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> L3S2P21 terminator <400> 3 ctcggtacca aattccagaa aagaggcctc ccgaaagggg ggcctttttt cgttttggtc 60 c 61 <210> 4 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> 35S terminator <400> 4 ctctagctag agtcgatcga caagctcgag tttctccata ataatgtgtg agtagttccc 60 agataaggga attagggttc ctatagggtt tcgctcatgt gttgagcata taagaaaccc 120 ttagtatgta tttgtatttg taaaatactt ctatcaataa aatttctaat tcctaaaacc 180 aaaatccagt actaaaatcc agat 204 <210> 5 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Ec1 promoter <400> 5 tttacagcta gctcagtcct aggtataatg ctagcagatc taataatttt gtttaacttt 60 gggaggata 69 <210> 6 <211> 129 <212> DNA <213> Artificial Sequence <220> <223> BBa_B0015 terminator <400> 6 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt 60 gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct 120 gcgtttata 129 <210> 7 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> AtU6 promoter <400> 7 tgatcaaaag tcccacatcg atcaggtgat atatagcagc ttagtttata taatgataga 60 gtcgacatag cg 72 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> J23119 promoter <400> 8 ttgacagcta gctcagtcct aggtataata ctagt 35 <210> 9 <211> 4104 <212> DNA <213> Artificial Sequence <220> <223> nCas9(D10A) <400> 9 atggataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 60 ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 120 cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 180 gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 240 tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 300 ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 360 aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 420 aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 480 atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 540 gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 600 ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 660 cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 720 cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 780 gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 840 caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 900 ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 960 atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 1020 cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 1080 ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 1140 gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 1200 aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 1260 gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 1320 gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 1380 cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 1440 gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 1500 aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 1560 tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 1620 agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 1680 gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 1740 tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 1800 attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 1860 ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 1920 cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 1980 cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 2040 gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 2100 tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 2160 cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 2220 gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 2280 atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 2340 atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 2400 gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 2460 gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 2520 attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 2580 gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 2640 aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 2700 actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 2760 ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 2820 acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 2880 aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 2940 taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 3000 tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 3060 atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 3120 aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 3180 cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 3240 gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 3300 cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 3360 gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 3420 tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 3480 aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 3540 ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 3600 tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 3660 caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 3720 cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 3780 cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 3840 atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 3900 cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 3960 ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 4020 gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 4080 gatttgtcac agcttggggg tgac 4104 <210> 10 <211> 4104 <212> DNA <213> Artificial Sequence <220> <223> dCas9 <400> 10 atggataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 60 ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 120 cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 180 gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 240 tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 300 ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 360 aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 420 aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 480 atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 540 gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 600 ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 660 cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 720 cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 780 gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 840 caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 900 ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 960 atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 1020 cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 1080 ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 1140 gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 1200 aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 1260 gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 1320 gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 1380 cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 1440 gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 1500 aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 1560 tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 1620 agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 1680 gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 1740 tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 1800 attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 1860 ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 1920 cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 1980 cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 2040 gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 2100 tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 2160 cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 2220 gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 2280 atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 2340 atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 2400 gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 2460 gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 2520 attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 2580 gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 2640 aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 2700 actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 2760 ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 2820 acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 2880 aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 2940 taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 3000 tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 3060 atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 3120 aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 3180 cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 3240 gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 3300 cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 3360 gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 3420 tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 3480 aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 3540 ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 3600 tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 3660 caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 3720 cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 3780 cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 3840 atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 3900 cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 3960 ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 4020 gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 4080 gatttgtcac agcttggggg tgac 4104 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sgRNA1 <400> 11 acacacacac ttagaatatg ggg 23 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sgRNA2 <400> 12 cacacacaca ttagaatatg ggg 23 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> AtGL1 <400> 13 ggaaaagttg tagactgaga tgg 23 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> SIMlo1 <400> 14 gtacaaagtt aatcaagaat agg 23 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> SIPelo <400> 15 tccagcatca ttcagttgtg ggg 23 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> inactive 1 <400> 16 ggtgaagcag cggacagcag tgg 23 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> inactive 2 <400> 17 gggatgagca tcgggtagcc tgg 23 <110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Recombinant vector for activity test of guide RNA for CRISPR-based base editing and uses its <130> PN22020 <160> 17 <170> KoPatentIn 3.0 <210> 1 <211 > 166 <212> DNA <213> Artificial Sequence <220> <223> GlpT promoter <400> 1 gaaagtgaaa cgtgatttca tgcgtcattt tgaacatttt gtaaatctta tttaataatg 60 tgtgcggcaa ttcacattta atttatgaat gttttcttaa catcgcggca actcaagaaa 120 cggcaggttc ggatcttagc tactagagaa agaggagaaa tactag 166 <210> 2 <211> 1729 <212> DNA <213> Artificial Sequence <220> <223> 35S promoter <400> 2 gaattccaat cccacaaaaa tctgagctta acagcacagt tgctcctctc agagcagaat 60 cgggtattca acaccctcat atcaactact acgttgtgta taacggtcca catgccggta 1 20 tatacgatga ctggggttgt acaaaggcgg caacaaacgg cgttcccgga gttgcacaca 180 agaaatttgc cactattaca gaggcaagag cagcagctga cgcgtacaca acaagtcagc 240 aaacagacag gttgaacttc atccccaaag gagaagctca actcaagccc aagagctttg 300 ctaaggccct aacaagccca ccaaagcaaa aagcccactg gctcacgcta ggaaccaaaa 360 ggcccagcag tgatccagcc ccaaaagaga tctcctttgc cccggagatt acaatggacg 420 att tcctcta tctttacgat ctaggaagga agttcgaagg tgaaggtgac gacactatgt 480 tcaccactga taatgagaag gttagcctct tcaatttcag aaagaatgct gacccacaga 540 tggttagaga ggcctacgca gcaagtctca tcaagacgat ctacccgagt aacaatctcc 600 aggagat caa ataccttccc aagaaggtta aagatgcagt caaaagattc aggactaatt 660 gcatcaagaa cacagagaaa gacatatttc tcaagatcag aagtactatt ccagtatgga 720 cgattcaagg cttgcttcat aaaccaaggc aagtaataga gattggagtc tctaaaaagg 780 tagttcctac tgaatctaag gccatgcatg gagtctaaga ttcaaatcga ggatctaaca 840 gaactcgccg tca agactgg cgaacagttc atacagagtc ttttacgact caatgacaag 900 aagaaaatct tcgtcaacat ggtggagcac gacactctgg tctactccaa aaatgtcaaa 960 gatacagtct cagaagatca aagggctatt gagacttttc aacaaaggat aatttcggga 1020 aacctcctcg gattccattg c ccagctatc tgtcacttca tcgaaaggac agtagaaaag 1080 gaaggtggct cctacaaatg ccatcattgc gataaaggaa aggctatcat tcaagatctc 1140 tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa 1200 gaggttccaa ccacgtctac aaagcaagtg gattgatgtg acatctccac tgacgtaagg 1260 gatgacgcac a atcccacta tccttcgcaa gacccttcct ctatataagg aagttcattt 1320 catttggaga ggacacgctc gagtataagg taaatttctg tgttccttat tctctcaaaa 1380 tcttcgattt tgttttcgtt cgatcccaat ttcgtatatg ttctttggtt tagattctgt 1440 taatcttaga tcgaagatga ttttctgggt ttgatcgtta gatatcatct taattctcga 1500 ttagggtttc atagatatca tccgatttgt tcaaataatt tgagttttgt cgaataatta 1560 ctcttcgatt tgtgatttct atctagatct ggtgttagtt tctagtttgt gcgatcgaat 1620 ttgtcgatta atctgagttt ttctgattaa caggagctca tttttacaac aattaccaac 1680 aacaacaaac aacaaacaac attacaatta catttacaat tatcgatac 1729 <210> 3 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> L3S2P21 terminator < 400> 3 ctcggtacca aattccagaa aagaggcctc ccgaaagggg ggcctttttt cgttttggtc 60 c 61 <210> 4 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> 35S terminator <400> 4 ctctagctag agtc gatcga caagctcgag tttctccata ataatgtgtg agtagttccc 60 agataaggga attagggttc ctatagggtt tcgctcatgt gttgagcata taagaaaccc 120 ttagtatgta tttgtatttg taaaatactt ctatcaataa aatttctaat tcctaaaacc 180 aaaatccagt actaaaatcc agat 204 <210> 5 <211> 69 <212> DNA <213> Artificial Sequence <22 0> <223> Ec1 promoter <400> 5 tttacagcta gctcagtcct aggtataatg ctagcagatc taataatttt gtttaacttt 60 gggaggata 69 <210> 6 <211> 129 <212> DNA <213> Artificial Sequence <220> <223> BBa_B0015 terminator <400> 6 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt 60 gt ttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct 120 gcgtttata 129 < 210> 7 <211> 72 <212> DNA <213> Artificial sequence <220> <223> ATU6 Promoter <400> 7 tgatccacatcg Atccacatcg ATAGTAGC TTAGTTAGCAGA 60 GTCG Acatag CG 72 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> J23119 promoter <400> 8 ttgacagcta gctcagtcct aggtataata ctagt 35 <210> 9 <211> 4104 <212> DNA <213> Artificial Sequence <220> <223> nCas9(D10A) < 400> 9 atggataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 60 ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 120 cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacgg cagag 180 gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 240 tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 300 ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcac cc catctttgga 360 aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 420 aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 480 atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 540 gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgt ttga agagaaccct 600 ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 660 cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 720 cttatagcgc tctcactagg cctgacacca aattttaagt cgaact tcga cttagctgaa 780 gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 840 caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 900 ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 960 atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 1020 cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 1080 ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 1140 gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgc ga 1200 aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 1260 gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 1320 gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 1380 cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 1440 gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 1500 aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 1560 tacaatgaac tcacgaaagt taagtatgtc actgagggca tgc gtaaacc cgcctttcta 1620 agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 1680 gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 1740 tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 1800 attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 1860 ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 1920 cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 1980 cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 2040 gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 2100 tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 2160 cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 2220 gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 2280 atcgagatgg cacgc gaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 2340 atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 2400 gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 2460 gacatgtatg ttgat cagga actggacata aaccgtttat ctgattacga cgtcgatcac 2520 attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 2580 gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 2640 aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 2700 actaaagctg agaggggtgg ctt gtctgaa cttgacaagg ccggatttat taaacgtcag 2760 ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 2820 acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 2880 aaattggtgt cggact tcag aaaggatttt caattctata aagttaggga gataaataac 2940 taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 3000 tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 3060 atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 3120 aacattatga atttctttaa gacggaaatc actct ggcaa acggagagat acgcaaacga 3180 cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 3240 gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 3300 cagaccggag ggttttcaaa ggaatc gatt cttccaaaaa ggaatagtga taagctcatc 3360 gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 3420 tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 3480 aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 3540 ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa act accaaag 3600 tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 3660 caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 3720 cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagca act ttttgttgag 3780 cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 3840 atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 3900 cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 3960 ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctacca ag 4020 gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 4080 gatttgtcac agcttggggg tgac 4104 <210> 10 <211> 4104 <212> DNA <213> Artificial Sequence <220> <223> dCas9 <400> 10 atggataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 60 ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 120 cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 180 gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 240 tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 300 ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 360 aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 420 aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 480 atgataaagt tccgtgggca ctttctcatt gagggtgatc taaat ccgga caactcggat 540 gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 600 ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 660 cggctagaaa acctgatcgc acaattaccc ggagaga aga aaaatgggtt gttcggtaac 720 cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 780 gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 840 caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 900 ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccg cttca 960 atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 1020 cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 1080 ggttatattg acggcggagc gagtcaagag gaattctaca agtttat caa acccatatta 1140 gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 1200 aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 1260 gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 1320 gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaact ct 1380 cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 1440 gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 1500 aatttaccga acgaaaaagt attgcctaag cacagtttac t ttacgagta tttcacagtg 1560 tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 1620 agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 1680 gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 1740 tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 18 00 attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 1860 ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 1920 cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 1980 cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 2040 gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 2100 tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 2160 cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 2220 gtcaa agtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 2280 atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 2340 atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 2400 gt ggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 2460 gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 2520 attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 2580 gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 2640 aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 2700 actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 2760 ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 2820 acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 2880 aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 2940 taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 3000 tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 3060 atgatcgcga aaagcgaaca ggagata ggc aaggctacag ccaaatactt cttttattct 3120 aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 3180 cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 3240 gcgacggtga gaaaagtttt g tccatgccc caagtcaaca tagtaaagaa aactgaggtg 3300 cagaccggag ggttttcaaa ggaatcgatt cttccaaaaaa ggaatagtga taagctcatc 3360 gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 3420 tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 3480 aaagaattat tggggataac gattatggag cgctcgtct t ttgaaaagaa ccccatcgac 3540 ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 3600 tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 3660 caaaagggga acgaactcgc actaccgtct aaatacgtga at ttcctgta tttagcgtcc 3720 cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 3780 cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 3840 atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca caggggataaa 3900 cccatacgtg agcaggcgga aaatattatc catttgttta ctctt accaa cctcggcgct 3960 ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 4020 gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 4080 gatttgtcac agcttggggg tgac 4104 <210> 11 <211> 23 < 212> DNA <213> Artificial Sequence <220> <223> sgRNA1 <400> 11 acacacaacac ttagaatatg ggg 23 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sgRNA2 <400> 12 cacacacaca ttagaatatg ggg 23 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> AtGL1 <400> 13 ggaaaagttg tagactgaga tgg 23 <210> 14 <211> 23 <212> DNA < 213> Artificial Sequence <220> <223> SIMlo1 <400> 14 gtacaaagtt aatcaagaat agg 23 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> SIPelo <400> 15 tccagcatca ttcagttgtg ggg 23 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> inactive 1 <400> 16 ggtgaagcag cggacagcag tgg 23 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> inactive 2<400> 17 gggatgagca tcgggtagcc tgg 23
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