KR102497690B1 - Novel CRISPR Associated Protein and Use thereof - Google Patents

Novel CRISPR Associated Protein and Use thereof Download PDF

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KR102497690B1
KR102497690B1 KR1020200121989A KR20200121989A KR102497690B1 KR 102497690 B1 KR102497690 B1 KR 102497690B1 KR 1020200121989 A KR1020200121989 A KR 1020200121989A KR 20200121989 A KR20200121989 A KR 20200121989A KR 102497690 B1 KR102497690 B1 KR 102497690B1
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Abstract

본 발명은 신규한 CRISPR 연관 단백질 및 이의 용도에 관한 것으로, 가이드 RNA와 결합된 세포 내 핵산 서열을 인식하여 절단하는 엔도뉴클레아제 활성이 종래 사용되는 AsCpf1 단백질보다도 현저히 우수하므로, CRISPR-Cas 시스템에서 인델 효율이 우수한 뉴클레아제로써 효과적으로 활용될 수 있다.The present invention relates to a novel CRISPR-associated protein and its use. Since the endonuclease activity for recognizing and cutting intracellular nucleic acid sequences bound to guide RNA is significantly superior to AsCpf1 protein previously used, in the CRISPR-Cas system It can be effectively utilized as a nuclease with excellent indel efficiency.

Description

신규한 CRISPR 연관 단백질 및 이의 용도{Novel CRISPR Associated Protein and Use thereof}Novel CRISPR Associated Protein and Use thereof {Novel CRISPR Associated Protein and Use thereof}

본 발명은 신규한 CRISPR 연관 단백질 및 이의 용도에 관한 것이다.The present invention relates to novel CRISPR-associated proteins and uses thereof.

유전체 편집(Genome Editing)이란 생명체의 유전정보를 자유롭게 교정하는 기술을 말한다. 생명과학분야의 진보와 유전체 서열 분석 기술의 발전을 통해 우리는 다양한 유전정보에 대해 폭넓게 이해할 수 있게 되었다. 예를 들어 동식물의 번식, 질병과 성장, 다양한 인간 유전질병을 일으키는 유전자 변이, 바이오연료의 생산 등을 위한 유전자에 대한 이해는 이미 확보된 상황이지만 이를 직접적으로 활용하여 생명체를 개선하고, 인간 질병을 치료하는 수준에까지 이르기 위해서는 그 이상의 기술 진보가 필수적이다.Genome editing is a technology that freely corrects the genetic information of living organisms. Advances in the field of life science and advances in genome sequencing technology have enabled us to broadly understand various genetic information. For example, although understanding of genes for animal and plant reproduction, disease and growth, genetic mutations that cause various human genetic diseases, and production of biofuels has already been secured, it is possible to directly utilize them to improve living organisms and prevent human diseases. Further technological advances are essential to reach the level of treatment.

유전체 편집 기술은 인간을 포함하여 동물, 식물, 미생물의 유전정보를 변화시켜 그 활용범위를 획기적으로 확장시킬 수 있다. 유전자 가위는 원하는 유전정보를 정확히 자를 수 있도록 설계되어 만들어지는 분자 도구로 유전체 편집 기술에서 핵심역할을 하고 있다. 유전자 서열분석 분야를 한 차원 발전시켰던 차세대시퀀싱(Next generation sequencing) 기술과 같이, 유전자 가위는 유전정보 활용의 속도와 그 범위를 확장시키고 새로운 산업 분야를 창출해 내는 핵심 기술이 되고 있다.Genome editing technology can change the genetic information of animals, plants, and microorganisms, including humans, to dramatically expand its application range. Genetic scissors are molecular tools designed and made to accurately cut the desired genetic information, and play a key role in genome editing technology. Like the next-generation sequencing technology that advanced the genetic sequencing field to a new level, genetic scissors are becoming a key technology that expands the speed and scope of genetic information utilization and creates new industrial fields.

지금까지 개발된 유전자 가위는 그 순서에 따라 3세대로 나눌 수 있다. 1세대 유전자 가위는 ZFN(Zinc Finger Nuclease), 2세대 유전자 가위는 TALEN(Transcription Activator-Like Effector Nuclease), 가장 최근에 연구된 CRISPR(Clustered regularly interspaced short palindromic repeat)-Cas(CRISPR-associated)9은 3세대 유전자 가위다.The genetic scissors developed so far can be divided into three generations according to the order. ZFN (Zinc Finger Nuclease) is the first generation gene scissors, Transcription Activator-Like Effector Nuclease (TALEN) is the second generation gene scissors, and CRISPR (Clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated)9, which has been studied most recently, is 3rd generation genetic scissors.

CRISPRs는 유전자 서열이 밝혀진 박테리아의 대략 40% 및 유전자 서열이 밝혀진 고세균의 90%의 유전체에서 발견되는 여러 짧은 직접 반복을 포함하는 좌위이다. Cas 단백질은 CRISPR RNA(crRNA) 및 trans-activating crRNA(tracrRNA)로 명명된 두 개의 RNA와 복합체를 형성했을 때, 활성 엔도뉴클레아제(endonuclease)를 형성하고, 그렇게 함으로써 파지 또는 플라스미드의 침입에서 외부 유전적 요소를 묵살하여 숙주 세포를 보호한다. crRNA는 전달에 외부 침입자로부터 점유되었던 숙주 유전체의 CRISPR 요소로부터 전사된다.CRISPRs are loci containing multiple short direct repeats found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. When the Cas protein complexes with two RNAs, termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), it forms an active endonuclease, thereby preventing invasion by phages or plasmids. It protects the host cell by ignoring the genetic component. The crRNA is transcribed from the CRISPR element of the host genome that was occupied by the foreign invader upon transfer.

이러한 CRISPR-Cas 시스템 유래의 RNA-가이드 뉴클레아제(RNA-guided nuclease)는 유전체를 편집할 수 있는 수단을 제공해준다. 특히, 단일 가이드 RNA(sgRNA)와 Cas 단백질을 이용하여 세포 및 기관의 유전체를 편집할 수 있는 기술과 관련된 연구들이 활발히 진행되어왔다. 최근, Cpf1(Cas12a) 단백질(Prevotella 및 Francisella 1 유래)이 CRISPR-Cas 시스템의 또 다른 뉴클레아제 단백질로서 보고되었고(B Zetsche, et al, 2015), 이에 따라 유전체 편집에 있어서 선택의 폭이 넓어졌다.RNA-guided nucleases from the CRISPR-Cas system provide a means to edit the genome. In particular, studies related to technology capable of editing the genome of cells and organs using single guide RNA (sgRNA) and Cas protein have been actively conducted. Recently, the Cpf1 (Cas12a) protein (derived from Prevotella and Francisella 1) was reported as another nuclease protein of the CRISPR-Cas system (B Zetsche, et al, 2015), thus providing a wide range of options for genome editing. lost.

다만, Cpf1 단백질 기반의 CRISPR-Cas 시스템 역시 종래 Cas9 단백질 기반 유전자 가위의 표적이탈(off-target) 문제를 여전히 나타낸다. 표적이탈 효과에 의한 비표적 DNA 절단은 전암유전자(proto-oncogene) 및 암억제유전자(tumor suppressor gene)와 같이 원치 않는 유전자에서 돌연변이를 야기할 수 있고, 전위(translocation), 결실(deletion), 및 역위(inversion)와 같은 유전체 재조합을 증가시킬 수 있어, 연구 분야 및 의학 분야 등에서 유전자 가위를 이용하는데 심각한 문제가 된다. 따라서, 최근 유전자 가위의 표적이탈을 감소시키기 위하여, 인델(indel) 효율이 우수한 유전자 가위를 개발하기 위한 연구가 수행되고 있으나, 전체 유전체 수준에서 표적이탈 효과 없이 표적 위치에만 특이적으로 작동하는 유전자 가위는 아직까지 보고된 바 없다.However, the CRISPR-Cas system based on the Cpf1 protein still exhibits the off-target problem of conventional Cas9 protein-based genetic scissors. Off-target DNA cleavage by off-target effects can lead to mutations in unwanted genes, such as proto-oncogenes and tumor suppressor genes, resulting in translocation, deletion, and It can increase genetic recombination such as inversion, which is a serious problem in using genetic scissors in research fields and medical fields. Therefore, recently, in order to reduce the off-target of genetic scissors, research is being conducted to develop genetic scissors with excellent indel efficiency, but gene scissors that operate specifically on the target site without off-target effects at the whole genome level. has not yet been reported

이에, 본 발명자들은 유전자 가위에 효과적으로 적용될 수 있는 신규 CRISPR 연관 단백질을 개발하기 위한 연구를 수행하여 본 발명을 완성하였다.Accordingly, the present inventors completed the present invention by conducting research to develop a novel CRISPR-associated protein that can be effectively applied to genetic scissors.

대한민국 특허등록 제10-1828933호Republic of Korea Patent Registration No. 10-1828933

본 발명의 하나의 목적은 서열번호 1의 아미노산 서열로 이루어진 단백질; 및 상기 단백질의 C-말단에 서열번호 4로 이루어진 myc-NLS(nuclear localization sequences)가 연결된 Cas12a 단백질을 제공하는 것이다.One object of the present invention is a protein consisting of the amino acid sequence of SEQ ID NO: 1; and a Cas12a protein in which myc-NLS (nuclear localization sequences) consisting of SEQ ID NO: 4 is linked to the C-terminus of the protein.

본 발명의 다른 목적은 상기 Cas12a 단백질 또는 이를 암호화하는 DNA; 및 표적 뉴클레오티드(target nucleotide) 서열과 혼성화 가능한 뉴클레오티드 서열을 포함하는 가이드 RNA 또는 이를 암호화하는 DNA를 포함하는 유전체 편집용 조성물을 제공하는 것이다.Another object of the present invention is the Cas12a protein or DNA encoding it; and a guide RNA comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the guide RNA.

본 발명의 또 다른 목적은 상기 유전체 편집용 조성물을 분리된 세포 또는 유기체에 도입하는 단계를 포함하는 유전체 편집 방법을 제공하는 것이다.Another object of the present invention is to provide a genome editing method comprising introducing the genome editing composition into a separated cell or organism.

본 발명의 또 다른 목적은 상기 유전체 편집용 조성물을 분리된 세포 또는 인간을 제외한 유기체에 도입하는 단계를 포함하는 형질 전환체의 제조 방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing a transformant comprising the step of introducing the composition for genome editing into isolated cells or organisms other than humans.

본 발명의 일 양상은 서열번호 1의 아미노산 서열로 이루어진 단백질; 및 상기 단백질의 C-말단에 서열번호 4로 이루어진 myc-NLS(nuclear localization sequences)가 연결된 Cas12a 단백질을 제공한다.One aspect of the present invention is a protein consisting of the amino acid sequence of SEQ ID NO: 1; and a Cas12a protein in which myc-NLS (nuclear localization sequences) consisting of SEQ ID NO: 4 is linked to the C-terminus of the protein.

본 명세서에서 사용한 용어 "Cas12a"는 CRISPR 연관 단백질로써, Cpf1로도 지칭될 수 있는 type V CRISPR 시스템 단백질이다. Cas12a는 단일 단백질로써, crRNA과 결합하여 표적 유전자를 절단한다는 점은 type Ⅱ CRISPR 시스템 단백질인 Cas9와 유사하나, 그 작동 방식에서 차이가 있다. Cas12a 단백질은 하나의 crRNA로 작동하므로, Cas9의 경우와 같이 crRNA와 trans-activating crRNA(tracrRNA)를 동시에 사용하거나, 인위적으로 tracrRNA와 crRNA를 합친 single guide RNA(sgRNA)를 제작할 필요가 없다.As used herein, the term "Cas12a" is a CRISPR-associated protein and is a type V CRISPR system protein that can also be referred to as Cpf1. Cas12a is a single protein, similar to Cas9, a type II CRISPR system protein, in that it binds to crRNA and cleave a target gene, but differs in its mode of operation. Since the Cas12a protein works as a single crRNA, there is no need to use crRNA and trans-activating crRNA (tracrRNA) at the same time, as in the case of Cas9, or to artificially create a single guide RNA (sgRNA) combining tracrRNA and crRNA.

또한, Cas12a 시스템은 Cas9와 달리 PAM이 표적 서열의 5' 위치에 존재한다. 또한, Cas12a 시스템은 표적을 결정하는 guide RNA(gRNA)의 길이도 Cas9에 비해 짧다. 또한, Cas12a는 표적 DNA가 절단된 위치에 blunt-end가 아닌 5' overhang(sticky end)을 발생시키므로, 보다 정확하고 다양한 유전자 편집이 가능하다는 장점을 갖는다.In addition, in the Cas12a system, unlike Cas9, the PAM is present at the 5' position of the target sequence. In addition, the length of the guide RNA (gRNA) that determines the target in the Cas12a system is shorter than that of Cas9. In addition, since Cas12a generates a 5' overhang (sticky end) rather than a blunt-end at the site where the target DNA was cut, more accurate and diverse gene editing is possible.

mgCas12a는 AsCpf1 및 LbCpf1 대비 인델 효율이 낮아, CRISPR-Cas 시스템에 적용되기 어려웠다. 다만, 본 발명에서는 mgCas12a-1의 C-말단에 핵위치 신호인 myc-NLS를 반복연결한 mgCas12a-1-6XNLS에서 현저히 우수한 인델 효율이 확인되어, 종래 CRISPR-Cas 시스템의 유전자 편집 효율을 개선하기 위해 mgCas12a-1-6XNLS이 유용하게 활용될 수 있음이 확인되었다.mgCas12a has lower indel efficiency than AsCpf1 and LbCpf1, making it difficult to apply to the CRISPR-Cas system. However, in the present invention, significantly superior indel efficiency was confirmed in mgCas12a-1-6XNLS, in which myc-NLS, a nuclear localization signal, was repeatedly linked to the C-terminus of mgCas12a-1, so that the gene editing efficiency of the conventional CRISPR-Cas system could be improved. It was confirmed that mgCas12a-1-6XNLS can be usefully utilized for this purpose.

본 발명의 일 구체예에 따르면, 상기 연결은 Gly-Gly-Ser 아미노산 서열에 의한 것일 수 있다.According to one embodiment of the present invention, the connection may be by a Gly-Gly-Ser amino acid sequence.

myc-NLS는 Gly-Gly-Ser 아미노산 서열에 의해 mgCas12a-1의 C-말단에 연결되는 것이 바람직하다.The myc-NLS is preferably linked to the C-terminus of mgCas12a-1 by the Gly-Gly-Ser amino acid sequence.

본 발명의 일 구체예에 따르면, 상기 myc-NLS는 2 내지 10개가 반복연결된 것일 수 있다.According to one embodiment of the present invention, 2 to 10 myc-NLSs may be repeatedly linked.

본 발명의 일 구체예에 따르면, 상기 myc-NLS는 6개가 반복연결된 것일 수 있다.According to one embodiment of the present invention, six myc-NLSs may be repeatedly linked.

본 발명의 일 구체예에 따르면, 상기 myc-NLS는 각각 Gly-Gly-Ser 아미노산 서열로 연결된 것일 수 있다.According to one embodiment of the present invention, each of the myc-NLSs may be linked by a Gly-Gly-Ser amino acid sequence.

mgCas12a-1의 C-말단에 연결되는 myc-NLS는 복수개가 반복연결된 것일 수 있고, 바람직하게는 2 내지 10개가 반복연결된 것일 수 있으며, 가장 바람직하게는 6개가 반복연결된 것일 수 있다. myc-NLS는 복수개의 mgCas12a-1 C-말단 반복연결에 의하여, mgCas12a-1의 유전자 편집 효율이 현저히 개선될 수 있으며, myc-NLS는 Gly-Gly-Ser 아미노산 서열에 의해 서로 연결될 수 있다.The myc-NLS linked to the C-terminus of mgCas12a-1 may be a plurality of repeats, preferably 2 to 10 repeats, and most preferably 6 repeats. The myc-NLS can significantly improve the gene editing efficiency of mgCas12a-1 by connecting a plurality of mgCas12a-1 C-terminal repeats, and the myc-NLS can be linked to each other by the Gly-Gly-Ser amino acid sequence.

본 발명의 일 구체예에 따르면, 상기 서열번호 1의 아미노산 서열 중 169번째 위치의 라이신(Lys)이 아르기닌(Arg)으로 치환된 것일 수 있다.According to one embodiment of the present invention, lysine (Lys) at position 169 in the amino acid sequence of SEQ ID NO: 1 may be substituted with arginine (Arg).

본 발명의 일 구체예에 따르면, 상기 서열번호 1의 아미노산 서열 중 529번째 위치의 아스파르트산(Asp)이 아르기닌(Arg)으로 치환된 것일 수 있다.According to one embodiment of the present invention, aspartic acid (Asp) at position 529 in the amino acid sequence of SEQ ID NO: 1 may be substituted with arginine (Arg).

특히, 본 발명의 mgCas12a-1-6XNLS의 아미노산 서열 중 169번째 위치의 라이신(Lys)이 아르기닌(Arg)으로 치환 및 529번째 위치의 아스파르트산(Asp)이 아르기닌(Arg)으로 치환에 의해 최적화된 opmgCas12a-1-6XNLS에 의하여 효과적인 유전자 편집이 이루어질 수 있다.In particular, in the amino acid sequence of mgCas12a-1-6XNLS of the present invention, lysine (Lys) at position 169 is substituted with arginine (Arg) and aspartic acid (Asp) at position 529 is replaced with arginine (Arg). Effective gene editing can be achieved by opmgCas12a-1-6XNLS.

본 발명의 일 구체예에 따르면, 상기 Cas12a 단백질은 서열번호 19의 아미노산 서열로 이루어진 것일 수 있다.According to one embodiment of the present invention, the Cas12a protein may consist of the amino acid sequence of SEQ ID NO: 19.

본 발명의 다른 양상은 상기 Cas12a 단백질 또는 이를 암호화하는 DNA; 및 표적 뉴클레오티드(target nucleotide) 서열과 혼성화 가능한 뉴클레오티드 서열을 포함하는 가이드 RNA 또는 이를 암호화하는 DNA를 포함하는 유전체 편집용 조성물을 제공한다.Another aspect of the present invention is the Cas12a protein or DNA encoding the same; and a guide RNA comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the guide RNA.

본 발명에서 사용되는 용어, "가이드 RNA(gRNA)"는 표적 DNA에 상보적으로 결합할 수 있는 염기서열을 포함하는 RNA로, gRNA는 mgCas12a-1-6XNLS 단백질과 복합체를 형성할 수 있고, mgCas12a-1-6XNLS 단백질을 표적 DNA에 가져올 수 있는 단일 사슬 RNA를 말한다. 본 발명에서 가이드 RNA는 절단하고자 하는 어떠한 표적에 특이적이 되도록 제조될 수 있다.As used herein, the term "guide RNA (gRNA)" is RNA containing a nucleotide sequence capable of complementary binding to target DNA, and gRNA can form a complex with mgCas12a-1-6XNLS protein, and mgCas12a -1-6XNLS Refers to single-stranded RNA capable of bringing proteins to target DNA. Guide RNA in the present invention can be prepared to be specific to any target to be cleaved.

본 발명의 mgCas12a-1-6XNLS 단백질은 세포 내로 도입되기에 용이한 형태일 수 있다. 예를 들면, mgCas12a-1-6XNLS 단백질은 세포 투과 펩타이드 또는 단백질 전달 도메인(protein transduction domain)과 연결된 것일 수 있다. 상기 단백질 전달 도메인은 폴리-아르기닌 또는 HIV 유래의 TAT 단백질일 수 있으나, 이에 한정되는 것은 아니다.The mgCas12a-1-6XNLS protein of the present invention may be in a form that is easily introduced into cells. For example, the mgCas12a-1-6XNLS protein may be linked to a cell penetrating peptide or a protein transduction domain. The protein transduction domain may be poly-arginine or HIV-derived TAT protein, but is not limited thereto.

가이드 RNA의 표적 DNA 서열의 상보적 사슬과 염기 쌍을 형성할 수 있는 서열의 길이는 17 내지 23bp, 18 내지 23bp, 19 내지 23bp, 보다 구체적으로 20 내지 23bp, 보다 더 구체적으로, 21 내지 23bp 일 수 있으나, 이에 한정되는 것은 아니다.The length of the sequence capable of forming a base pair with the complementary chain of the target DNA sequence of the guide RNA is 17 to 23 bp, 18 to 23 bp, 19 to 23 bp, more specifically 20 to 23 bp, still more specifically 21 to 23 bp It may, but is not limited thereto.

가이드 RNA에 의해 표적화되는 DNA 서열은 5'-말단 부위 상류에 3 내지 4 개의 추가적인 뉴클레오티드인 PAM(protospacer adjacent motif) 서열을 포함하며, 구체적으로 상기 PAM 서열은 5'-TTTG-3' 또는 5'-TTTA-3'인 것이 바람직하다.The DNA sequence targeted by the guide RNA includes a protospacer adjacent motif (PAM) sequence of 3 to 4 additional nucleotides upstream of the 5'-terminal region, specifically, the PAM sequence is 5'-TTTG-3' or 5' -TTTA-3' is preferred.

mgCas12a-1-6XNLS 단백질은 분리 및/또는 정제에 유리한 태그와 연결될 수 있다. 예를 들어, His 태그, Flag 태그, S 태그 등과 같은 작은 펩타이드 태그, 또는 GST(Glutathione S-transferase) 태그, MBP(Maltose binding protein) 태그 등이 목적에 따라 사용될 수 있으나, 이에 한정되는 것은 아니다.The mgCas12a-1-6XNLS protein can be linked with tags advantageous for isolation and/or purification. For example, a small peptide tag such as a His tag, a Flag tag, an S tag, or a Glutathione S-transferase (GST) tag or a Maltose binding protein (MBP) tag may be used depending on the purpose, but is not limited thereto.

본 발명의 CRISPR/mgCas12a-1-6XNLS 시스템은 가이드 RNA를 코딩하는 DNA 및 Cas 단백질을 코딩하는 핵산과 같은 분리된 핵산 자체로 이용될 수 있고, 또한 가이드 RNA, 또는/및 Cas 단백질을 발현하기 위한 발현 카세트를 포함하는 재조합 발현 벡터 형태로도 사용될 수 있다.The CRISPR/mgCas12a-1-6XNLS system of the present invention can be used as an isolated nucleic acid itself, such as DNA encoding guide RNA and nucleic acid encoding Cas protein, and can also be used for expressing guide RNA, or/and Cas protein. It can also be used in the form of a recombinant expression vector containing an expression cassette.

본 발명에서 사용되는 용어, "재조합 발현벡터"는 목적한 코딩 서열과, 특정 숙주 생물에서 작동가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. 진핵세포에서 이용 가능한 프로모터, 인핸서, 종결신호 및 폴리아데닐레이션 신호는 공지되어 있다.As used herein, the term "recombinant expression vector" refers to a recombinant DNA molecule containing a desired coding sequence and an appropriate nucleic acid sequence essential for expressing the operably linked coding sequence in a specific host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

본 발명에서 사용되는 용어, "작동가능하게 연결된"은 유전자 발현 조절 서열과 다른 뉴클레오티드 서열사이의 기능적인 결합을 의미한다. 상기 유전자 발현 조절 서열은 복제원점(replication origin), 프로모터 및 전사 종결 서열(terminator) 등으로 이루어진 군으로부터 선택되는 1종 이상일 수 있다. 전사 종결 서열은 폴리아데닐화 서열(pA)일 수 있으며, 복제 원점은 f1 복제원점, SV40 복제원점, pMB1 복제원점, 아데노 복제원점, AAV 복제원점 또는 BBV 복제원점 등일 수 있으나, 이에 한정되는 것은 아니다.As used herein, the term "operably linked" means a functional linkage between a gene expression control sequence and another nucleotide sequence. The gene expression control sequence may be one or more selected from the group consisting of a replication origin, a promoter, and a transcription termination sequence. The transcription termination sequence may be a polyadenylation sequence (pA), and the origin of replication may be the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, or the BBV origin of replication, but is not limited thereto. .

본 발명에서 사용되는 용어, "프로모터"는 구조 유전자로부터의 DNA 업스트림의 영역을 의미하며, 전사를 개시하기 위하여 RNA 폴리머라아제가 결합하는 DNA 분자를 말한다.As used herein, the term "promoter" refers to a region of DNA upstream from a structural gene, and refers to a DNA molecule to which RNA polymerase binds to initiate transcription.

본 발명의 일 구체예에 따른 프로모터는 특정 유전자의 전사 개시를 조절하는 전사 조절 서열 중 하나로, 약 100bp 내지 약 2500bp 길이의 폴리뉴클레오티드 단편일 수 있다. 프로모터는 세포, 예를 들어, 진핵 세포(예컨대, 식물 세포, 또는 동물 세포(예를 들어, 인간, 마우스 등의 포유류 세포 등) 등)에서 전사 개시를 조절할 수 있으면, 제한 없이 사용 가능하다. 예를 들어, 프로모터는 CMV 프로모터(cytomegalovirus promoter(예를 들어, 인간 또는 마우스 CMV immediate-early 프로모터), U6 프로모터, EF1-alpha(elongation factor 1-a) 프로모터, EF1-alpha short(EFS) 프로모터, SV40 프로모터, 아데노바이러스 프로모터(major late promoter), pL λ 프로모터, trp 프로모터, lac 프로모터, tac 프로모터, T7 프로모터, 백시니아 바이러스 7.5K 프로모터, HSV의 tk 프로모터, SV40E1 프로모터, 호흡기 세포융합 바이러스(Respiratory syncytial virus; RSV) 프로모터, 메탈로티오닌 프로모터(metallothionin promoter), β-액틴 프로모터, 유비퀴틴 C 프로모터, 인간 IL-2(human interleukin-2) 유전자 프로모터, 인간 림포톡신(human lymphotoxin) 유전자 프로모터 및 인간 GM-CSF(human granulocyte-macrophage colony stimulating factor) 유전자 프로모터로 이루어진 군으로부터 선택되는 것일 수 있으나, 이에 한정되는 것은 아니다. A promoter according to one embodiment of the present invention is one of the transcription control sequences that control the initiation of transcription of a specific gene, and may be a polynucleotide fragment with a length of about 100 bp to about 2500 bp. A promoter can be used without limitation as long as it can regulate transcription initiation in cells, for example, eukaryotic cells (eg, plant cells, or animal cells (eg, mammalian cells such as humans and mice), etc.). For example, the promoter may include a CMV promoter (cytomegalovirus promoter (eg, human or mouse CMV immediate-early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, SV40 promoter, adenovirus promoter (major late promoter), pL λ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, tk promoter of HSV, SV40E1 promoter, respiratory syncytial virus virus; RSV) promoter, metallothionein promoter, β-actin promoter, ubiquitin C promoter, human interleukin-2 (IL-2) gene promoter, human lymphotoxin gene promoter and human GM -CSF (human granulocyte-macrophage colony stimulating factor) may be selected from the group consisting of gene promoters, but is not limited thereto.

본 발명의 일 구체예에 따른 재조합 발현벡터는 플라스미드 벡터, 코즈미드 벡터 및 박테리오파아지 벡터, 아데노바이러스 벡터, 레트로바이러스 벡터 및 아데노-연관 바이러스 벡터와 같은 바이러스 벡터로 이루어진 군으로부터 선택되는 것일 수 있다. 재조합 발현벡터로 사용될 수 있는 벡터는 당업계에서 사용되는 플라스미드(예를 들어, pcDNA 시리즈, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX 시리즈, pET 시리즈, pUC19 등), 파지(예를 들어, λgt4λB, λ-Charon, λΔz1, M13 등) 또는 바이러스 벡터(예를 들어, 아데노-연관 바이러스(AAV) 벡터 등) 등을 기본으로 하여 제작될 수 있으나, 이에 한정되는 것은 아니다.The recombinant expression vector according to one embodiment of the present invention may be selected from the group consisting of plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retrovirus vectors and adeno-associated virus vectors. Vectors that can be used as recombinant expression vectors include plasmids used in the art (eg, pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1 , pHV14, pGEX series, pET series, pUC19, etc.), phage (eg, λgt4λB, λ-Charon, λΔz1, M13, etc.) or viral vectors (eg, adeno-associated virus (AAV) vectors, etc.) It may be manufactured based on, but is not limited thereto.

본 발명의 재조합 발현벡터는 하나 이상의 선택성 마커를 더 포함할 수 있다. 상기 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질주입된 세포를 비형질주입 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 예를 들어, 글리포세이트(glyphosate), 글루포시네이트암모늄(glufosinate ammonium) 또는 포스피노트리신(phosphinothricin)과 같은 제초제 저항성 유전자, 암피실린(ampicillin), 카나마이신(kanamycin), G418, 블레오마이신(Bleomycin), 하이그로마이신(hygromycin), 클로람페니콜(chloramphenicol)과 같은 항생제 내성 유전자일 수 있으나, 이에 한정되는 것은 아니다.The recombinant expression vector of the present invention may further include one or more selectable markers. The marker is a nucleic acid sequence having characteristics that can be selected by conventional chemical methods, and includes all genes capable of distinguishing transfected cells from non-transfected cells. For example, herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, ampicillin, kanamycin, G418, bleomycin , hygromycin (hygromycin), may be an antibiotic resistance gene such as chloramphenicol (chloramphenicol), but is not limited thereto.

본 발명의 재조합 발현벡터의 제작은 당해 기술 분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용하여 수행될 수 있다.The recombinant expression vector of the present invention can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cutting and linking can be performed using enzymes generally known in the art. there is.

본 발명의 또 다른 양상은 상기 유전체 편집용 조성물을 분리된 세포 또는 유기체에 도입하는 단계를 포함하는 유전체 편집 방법을 제공한다.Another aspect of the present invention provides a genome editing method comprising introducing the genome editing composition into a separated cell or organism.

본 발명의 또 다른 양상은 상기 유전체 편집용 조성물을 분리된 세포 또는 인간을 제외한 유기체에 도입하는 단계를 포함하는 형질 전환체의 제조 방법을 제공한다.Another aspect of the present invention provides a method for producing a transformant comprising the step of introducing the genome editing composition into isolated cells or organisms other than humans.

본 발명의 유전체 편집용 조성물은 핵산 분자를 유기체, 세포, 조직 또는 기관에 도입하는 당 분야에서 공지된 방법에 의해 세포 또는 유기체에 도입될 수 있으며, 당 분야에서 공지된 바와 같이 숙주 세포에 따라 적합한 표준 기술을 선택하여 수행할 수 있다. 이런 방법에는 예를 들어, 전기천공법(electroporation), 인산칼슘(CaPO4) 침전, 염화칼슘(CaCl2) 침전, 미세주입법(microinjection), 폴리에틸렌글리콜(PEG)법, DEAE-덱스트란법, 양이온성 리포좀법, 및 초산 리튬-DMSO법 등이 포함될 수 있으나, 이에 한정되는 것은 아니다.The composition for genome editing of the present invention can be introduced into cells or organisms by methods known in the art for introducing nucleic acid molecules into organisms, cells, tissues or organs, and as known in the art, suitable for host cells This can be done with a selection of standard techniques. These methods include, for example, electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic A liposome method, a lithium acetate-DMSO method, and the like may be included, but are not limited thereto.

신규한 CRISPR 연관 단백질 및 이의 용도에 따르면, 가이드 RNA와 결합된 세포 내 핵산 서열을 인식하여 절단하는 엔도뉴클레아제 활성이 종래 사용되는 AsCpf1 단백질보다도 현저히 우수하므로, CRISPR-Cas 시스템에서 인델 효율이 우수한 뉴클레아제로써 효과적으로 활용될 수 있다.According to the novel CRISPR-associated protein and its uses, the endonuclease activity that recognizes and cuts intracellular nucleic acid sequences bound to guide RNA is significantly superior to that of the previously used AsCpf1 protein, resulting in excellent indel efficiency in the CRISPR-Cas system. It can be effectively utilized as a nuclease.

도 1은 pET28a-His-opmgCas12a-1-6XNLS-His 재조합 발현 벡터의 구조를 나타낸 그림이다.
도 2는 SDS-PAGE에 의하여 opmgCas12a-1-6XNLS 단백질의 최적 발현 조건이 확인된 사진이다.
도 3은 His 컬럼을 사용하여 opmgCas12a-1-6XNLS를 확인한 FPLC 크로마토그래피 결과를 나타낸 그래프이다.
도 4는 탈염(desalting) 컬럼을 사용하여 opmgCas12a-1-6XNLS를 확인한 FPLC 크로마토그래피 결과를 나타낸 그래프이다.
도 5는 opmgCas12a-1-6XNLS 분획에 대한 SDS-PAGE의 쿠마시 염색 결과를 나타낸 사진이다.
도 6은 HEK293T에서 HPRT1 유전자에 대한 AsCas12a, mgCas12a1, opmgCas12a-1-6XNLS 및 mgCas12a1-GFP의 녹아웃(knockout) 효율을 비교한 사진이다.
도 7은 HEK293T에서 HPRT1 유전자에 대한 AsCas12a, mgCas12a1, opmgCas12a-1-6XNLS 및 mgCas12a1-GFP의 녹아웃(knockout) 효율을 비교한 그래프이다.1 is a diagram showing the structure of the pET28a-His-opmgCas12a-1-6XNLS-His recombinant expression vector.
2 is a photograph confirming the optimal expression conditions of the opmgCas12a-1-6XNLS protein by SDS-PAGE.
3 is a graph showing the results of FPLC chromatography confirming opmgCas12a-1-6XNLS using a His column.
4 is a graph showing the results of FPLC chromatography confirming opmgCas12a-1-6XNLS using a desalting column.
Figure 5 is a photograph showing the Coomassie staining result of SDS-PAGE for the opmgCas12a-1-6XNLS fraction.
6 is a photograph comparing knockout efficiencies of AsCas12a, mgCas12a1, opmgCas12a-1-6XNLS, and mgCas12a1-GFP for the HPRT1 gene in HEK293T.
7 is a graph comparing knockout efficiencies of AsCas12a, mgCas12a1, opmgCas12a-1-6XNLS, and mgCas12a1-GFP against the HPRT1 gene in HEK293T.

이하 본 발명을 하나 이상의 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through one or more embodiments. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

실시예 1. 유전자 편집 효율이 증가된 mgCas12a-1 변이체 재조합 벡터 제작Example 1. Production of mgCas12a-1 mutant recombinant vector with increased gene editing efficiency

1-1. pET28a-His-mgCas12a-1-6XNLS-His 재조합 벡터1-1. pET28a-His-mgCas12a-1-6XNLS-His recombinant vector

서열번호 2의 염기서열에 의해 암호화되는 서열번호 1의 아미노산 서열을 갖는 단백질(mgCas12a-1)의 유전자 편집 효율을 증가시키기 위하여, mgCas12a-1의 C-말단에 표 1의 myc-NLS(nuclear localization sequences)를 6번 반복하여 추가한 단백질 변이체(mgCas12a-1-6XNLS) 재조합 벡터를 제작하였다.In order to increase the gene editing efficiency of the protein (mgCas12a-1) having the amino acid sequence of SEQ ID NO: 1 encoded by the nucleotide sequence of SEQ ID NO: 2, the myc-NLS (nuclear localization of Table 1) is attached to the C-terminus of mgCas12a-1. sequences) were repeated 6 times to construct a recombinant vector with an added protein variant (mgCas12a-1-6XNLS).

명칭designation 구분division 서열order 서열번호sequence number myc-NLSmyc-NLS 염기서열(5'→3')Base sequence (5'→3') ccggcagccaagagagtcaaactcgacccggcagccaagagagtcaaactcgac 서열번호 3SEQ ID NO: 3 아미노산서열(N→C)Amino acid sequence (N→C) PAAKRVKLDPAAKRVKLD 서열번호 4SEQ ID NO: 4

구체적으로, 표 2의 Core region(130bp)을 프라이머 형태로 주문하여 어닐링(annealing)한 후, 표 3의 신장(extension) 프라이머를 사용하여 PCR 증폭함으로써, 인서트(Insert)를 준비하였다. Specifically, inserts were prepared by ordering and annealing the core region (130bp) of Table 2 in the form of primers, and then PCR amplification using the extension primers of Table 3.

명칭designation 염기서열(5'→3')Base sequence (5'→3') 서열번호sequence number Core regionCore region agccaagagagtcaaactcgacggcggctccccagcggcaaaaagggtgaaactagacgggggtagccccgccgcgaagcgtgtaaagctggatggaggatcgcctgcggctaagcgagtcaaattagacagccaagagagtcaaactcgacggcggctccccagcggcaaaaagggtgaaactagacgggggtagccccgccgcgaagcgtgtaaagctggatggaggatcgcctgcggctaagcgagtcaaattagac 서열번호 5SEQ ID NO: 5

프라이머primer 프라이머 염기서열(5'→3')Primer nucleotide sequence (5'→3') 서열번호sequence number extension primerextension primer forwardforward ttgacttcattcaaaataagcggtatctgggcggctcccctgctgctaaacgtgttaagcttgatgggggtagcccggcagccaagagagtcaaactcgttgacttcattcaaaataagcggtatctgggcggctcccctgctgctaaacgtgttaagcttgatgggggtagcccggcagccaagagagtcaaactcg 서열번호 6SEQ ID NO: 6 reversereverse agccggatctcagtggtggtggtggtggtggctgccgctagcatccaatttgacgcgctttgcagccggtgacccaccgtctaatttgactcgcttagccagccggatctcagtggtggtggtggtggtggctgccgctagcatccaatttgacgcgctttgcagccggtgacccaccgtctaatttgactcgcttagcc 서열번호 7SEQ ID NO: 7

한편, 종래 사용되고 있는 pET28a-6XHis-mgCas12a-1 플라스미드를 NotI 및 SalI으로 이중 절단(double cut)하여 벡터를 준비하였다. 이후, 깁슨 어셈블리(Gibson assembly)를 수행하여, 벡터에 인서트를 클로닝한 결과, 3개의 콜로니(colony)에서 pET28a-His-mgCas12a-1-6XNLS-His 구조(construction)가 제작된 것으로 확인되었다. mgCas12a-1-6XNLS 단백질은 서열번호 9의 염기서열로 코딩되는 서열번호 8의 아미노산 서열을 갖는다.On the other hand, a vector was prepared by double-cutting the pET28a-6XHis-mgCas12a-1 plasmid, which is conventionally used, with NotI and SalI. Subsequently, as a result of cloning the insert into the vector by performing Gibson assembly, it was confirmed that the pET28a-His-mgCas12a-1-6XNLS-His construction was constructed in three colonies. The mgCas12a-1-6XNLS protein has an amino acid sequence of SEQ ID NO: 8 encoded by the nucleotide sequence of SEQ ID NO: 9.

1-2. pET28a-His-opmgCas12a-1-6XNLS-His 재조합 벡터1-2. pET28a-His-opmgCas12a-1-6XNLS-His recombinant vector

실시예 1-1의 mgCas12a-1-6XNLS의 재조합 벡터에서, 점돌연변이(point mutation)에 의해 코돈 최적화된 opmgCas12a-1-6XNLS 재조합 벡터를 제작하였다.From the mgCas12a-1-6XNLS recombinant vector of Example 1-1, a codon-optimized opmgCas12a-1-6XNLS recombinant vector was constructed by point mutation.

구체적으로, 인서트간의 상동성 재조합(homologous recombination, HR) 부위에 점돌연변이(K169R 및 D529R)가 생성되도록 디자인하였다(도 1). 그 후, PCR 증폭한 후, DpnI 처리 및 PCR 산물을 수득하여 3가지의 인서트를 각각 준비하였다.Specifically, it was designed to generate point mutations (K169R and D529R) at homologous recombination (HR) sites between inserts (FIG. 1). Then, after PCR amplification, DpnI treatment and PCR products were obtained to prepare three inserts, respectively.

인서트 염기서열(5'→3')Insert sequence (5'→3') Forward primerForward primer Reverse primerReverse primer 1One 서열번호 10SEQ ID NO: 10 서열번호 11SEQ ID NO: 11 서열번호 12SEQ ID NO: 12 gcgccaaattgattagtgacatcctgccggaattcgttattcacaataacaattactctgctagcgagaaggaagagaaaacccaagtcataaagctcttttcccggttcgccacttcatttaaagattatttccgcaaccgcgcaaattgctttagcgccgacgcgccaaattgattagtgacatcctgccggaattcgttattcacaataacaattactctgctagcgagaaggaagagaaaacccaagtcataaagctcttttcccggttcgccacttcatttaaagattatttccgcaaccgcgcaaattgctttagcgccgac gcgccaaattgattagtgacatcctgccggaattcgttattcacaataacaatgcgccaaattgattagtgacatcctgccggaattcgttattcacaataacaat gtcggcgctaaagcaatttgcgcggttgcggaaataatctttaaatgaagtggcgtcggcgctaaagcaatttgcgcggttgcggaaataatctttaaatgaagtggc 22 서열번호 13SEQ ID NO: 13 서열번호 14SEQ ID NO: 14 서열번호 15SEQ ID NO: 15 cgcaaccgcgcaaattgctttagcgccgacgatatcagttctagctcctgtcatcggattgtgaacgacaatgctgaaatcttcttttcaaacgcccttgtataccgccggattgtgaaaaatctgagcaacgatgacataaataagatcagtggagatattaaagactctttgaaggagatgagcctggaagagatctattcctacgaaaaatatggggagttcattacccaggaaggcatatcattttacaacgatatctgcggtaaggttaatagcttcatgaacctctattgtcagaaaaataaggagaacaaaaatctttacaagctgcgcaaattgcacaagcaaattctgtgcatcgcagacacaagttatgaagtcccttacaaatttgagtctgatgaagaggtgtatcagagcgtaaacggcttcctcgacaatatttcctcaaagcatatagtggaacggcttcgcaaaatcggagataactacaatgggtataacctggacaagatttacatcgttagcaaattttatgagagtgtctctcagaagacctaccgggattgggaaactattaataccgccttggagatacactataacaatatcctgcccggcaacggtaaaagcaaggctgacaaagtgaagaaagccgtaaagaatgatctccaaaaatccattacagaaatcaacgagcttgtgtcaaattacaagctgtgtccggacgataacattaaagcagaaacctatatacatgagatcagccacattttgaataacttcgaagcccaggagctgaagtacaatccagaaatccatctcgttgagagtgaacttaaagcttctgagctgaagaacgtcttggacgtgattatgaatgcctttcactggtgcagcgtattcatgactgaagagctggtggataaagacaacaatttttatgcagaactcgaggaaatatacgatgagatctataccgttatttccctttacaacctggtccgcaattatgtgacacagaagccctactcaaccaaaaagatcaaattgaacttcggcattccgactctggcccgccgcaaccgcgcaaattgctttagcgccgacgatatcagttctagctcctgtcatcggattgtgaacgacaatgctgaaatcttcttttcaaacgcccttgtataccgccggattgtgaaaaatctgagcaacgatgacataaataagatcagtggagatattaaagactctttgaaggagatgagcctggaagagatctattcctacgaaaaatatggggagttcattacccaggaaggcatatcattttacaacgatatctgcggtaaggttaatagcttcatgaacctctattgtcagaaaaataaggagaacaaaaatctttacaagctgcgcaaattgcacaagcaaattctgtgcatcgcagacacaagttatgaagtcccttacaaatttgagtctgatgaagaggtgtatcagagcgtaaacggcttcctcgacaatatttcctcaaagcatatagtggaacggcttcgcaaaatcggagataactacaatgggtataacctggacaagatttacatcgttagcaaattttatgagagtgtctctcagaagacctaccgggattgggaaactattaataccgccttggagatacactataacaatatcctgcccggcaacggtaaaagcaaggctgacaaagtgaagaaagccgtaaagaatgatctccaaaaatccattacagaaatcaacgagcttgtgtcaaattacaagctgtgtccggacgataacattaaagcagaaacctatatacatgagatcagccacattttgaataacttcgaagcccaggagctgaagtacaatccagaaatccatctcgttgagagtgaacttaaagcttctgagctgaagaacgtcttggacgtgattatgaatgcctttcactggtgcagcgtattcatgactgaagagctggtggataaagacaacaatttttatgcagaactcgaggaaatatacgatgagatctataccgttatttccctttaca acctggtccgcaattatgtgacacagaagccctactcaaccaaaaagatcaaattgaacttcggcattccgactctggcccgc cgcaaccgcgcaaattgctttagcgccgaccgcaaccgcgcaaattgctttagcgccgac gcgggccagagtcggaatgccgaagttcaagcgggccagagtcggaatgccgaagttcaa 33 서열번호 16SEQ ID NO: 16 서열번호 17SEQ ID NO: 17 서열번호 18SEQ ID NO: 18 ttgaacttcggcattccgactctggcccgcggatggagcaagagtaaagaatattctaataacgctataatcctcatgcgggataatctttactatctggggatttttaacgccaagaataaacctgacaagaaaatcattgagggcaacaccagcgaaaataagggtgattacaaaaagatgatatataacttgctgcccggcccgaataaaatgatcccaaaggtattcctctcctcaaaaacaggagtggagacctacaagcccagcgcatatattctttgaacttcggcattccgactctggcccgcggatggagcaagagtaaagaatattctaataacgctataatcctcatgcgggataatctttactatctggggatttttaacgccaagaataaacctgacaagaaaatcattgagggcaacaccagcgaaaataagggtgattacaaaaagatgatatataacttgctgcccggcccgaataaaatgatcccaaaggtattcctctcctcaaaaacaggagtggagacctacaagcccagcgcatatattct ttgaacttcggcattccgactctggcccgcggatggagcaagagtaaagattgaacttcggcattccgactctggcccgcggatggagcaagagtaaaga agaatatatgcgctgggcttgtaggtctccactcctgtttttgaggagagagaatatatgcgctgggcttgtaggtctccactcctgtttttgaggagag

한편, 실시예 1-1의 pET28a-His-mgCas12a-1-6XNLS-His 벡터를 EcorI과 BsaI을 사용하여 이중 절단하여 벡터를 준비하였다. 이후, 깁슨 어셈블리를 수행하여 벡터에 인서트를 클로닝함으로써, opmgCas12a-1이 클로닝된 pET28a-His-opmgCas12a-1-6XNLS-His 재조합 벡터를 제작하였다. opmgCas12a-1-6XNLS 단백질은 서열번호 20의 염기서열로 코딩되는 서열번호 19의 아미노산 서열을 갖는다.On the other hand, the pET28a-His-mgCas12a-1-6XNLS-His vector of Example 1-1 was double digested using EcorI and BsaI to prepare a vector. Thereafter, by performing Gibson assembly and cloning the insert into the vector, a pET28a-His-opmgCas12a-1-6XNLS-His recombinant vector in which opmgCas12a-1 was cloned was constructed. The opmgCas12a-1-6XNLS protein has an amino acid sequence of SEQ ID NO: 19 encoded by the nucleotide sequence of SEQ ID NO: 20.

실시예 2. opmgCas12a-1-6XNLS 단백질 발현 최적조건 확인Example 2. Confirmation of optimal conditions for opmgCas12a-1-6XNLS protein expression

2-1. 형질전환2-1. transformation

실시예 1-2에서 제작한 opmgCas12a-1-6XNLS 발현 재조합 벡터를 컴피턴트 세포(competent cell)인 Rosetta(DE3) 또는 Rosetta2(DE3)pLysS에 각각에 각각 형질전환하였다.The opmgCas12a-1-6XNLS expression recombinant vector prepared in Example 1-2 was transformed into competent cells, Rosetta (DE3) or Rosetta2 (DE3) pLysS, respectively.

구체적으로, Rosetta(DE3) 또는 Rosetta2(DE3)pLysS 100㎕에 실시예 1-2의 재조합 벡터 1㎕를 넣은 후 얼음상에서 30분 동안 반응시켰다. 그 후, 42℃에서 45초간 열 충격(heat shock)을 가한 후 재빨리 얼음으로 옮겨 2분 동안 반응시켰다. LB 배지 1㎖을 넣은 후, 37℃에서 1시간 동안 진탕배양(shaking incubation)한 다음, 13,000rpm에서 3분간 원심분리하여 세포를 침전시키고, 상층액 100㎕를 남긴 후 재현탁(resuspension)하여, 카나마이신이 함유된 LB 플레이트에 스프레딩(spreading)한 다음, 37℃에서 밤새 배양하여 형질전환하였다.Specifically, 1 μl of the recombinant vector of Example 1-2 was added to 100 μl of Rosetta (DE3) or Rosetta2 (DE3) pLysS, followed by reaction on ice for 30 minutes. Thereafter, heat shock was applied at 42° C. for 45 seconds, and then quickly transferred to ice and allowed to react for 2 minutes. After adding 1 ml of LB medium, shaking incubation at 37 ° C. for 1 hour, followed by centrifugation at 13,000 rpm for 3 minutes to precipitate the cells, leaving 100 μl of the supernatant and resuspending, It was spread on an LB plate containing kanamycin, and then incubated overnight at 37° C. to transform it.

2-2. 단백질 발현량 확인2-2. Check protein expression level

opmgCas12a-1-6XNLS 단백질 발현량을 확인하기 위하여, 실시예 2-1에서 형질전환된 Rosetta(DE3) 및 Rosetta2(DE3)pLysS의 발현 최적조건을 분석하였다.In order to confirm the opmgCas12a-1-6XNLS protein expression level, the optimal expression conditions of Rosetta (DE3) and Rosetta2 (DE3)pLysS transformed in Example 2-1 were analyzed.

구체적으로, 실시예 2-1에서 형질전환된 Rosetta(DE3) 또는 Rosetta2(DE3)pLysS를 하룻밤 동안 배양하고, 100㎎/㎖의 카나마이신 항생제가 추가된 500㎖의 액체 TB 배지에 5㎖를 접종한 후, 37℃ 배양기에서 0.6 OD600이 될 때까지 배양하였다. 0.5mM의 IPTG(Isopropyl β-D-1-thiogalactopyranoside)를 처리하여 opmgCas12a-1-6XNLS 단백질 발현을 유도한 후, 18℃ 또는 28℃에서 18시간 동안 배양하였다. 원심분리 후 수득한 세포를 10㎖의 용해 버퍼(20mM HEPES pH7.5, 100mM KCl, 20mM 이미다졸, 10% 글리세롤 및 EDTA-프리 프로테아제 인히비터 칵테일)와 혼합한 후, 초음파를 처리하여 세포를 분쇄하였다. 분쇄물을 6,000rpm에서 20분간 3회 원심분리한 후 0.22 마이크론 필터로 여과하였다. 이후, 니켈 컬럼(HisTrap FF 5㎖) 및 300mM 이미다졸 버퍼를 이용하여 세척 및 용출한 다음, SDS-PAGE 전기영동 및 쿠마시 염색(coomassie staning)을 수행하여 opmgCas12a-1-6XNLS 단백질을 확인하였다.Specifically, Rosetta (DE3) or Rosetta2 (DE3) pLysS transformed in Example 2-1 was cultured overnight, and 5 ml was inoculated into 500 ml of liquid TB medium supplemented with 100 mg / ml kanamycin antibiotic. After that, it was cultured in a 37° C. incubator until OD 600 of 0.6. opmgCas12a-1-6XNLS by treatment with 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) After inducing protein expression, it was cultured at 18°C or 28°C for 18 hours. Cells obtained after centrifugation were mixed with 10 ml of lysis buffer (20 mM HEPES pH7.5, 100 mM KCl, 20 mM imidazole, 10% glycerol and EDTA-free protease inhibitor cocktail), and then treated with sonication to disrupt the cells. did The pulverized material was centrifuged 3 times for 20 minutes at 6,000 rpm and filtered through a 0.22 micron filter. Thereafter, after washing and elution using a nickel column (HisTrap FF 5 ml) and 300 mM imidazole buffer, SDS-PAGE electrophoresis and coomassie staining were performed to confirm the opmgCas12a-1-6XNLS protein.

그 결과, Rosetta2(DE3)pLysS를 사용한 18℃의 18시간 배양 조건이 opmgCas12a-1-6XNLS 단백질의 발현에 최적 조건인 것으로 확인되었다(도 2).As a result, it was confirmed that the incubation condition at 18° C. for 18 hours using Rosetta2(DE3)pLysS was optimal for the expression of the opmgCas12a-1-6XNLS protein (FIG. 2).

실시예 3. opmgCas12a-1-6XNLS 단백질 정제Example 3. opmgCas12a-1-6XNLS protein purification

실시예 2의 방법으로 세척 및 용출한 opmgCas12a-1-6XNLS 단백질을 크로마토그래피 방법으로 정제하였다.The opmgCas12a-1-6XNLS protein washed and eluted in Example 2 was purified by chromatography.

구체적으로, opmgCas12a-1-6XNLS 단백질을 His 컬럼(column) 또는 탈염(desalting) 칼럼을 사용한 FPLC 크로마토그래피 방법으로 정제하였다. 투석 버퍼(20mM HEPES pH 7.5, 100mM KCl, 1mM DTT, 10% 글리세롤)로 하룻밤 동안 투석하고, 단백질 크기에 맞춰 선택적으로 여과 및 농축(Amicon Ultra Centrifugal Filter 100,000 MWCO)하여 opmgCas12a-1-6XNLS 단백질을 정제하였다. Specifically, the opmgCas12a-1-6XNLS protein was purified by FPLC chromatography using a His column or a desalting column. Purify opmgCas12a-1-6XNLS protein by dialysis overnight with dialysis buffer (20 mM HEPES pH 7.5, 100 mM KCl, 1 mM DTT, 10% glycerol), followed by selective filtration and concentration according to protein size (Amicon Ultra Centrifugal Filter 100,000 MWCO) did

SDS-PAGE 전기영동 및 쿠마시 염색을 수행하여 opmgCas12a-1-6XNLS 단백질의 정제 수율을 확인한 결과, 단백질 농도는 35.54㎎/㎖ 및 단백질 부피는 1.6㎖로써, 56.9㎎/ℓ의 opmgCas12a-1-6XNLS 단백질 정제 수율이 확인되었다(도 3 내지 도 5).As a result of confirming the purification yield of the opmgCas12a-1-6XNLS protein by performing SDS-PAGE electrophoresis and Coomassie staining, the protein concentration was 35.54 mg/mL and the protein volume was 1.6 mL, resulting in 56.9 mg/L of opmgCas12a-1-6XNLS. The protein purification yield was confirmed (Figs. 3 to 5).

이후, 정제된 opmgCas12a-1-6XNLS 단백질은 브래드포드 정량법으로 농도를 측정하고, -80℃에서 보관하며 사용하였다.Then, the purified opmgCas12a-1-6XNLS protein The concentration was measured by the Bradford quantification method, and stored at -80 ° C for use.

실시예 4. opmgCas12a-1-6XNLS의 향상된 유전자 편집 효율 확인Example 4. Confirmation of improved gene editing efficiency of opmgCas12a-1-6XNLS

4-1. HPRT1 유전자 편집을 위한 opmgCas12a-1-6XNLS를 포함한 RNP 제작4-1. Construction of RNPs with opmgCas12a-1-6XNLS for HPRT1 gene editing

HEK 293T 세포를 10% 태아 소혈청인 FBS와 P/S(penicillin-streptomycin)가 함유된 DMEM(Dulbecco Modified Eagle Medium)에서 37℃의 온도로 5% CO2 배양기에서 배양하였다. 126pmol의 opmgCas12a-1-6XNLS 단백질 5μM과 HPRT1을 표적화하는 160pmol의 crRNA(표 4) 6.4μM를 상온에서 20분간 배양하여 리보핵산단백질(RNP)을 제작하였다. 사용된 HPRT1의 crRNA 서열은 IDT(Integrated DNA Technologies)로부터 합성되었다.HEK 293T cells were cultured in DMEM (Dulbecco Modified Eagle Medium) containing 10% fetal bovine serum FBS and penicillin-streptomycin (P/S) at 37°C in a 5% CO 2 incubator. Ribonucleic acid protein (RNP) was prepared by incubating 5 μM of 126 pmol of opmgCas12a-1-6XNLS protein and 6.4 μM of 160 pmol of crRNA (Table 4) targeting HPRT1 at room temperature for 20 minutes. The crRNA sequence of HPRT1 used was synthesized from IDT (Integrated DNA Technologies).

유전자gene crRNA sequence(5'→3')crRNA sequence (5'→3') 서열번호sequence number HPRT1HPRT1 GGTTAAAGATGGTTAAATGATGGTTAAAGATGGTTAAATGAT 서열번호 21SEQ ID NO: 21

HEK293T 세포 2×105개를 20㎕의 뉴클레오펙션(nucleofection) 시약과 혼합하고, 10㎕의 RNP와 혼합한 다음, 4D-Nucleofector 기기(Lonza)를 사용하여 세포 내로 RNP를 전기천공(electroporation) 방법으로 도입하였다. 형질전환 48시간 후, PureLinkTM Genomic DNA Mini Kit(invitrogen)를 사용하여 세포로부터 게놈 DNA를 추출하였다.2×10 5 HEK293T cells were mixed with 20 μl of nucleofection reagent, mixed with 10 μl of RNPs, and then electroporated RNPs into cells using a 4D-Nucleofector instrument (Lonza) introduced in this way. After 48 hours of transformation, genomic DNA was extracted from the cells using the PureLink ™ Genomic DNA Mini Kit (invitrogen).

4-2. 표적 부위에 대한 염기서열 분석4-2. Sequencing analysis of the target site

실시예 4-1에서 추출한 게놈 DNA를 일루미나(Illumina)의 프로토콜에 따라 정제 및 시퀀싱 라이브러리를 제조하였으며, MiniSeq 장비를 이용하여 표적 부위에 대한 딥시퀀싱(deep sequencing) 분석을 진행하였다.The genomic DNA extracted in Example 4-1 was purified and a sequencing library was prepared according to Illumina's protocol, and deep sequencing analysis was performed on the target site using MiniSeq equipment.

그 결과, mgCas12a-1 대비 opmgCas12a-1-6XNLS의 HPRT1 유전자의 녹아웃 효율이 약 4배 우수한 것으로 나타났으며, 종래 일반적으로 사용되는 AsCpf1보다도 녹아웃 효율이 약 2배 우수한 것으로 나타나, opmgCas12a-1-6XNLS 단백질에 의한 우수한 유전자 편집 효율이 확인되었다(도 6 및 도 7).As a result, the knockout efficiency of the HPRT1 gene of opmgCas12a-1-6XNLS compared to mgCas12a-1 was found to be about 4 times better, and the knockout efficiency was about 2 times better than the commonly used AsCpf1, opmgCas12a-1-6XNLS Excellent gene editing efficiency by the protein was confirmed (FIGS. 6 and 7).

이제까지 본 발명에 대하여 그 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 청구범위에 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 the embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can 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 limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be construed as being included in the present invention.

<110> G+FLAS Life Sciences <120> Novel CRISPR Associated Protein and Use thereof <130> SP20-0124KR <160> 21 <170> KoPatentIn 3.0 <210> 1 <211> 1330 <212> PRT <213> Artificial Sequence <220> <223> mgCas12a-1 <400> 1 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Asn Asn Gly Thr Asn Asn Phe Gln Asn Phe Ile Gly Ile 35 40 45 Ser Ser Leu Gln Lys Thr Leu Arg Asn Ala Leu Ile Pro Thr Glu Thr 50 55 60 Thr Gln Gln Phe Ile Val Lys Asn Gly Ile Ile Lys Glu Asp Glu Leu 65 70 75 80 Arg Gly Glu Asn Arg Gln Ile Leu Lys Asp Ile Met Asp Asp Tyr Tyr 85 90 95 Arg Gly Phe Ile Ser Glu Thr Leu Ser Ser Ile Asp Asp Ile Asp Trp 100 105 110 Thr Ser Leu Phe Glu Lys Met Glu Ile Gln Leu Lys Asn Gly Asp Asn 115 120 125 Lys Asp Thr Leu Ile Lys Glu Gln Ala Glu Lys Arg Lys Ala Ile Tyr 130 135 140 Lys Lys Phe Ala Asp Asp Asp Arg Phe Lys Asn Met Phe Ser Ala Lys 145 150 155 160 Leu Ile Ser Asp Ile Leu Pro Glu Phe Val Ile His Asn Asn Asn Tyr 165 170 175 Ser Ala Ser Glu Lys Glu Glu Lys Thr Gln Val Ile Lys Leu Phe Ser 180 185 190 Arg Phe Ala Thr Ser Phe Lys Asp Tyr Phe Lys Asn Arg Ala Asn Cys 195 200 205 Phe Ser Ala Asp Asp Ile Ser Ser Ser Ser Cys His Arg Ile Val Asn 210 215 220 Asp Asn Ala Glu Ile Phe Phe Ser Asn Ala Leu Val Tyr Arg Arg Ile 225 230 235 240 Val Lys Asn Leu Ser Asn Asp Asp Ile Asn Lys Ile Ser Gly Asp Ile 245 250 255 Lys Asp Ser Leu Lys Glu Met Ser Leu Glu Glu Ile Tyr Ser Tyr Glu 260 265 270 Lys Tyr Gly Glu Phe Ile Thr Gln Glu Gly Ile Ser Phe Tyr Asn Asp 275 280 285 Ile Cys Gly Lys Val Asn Ser Phe Met Asn Leu Tyr Cys Gln Lys Asn 290 295 300 Lys Glu Asn Lys Asn Leu Tyr Lys Leu Arg Lys Leu His Lys Gln Ile 305 310 315 320 Leu Cys Ile Ala Asp Thr Ser Tyr Glu Val Pro Tyr Lys Phe Glu Ser 325 330 335 Asp Glu Glu Val Tyr Gln Ser Val Asn Gly Phe Leu Asp Asn Ile Ser 340 345 350 Ser Lys His Ile Val Glu Arg Leu Arg Lys Ile Gly Asp Asn Tyr Asn 355 360 365 Gly Tyr Asn Leu Asp Lys Ile Tyr Ile Val Ser Lys Phe Tyr Glu Ser 370 375 380 Val Ser Gln Lys Thr Tyr Arg Asp Trp Glu Thr Ile Asn Thr Ala Leu 385 390 395 400 Glu Ile His Tyr Asn Asn Ile Leu Pro Gly Asn Gly Lys Ser Lys Ala 405 410 415 Asp Lys Val Lys Lys Ala Val Lys Asn Asp Leu Gln Lys Ser Ile Thr 420 425 430 Glu Ile Asn Glu Leu Val Ser Asn Tyr Lys Leu Cys Pro Asp Asp Asn 435 440 445 Ile Lys Ala Glu Thr Tyr Ile His Glu Ile Ser His Ile Leu Asn Asn 450 455 460 Phe Glu Ala Gln Glu Leu Lys Tyr Asn Pro Glu Ile His Leu Val Glu 465 470 475 480 Ser Glu Leu Lys Ala Ser Glu Leu Lys Asn Val Leu Asp Val Ile Met 485 490 495 Asn Ala Phe His Trp Cys Ser Val Phe Met Thr Glu Glu Leu Val Asp 500 505 510 Lys Asp Asn Asn Phe Tyr Ala Glu Leu Glu Glu Ile Tyr Asp Glu Ile 515 520 525 Tyr Thr Val Ile Ser Leu Tyr Asn Leu Val Arg Asn Tyr Val Thr Gln 530 535 540 Lys Pro Tyr Ser Thr Lys Lys Ile Lys Leu Asn Phe Gly Ile Pro Thr 545 550 555 560 Leu Ala Asp Gly Trp Ser Lys Ser Lys Glu Tyr Ser Asn Asn Ala Ile 565 570 575 Ile Leu Met Arg Asp Asn Leu Tyr Tyr Leu Gly Ile Phe Asn Ala Lys 580 585 590 Asn Lys Pro Asp Lys Lys Ile Ile Glu Gly Asn Thr Ser Glu Asn Lys 595 600 605 Gly Asp Tyr Lys Lys Met Ile Tyr Asn Leu Leu Pro Gly Pro Asn Lys 610 615 620 Met Ile Pro Lys Val Phe Leu Ser Ser Lys Thr Gly Val Glu Thr Tyr 625 630 635 640 Lys Pro Ser Ala Tyr Ile Leu Glu Gly Tyr Lys Gln Asn Lys His Leu 645 650 655 Lys Ser Ser Lys Asp Phe Asp Ile Thr Phe Cys His Asp Leu Ile Asp 660 665 670 Tyr Phe Lys Asn Cys Ile Ala Ile His Pro Glu Trp Lys Asn Phe Gly 675 680 685 Phe Asp Phe Ser Asp Thr Ser Thr Tyr Glu Asp Ile Ser Gly Phe Tyr 690 695 700 Arg Glu Val Glu Leu Gln Gly Tyr Lys Ile Asp Trp Thr Tyr Ile Ser 705 710 715 720 Glu Lys Asp Ile Asp Leu Leu Gln Glu Lys Gly Gln Leu Tyr Leu Phe 725 730 735 Gln Ile Tyr Asn Lys Asp Phe Ser Lys Lys Ser Thr Gly Asn Asp Asn 740 745 750 Leu His Thr Met Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Lys 755 760 765 Asp Ile Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys 770 775 780 Ser Ser Ile Lys Asn Pro Ile Ile His Lys Lys Gly Ser Ile Leu Val 785 790 795 800 Asn Arg Thr Tyr Glu Ala Glu Glu Lys Asp Gln Phe Gly Asn Ile Gln 805 810 815 Ile Val Arg Lys Thr Ile Pro Glu Asn Ile Tyr Gln Glu Leu Tyr Lys 820 825 830 Tyr Phe Asn Asp Lys Ser Asp Lys Glu Leu Ser Asp Glu Ala Ala Lys 835 840 845 Leu Lys Asn Val Val Gly His His Glu Ala Ala Thr Asn Ile Val Lys 850 855 860 Asp Tyr Arg Tyr Thr Tyr Asp Lys Tyr Phe Leu His Met Pro Ile Thr 865 870 875 880 Ile Asn Phe Lys Ala Asn Lys Thr Ser Phe Ile Asn Asp Arg Ile Leu 885 890 895 Gln Tyr Ile Ala Lys Glu Lys Asn Leu His Val Ile Gly Ile Asp Arg 900 905 910 Gly Glu Arg Asn Leu Ile Tyr Val Ser Val Ile Asp Thr Cys Gly Asn 915 920 925 Ile Val Glu Gln Lys Ser Phe Asn Ile Val Asn Gly Tyr Asp Tyr Gln 930 935 940 Ile Lys Leu Lys Gln Gln Glu Gly Ala Arg Gln Ile Ala Arg Lys Glu 945 950 955 960 Trp Lys Glu Ile Gly Lys Ile Lys Glu Ile Lys Glu Gly Tyr Leu Ser 965 970 975 Leu Val Ile His Glu Ile Ser Lys Met Val Ile Lys Tyr Asn Ala Ile 980 985 990 Ile Ala Met Glu Asp Leu Ser Tyr Gly Phe Lys Lys Gly Arg Phe Lys 995 1000 1005 Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu Ile Asn Lys 1010 1015 1020 Leu Asn Tyr Leu Val Phe Lys Asp Ile Ser Ile Thr Glu Asn Gly Gly 1025 1030 1035 1040 Leu Leu Lys Gly Tyr Gln Leu Thr Tyr Ile Pro Asp Lys Leu Lys Asn 1045 1050 1055 Val Gly His Gln Cys Gly Cys Ile Phe Tyr Val Pro Ala Ala Tyr Thr 1060 1065 1070 Ser Lys Ile Asp Pro Thr Thr Gly Phe Val Asn Ile Phe Lys Phe Lys 1075 1080 1085 Asp Leu Thr Val Asp Ala Lys Arg Glu Phe Ile Lys Lys Phe Asp Ser 1090 1095 1100 Ile Arg Tyr Asp Ser Glu Lys Lys Leu Phe Cys Phe Thr Phe Asp Tyr 1105 1110 1115 1120 Asn Asn Phe Ile Thr Gln Asn Thr Val Met Ser Lys Ser Ser Trp Ser 1125 1130 1135 Val Tyr Thr Tyr Gly Val Arg Ile Lys Arg Arg Phe Val Asn Gly Arg 1140 1145 1150 Phe Ser Asn Glu Ser Asp Thr Ile Asp Ile Thr Lys Asp Met Glu Lys 1155 1160 1165 Thr Leu Glu Met Thr Asp Ile Asn Trp Arg Asp Gly His Asp Leu Arg 1170 1175 1180 Gln Asp Ile Ile Asp Tyr Glu Ile Val Gln His Ile Phe Glu Ile Phe 1185 1190 1195 1200 Arg Leu Thr Val Gln Met Arg Asn Ser Leu Ser Glu Leu Glu Asp Arg 1205 1210 1215 Asp Tyr Asp Arg Leu Ile Ser Pro Val Leu Asn Glu Asn Asn Ile Phe 1220 1225 1230 Tyr Asp Ser Ala Lys Ala Gly Asp Ala Leu Pro Lys Asp Ala Asp Ala 1235 1240 1245 Asn Gly Ala Tyr Cys Ile Ala Leu Lys Gly Leu Tyr Glu Ile Lys Gln 1250 1255 1260 Ile Thr Glu Asn Trp Lys Glu Asp Gly Lys Phe Ser Arg Asp Lys Leu 1265 1270 1275 1280 Lys Ile Ser Asn Lys Asp Trp Phe Asp Phe Ile Gln Asn Lys Arg Tyr 1285 1290 1295 Leu Ala Ala Ala Leu Glu Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly 1300 1305 1310 Gln Ala Lys Lys Lys Lys Ser Thr Pro Pro Pro Pro Pro Leu Arg Ser 1315 1320 1325 Gly Cys 1330 <210> 2 <211> 3993 <212> DNA <213> Artificial Sequence <220> <223> mgCas12a-1 <400> 2 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgaacaa tggcaccaac 120 aatttccaga actttatcgg aattagcagt ctgcaaaaga ctctccggaa tgcccttata 180 cccaccgaga caacccagca gttcatcgtg aaaaacggga ttatcaagga agacgagctg 240 cgcggcgaaa atcggcaaat tttgaaagat ataatggacg attattaccg cggttttatc 300 tctgagactc tgagctccat tgacgatatc gactggacct cactcttcga aaagatggag 360 attcagctta aaaacggcga taataaggac acactgataa aagaacaggc tgagaagcgg 420 aaagccatct ataagaaatt tgcagatgac gatcgcttca agaacatgtt tagcgccaaa 480 ttgattagtg acatcctgcc ggaattcgtt attcacaata acaattactc tgctagcgag 540 aaggaagaga aaacccaagt cataaagctc ttttcccggt tcgccacttc atttaaagat 600 tatttcaaga accgcgcaaa ttgctttagc gccgacgata tcagttctag ctcctgtcat 660 cggattgtga acgacaatgc tgaaatcttc ttttcaaacg cccttgtata ccgccggatt 720 gtgaaaaatc tgagcaacga tgacataaat aagatcagtg gagatattaa agactctttg 780 aaggagatga gcctggaaga gatctattcc tacgaaaaat atggggagtt cattacccag 840 gaaggcatat cattttacaa cgatatctgc ggtaaggtta atagcttcat gaacctctat 900 tgtcagaaaa ataaggagaa caaaaatctt tacaagctgc gcaaattgca caagcaaatt 960 ctgtgcatcg cagacacaag ttatgaagtc ccttacaaat ttgagtctga tgaagaggtg 1020 tatcagagcg taaacggctt cctcgacaat atttcctcaa agcatatagt ggaacggctt 1080 cgcaaaatcg gagataacta caatgggtat aacctggaca agatttacat cgttagcaaa 1140 ttttatgaga gtgtctctca gaagacctac cgggattggg aaactattaa taccgccttg 1200 gagatacact ataacaatat cctgcccggc aacggtaaaa gcaaggctga caaagtgaag 1260 aaagccgtaa agaatgatct ccaaaaatcc attacagaaa tcaacgagct tgtgtcaaat 1320 tacaagctgt gtccggacga taacattaaa gcagaaacct atatacatga gatcagccac 1380 attttgaata acttcgaagc ccaggagctg aagtacaatc cagaaatcca tctcgttgag 1440 agtgaactta aagcttctga gctgaagaac gtcttggacg tgattatgaa tgcctttcac 1500 tggtgcagcg tattcatgac tgaagagctg gtggataaag acaacaattt ttatgcagaa 1560 ctcgaggaaa tatacgatga gatctatacc gttatttccc tttacaacct ggtccgcaat 1620 tatgtgacac agaagcccta ctcaaccaaa aagatcaaat tgaacttcgg cattccgact 1680 ctggccgacg gatggagcaa gagtaaagaa tattctaata acgctataat cctcatgcgg 1740 gataatcttt actatctggg gatttttaac gccaagaata aacctgacaa gaaaatcatt 1800 gagggcaaca ccagcgaaaa taagggtgat tacaaaaaga tgatatataa cttgctgccc 1860 ggcccgaata aaatgatccc aaaggtattc ctctcctcaa aaacaggagt ggagacctac 1920 aagcccagcg catatattct tgaagggtac aaacaaaaca agcatctgaa aagttctaag 1980 gactttgata tcactttctg tcacgacttg attgattatt ttaaaaattg catagccatc 2040 catccggagt ggaagaactt cggctttgac ttcagcgata cctccacata cgaagacatt 2100 tcaggttttt atcgcgaggt tgaactgcag ggctacaaaa tcgattggac ctatattagc 2160 gagaaggaca tagatctcct tcaggaaaaa ggacaactgt acttgttcca gatctataat 2220 aaggacttta gtaaaaagtc tactgggaac gataatctgc acaccatgta cctcaaaaac 2280 cttttcagcg aggaaaatct gaaggacatt gtcttgaaac tgaacggcga ggctgaaatc 2340 tttttccgga agtcctcaat taaaaatcct ataatccata agaaaggtag cattctcgtg 2400 aaccgcacat atgaggccga agagaaggat cagtttggca atatccaaat tgtacggaaa 2460 accatacccg aaaacatcta ccaggagctt tataagtact tcaatgacaa aagtgataag 2520 gaactgtctg acgaggcagc caaattgaag aacgtggttg gacaccatga agctgccact 2580 aatattgtca aagattatcg ctacacctat gacaagtact ttctgcacat gccgatcaca 2640 attaacttca aagcaaataa gaccagcttt ataaacgatc ggattctcca gtatattgcc 2700 aaagagaaga atcttcatgt gatcgggatt gaccgcggcg aacggaacct gatatacgta 2760 tccgtgatcg atacttgtgg taatattgtt gagcaaaaat cattcaacat cgtcaatggc 2820 tatgactacc agattaagtt gaaacagcaa gaaggagctc gccagatagc ccggaaggag 2880 tggaaggaaa tcgggaaaat taaggagatc aaagaaggct atctgagcct cgtgattcac 2940 gagataagta agatggtaat caaatacaac gcaattatcg ccatggaaga tctttcttat 3000 ggttttaaga aaggccgctt caaggtggag cggcaagttt accagaaatt tgaaaccatg 3060 ctgattaata agttgaacta tctggtcttc aaagacataa gcatcacaga gaatggaggg 3120 ctccttaagg gctaccagct gacctatatt ccagataaat tgaagaacgt gggtcatcaa 3180 tgcggctgta tcttttacgt acccgctgcc tatacttcca aaattgaccc gaccacagga 3240 ttcgtgaata tatttaagtt caaagatctg accgttgacg caaagcgcga atttatcaaa 3300 aagttcgatt caattcggta cgacagcgag aaaaagctct tttgcttcac ttttgattat 3360 aacaatttca tcacccagaa cacagtcatg agtaaatcta gctggtccgt gtacacctat 3420 ggggtacgca ttaagcggcg ctttgtgaat ggccggttct caaacgaaag cgacactata 3480 gatatcacca aagacatgga gaagacactt gaaatgaccg atattaattg gcgcgacggt 3540 cacgatctgc ggcaggacat cattgattac gagatagttc aacatatctt tgaaattttc 3600 cgcttgactg tccagatgcg gaacagtctg tctgagctcg aagaccgcga ttatgaccgg 3660 cttatcagcc ctgtgctgaa tgagaacaat attttttacg attccgccaa agctggcgac 3720 gccttgccca aggatgcaga cgccaacgga gcttattgta tagccctgaa agggctctac 3780 gaaatcaagc agattaccga gaattggaaa gaagatggca agttctcacg cgacaaactt 3840 aagatcagca acaaagattg gtttgacttc attcaaaata agcggtatct ggcggccgca 3900 ctcgagaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtcgaca 3960 ccaccaccac caccactgag atccggctgc taa 3993 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> myc-NLS <400> 3 ccggcagcca agagagtcaa actcgac 27 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> myc-NLS <400> 4 Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5 <210> 5 <211> 130 <212> DNA <213> Artificial Sequence <220> <223> Core region <400> 5 agccaagaga gtcaaactcg acggcggctc cccagcggca aaaagggtga aactagacgg 60 gggtagcccc gccgcgaagc gtgtaaagct ggatggagga tcgcctgcgg ctaagcgagt 120 caaattagac 130 <210> 6 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> extension primer_forward <400> 6 ttgacttcat tcaaaataag cggtatctgg gcggctcccc tgctgctaaa cgtgttaagc 60 ttgatggggg tagcccggca gccaagagag tcaaactcg 99 <210> 7 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> extension primer_reverse <400> 7 agccggatct cagtggtggt ggtggtggtg gctgccgcta gcatccaatt tgacgcgctt 60 tgcagccggt gacccaccgt ctaatttgac tcgcttagcc 100 <210> 8 <211> 1379 <212> PRT <213> Artificial Sequence <220> <223> mgCas12a-1-6XNLS <400> 8 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Asn Asn Gly Thr Asn Asn Phe Gln Asn Phe Ile Gly Ile 35 40 45 Ser Ser Leu Gln Lys Thr Leu Arg Asn Ala Leu Ile Pro Thr Glu Thr 50 55 60 Thr Gln Gln Phe Ile Val Lys Asn Gly Ile Ile Lys Glu Asp Glu Leu 65 70 75 80 Arg Gly Glu Asn Arg Gln Ile Leu Lys Asp Ile Met Asp Asp Tyr Tyr 85 90 95 Arg Gly Phe Ile Ser Glu Thr Leu Ser Ser Ile Asp Asp Ile Asp Trp 100 105 110 Thr Ser Leu Phe Glu Lys Met Glu Ile Gln Leu Lys Asn Gly Asp Asn 115 120 125 Lys Asp Thr Leu Ile Lys Glu Gln Ala Glu Lys Arg Lys Ala Ile Tyr 130 135 140 Lys Lys Phe Ala Asp Asp Asp Arg Phe Lys Asn Met Phe Ser Ala Lys 145 150 155 160 Leu Ile Ser Asp Ile Leu Pro Glu Phe Val Ile His Asn Asn Asn Tyr 165 170 175 Ser Ala Ser Glu Lys Glu Glu Lys Thr Gln Val Ile Lys Leu Phe Ser 180 185 190 Arg Phe Ala Thr Ser Phe Lys Asp Tyr Phe Lys Asn Arg Ala Asn Cys 195 200 205 Phe Ser Ala Asp Asp Ile Ser Ser Ser Ser Cys His Arg Ile Val Asn 210 215 220 Asp Asn Ala Glu Ile Phe Phe Ser Asn Ala Leu Val Tyr Arg Arg Ile 225 230 235 240 Val Lys Asn Leu Ser Asn Asp Asp Ile Asn Lys Ile Ser Gly Asp Ile 245 250 255 Lys Asp Ser Leu Lys Glu Met Ser Leu Glu Glu Ile Tyr Ser Tyr Glu 260 265 270 Lys Tyr Gly Glu Phe Ile Thr Gln Glu Gly Ile Ser Phe Tyr Asn Asp 275 280 285 Ile Cys Gly Lys Val Asn Ser Phe Met Asn Leu Tyr Cys Gln Lys Asn 290 295 300 Lys Glu Asn Lys Asn Leu Tyr Lys Leu Arg Lys Leu His Lys Gln Ile 305 310 315 320 Leu Cys Ile Ala Asp Thr Ser Tyr Glu Val Pro Tyr Lys Phe Glu Ser 325 330 335 Asp Glu Glu Val Tyr Gln Ser Val Asn Gly Phe Leu Asp Asn Ile Ser 340 345 350 Ser Lys His Ile Val Glu Arg Leu Arg Lys Ile Gly Asp Asn Tyr Asn 355 360 365 Gly Tyr Asn Leu Asp Lys Ile Tyr Ile Val Ser Lys Phe Tyr Glu Ser 370 375 380 Val Ser Gln Lys Thr Tyr Arg Asp Trp Glu Thr Ile Asn Thr Ala Leu 385 390 395 400 Glu Ile His Tyr Asn Asn Ile Leu Pro Gly Asn Gly Lys Ser Lys Ala 405 410 415 Asp Lys Val Lys Lys Ala Val Lys Asn Asp Leu Gln Lys Ser Ile Thr 420 425 430 Glu Ile Asn Glu Leu Val Ser Asn Tyr Lys Leu Cys Pro Asp Asp Asn 435 440 445 Ile Lys Ala Glu Thr Tyr Ile His Glu Ile Ser His Ile Leu Asn Asn 450 455 460 Phe Glu Ala Gln Glu Leu Lys Tyr Asn Pro Glu Ile His Leu Val Glu 465 470 475 480 Ser Glu Leu Lys Ala Ser Glu Leu Lys Asn Val Leu Asp Val Ile Met 485 490 495 Asn Ala Phe His Trp Cys Ser Val Phe Met Thr Glu Glu Leu Val Asp 500 505 510 Lys Asp Asn Asn Phe Tyr Ala Glu Leu Glu Glu Ile Tyr Asp Glu Ile 515 520 525 Tyr Thr Val Ile Ser Leu Tyr Asn Leu Val Arg Asn Tyr Val Thr Gln 530 535 540 Lys Pro Tyr Ser Thr Lys Lys Ile Lys Leu Asn Phe Gly Ile Pro Thr 545 550 555 560 Leu Ala Asp Gly Trp Ser Lys Ser Lys Glu Tyr Ser Asn Asn Ala Ile 565 570 575 Ile Leu Met Arg Asp Asn Leu Tyr Tyr Leu Gly Ile Phe Asn Ala Lys 580 585 590 Asn Lys Pro Asp Lys Lys Ile Ile Glu Gly Asn Thr Ser Glu Asn Lys 595 600 605 Gly Asp Tyr Lys Lys Met Ile Tyr Asn Leu Leu Pro Gly Pro Asn Lys 610 615 620 Met Ile Pro Lys Val Phe Leu Ser Ser Lys Thr Gly Val Glu Thr Tyr 625 630 635 640 Lys Pro Ser Ala Tyr Ile Leu Glu Gly Tyr Lys Gln Asn Lys His Leu 645 650 655 Lys Ser Ser Lys Asp Phe Asp Ile Thr Phe Cys His Asp Leu Ile Asp 660 665 670 Tyr Phe Lys Asn Cys Ile Ala Ile His Pro Glu Trp Lys Asn Phe Gly 675 680 685 Phe Asp Phe Ser Asp Thr Ser Thr Tyr Glu Asp Ile Ser Gly Phe Tyr 690 695 700 Arg Glu Val Glu Leu Gln Gly Tyr Lys Ile Asp Trp Thr Tyr Ile Ser 705 710 715 720 Glu Lys Asp Ile Asp Leu Leu Gln Glu Lys Gly Gln Leu Tyr Leu Phe 725 730 735 Gln Ile Tyr Asn Lys Asp Phe Ser Lys Lys Ser Thr Gly Asn Asp Asn 740 745 750 Leu His Thr Met Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Lys 755 760 765 Asp Ile Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys 770 775 780 Ser Ser Ile Lys Asn Pro Ile Ile His Lys Lys Gly Ser Ile Leu Val 785 790 795 800 Asn Arg Thr Tyr Glu Ala Glu Glu Lys Asp Gln Phe Gly Asn Ile Gln 805 810 815 Ile Val Arg Lys Thr Ile Pro Glu Asn Ile Tyr Gln Glu Leu Tyr Lys 820 825 830 Tyr Phe Asn Asp Lys Ser Asp Lys Glu Leu Ser Asp Glu Ala Ala Lys 835 840 845 Leu Lys Asn Val Val Gly His His Glu Ala Ala Thr Asn Ile Val Lys 850 855 860 Asp Tyr Arg Tyr Thr Tyr Asp Lys Tyr Phe Leu His Met Pro Ile Thr 865 870 875 880 Ile Asn Phe Lys Ala Asn Lys Thr Ser Phe Ile Asn Asp Arg Ile Leu 885 890 895 Gln Tyr Ile Ala Lys Glu Lys Asn Leu His Val Ile Gly Ile Asp Arg 900 905 910 Gly Glu Arg Asn Leu Ile Tyr Val Ser Val Ile Asp Thr Cys Gly Asn 915 920 925 Ile Val Glu Gln Lys Ser Phe Asn Ile Val Asn Gly Tyr Asp Tyr Gln 930 935 940 Ile Lys Leu Lys Gln Gln Glu Gly Ala Arg Gln Ile Ala Arg Lys Glu 945 950 955 960 Trp Lys Glu Ile Gly Lys Ile Lys Glu Ile Lys Glu Gly Tyr Leu Ser 965 970 975 Leu Val Ile His Glu Ile Ser Lys Met Val Ile Lys Tyr Asn Ala Ile 980 985 990 Ile Ala Met Glu Asp Leu Ser Tyr Gly Phe Lys Lys Gly Arg Phe Lys 995 1000 1005 Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu Ile Asn Lys 1010 1015 1020 Leu Asn Tyr Leu Val Phe Lys Asp Ile Ser Ile Thr Glu Asn Gly Gly 1025 1030 1035 1040 Leu Leu Lys Gly Tyr Gln Leu Thr Tyr Ile Pro Asp Lys Leu Lys Asn 1045 1050 1055 Val Gly His Gln Cys Gly Cys Ile Phe Tyr Val Pro Ala Ala Tyr Thr 1060 1065 1070 Ser Lys Ile Asp Pro Thr Thr Gly Phe Val Asn Ile Phe Lys Phe Lys 1075 1080 1085 Asp Leu Thr Val Asp Ala Lys Arg Glu Phe Ile Lys Lys Phe Asp Ser 1090 1095 1100 Ile Arg Tyr Asp Ser Glu Lys Lys Leu Phe Cys Phe Thr Phe Asp Tyr 1105 1110 1115 1120 Asn Asn Phe Ile Thr Gln Asn Thr Val Met Ser Lys Ser Ser Trp Ser 1125 1130 1135 Val Tyr Thr Tyr Gly Val Arg Ile Lys Arg Arg Phe Val Asn Gly Arg 1140 1145 1150 Phe Ser Asn Glu Ser Asp Thr Ile Asp Ile Thr Lys Asp Met Glu Lys 1155 1160 1165 Thr Leu Glu Met Thr Asp Ile Asn Trp Arg Asp Gly His Asp Leu Arg 1170 1175 1180 Gln Asp Ile Ile Asp Tyr Glu Ile Val Gln His Ile Phe Glu Ile Phe 1185 1190 1195 1200 Arg Leu Thr Val Gln Met Arg Asn Ser Leu Ser Glu Leu Glu Asp Arg 1205 1210 1215 Asp Tyr Asp Arg Leu Ile Ser Pro Val Leu Asn Glu Asn Asn Ile Phe 1220 1225 1230 Tyr Asp Ser Ala Lys Ala Gly Asp Ala Leu Pro Lys Asp Ala Asp Ala 1235 1240 1245 Asn Gly Ala Tyr Cys Ile Ala Leu Lys Gly Leu Tyr Glu Ile Lys Gln 1250 1255 1260 Ile Thr Glu Asn Trp Lys Glu Asp Gly Lys Phe Ser Arg Asp Lys Leu 1265 1270 1275 1280 Lys Ile Ser Asn Lys Asp Trp Phe Asp Phe Ile Gln Asn Lys Arg Tyr 1285 1290 1295 Leu Gly Gly Ser Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Gly Ser 1300 1305 1310 Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Gly Ser Pro Ala Ala Lys 1315 1320 1325 Arg Val Lys Leu Asp Gly Gly Ser Pro Ala Ala Lys Arg Val Lys Leu 1330 1335 1340 Asp Gly Gly Ser Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Gly Ser 1345 1350 1355 1360 Pro Ala Ala Lys Arg Val Lys Leu Asp Ala Ser Gly Ser His His His 1365 1370 1375 His His His <210> 9 <211> 4140 <212> DNA <213> Artificial Sequence <220> <223> mgCas12a-1-6XNLS <400> 9 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgaacaa tggcaccaac 120 aatttccaga actttatcgg aattagcagt ctgcaaaaga ctctccggaa tgcccttata 180 cccaccgaga caacccagca gttcatcgtg aaaaacggga ttatcaagga agacgagctg 240 cgcggcgaaa atcggcaaat tttgaaagat ataatggacg attattaccg cggttttatc 300 tctgagactc tgagctccat tgacgatatc gactggacct cactcttcga aaagatggag 360 attcagctta aaaacggcga taataaggac acactgataa aagaacaggc tgagaagcgg 420 aaagccatct ataagaaatt tgcagatgac gatcgcttca agaacatgtt tagcgccaaa 480 ttgattagtg acatcctgcc ggaattcgtt attcacaata acaattactc tgctagcgag 540 aaggaagaga aaacccaagt cataaagctc ttttcccggt tcgccacttc atttaaagat 600 tatttcaaga accgcgcaaa ttgctttagc gccgacgata tcagttctag ctcctgtcat 660 cggattgtga acgacaatgc tgaaatcttc ttttcaaacg cccttgtata ccgccggatt 720 gtgaaaaatc tgagcaacga tgacataaat aagatcagtg gagatattaa agactctttg 780 aaggagatga gcctggaaga gatctattcc tacgaaaaat atggggagtt cattacccag 840 gaaggcatat cattttacaa cgatatctgc ggtaaggtta atagcttcat gaacctctat 900 tgtcagaaaa ataaggagaa caaaaatctt tacaagctgc gcaaattgca caagcaaatt 960 ctgtgcatcg cagacacaag ttatgaagtc ccttacaaat ttgagtctga tgaagaggtg 1020 tatcagagcg taaacggctt cctcgacaat atttcctcaa agcatatagt ggaacggctt 1080 cgcaaaatcg gagataacta caatgggtat aacctggaca agatttacat cgttagcaaa 1140 ttttatgaga gtgtctctca gaagacctac cgggattggg aaactattaa taccgccttg 1200 gagatacact ataacaatat cctgcccggc aacggtaaaa gcaaggctga caaagtgaag 1260 aaagccgtaa agaatgatct ccaaaaatcc attacagaaa tcaacgagct tgtgtcaaat 1320 tacaagctgt gtccggacga taacattaaa gcagaaacct atatacatga gatcagccac 1380 attttgaata acttcgaagc ccaggagctg aagtacaatc cagaaatcca tctcgttgag 1440 agtgaactta aagcttctga gctgaagaac gtcttggacg tgattatgaa tgcctttcac 1500 tggtgcagcg tattcatgac tgaagagctg gtggataaag acaacaattt ttatgcagaa 1560 ctcgaggaaa tatacgatga gatctatacc gttatttccc tttacaacct ggtccgcaat 1620 tatgtgacac agaagcccta ctcaaccaaa aagatcaaat tgaacttcgg cattccgact 1680 ctggccgacg gatggagcaa gagtaaagaa tattctaata acgctataat cctcatgcgg 1740 gataatcttt actatctggg gatttttaac gccaagaata aacctgacaa gaaaatcatt 1800 gagggcaaca ccagcgaaaa taagggtgat tacaaaaaga tgatatataa cttgctgccc 1860 ggcccgaata aaatgatccc aaaggtattc ctctcctcaa aaacaggagt ggagacctac 1920 aagcccagcg catatattct tgaagggtac aaacaaaaca agcatctgaa aagttctaag 1980 gactttgata tcactttctg tcacgacttg attgattatt ttaaaaattg catagccatc 2040 catccggagt ggaagaactt cggctttgac ttcagcgata cctccacata cgaagacatt 2100 tcaggttttt atcgcgaggt tgaactgcag ggctacaaaa tcgattggac ctatattagc 2160 gagaaggaca tagatctcct tcaggaaaaa ggacaactgt acttgttcca gatctataat 2220 aaggacttta gtaaaaagtc tactgggaac gataatctgc acaccatgta cctcaaaaac 2280 cttttcagcg aggaaaatct gaaggacatt gtcttgaaac tgaacggcga ggctgaaatc 2340 tttttccgga agtcctcaat taaaaatcct ataatccata agaaaggtag cattctcgtg 2400 aaccgcacat atgaggccga agagaaggat cagtttggca atatccaaat tgtacggaaa 2460 accatacccg aaaacatcta ccaggagctt tataagtact tcaatgacaa aagtgataag 2520 gaactgtctg acgaggcagc caaattgaag aacgtggttg gacaccatga agctgccact 2580 aatattgtca aagattatcg ctacacctat gacaagtact ttctgcacat gccgatcaca 2640 attaacttca aagcaaataa gaccagcttt ataaacgatc ggattctcca gtatattgcc 2700 aaagagaaga atcttcatgt gatcgggatt gaccgcggcg aacggaacct gatatacgta 2760 tccgtgatcg atacttgtgg taatattgtt gagcaaaaat cattcaacat cgtcaatggc 2820 tatgactacc agattaagtt gaaacagcaa gaaggagctc gccagatagc ccggaaggag 2880 tggaaggaaa tcgggaaaat taaggagatc aaagaaggct atctgagcct cgtgattcac 2940 gagataagta agatggtaat caaatacaac gcaattatcg ccatggaaga tctttcttat 3000 ggttttaaga aaggccgctt caaggtggag cggcaagttt accagaaatt tgaaaccatg 3060 ctgattaata agttgaacta tctggtcttc aaagacataa gcatcacaga gaatggaggg 3120 ctccttaagg gctaccagct gacctatatt ccagataaat tgaagaacgt gggtcatcaa 3180 tgcggctgta tcttttacgt acccgctgcc tatacttcca aaattgaccc gaccacagga 3240 ttcgtgaata tatttaagtt caaagatctg accgttgacg caaagcgcga atttatcaaa 3300 aagttcgatt caattcggta cgacagcgag aaaaagctct tttgcttcac ttttgattat 3360 aacaatttca tcacccagaa cacagtcatg agtaaatcta gctggtccgt gtacacctat 3420 ggggtacgca ttaagcggcg ctttgtgaat ggccggttct caaacgaaag cgacactata 3480 gatatcacca aagacatgga gaagacactt gaaatgaccg atattaattg gcgcgacggt 3540 cacgatctgc ggcaggacat cattgattac gagatagttc aacatatctt tgaaattttc 3600 cgcttgactg tccagatgcg gaacagtctg tctgagctcg aagaccgcga ttatgaccgg 3660 cttatcagcc ctgtgctgaa tgagaacaat attttttacg attccgccaa agctggcgac 3720 gccttgccca aggatgcaga cgccaacgga gcttattgta tagccctgaa agggctctac 3780 gaaatcaagc agattaccga gaattggaaa gaagatggca agttctcacg cgacaaactt 3840 aagatcagca acaaagattg gtttgacttc attcaaaata agcggtatct gggcggctcc 3900 cctgctgcta aacgtgttaa gcttgatggg ggtagcccgg cagccaagag agtcaaactc 3960 gacggcggct ccccagcggc aaaaagggtg aaactagacg ggggtagccc cgccgcgaag 4020 cgtgtaaagc tggatggagg atcgcctgcg gctaagcgag tcaaattaga cggtgggtca 4080 ccggctgcaa agcgcgtcaa attggatgct agcggcagcc accaccacca ccaccactga 4140 4140 <210> 10 <211> 164 <212> DNA <213> Artificial Sequence <220> <223> insert1 <400> 10 gcgccaaatt gattagtgac atcctgccgg aattcgttat tcacaataac aattactctg 60 ctagcgagaa ggaagagaaa acccaagtca taaagctctt ttcccggttc gccacttcat 120 ttaaagatta tttccgcaac cgcgcaaatt gctttagcgc cgac 164 <210> 11 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> insert1_primer_forward <400> 11 gcgccaaatt gattagtgac atcctgccgg aattcgttat tcacaataac aat 53 <210> 12 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> insert1_primer_reverse <400> 12 gtcggcgcta aagcaatttg cgcggttgcg gaaataatct ttaaatgaag tggc 54 <210> 13 <211> 1083 <212> DNA <213> Artificial Sequence <220> <223> insert2 <400> 13 cgcaaccgcg caaattgctt tagcgccgac gatatcagtt ctagctcctg tcatcggatt 60 gtgaacgaca atgctgaaat cttcttttca aacgcccttg tataccgccg gattgtgaaa 120 aatctgagca acgatgacat aaataagatc agtggagata ttaaagactc tttgaaggag 180 atgagcctgg aagagatcta ttcctacgaa aaatatgggg agttcattac ccaggaaggc 240 atatcatttt acaacgatat ctgcggtaag gttaatagct tcatgaacct ctattgtcag 300 aaaaataagg agaacaaaaa tctttacaag ctgcgcaaat tgcacaagca aattctgtgc 360 atcgcagaca caagttatga agtcccttac aaatttgagt ctgatgaaga ggtgtatcag 420 agcgtaaacg gcttcctcga caatatttcc tcaaagcata tagtggaacg gcttcgcaaa 480 atcggagata actacaatgg gtataacctg gacaagattt acatcgttag caaattttat 540 gagagtgtct ctcagaagac ctaccgggat tgggaaacta ttaataccgc cttggagata 600 cactataaca atatcctgcc cggcaacggt aaaagcaagg ctgacaaagt gaagaaagcc 660 gtaaagaatg atctccaaaa atccattaca gaaatcaacg agcttgtgtc aaattacaag 720 ctgtgtccgg acgataacat taaagcagaa acctatatac atgagatcag ccacattttg 780 aataacttcg aagcccagga gctgaagtac aatccagaaa tccatctcgt tgagagtgaa 840 cttaaagctt ctgagctgaa gaacgtcttg gacgtgatta tgaatgcctt tcactggtgc 900 agcgtattca tgactgaaga gctggtggat aaagacaaca atttttatgc agaactcgag 960 gaaatatacg atgagatcta taccgttatt tccctttaca acctggtccg caattatgtg 1020 acacagaagc cctactcaac caaaaagatc aaattgaact tcggcattcc gactctggcc 1080 cgc 1083 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> insert2_primer_forward <400> 14 cgcaaccgcg caaattgctt tagcgccgac 30 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> insert2_primer_reverse <400> 15 gcgggccaga gtcggaatgc cgaagttcaa 30 <210> 16 <211> 281 <212> DNA <213> Artificial Sequence <220> <223> insert3 <400> 16 ttgaacttcg gcattccgac tctggcccgc ggatggagca agagtaaaga atattctaat 60 aacgctataa tcctcatgcg ggataatctt tactatctgg ggatttttaa cgccaagaat 120 aaacctgaca agaaaatcat tgagggcaac accagcgaaa ataagggtga ttacaaaaag 180 atgatatata acttgctgcc cggcccgaat aaaatgatcc caaaggtatt cctctcctca 240 aaaacaggag tggagaccta caagcccagc gcatatattc t 281 <210> 17 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> insert3_primer_forward <400> 17 ttgaacttcg gcattccgac tctggcccgc ggatggagca agagtaaaga 50 <210> 18 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> insert3_primer_reverse <400> 18 agaatatatg cgctgggctt gtaggtctcc actcctgttt ttgaggagag 50 <210> 19 <211> 1379 <212> PRT <213> Artificial Sequence <220> <223> opmgCas12a-1-6XNLS <400> 19 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Asn Asn Gly Thr Asn Asn Phe Gln Asn Phe Ile Gly Ile 35 40 45 Ser Ser Leu Gln Lys Thr Leu Arg Asn Ala Leu Ile Pro Thr Glu Thr 50 55 60 Thr Gln Gln Phe Ile Val Lys Asn Gly Ile Ile Lys Glu Asp Glu Leu 65 70 75 80 Arg Gly Glu Asn Arg Gln Ile Leu Lys Asp Ile Met Asp Asp Tyr Tyr 85 90 95 Arg Gly Phe Ile Ser Glu Thr Leu Ser Ser Ile Asp Asp Ile Asp Trp 100 105 110 Thr Ser Leu Phe Glu Lys Met Glu Ile Gln Leu Lys Asn Gly Asp Asn 115 120 125 Lys Asp Thr Leu Ile Lys Glu Gln Ala Glu Lys Arg Lys Ala Ile Tyr 130 135 140 Lys Lys Phe Ala Asp Asp Asp Arg Phe Lys Asn Met Phe Ser Ala Lys 145 150 155 160 Leu Ile Ser Asp Ile Leu Pro Glu Phe Val Ile His Asn Asn Asn Tyr 165 170 175 Ser Ala Ser Glu Lys Glu Glu Lys Thr Gln Val Ile Lys Leu Phe Ser 180 185 190 Arg Phe Ala Thr Ser Phe Lys Asp Tyr Phe Arg Asn Arg Ala Asn Cys 195 200 205 Phe Ser Ala Asp Asp Ile Ser Ser Ser Ser Cys His Arg Ile Val Asn 210 215 220 Asp Asn Ala Glu Ile Phe Phe Ser Asn Ala Leu Val Tyr Arg Arg Ile 225 230 235 240 Val Lys Asn Leu Ser Asn Asp Asp Ile Asn Lys Ile Ser Gly Asp Ile 245 250 255 Lys Asp Ser Leu Lys Glu Met Ser Leu Glu Glu Ile Tyr Ser Tyr Glu 260 265 270 Lys Tyr Gly Glu Phe Ile Thr Gln Glu Gly Ile Ser Phe Tyr Asn Asp 275 280 285 Ile Cys Gly Lys Val Asn Ser Phe Met Asn Leu Tyr Cys Gln Lys Asn 290 295 300 Lys Glu Asn Lys Asn Leu Tyr Lys Leu Arg Lys Leu His Lys Gln Ile 305 310 315 320 Leu Cys Ile Ala Asp Thr Ser Tyr Glu Val Pro Tyr Lys Phe Glu Ser 325 330 335 Asp Glu Glu Val Tyr Gln Ser Val Asn Gly Phe Leu Asp Asn Ile Ser 340 345 350 Ser Lys His Ile Val Glu Arg Leu Arg Lys Ile Gly Asp Asn Tyr Asn 355 360 365 Gly Tyr Asn Leu Asp Lys Ile Tyr Ile Val Ser Lys Phe Tyr Glu Ser 370 375 380 Val Ser Gln Lys Thr Tyr Arg Asp Trp Glu Thr Ile Asn Thr Ala Leu 385 390 395 400 Glu Ile His Tyr Asn Asn Ile Leu Pro Gly Asn Gly Lys Ser Lys Ala 405 410 415 Asp Lys Val Lys Lys Ala Val Lys Asn Asp Leu Gln Lys Ser Ile Thr 420 425 430 Glu Ile Asn Glu Leu Val Ser Asn Tyr Lys Leu Cys Pro Asp Asp Asn 435 440 445 Ile Lys Ala Glu Thr Tyr Ile His Glu Ile Ser His Ile Leu Asn Asn 450 455 460 Phe Glu Ala Gln Glu Leu Lys Tyr Asn Pro Glu Ile His Leu Val Glu 465 470 475 480 Ser Glu Leu Lys Ala Ser Glu Leu Lys Asn Val Leu Asp Val Ile Met 485 490 495 Asn Ala Phe His Trp Cys Ser Val Phe Met Thr Glu Glu Leu Val Asp 500 505 510 Lys Asp Asn Asn Phe Tyr Ala Glu Leu Glu Glu Ile Tyr Asp Glu Ile 515 520 525 Tyr Thr Val Ile Ser Leu Tyr Asn Leu Val Arg Asn Tyr Val Thr Gln 530 535 540 Lys Pro Tyr Ser Thr Lys Lys Ile Lys Leu Asn Phe Gly Ile Pro Thr 545 550 555 560 Leu Ala Arg Gly Trp Ser Lys Ser Lys Glu Tyr Ser Asn Asn Ala Ile 565 570 575 Ile Leu Met Arg Asp Asn Leu Tyr Tyr Leu Gly Ile Phe Asn Ala Lys 580 585 590 Asn Lys Pro Asp Lys Lys Ile Ile Glu Gly Asn Thr Ser Glu Asn Lys 595 600 605 Gly Asp Tyr Lys Lys Met Ile Tyr Asn Leu Leu Pro Gly Pro Asn Lys 610 615 620 Met Ile Pro Lys Val Phe Leu Ser Ser Lys Thr Gly Val Glu Thr Tyr 625 630 635 640 Lys Pro Ser Ala Tyr Ile Leu Glu Gly Tyr Lys Gln Asn Lys His Leu 645 650 655 Lys Ser Ser Lys Asp Phe Asp Ile Thr Phe Cys His Asp Leu Ile Asp 660 665 670 Tyr Phe Lys Asn Cys Ile Ala Ile His Pro Glu Trp Lys Asn Phe Gly 675 680 685 Phe Asp Phe Ser Asp Thr Ser Thr Tyr Glu Asp Ile Ser Gly Phe Tyr 690 695 700 Arg Glu Val Glu Leu Gln Gly Tyr Lys Ile Asp Trp Thr Tyr Ile Ser 705 710 715 720 Glu Lys Asp Ile Asp Leu Leu Gln Glu Lys Gly Gln Leu Tyr Leu Phe 725 730 735 Gln Ile Tyr Asn Lys Asp Phe Ser Lys Lys Ser Thr Gly Asn Asp Asn 740 745 750 Leu His Thr Met Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Lys 755 760 765 Asp Ile Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys 770 775 780 Ser Ser Ile Lys Asn Pro Ile Ile His Lys Lys Gly Ser Ile Leu Val 785 790 795 800 Asn Arg Thr Tyr Glu Ala Glu Glu Lys Asp Gln Phe Gly Asn Ile Gln 805 810 815 Ile Val Arg Lys Thr Ile Pro Glu Asn Ile Tyr Gln Glu Leu Tyr Lys 820 825 830 Tyr Phe Asn Asp Lys Ser Asp Lys Glu Leu Ser Asp Glu Ala Ala Lys 835 840 845 Leu Lys Asn Val Val Gly His His Glu Ala Ala Thr Asn Ile Val Lys 850 855 860 Asp Tyr Arg Tyr Thr Tyr Asp Lys Tyr Phe Leu His Met Pro Ile Thr 865 870 875 880 Ile Asn Phe Lys Ala Asn Lys Thr Ser Phe Ile Asn Asp Arg Ile Leu 885 890 895 Gln Tyr Ile Ala Lys Glu Lys Asn Leu His Val Ile Gly Ile Asp Arg 900 905 910 Gly Glu Arg Asn Leu Ile Tyr Val Ser Val Ile Asp Thr Cys Gly Asn 915 920 925 Ile Val Glu Gln Lys Ser Phe Asn Ile Val Asn Gly Tyr Asp Tyr Gln 930 935 940 Ile Lys Leu Lys Gln Gln Glu Gly Ala Arg Gln Ile Ala Arg Lys Glu 945 950 955 960 Trp Lys Glu Ile Gly Lys Ile Lys Glu Ile Lys Glu Gly Tyr Leu Ser 965 970 975 Leu Val Ile His Glu Ile Ser Lys Met Val Ile Lys Tyr Asn Ala Ile 980 985 990 Ile Ala Met Glu Asp Leu Ser Tyr Gly Phe Lys Lys Gly Arg Phe Lys 995 1000 1005 Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu Ile Asn Lys 1010 1015 1020 Leu Asn Tyr Leu Val Phe Lys Asp Ile Ser Ile Thr Glu Asn Gly Gly 1025 1030 1035 1040 Leu Leu Lys Gly Tyr Gln Leu Thr Tyr Ile Pro Asp Lys Leu Lys Asn 1045 1050 1055 Val Gly His Gln Cys Gly Cys Ile Phe Tyr Val Pro Ala Ala Tyr Thr 1060 1065 1070 Ser Lys Ile Asp Pro Thr Thr Gly Phe Val Asn Ile Phe Lys Phe Lys 1075 1080 1085 Asp Leu Thr Val Asp Ala Lys Arg Glu Phe Ile Lys Lys Phe Asp Ser 1090 1095 1100 Ile Arg Tyr Asp Ser Glu Lys Lys Leu Phe Cys Phe Thr Phe Asp Tyr 1105 1110 1115 1120 Asn Asn Phe Ile Thr Gln Asn Thr Val Met Ser Lys Ser Ser Trp Ser 1125 1130 1135 Val Tyr Thr Tyr Gly Val Arg Ile Lys Arg Arg Phe Val Asn Gly Arg 1140 1145 1150 Phe Ser Asn Glu Ser Asp Thr Ile Asp Ile Thr Lys Asp Met Glu Lys 1155 1160 1165 Thr Leu Glu Met Thr Asp Ile Asn Trp Arg Asp Gly His Asp Leu Arg 1170 1175 1180 Gln Asp Ile Ile Asp Tyr Glu Ile Val Gln His Ile Phe Glu Ile Phe 1185 1190 1195 1200 Arg Leu Thr Val Gln Met Arg Asn Ser Leu Ser Glu Leu Glu Asp Arg 1205 1210 1215 Asp Tyr Asp Arg Leu Ile Ser Pro Val Leu Asn Glu Asn Asn Ile Phe 1220 1225 1230 Tyr Asp Ser Ala Lys Ala Gly Asp Ala Leu Pro Lys Asp Ala Asp Ala 1235 1240 1245 Asn Gly Ala Tyr Cys Ile Ala Leu Lys Gly Leu Tyr Glu Ile Lys Gln 1250 1255 1260 Ile Thr Glu Asn Trp Lys Glu Asp Gly Lys Phe Ser Arg Asp Lys Leu 1265 1270 1275 1280 Lys Ile Ser Asn Lys Asp Trp Phe Asp Phe Ile Gln Asn Lys Arg Tyr 1285 1290 1295 Leu Gly Gly Ser Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Gly Ser 1300 1305 1310 Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Gly Ser Pro Ala Ala Lys 1315 1320 1325 Arg Val Lys Leu Asp Gly Gly Ser Pro Ala Ala Lys Arg Val Lys Leu 1330 1335 1340 Asp Gly Gly Ser Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Gly Ser 1345 1350 1355 1360 Pro Ala Ala Lys Arg Val Lys Leu Asp Ala Ser Gly Ser His His His 1365 1370 1375 His His His <210> 20 <211> 4140 <212> DNA <213> Artificial Sequence <220> <223> opmgCas12a-1-6XNLS <400> 20 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgaacaa tggcaccaac 120 aatttccaga actttatcgg aattagcagt ctgcaaaaga ctctccggaa tgcccttata 180 cccaccgaga caacccagca gttcatcgtg aaaaacggga ttatcaagga agacgagctg 240 cgcggcgaaa atcggcaaat tttgaaagat ataatggacg attattaccg cggttttatc 300 tctgagactc tgagctccat tgacgatatc gactggacct cactcttcga aaagatggag 360 attcagctta aaaacggcga taataaggac acactgataa aagaacaggc tgagaagcgg 420 aaagccatct ataagaaatt tgcagatgac gatcgcttca agaacatgtt tagcgccaaa 480 ttgattagtg acatcctgcc ggaattcgtt attcacaata acaattactc tgctagcgag 540 aaggaagaga aaacccaagt cataaagctc ttttcccggt tcgccacttc atttaaagat 600 tatttccgca accgcgcaaa ttgctttagc gccgacgata tcagttctag ctcctgtcat 660 cggattgtga acgacaatgc tgaaatcttc ttttcaaacg cccttgtata ccgccggatt 720 gtgaaaaatc tgagcaacga tgacataaat aagatcagtg gagatattaa agactctttg 780 aaggagatga gcctggaaga gatctattcc tacgaaaaat atggggagtt cattacccag 840 gaaggcatat cattttacaa cgatatctgc ggtaaggtta atagcttcat gaacctctat 900 tgtcagaaaa ataaggagaa caaaaatctt tacaagctgc gcaaattgca caagcaaatt 960 ctgtgcatcg cagacacaag ttatgaagtc ccttacaaat ttgagtctga tgaagaggtg 1020 tatcagagcg taaacggctt cctcgacaat atttcctcaa agcatatagt ggaacggctt 1080 cgcaaaatcg gagataacta caatgggtat aacctggaca agatttacat cgttagcaaa 1140 ttttatgaga gtgtctctca gaagacctac cgggattggg aaactattaa taccgccttg 1200 gagatacact ataacaatat cctgcccggc aacggtaaaa gcaaggctga caaagtgaag 1260 aaagccgtaa agaatgatct ccaaaaatcc attacagaaa tcaacgagct tgtgtcaaat 1320 tacaagctgt gtccggacga taacattaaa gcagaaacct atatacatga gatcagccac 1380 attttgaata acttcgaagc ccaggagctg aagtacaatc cagaaatcca tctcgttgag 1440 agtgaactta aagcttctga gctgaagaac gtcttggacg tgattatgaa tgcctttcac 1500 tggtgcagcg tattcatgac tgaagagctg gtggataaag acaacaattt ttatgcagaa 1560 ctcgaggaaa tatacgatga gatctatacc gttatttccc tttacaacct ggtccgcaat 1620 tatgtgacac agaagcccta ctcaaccaaa aagatcaaat tgaacttcgg cattccgact 1680 ctggcccgcg gatggagcaa gagtaaagaa tattctaata acgctataat cctcatgcgg 1740 gataatcttt actatctggg gatttttaac gccaagaata aacctgacaa gaaaatcatt 1800 gagggcaaca ccagcgaaaa taagggtgat tacaaaaaga tgatatataa cttgctgccc 1860 ggcccgaata aaatgatccc aaaggtattc ctctcctcaa aaacaggagt ggagacctac 1920 aagcccagcg catatattct tgaagggtac aaacaaaaca agcatctgaa aagttctaag 1980 gactttgata tcactttctg tcacgacttg attgattatt ttaaaaattg catagccatc 2040 catccggagt ggaagaactt cggctttgac ttcagcgata cctccacata cgaagacatt 2100 tcaggttttt atcgcgaggt tgaactgcag ggctacaaaa tcgattggac ctatattagc 2160 gagaaggaca tagatctcct tcaggaaaaa ggacaactgt acttgttcca gatctataat 2220 aaggacttta gtaaaaagtc tactgggaac gataatctgc acaccatgta cctcaaaaac 2280 cttttcagcg aggaaaatct gaaggacatt gtcttgaaac tgaacggcga ggctgaaatc 2340 tttttccgga agtcctcaat taaaaatcct ataatccata agaaaggtag cattctcgtg 2400 aaccgcacat atgaggccga agagaaggat cagtttggca atatccaaat tgtacggaaa 2460 accatacccg aaaacatcta ccaggagctt tataagtact tcaatgacaa aagtgataag 2520 gaactgtctg acgaggcagc caaattgaag aacgtggttg gacaccatga agctgccact 2580 aatattgtca aagattatcg ctacacctat gacaagtact ttctgcacat gccgatcaca 2640 attaacttca aagcaaataa gaccagcttt ataaacgatc ggattctcca gtatattgcc 2700 aaagagaaga atcttcatgt gatcgggatt gaccgcggcg aacggaacct gatatacgta 2760 tccgtgatcg atacttgtgg taatattgtt gagcaaaaat cattcaacat cgtcaatggc 2820 tatgactacc agattaagtt gaaacagcaa gaaggagctc gccagatagc ccggaaggag 2880 tggaaggaaa tcgggaaaat taaggagatc aaagaaggct atctgagcct cgtgattcac 2940 gagataagta agatggtaat caaatacaac gcaattatcg ccatggaaga tctttcttat 3000 ggttttaaga aaggccgctt caaggtggag cggcaagttt accagaaatt tgaaaccatg 3060 ctgattaata agttgaacta tctggtcttc aaagacataa gcatcacaga gaatggaggg 3120 ctccttaagg gctaccagct gacctatatt ccagataaat tgaagaacgt gggtcatcaa 3180 tgcggctgta tcttttacgt acccgctgcc tatacttcca aaattgaccc gaccacagga 3240 ttcgtgaata tatttaagtt caaagatctg accgttgacg caaagcgcga atttatcaaa 3300 aagttcgatt caattcggta cgacagcgag aaaaagctct tttgcttcac ttttgattat 3360 aacaatttca tcacccagaa cacagtcatg agtaaatcta gctggtccgt gtacacctat 3420 ggggtacgca ttaagcggcg ctttgtgaat ggccggttct caaacgaaag cgacactata 3480 gatatcacca aagacatgga gaagacactt gaaatgaccg atattaattg gcgcgacggt 3540 cacgatctgc ggcaggacat cattgattac gagatagttc aacatatctt tgaaattttc 3600 cgcttgactg tccagatgcg gaacagtctg tctgagctcg aagaccgcga ttatgaccgg 3660 cttatcagcc ctgtgctgaa tgagaacaat attttttacg attccgccaa agctggcgac 3720 gccttgccca aggatgcaga cgccaacgga gcttattgta tagccctgaa agggctctac 3780 gaaatcaagc agattaccga gaattggaaa gaagatggca agttctcacg cgacaaactt 3840 aagatcagca acaaagattg gtttgacttc attcaaaata agcggtatct gggcggctcc 3900 cctgctgcta aacgtgttaa gcttgatggg ggtagcccgg cagccaagag agtcaaactc 3960 gacggcggct ccccagcggc aaaaagggtg aaactagacg ggggtagccc cgccgcgaag 4020 cgtgtaaagc tggatggagg atcgcctgcg gctaagcgag tcaaattaga cggtgggtca 4080 ccggctgcaa agcgcgtcaa attggatgct agcggcagcc accaccacca ccaccactga 4140 4140 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HPRT1 <400> 21 ggttaaagat ggttaaatga t 21 <110> G+FLAS Life Sciences <120> Novel CRISPR Associated Protein and Use thereof <130> SP20-0124KR <160> 21 <170> KoPatentIn 3.0 <210> 1 <211> 1330 <212> PRT <213> Artificial Sequence <220> <223> mgCas12a-1 <400> 1 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Asn Asn Gly Thr Asn Asn Phe Gln Asn Phe Ile Gly Ile 35 40 45 Ser Ser Leu Gln Lys Thr Leu Arg Asn Ala Leu Ile Pro Thr Glu Thr 50 55 60 Thr Gln Gln Phe Ile Val Lys Asn Gly Ile Ile Lys Glu Asp Glu Leu 65 70 75 80 Arg Gly Glu Asn Arg Gln Ile Leu Lys Asp Ile Met Asp Asp Tyr Tyr 85 90 95 Arg Gly Phe Ile Ser Glu Thr Leu Ser Ser Ile Asp Asp Ile Asp Trp 100 105 110 Thr Ser Leu Phe Glu Lys Met Glu Ile Gln Leu Lys Asn Gly Asp Asn 115 120 125 Lys Asp Thr Leu Ile Lys Glu Gln Ala Glu Lys Arg Lys Ala Ile Tyr 130 135 140 Lys L ys Phe Ala Asp Asp Asp Arg Phe Lys Asn Met Phe Ser Ala Lys 145 150 155 160 Leu Ile Ser Asp Ile Leu Pro Glu Phe Val Ile His Asn Asn Asn Tyr 165 170 175 Ser Ala Ser Glu Lys Glu Glu Lys Thr Gln Val Ile Lys Leu Phe Ser 180 185 190 Arg Phe Ala Thr Ser Phe Lys Asp Tyr Phe Lys Asn Arg Ala Asn Cys 195 200 205 Phe Ser Ala Asp Asp Ile Ser Ser Ser Ser Cys His Arg Ile Val Asn 210 215 220 Asp Asn Ala Glu Ile Phe Phe Ser Asn Ala Leu Val Tyr Arg Arg Ile 225 230 235 240 Val Lys Asn Leu Ser Asn Asp Asp Ile Asn Lys Ile Ser Gly Asp Ile 245 250 255 Lys Asp Ser Leu Lys Glu Met Ser Leu Glu Glu Tyr Ser Tyr Glu 260 265 270 Lys Tyr Gly Glu Phe Ile Thr Gln Glu Gly Ile Ser Phe Tyr Asn Asp 275 280 285 Ile Cys Gly Lys Val Asn Ser Phe Met Asn Leu Tyr Cys Gln Lys Asn 290 295 300 Lys Glu Asn Lys Asn Leu Tyr Lys Leu Arg Lys Leu His Lys Gln Ile 305 310 315 320 Leu Cys Ile Ala Asp Thr Ser Tyr Glu Val Pro Tyr Lys Phe Glu Ser 325 330 335 Asp Glu Glu Val Tyr Gln Ser Val Asn Gly Phe Leu Asp Asn Ile Ser 340 345 350 Ser Lys His Ile Val Glu Arg Leu Arg Lys Ile Gly Asp Asn Tyr Asn 355 360 365 Gly Tyr Asn Leu Asp Lys Ile Tyr Ile Val Ser Lys Phe Tyr Glu Ser 370 375 380 Val Ser Gln Lys Thr Tyr Arg Asp Trp Glu Thr Ile Asn Thr Ala Leu 385 390 395 400 Glu Ile His Tyr Asn Asn Ile Leu Pro Gly Asn Gly Lys Ser Lys Ala 405 410 415 Asp Lys Val Lys Lys Ala Val Lys Asn Asp Leu Gln Lys Ser Ile Thr 420 425 430 Glu Ile Asn Glu Leu Val Ser Asn Tyr Lys Leu Cys Pro Asp Asp Asn 435 440 445 Ile Lys Ala Glu Thr Tyr Ile His Glu Ile Ser His Ile Leu Asn Asn 450 455 460 Phe Glu Ala Gln Glu Leu Lys Tyr Asn Pro Glu Ile His Leu Val Glu 465 470 475 480 Ser Glu Leu Lys Ala Ser Glu Leu Lys Asn Val Leu Asp Val Ile Met 485 490 495 Asn Ala Phe His Trp Cys Ser Val Phe Met Thr Glu Glu Leu Val Asp 500 505 510 Lys Asp Asn Asn Phe Tyr Ala Glu Leu Glu Glu Ile Tyr Asp Glu Ile 515 520 525 Tyr Thr Val Ile Ser Leu Tyr Asn Leu Val Arg Asn Tyr Val Thr Gln 530 535 540 Lys Pro Tyr Ser Thr Lys Lys Ile Lys Leu Asn Phe Gly Ile Pro Thr 545 550 555 560 Leu Ala Asp Gly Trp Ser Lys Ser Lys Glu Tyr Ser Asn Asn Ala Ile 565 570 575 Ile Leu Met Arg Asp Asn Leu Tyr Tyr Leu Gly Ile Phe Asn Ala Lys 580 585 590 Asn Lys Pro Asp Lys Lys Ile Ile Glu Gly Asn Thr Ser Glu Asn Lys 595 600 605 Gly Asp Tyr Lys Lys Met Ile Tyr Asn Leu Leu Pro Gly Pro Asn Lys 610 615 620 Met Ile Pro Lys Val Phe Leu Ser Ser Lys Thr Gly Val Glu Thr Tyr 625 630 635 640 Lys Pro Ser Ala Tyr Ile Leu Glu Gly Tyr Lys Gln Asn Lys His Leu 645 650 655 Lys Ser Ser Lys Asp Phe Asp Ile Thr Phe Cys His Asp Leu Ile Asp 660 665 670 Tyr Phe Lys Asn Cys Ile Ala Ile His Pro Glu Trp Lys Asn Phe Gly 675 680 685 Phe Asp Phe Ser Asp Thr Ser Thr Ser Tyr Glu Asp Ile Ser Gly Phe Tyr 690 695 700 Arg Glu Val Glu Leu Gln Gly Tyr Lys Ile Asp Trp Thr Tyr Ile Ser 705 7 10 715 720 Glu Lys Asp Ile Asp Leu Leu Gln Glu Lys Gly Gln Leu Tyr Leu Phe 725 730 735 Gln Ile Tyr Asn Lys Asp Phe Ser Lys Lys Ser Thr Gly Asn Asp Asn 740 745 750 Leu His Thr Met Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Lys 755 760 765 Asp Ile Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys 770 775 780 Ser Ser Ile Lys Asn Pro Ile Ile His Lys Lys Gly Ser Ile Leu Val 785 790 795 800 Asn Arg Thr Tyr Glu Ala Glu Glu Lys Asp Gln Phe Gly Asn Ile Gln 805 810 815 Ile Val Arg Lys Thr Ile Pro Glu Asn Ile Tyr Gln Glu Leu Tyr Lys 820 825 830 Tyr Phe Asn Asp Lys Ser Asp Lys Glu Leu Ser Asp Glu Ala Ala Lys 835 840 845 Leu Lys Asn Val Val Gly His Glu Ala Ala Thr Asn Ile Val Lys 850 855 860 Asp Tyr Arg Tyr Thr Tyr Asp Lys Tyr Phe Leu His Met Pro Ile Thr 865 870 875 880 Ile Asn Phe Lys Ala Asn Lys Thr Ser Phe Ile Asn Asp Arg Ile Leu 885 890 895 Gln Tyr Ile Ala Lys Glu Lys Asn Leu His Val Ile Gly Ile Asp Arg 900 905 910 Gly Glu Arg Asn Leu Ile Tyr Val Ser Val Ile Asp Thr Cys Gly Asn 915 920 925 Ile Val Glu Gln Lys Ser Phe Asn Ile Val Asn Gly Tyr Asp Tyr Gln 930 935 940 Ile Lys Leu Lys Gln Gln Glu Gly Ala Arg Gln Ile Ala Arg Lys Glu 945 950 955 960 Trp Lys Glu Ile Gly Lys Ile Lys Glu Ile Lys Glu Gly Tyr Leu Ser 965 970 975 Leu Val Ile His Glu Ile Ser Lys Met Val Ile Lys Tyr Asn Ala Ile 980 985 990 Ile Ala Met Glu Asp Leu Ser Tyr Gly Phe Lys Lys Gly Arg Phe L ys 995 1000 1005 Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu Ile Asn Lys 1010 1015 1020 Leu Asn Tyr Leu Val Phe Lys Asp Ile Ser Ile Thr Glu Asn Gly Gly 1025 1030 1035 1040 Leu Leu Lys Gly Tyr Gln Leu Thr Tyr Ile Pro Asp Lys Leu Lys Asn 1045 1050 1055 Val Gly His Gln Cys Gly Cys Ile Phe Tyr Val Pro Ala Ala Tyr Thr 1060 1065 1070 Ser Lys Ile Asp Pro Thr Gly Phe Val Asn Ile Phe Lys Phe Lys 1075 1080 1085 Asp Leu Thr Val Asp Ala Lys Arg Glu Phe Ile Lys Lys Phe Asp Ser 1090 1095 1100 Ile Arg Tyr Asp Ser Glu Lys Lys Leu Phe Cys Phe Thr Phe Asp Tyr 1105 1110 1115 1120 Asn Asn Phe Ile Thr Gln Asn Thr Val Met Ser Lys Ser Ser Trp Ser 1125 1130 1135 Val Tyr Thr Tyr Gly Val Arg Ile Lys Arg Arg Phe Val Asn Gly Arg 1140 1145 1150 Phe Ser Asn Glu Ser Asp Thr Ile Asp Ile Thr Lys Asp Met Glu Lys 1155 1160 1165 Thr Leu Glu Met Thr Asp Ile Asn Trp Arg Asp Gly His Asp Leu Arg 1170 1175 1180 Gln Asp Ile Ile Ile Asp Tyr Glu Ile Val Gln His Ile Phe Glu Ile Phe 1185 1190 1195 1200 Arg Leu Thr Val Gln Met Arg Asn Ser Leu Ser Glu Leu Glu Asp Arg 1205 1210 1215 Asp Tyr Asp Arg Leu Ile Ser Pro Val Leu Asn Glu Asn Asn Ile Phe 1220 1225 1230 Tyr Asp Ser Ala Lys Ala Gly Asp Ala Leu Pro Lys Asp Ala Asp Ala 1235 1240 1245 Asn Gly Ala Tyr Cys Ile Ala Leu Lys Gly Leu Tyr Glu Ile Lys Gln 1250 1255 1260 Ile Thr Glu Asn Trp Lys Glu Asp Gly Lys Phe Ser Arg Asp Lys Leu 1265 1270 1275 1280 Lys Ile Ser Asn Lys Asp Trp Phe Asp Phe Ile Gln Asn Lys Arg Tyr 1285 1290 1295 Leu Ala Ala Ala Leu Glu Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly 1300 1305 1310 Gln Ala Lys Lys Lys Lys Ser Thr Pro Pro Pro Pro Pro Pro Leu Arg Ser 1315 1320 1325 Gly Cys 1330 <210> 2 <211> 3993 <212> DNA <213> Artificial Sequence <220> <223> mgCas12a-1 <400> 2 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgaacaa tggcaccaac 120 aatttccaga actttatcgg aattagcagt ctgcaaaaga ctctccggaa tgcccttata 180 cccaccgaga caacccagca gttcatcgtg aaaaacggga ttatcaagga agacgagctg 240 cgcgg cgaaa atcggcaaat tttgaaagat ataatggacg attattaccg cggttttatc 300 tctgagactc tgagctccat tgacgatatc gactggacct cactcttcga aaagatggag 360 attcagctta aaaacggcga taataaggac acactgataa aagaacaggc tgagaagcgg 420 aaagccatct ataagaaatt tgcagatgac gatcgcttca agaacatgtt tagcgccaaa 480 ttgattagtg acatcctgcc ggaattcgtt attcacaata acaattactc tgctagcgag 540 aaggaagaga aaacccaagt cataaagctc ttttcccggt tcgccacttc atttaaagat 600 tatttcaaga accgcgcaaa ttgctttagc gccgacgata tcagttctag ctcctgtcat 660 cggattgtga acgacaatgc tgaaatcttc ttttcaaacg cccttgtata ccgccggatt 720 gtgaaaaatc tgagcaacga tgacataaat aagatcagtg gagatattaa agactctttg 780 aaggagatga gcctggaaga gatctattcc tacgaaaaat atggggagtt cattacccag 840 gaaggcatat cattttacaa cgatatctgc ggtaaggtta atagcttcat gaacctctat 900 tgtcagaaaa ataaggagaa caaaaatctt tacaagctgc gcaaattgca caagcaaatt 960 ctgtgcatcg cagacacaag ttatgaagtc ccttacaaat ttgagtctga tgaagaggtg 1020 tatcagagcg taaacggctt cctcgacaat atttcctcaa agcatatagt ggaacggctt 1080 cgcaaaatcg gagataacta c aatgggtat aacctggaca agatttacat cgttagcaaa 1140 ttttatgaga gtgtctctca gaagacctac cgggattggg aaactattaa taccgccttg 1200 gagatacact ataacaatat cctgcccggc aacggtaaaa gcaaggctga caaagtgaag 1260 aaagccgtaa agaatgatct ccaaaaatcc attacagaaa tcaacgagct tgtgtcaaat 1320 tacaagctgt gtccggacga taacattaaa gcagaaacct atatacatga gatcagccac 1380 attttgaata acttcgaagc ccaggagctg aagtacaatc cagaaatcca tctcgttgag 1440 agtgaactta aagcttctga gctgaagaac gtcttggacg tgattatgaa tgcctttcac 1500 tggtgcagcg tattcatgac tgaagagctg gtggataaag acaacaattt ttatgcagaa 1560 ctcgaggaaa tatacgatga gatctatacc gttatttccc tttacaacct ggtccgcaat 1620 tatgtgacac agaagcccta ctcaaccaaa aagatcaaat tgaacttcgg cattccgact 1680 ctggccgacg gatggagcaa gagtaaagaa tattctaata acgctataat cctcatgcgg 1740 gataatcttt actatctggg gatttttaac gccaagaata aacctgacaa gaaaatcatt 1800 gagggcaaca ccagcgaaaa taagggtgat tacaaaaaga tgatatataa cttgctgccc 1860 ggcccgaata aaatgatccc aaaggtattc ctctcctcaa aaacaggagt ggagacctac 1920 aagcccagcg catatattct tgaaggg tac aaacaaaaca agcatctgaa aagttctaag 1980 gactttgata tcactttctg tcacgacttg attgattatt ttaaaaattg catagccatc 2040 catccggagt ggaagaactt cggctttgac ttcagcgata cctccacata cgaagacatt 2100 tcaggttttt atcgcgaggt tgaactgcag ggctacaaaa tcgattggac ctatattagc 2160 gagaaggaca tagatctcct tcaggaaaaa ggacaactgt acttgttcca gatctataat 2220 aaggacttta gtaaaaagtc tactgggaac gataatctgc acaccatgta cctcaaaaac 2280 cttttcagcg aggaaaatct gaaggacatt gtcttgaaac tgaacggcga ggctgaaatc 2340 tttttccgga agtcctcaat taaaaatcct ataatccata agaaaggtag cattctcgtg 2400 aaccgcacat atgaggccga agagaaggat cagtttggca atatccaaat tgtacggaaa 2460 accatacccg aaaacatcta ccaggagctt tataagtact tcaatgacaa aagtgataag 2520 gaactgtctg acgaggcagc caaattgaag aacgtggttg gacaccatga agctgccact 2580 aatattgtca aagattatcg ctacacctat gacaagtact ttctgcacat gccgatcaca 2640 attaacttca aagcaaataa gaccagcttt ataaacgatc ggattctcca gtatattgcc 2700 aaagagaaga atcttcatgt gatcgggatt gaccgcggcg aacggaacct gatatacgta 2760 tccgtgatcg atacttgtgg taatattgtt ga gcaaaaat cattcaacat cgtcaatggc 2820 tatgactacc agattaagtt gaaacagcaa gaaggagctc gccagatagc ccggaaggag 2880 tggaaggaaa tcgggaaaat taaggagatc aaagaaggct atctgagcct cgtgattcac 2940 gagataagta agatggtaat caaatacaac gcaattatcg ccatggaaga tctttcttat 3000 ggttttaaga aaggccgctt caaggtggag cggcaagttt accagaaatt tgaaaccatg 3060 ctgattaata agttgaacta tctggtcttc aaagacataa gcatcacaga gaatggaggg 3120 ctccttaagg gctaccagct gacctatatt ccagataaat tgaagaacgt gggtcatcaa 3180 tgcggctgta tcttttacgt acccgctgcc tatacttcca aaattgaccc gaccacagga 3240 ttcgtgaata tatttaagtt caaagatctg accgttgacg caaagcgcga atttatcaaa 3300 aagttcgatt caattcggta cgacagcgag aaaaagctct tttgcttcac ttttgattat 3360 aacaatttca tcacccagaa cacagtcatg agtaaatcta gctggtccgt gtacacctat 3420 ggggtacgca ttaagcggcg ctttgtgaat ggccggttct caaacgaaag cgacactata 3480 gatatcacca aagacatgga gaagacactt gaaatgaccg atattaattg gcgcgacggt 3540 cacgatctgc ggcaggacat cattgattac gagatagttc aacatatctt tgaaattttc 3600 cgcttgactg tccagatgcg gaacagtctg tctgagct cg aagaccgcga ttatgaccgg 3660 cttatcagcc ctgtgctgaa tgagaacaat attttttacg attccgccaa agctggcgac 3720 gccttgccca aggatgcaga cgccaacgga gcttattgta tagccctgaa agggctctac 3780 gaaatcaagc agattaccga gaattggaaa gaagatggca agttctcacg cgacaaactt 3840 aagatcagca acaaagattg gtttgacttc attcaaaata agcggtatct ggcggccgca 3900 ctcgagaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtcgaca 3960 ccaccaccac caccactgag atccggctgc taa 3993 <210> 3 <211 > 27 <212> DNA <213> Artificial Sequence <220> <223> myc-NLS <400> 3 ccggcagcca agagagtcaa actcgac 27 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223 > myc-NLS <400> 4 Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5 <210> 5 <211> 130 <212> DNA <213> Artificial Sequence <220> <223> Core region <400> 5 agccaagaga gtcaaactcg acggcggctc cccagcggca aaaagggtga aactagacgg 60 gggtagcccc gccgcgaagc gtgtaaagct ggatggagga tcgcctgcgg ctaagcgagt 120 caaattagac 130 <210> 6 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> primer extension_for ward <400> 6 ttgacttcat tcaaaataag cggtatctgg gcggctcccc tgctgctaaa cgtgttaagc 60 ttgatggggg tagcccggca gccaagagag tcaaactcg 99 <210> 7 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> extension primer_reverse <400> 7 agccggatct cagtggtggt ggtggtggtg gctgccgcta gcatccaatt tgacgcgctt 60 tgcagccggt gacccaccgt ctaatttgac tcgcttagcc 100 <210> 8 <211> 1379 <212> PRT <213> Artificial Sequence <220> <223> mgCas12a-1-6XNLS <400> 8 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Asn Asn Gly Thr Asn Asn Phe Gln Asn Phe Ile Gly Ile 35 40 45 Ser Ser Leu Gln Lys Thr Leu Arg Asn Ala Leu Ile Pro Thr Glu Thr 50 55 60 Thr Gln Gln Phe Ile Val Lys Asn Gly Ile Ile Lys Glu Asp Glu Leu 65 70 75 80 Arg Gly Glu Asn Arg Gln Ile Leu Lys Asp Ile Met Asp Asp Tyr Tyr 85 90 95 Arg Gly Phe Ile Ser Glu Thr Leu Ser Ser Ile Asp Asp Ile Asp Trp 100 105 110 Thr Ser Leu Phe Glu Lys Met Glu Ile Gln Leu Lys Asn Gly Asp Asn 115 120 125 Lys Asp Thr Leu Ile Lys Glu Gln Ala Glu Lys Arg Lys Ala Ile Tyr 130 135 140 Lys Lys Phe Ala Asp Asp Asp Arg Phe Lys Asn Met Phe Ser Ala Lys 145 150 155 160 Leu Ile Ser Asp Ile Leu Pro Glu Phe Val Ile His Asn Asn Asn Tyr 165 170 175 Ser Ala Ser Glu Lys Glu Glu Lys Thr Gln Val Ile Lys Leu Phe Ser 180 185 190 Arg Phe Ala Thr Ser Phe Lys Asp Tyr Phe Lys Asn Arg Ala Asn Cys 195 200 205 Phe Ser Ala Asp Asp Ile Ser Ser Ser Ser Cys His Arg Ile Val Asn 210 215 220 Asp Asn Ala Glu Ile Phe Phe Ser Asn Ala Leu Val Tyr Arg Arg Ile 225 230 235 240 Val Lys Asn Leu Ser Asn Asp Asp Ile Asn Lys Ile Ser Gly Asp Ile 245 250 255 Lys Asp Ser Leu Lys Glu Met Ser Leu Glu Glu Ile Tyr Ser Tyr Glu 260 265 270 Lys Tyr Gly Glu Phe Ile Thr Gln Glu Gly Ile Ser Phe Tyr Asn Asp 275 280 285 Ile Cys Gly Lys Val Asn Ser Phe Met Asn Leu Tyr Cys Gln Lys Asn 290 295 300 Lys Glu Asn Lys Asn Leu Tyr Lys Leu Arg Lys Leu His Lys Gln Ile 305 310 315 320 Leu Cys Ile Ala Asp Thr Ser Tyr Glu Val Pro Tyr Lys Phe Glu Ser 325 330 335 Asp Glu Glu Val Tyr Gln Ser Val Asn Gly Phe Leu Asp Asn Ile Ser 340 345 350 Ser Lys His Ile Val Glu Arg Leu Arg Lys Ile Gly Asp Asn Tyr Asn 355 360 365 Gly Tyr Asn Leu Asp Lys Ile Tyr Ile Val Ser Lys Phe Tyr Glu Ser 370 375 380 Val Ser Gln Lys Thr Tyr Arg Asp Trp Glu Thr Ile Asn Thr Ala Leu 385 390 395 400 Glu Ile His Tyr Asn Asn Ile Leu Pro Gly Asn Gly Lys Ser Lys Ala 405 410 415 Asp Lys Val Lys Lys Ala Val Lys Asn Asp Leu Gln Lys Ser Ile Thr 420 425 430 Glu Ile Asn Glu Leu Val Ser Asn Tyr Lys Leu Cys Pro Asp Asp Asn 435 440 445 Ile Lys Ala Glu Thr Tyr Ile His Glu Ile Ser His Ile Leu Asn Asn 450 455 460 Phe Glu Ala Gln Glu Leu Lys Tyr Asn Pro Glu Ile His Leu Val Glu 465 470 475 480 Ser Glu Leu Lys Ala Ser Glu Leu Lys Asn Val Leu Asp Val Ile Met 485 490 495 Asn Ala Phe His Trp Cys Ser Val Phe Met Thr Glu Glu Leu Val Asp 500 505 510 Lys Asp Asn Asn Phe Tyr Ala Glu Leu Glu Glu Ile Tyr Asp Glu Ile 515 520 525 Tyr Thr Val Ile Ser Leu Tyr Asn Leu Val Arg Asn Tyr Val Thr Gln 530 535 540 Lys Pro Tyr Ser Thr Lys Lys Ile Lys Leu Asn Phe Gly Ile Pro Thr 545 550 555 560 Leu Ala Asp Gly Trp Ser Lys Ser Lys Glu Tyr Ser Asn Asn Ala Ile 565 570 575 Ile Leu Met Arg Asp Asn Leu Tyr Tyr Leu Gly Ile Phe Asn Ala Lys 580 585 590 Asn Lys Pro Asp Lys Lys Ile Ile Glu Gly Asn Thr Ser Glu Asn Lys 595 600 605 Gly Asp Tyr Lys Lys Met Ile Tyr Asn Leu Leu Pro Gly Pro Asn Lys 610 615 620 Met Ile Pro Lys Val Phe Leu Ser Ser Lys Thr Gly Val Glu Thr Tyr 625 630 635 640 Lys Pro Ser Ala Tyr Ile Leu Glu Gly Tyr Lys Gln Asn Lys His Leu 645 650 655 Lys Ser Ser Lys Asp Phe Asp Ile Thr Phe Cys His Asp Leu Ile Asp 660 665 670 Tyr Phe Lys Asn Cys Ile Ala Ile His Pro Glu Trp Lys Asn Phe Gly 675 680 685 Phe Asp Phe Ser Asp Thr Ser Thr Ser Tyr Glu Asp Ile Ser Gly Phe Tyr 690 695 700 Arg Glu Val Glu Leu Gln Gly Tyr Lys Ile Asp Trp Thr Tyr Ile Ser 705 710 715 720 Glu Lys Asp Ile Asp Leu Leu Gln Glu Lys Gly Gln Leu Tyr Leu Phe 725 730 735 Gln Ile Tyr Asn Lys Asp Phe Ser Lys Lys Ser Thr Gly Asn Asp Asn 740 745 750 Leu His Thr Met Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Lys 755 760 765 Asp Ile Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys 770 775 780 Ser Ser Ile Lys Asn Pro Ile Ile His Lys Lys Gly Ser Ile Leu Val 785 790 795 800 Asn Arg Thr Tyr Glu Ala Glu Glu Lys Asp Gln Phe Gly Asn Ile Gln 805 810 815 Ile Val Arg Lys Thr Ile Pro Glu Asn Ile Tyr Gln Glu Leu Tyr Lys 820 825 830 Tyr Phe Asn Asp Lys Ser Asp Lys Glu Leu Ser Asp Glu Ala Ala Lys 835 840 845 Leu Lys Asn Val Val Gly His Glu Ala Ala Thr Asn Ile Val Lys 850 855 860 Asp Tyr Arg Tyr Thr Tyr Asp Lys Tyr Phe Leu His Met Pro Ile Thr 865 870 875 880 Ile Asn Phe Lys Ala Asn Lys Thr Ser Phe Ile Asn Asp Arg Ile Leu 885 890 895 Gln Tyr Ile Ala Lys Glu Lys Asn Leu His Val Ile Gly Ile Asp Arg 900 905 910 Gly Glu Arg Asn Leu Ile Tyr Val Ser Val Ile Asp Thr Cys Gly Asn 915 920 925 Ile Val Glu Gln Lys Ser Phe Asn Ile Val Asn Gly Tyr Asp Tyr Gln 930 935 940 Ile Lys Leu Lys Gln Gln Glu Gly Ala Arg Gln Ile Ala Arg Lys Glu 945 950 955 960 Trp Lys Glu Ile Gly Lys Ile Lys Glu Ile Lys Glu Gly Tyr Leu Ser 965 970 975 Leu Val Ile His Glu Ile Ser Lys Met Val Ile Lys Tyr Asn Ala Ile 980 985 990 Ile Ala Met Glu Asp Leu Ser Tyr Gly Phe Lys Lys Gly Arg Phe Lys 995 1000 1005 Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu Ile Asn Lys 1010 1015 1020 Leu Asn Tyr Leu Val Phe Lys Asp Ile Ser Ile Thr Glu Asn Gly Gly 1025 1030 1035 1040 Leu Leu Lys Gly Tyr Gln Leu Thr Tyr Asp Leu Thr Val Asp Ala Lys Arg Glu Phe Ile Lys Lys Phe Asp Ser 1090 1095 1100 Ile Arg Tyr Asp Ser Glu Lys Lys Leu Phe Cys Phe Thr Phe Asp Tyr 1105 1110 1115 1120 Asn Asn Phe Ile Thr Gln Asn Thr Val Met Ser Lys Ser Ser Trp Ser 1125 1130 1135 Val Tyr Thr Tyr Gly Val Arg Ile Lys Arg Arg Phe Val Asn Gly Arg 1140 1145 1150 Phe Ser Asn Glu Ser Asp Thr Ile Asp Ile Thr Lys Asp Met Glu Lys 1155 1160 1165 Thr Leu Glu Met Thr Asp Ile Asn Trp Arg Asp Gly His Asp Leu Arg 1170 1175 1180 Gln Asp Ile Ile Asp Tyr Glu Ile Val Gln His Ile Phe Glu Ile Phe 1185 1190 1195 1200 Arg Leu Thr Val Gln Met Arg Asn Ser Leu Ser Glu Leu Glu Asp Arg 1205 1210 1215 Asp Tyr Asp Arg Leu Ile Ser Pro Val Leu Asn Glu Asn Asn Ile Phe 1220 1225 1230 Tyr Asp Ser Ala Lys Ala Gly Asp Ala Leu Pro Lys Asp Ala Asp Ala 1235 1240 1245 Asn Gly Ala Tyr Cys Ile Ala Leu Lys Gly Leu Tyr Glu Ile Lys Gln 1250 1255 1260 Ile Thr Glu Asn Trp Lys Glu Asp Gly Lys Phe Ser Arg Asp Lys Leu 1265 1270 1275 1280 Lys Ile Ser Asn Lys Asp Trp Phe Asp Phe Ile Gln Asn Lys Arg Tyr 1285 1290 1295 Leu Gly Gly Ser Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Gly Ser 1300 1305 1310 Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Gly Ser Pro Ala Ala Lys 1315 1320 1325 Arg Val Lys Leu His His 1365 1370 1375 His His His <210> 9 <211> 4140 <212> DNA <213> Artificial Sequence <220> <223> mgCas12a-1-6XNLS <400> 9 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgaacaa tggcaccaac 120 aatttccaga actttatcgg aattagcagt ctgcaaaaga ctctccggaa tgcccttata 180 cccaccgaga caacccagca gttcatcgtg aaaaacggga ttatcaagga agacgagctg 240 cgcggcgaaa atcggcaaat tttgaaagat ataatggacg attattaccg cggttttatc 300 tctgagactc tgagctccat tgacgatatc gactggacct cactcttcga aaagatggag 360 attcagctta aaaacggcga taataaggac acactgataa aagaacaggc tgagaagcgg 420 aaagccatct ataagaaatt tgcagatgac gatcgcttca agaacatgtt tagcgccaaa 480 ttgattagtg acatcctgcc ggaattcgtt attcacaata acaattactc tgctagcgag 540 aaggaagaga aaacccaagt cataaagctc ttttcccggt tcgccacttc atttaaagat 600 tatttcaaga accgcgcaaa ttgctttagc gccgacgata tcagttctag ctcctgtcat 660 cggattgtga acgacaatgc tgaaatcttc ttttcaaacg cccttgtata ccgccggatt 720 gtgaaaaatc tgagcaacga tgacataaat aagatcagtg gagatattaa agactctttg 780 aaggagatga gcctggaaga gatctattcc tacgaaaaat atggggagtt cattacccag 840 gaaggcatat cattttacaa cgatatctgc ggtaaggtta atagcttcat gaacctctat 900 tgtcagaaaa ataaggagaa caaaaatctt tacaagctgc gcaaattgca caagcaaatt 960 ctgtgcatcg cagacacaag ttatgaagtc ccttacaaat ttgagtctga tgaagaggtg 1020 tatcagagcg taaacggctt cctcgacaat atttcctcaa agcatatagt ggaacggctt 1080 cgcaaaatcg gagataacta caatgggtat aacctggaca agatttacat cgttagcaaa 1140 ttttatgaga gtgtctctca gaagacctac cgggattggg aaactattaa taccgccttg 1200 gagatacact ataacaatat cctgcccggc aacggtaaaa gcaaggctga caaagtgaag 1260 aaagccgtaa agaatgatct ccaaaaatcc attacagaaa tcaacgagct tgtgtcaaat 1320 tacaagctgt gtccggacga taacattaaa gcagaaacct atatacatga gatcagccac 1380 attttgaata acttcgaagc ccaggagctg aagtacaatc cagaaatcca tctcgttgag 1440 agtgaactta aagcttctga gctgaagaac gtcttggacg tgattatgaa tgcctttcac 1500 tggtgcagcg tattcatgac tgaagagctg gtggataaag acaacaattt ttatgcagaa 1560 ctcgaggaaa tatacgatga gatctatacc gttatttccc tttacaacct ggtccgcaat 1620 tatgtgacac agaagcccta ctcaaccaaa aagatcaaat tgaacttcgg cattccgact 1680 ctggccgacg gatggagcaa gagtaaagaa tattctaata acgctataat cctcatgcgg 1740 ga taatcttt actatctggg gatttttaac gccaagaata aacctgacaa gaaaatcatt 1800 gagggcaaca ccagcgaaaa taagggtgat tacaaaaaga tgatatataa cttgctgccc 1860 ggcccgaata aaatgatccc aaaggtattc ctctcctcaa aaacaggagt ggagacctac 1920 aagcccagcg catatattct tgaagggtac aaacaaaaca agcatctgaa aagttctaag 1980 gactttgata tcactttctg tcacgacttg attgattatt ttaaaaattg catagccatc 2040 catccggagt ggaagaactt cggctttgac ttcagcgata cctccacata cgaagacatt 2100 tcaggttttt atcgcgaggt tgaactgcag ggctacaaaa tcgattggac ctatattagc 2160 gagaaggaca tagatctcct tcaggaaaaa ggacaactgt acttgttcca gatctataat 2220 aaggacttta gtaaaaagtc tactgggaac gataatctgc acaccatgta cctcaaaaac 2280 cttttcagcg aggaaaatct gaaggacatt gtcttgaaac tgaacggcga ggctgaaatc 2340 tttttccgga agtcctcaat taaaaatcct ataatccata agaaaggtag cattctcgtg 2400 aaccgcacat atgaggccga agagaaggat cagtttggca atatccaaat tgtacggaaa 2460 accatacccg aaaacatcta ccaggagctt tataagtact tcaatgacaa aagtgataag 2520 gaactgtctg acgaggcagc caaattgaag aacgtggttg gacaccatga agctgccact 2580 aatattgt ca aagattatcg ctacacctat gacaagtact ttctgcacat gccgatcaca 2640 attaacttca aagcaaataa gaccagcttt ataaacgatc ggattctcca gtatattgcc 2700 aaagagaaga atcttcatgt gatcgggatt gaccgcggcg aacggaacct gatatacgta 2760 tccgtgatcg atacttgtgg taatattgtt gagcaaaaat cattcaacat cgtcaatggc 2820 tatgactacc agattaagtt gaaacagcaa gaaggagctc gccagatagc ccggaaggag 2880 tggaaggaaa tcgggaaaat taaggagatc aaagaaggct atctgagcct cgtgattcac 2940 gagataagta agatggtaat caaatacaac gcaattatcg ccatggaaga tctttcttat 3000 ggttttaaga aaggccgctt caaggtggag cggcaagttt accagaaatt tgaaaccatg 3060 ctgattaata agttgaacta tctggtcttc aaagacataa gcatcacaga gaatggaggg 3120 ctccttaagg gctaccagct gacctatatt ccagataaat tgaagaacgt gggtcatcaa 3180 tgcggctgta tcttttacgt acccgctgcc tatacttcca aaattgaccc gaccacagga 3240 ttcgtgaata tatttaagtt caaagatctg accgttgacg caaagcgcga atttatcaaa 3300 aagttcgatt caattcggta cgacagcgag aaaaagctct tttgcttcac ttttgattat 3360 aacaatttca tcacccagaa cacagtcatg agtaaatcta gctggtccgt gtacacctat 3420 ggggtacgca tta agcggcg ctttgtgaat ggccggttct caaacgaaag cgacactata 3480 gatatcacca aagacatgga gaagacactt gaaatgaccg atattaattg gcgcgacggt 3540 cacgatctgc ggcaggacat cattgattac gagatagttc aacatatctt tgaaattttc 3600 cgcttgactg tccagatgcg gaacagtctg tctgagctcg aagaccgcga ttatgaccgg 3660 cttatcagcc ctgtgctgaa tgagaacaat attttttacg attccgccaa agctggcgac 3720 gccttgccca aggatgcaga cgccaacgga gcttattgta tagccctgaa agggctctac 3780 gaaatcaagc agattaccga gaattggaaa gaagatggca agttctcacg cgacaaactt 3840 aagatcagca acaaagattg gtttgacttc attcaaaata agcggtatct gggcggctcc 3900 cctgctgcta aacgtgttaa gcttgatggg ggtagcccgg cagccaagag agtcaaactc 3960 gacggcggct ccccagcggc aaaaagggtg aaactagacg ggggtagccc cgccgcgaag 4020 cgtgtaaagc tggatggagg atcgcctgcg gctaagcgag tcaaattaga cggtgggtca 4080 ccggctgcaa agcgcgtcaa attggatgct agcggcagcc accaccacca ccaccactga 4140 4140 <210> 10 <211> 164 <212> DNA <213> Artificial Sequence <220> <223> insert1 <400> 10 gcgccaaatt gattagtgac atcctgccgg aattcgttat tcacaataac aattactctg 60 ctag cgagaa ggaagagaaa acccaagtca taaagctctt ttcccggttc gccacttcat 120 ttaaagatta tttccgcaac cgcgcaaatt gctttagcgc cgac 164 <210> 11 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> insert1_primer_forward <400> 11 gcgccaaatt gattagtgac atcctgccgg aattcgttat tcacaataac aat 53 <210 > 12 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> insert1_primer_reverse <400> 12 gtcggcgcta aagcaatttg cgcggttgcg gAAataatct ttaaatgaag tggc 54 <210> 13 <211> 1083 <212> Artificial Sequence <220> <223> insert2 <400> 13 cgcaaccgcg caaattgctt tagcgccgac gatatcagtt ctagctcctg tcatcggatt 60 gtgaacgaca atgctgaaat cttcttttca aacgcccttg tataccgccg gattgtgaaa 120 aatctgagca acgatgacat aaataagatc agtggagata ttaaagactc tttgaaggag 180 atgagcctgg aagagatcta ttcctacgaa aaatatgggg agttcattac ccaggaaggc 240 atatcatttt acaacgatat ctgcggtaag gttaatagct tcatgaacct ctattgtcag 300 aaaaataagg agaacaaaaa tctttacaag ctgcgcaaat tgcacaagca aattctgtgc 360 atcgcagaca caagttatga agtcccttac aaatttgagt ctgatgaaga g gtgtatcag 420 agcgtaaacg gcttcctcga caatatttcc tcaaagcata tagtggaacg gcttcgcaaa 480 atcggagata actacaatgg gtataacctg gacaagattt acatcgttag caaattttat 540 gagagtgtct ctcagaagac ctaccgggat tgggaaacta ttaataccgc cttggagata 600 cactataaca atatcctgcc cggcaacggt aaaagcaagg ctgacaaagt gaagaaagcc 660 gtaaagaatg atctccaaaa atccattaca gaaatcaacg agcttgtgtc aaattacaag 720 ctgtgtccgg acgataacat taaagcagaa acctatatac atgagatcag ccacattttg 780 aataacttcg aagcccagga gctgaagtac aatccagaaa tccatctcgt tgagagtgaa 840 cttaaagctt ctgagctgaa gaacgtcttg gacgtgatta tgaatgcctt tcactggtgc 900 agcgtattca tgactgaaga gctggtggat aaagacaaca atttttatgc agaactcgag 960 gaaatatacg atgagatcta taccgttatt tccctttaca acctggtccg caattatgtg 1020 acacagaagc cctactcaac caaaaagatc aaattgaact tcggcattcc gactctggcc 1080 cgc 1083 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220 > <223> insert2_primer_forward <400> 14 cgcaaccgcg caaattgctt tagcgccgac 30 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> inser t2_primer_reverse <400> 15 gcgggccaga gtcggaatgc cgaagttcaa 30 <210> 16 <211> 281 <212> DNA <213> Artificial Sequence <220> <223> insert3 <400> 16 ttgaacttcg gcattccgac tctggcccgc ggatggagca agagtaaaga atattctaat 60 aacgctataa tcctcatgcg ggataatctt tactatctgg ggatttttaa cgccaagaat 120 aaacctgaca agaaaatcat tgagggcaac accagcgaaa ataagggtga ttacaaaaag 180 atgatatata acttgctgcc cggcccgaat aaaatgatcc caaaggtatt cctctcctca 240 aaaacaggag tggagaccta caagcccagc gcatatattc t 281 <210> 17 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> insert3_primer_forward <400> 17 ttgaacttcg gcattccgac tctggcccgc ggatggagca agagtaaaga 50 <210> 18 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> insert3_primer_reverse <400> 18 agaatatatg cgctgggctt gtaggtctcc actcctgttt ttgaggagag 50 <210> 12 <210> PRT <213> Artificial Sequence <220> <223> opmgCas12a-1-6XNLS <400> 19 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Asn Asn Gly Thr Asn Asn Phe Gln Asn Phe Ile Gly Ile 35 40 45 Ser Ser Leu Gln Lys Thr Leu Arg Asn Ala Leu Ile Pro Thr Glu Thr 50 55 60 Thr Gln Gln Phe Ile Val Lys Asn Gly Ile Ile Lys Glu Asp Glu Leu 65 70 75 80 Arg Gly Glu Asn Arg Gln Ile Leu Lys Asp Ile Met Asp Asp Tyr Tyr 85 90 95 Arg Gly Phe Ile Ser Glu Thr Leu Ser Ser Ile Asp Asp Ile Asp Trp 100 105 110 Thr Ser Leu Phe Glu Lys Met Glu Ile Gln Leu Lys Asn Gly Asp Asn 115 120 125 Lys Asp Thr Leu Ile Lys Glu Gln Ala Glu Lys Arg Lys Ala Ile Tyr 130 135 140 Lys Lys Phe Ala Asp Asp Asp Arg Phe Lys Asn Met Phe Ser Ala Lys 145 150 155 160 Leu Ile Ser Asp Ile Leu Pro Glu Phe Val Ile His Asn Asn Asn Tyr 165 170 175 Ser Ala Ser Glu Lys Glu Glu Lys Thr Gln Val Ile Lys Leu Phe Ser 180 185 190 Arg Phe Ala Thr Ser Phe Lys Asp Tyr Phe Arg Asn Arg Ala Asn Cys 195 200 205 Phe Ser Ala Asp Asp Ile Ser Ser Ser Ser Cys His Arg Ile Val Asn 210 215 220 Asp Asn Ala Glu Ile Phe Phe Ser Asn Ala Leu Val Tyr Arg Arg Ile 225 230 235 240 Val Lys Asn Leu Ser Asn Asp Asp Ile Asn Lys Ile Ser Gly Asp Ile 245 250 255 Lys Asp Ser Leu Lys Glu Met Ser Leu Glu Glu Ile Tyr Ser Tyr Glu 260 265 270 Lys Tyr Gly Glu Phe Ile Thr Gln Glu Gly Ile Ser Phe Tyr Asn Asp 275 280 285 Ile Cys Gly Lys Val Asn Ser Phe Met Asn Leu Tyr Cys Gln Lys Asn 290 295 300 Lys Glu Asn Lys Asn Leu Tyr Lys Leu Arg Lys Leu His Lys Gln Ile 305 310 315 320 Leu Cys Ile Ala Asp Thr Ser Tyr Glu Val Pro Tyr Lys Phe Glu Ser 325 330 335 Asp Glu Glu Val Tyr Gln Ser Val Asn Gly Phe Leu Asp Asn Ile Ser 340 345 350 Ser Lys His Ile Val Glu Arg Leu Arg Lys Ile Gly Asp Asn Tyr Asn 355 360 365 Gly Tyr Asn Leu Asp Lys Ile Tyr Ile Val Ser Lys Phe Tyr Glu Ser 370 375 380 Val Ser Gln Lys Thr Tyr Arg Asp Trp Glu Thr Ile Asn Thr Ala Leu 385 390 395 400 Glu Ile His Tyr Asn Asn Ile Leu Pro Gly Asn Gly Lys Ser Lys Ala 405 410 415 Asp Lys Val Lys Lys Ala Val Lys Asn Asp Leu Gln Lys Ser Ile Thr 420 425 430 Glu Ile Asn Glu Leu Val Ser Asn Tyr Lys Leu Cys Pro Asp Asp Asn 435 440 445 Ile Lys Ala Glu Thr Tyr Ile His Glu Ile Ser His Ile Leu Asn Asn 450 455 460 Phe Glu Ala Gln Glu Leu Lys Tyr Asn Pro Glu Ile His Leu Val Glu 465 470 475 480 Ser Glu Leu Lys Ala Ser Glu Leu Lys Asn Val Leu Asp Val Ile Met 485 490 495 Asn Ala Phe His Trp Cys Ser Val Phe Met Thr Glu Glu Leu Val Asp 500 505 510 Lys Asp Asn Asn Phe Tyr Ala Glu Leu Glu Glu Ile Tyr Asp Glu Ile 515 520 525 Tyr Thr Val Ile Ser Leu Tyr Asn Leu Val Arg Asn Tyr Val Thr Gln 530 535 540 Lys Pro Tyr Ser Thr Lys Lys Ile Lys Leu Asn Phe Gly Ile Pro Thr 545 550 555 560 Leu Ala Arg Gly Trp Ser Lys Ser Lys Glu Tyr Ser Asn Asn Ala Ile 565 570 575 Ile Leu Met Arg Asp Asn Leu Tyr Tyr Leu Gly Ile Phe Asn Ala Lys 580 585 590 Asn Lys Pro Asp Lys Lys Ile Ile Glu Gly Asn Thr Ser Glu Asn Lys 595 600 605 Gly Asp Tyr Lys Lys Met Ile Tyr Asn Leu Leu Pro Gly Pro Asn Lys 610 615 620 Met Ile Pro Lys Val Phe Leu Ser Ser Lys Thr Gly Val Glu Thr Tyr 625 630 635 640 Lys Pro Ser Ala Tyr Ile Leu Glu Gly Tyr Lys Gln Asn Lys His Leu 645 650 655 Lys Ser Ser Lys Asp Phe Asp Ile Thr Phe Cys His Asp Leu Ile Asp 660 665 670 Tyr Phe Lys Asn Cys Ile Ala Ile His Pro Glu Trp Lys Asn Phe Gly 675 680 685 Phe Asp Phe Ser Asp Thr Ser Thr Tyr Glu Asp Ile Ser Gly Phe Tyr 690 695 700 Arg Glu Val Glu Leu Gln Gly Tyr Lys Ile Asp Trp Thr Tyr Ile Ser 705 710 715 720 Glu Lys Asp Ile Asp Leu Leu Gln Glu Lys Gly Gln Leu Tyr Leu Phe 725 730 735 Gln Ile Tyr Asn Lys Asp Phe Ser Lys Lys Ser Thr Gly Asn Asp Asn 740 745 750 Leu His Thr Met Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Lys 755 760 765 Asp Ile Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys 770 775 780 Ser Ser Ile Lys Asn Pro Ile Ile His Lys Lys Gly Ser Ile Leu Val 785 790 795 800 Asn Arg Thr Tyr Glu Ala Glu Glu Lys Asp Gln Phe Gly Asn Ile Gln 805 810 815 Ile Val Arg Lys Thr Ile Pro Glu Asn Ile Tyr Gln Glu Leu Tyr Lys 820 825 830 Tyr Phe Asn Asp Lys Ser Asp Lys Glu Leu Ser Asp Glu Ala Ala Lys 835 840 845 Leu Lys Asn Val Val Gly His His Glu Ala Ala Thr Asn Ile Val Lys 850 855 860 Asp Tyr Arg Tyr Thr Tyr Asp Lys Tyr Phe Leu His Met Pro Ile Thr 865 870 875 880 Ile Asn Phe Lys Ala Asn Lys Thr Ser Phe Ile Asn Asp Arg Ile Leu 885 890 895 Gln Tyr Ile Ala Lys Glu Lys Asn Leu His Val Ile Gly Ile Asp Arg 900 905 910 Gly Glu Arg Asn Leu Ile Tyr Val Ser Val Ile Asp Thr Cys Gly Asn 915 920 925 Ile Val Glu Gln Lys Ser Phe Asn Ile Val Asn Gly Tyr Asp Tyr Gln 930 935 940 Ile Lys Leu Lys Gln Gln Glu Gly Ala Arg Gln Ile Ala Arg Lys Glu 945 950 955 960 Trp Lys Glu Ile Gly Lys Ile Lys Glu Ile Lys Glu Gly Tyr Leu Ser 965 970 975 Leu Val Ile His Glu Ile Ser Lys Met Val Ile Lys Tyr Asn Ala Ile 980 985 990 Ile Ala Met Glu Asp Leu Ser Tyr Gly Phe Lys Lys Gly Arg Phe Lys 995 1000 1005 Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu Ile Asn Lys 1010 1015 1020 Leu Asn Tyr Leu Val Phe Lys Asp Ile Ser Ile Thr Glu Asn Gly Gly 1025 1030 1035 1040 Leu Leu Lys Gly Tyr Gln Leu Thr Tyr Ile Pro Asp Lys Leu Lys Asn 1045 1050 1055 Val Gly His Gln Cys Gly Cys Ile Phe Tyr Val Pro Ala Ala Tyr Thr 1060 1065 1070 Ser Lys Ile Asp Pro Thr Thr Gly Phe Val Asn Ile Phe Lys Phe Lys 1075 1080 1085 Asp Leu Thr Val Asp Ala Lys Arg Glu Phe Ile Lys Lys Phe Asp Ser 1090 1095 1100 Ile Arg Tyr Asp Ser Glu Lys Lys Leu Phe Cys Phe Thr Phe Asp Tyr 1105 1110 1115 1120 Asn Asn Phe Ile Thr Gln Asn Thr Val Met Ser Lys Ser Ser Trp Ser 1125 1130 1135 Val Tyr Thr Tyr Gly Val Arg Ile Lys Arg Arg Phe Val Asn Gly Arg 1140 1145 1150 Phe Ser Asn Glu Ser Asp Thr Ile Asp Ile Thr Lys Asp Met Glu Lys 1155 1160 1165 Thr Leu Glu Met Thr Asp Ile Asn Trp Arg Asp Gly His Asp Leu Arg 1170 1175 1180 Gln Asp Ile Ile Asp Tyr Glu Ile Val Gln His Ile Phe Glu Ile Phe 1185 1190 1195 1200 Arg Leu Thr Val Gln Met Arg Asn Ser Leu Ser Glu Leu Glu Asp Arg 1205 1210 1215 Asp Tyr Asp Arg Leu Ile Ser Pro Val Leu Asn Glu Asn Asn Ile Phe 1220 1225 1230 Tyr Asp Ser Ala Lys Ala Gly Asp Ala Leu Pro Lys Asp Ala Asp Ala 1235 1240 1245 Asn Gly Ala Tyr Cys Ile Ala Leu Lys Gly Leu Tyr Glu Ile Lys Gln 1250 1255 1260 Ile Thr Glu Asn Trp Lys Glu Asp Gly Lys Phe Ser Arg Asp Lys Leu 1265 1270 1275 1280 Lys Ile Ser Asn Lys Asp Trp Phe Asp Phe Ile Gln Asn Lys Arg Tyr 1285 1290 1295 Leu Gly Gly Ser Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Gly Ser 1300 1305 1310 Pro Ala Ala Lys A rg Val Lys Leu Asp Gly Gly Ser Pro Ala Ala Lys 1315 1320 1325 Arg Val Lys Leu Asp Gly Gly Ser Pro Ala Ala Lys Arg Val Lys Leu 1330 1335 1340 Asp Gly Gly Ser Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Gly Ser 1345 1350 1355 1360 Pro Ala Ala Lys Arg Val Lys Leu Asp Ala Ser Gly Ser His His His 1365 1370 1375 His His His <210> 20 <211> 4140 <212> DNA <213> Artificial Sequence <220> <223> opmgCas12a -1-6XNLS <400> 20 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgaacaa tggcaccaac 120 aatttccaga actttatcgg aattagcagt ctgcaaaaga ctctccggaa tgcccttata 180 cccaccgaga caacccagca gttcatcgtg aaaaacggga ttatcaagga agacgagctg 240 cgcggcgaaa atcggcaaat tttgaaagat ataatggacg attattaccg cggttttatc 300 tctgagactc tgagctccat tgacgatatc gactggacct cactcttcga aaagatggag 360 attcagctta aaaacggcga taataaggac acactgataa aagaacaggc tgagaagcgg 420 aaagccatct ataagaaatt tgcagatgac gatcgcttca agaacatgtt tagcgccaaa 480 ttgattagtg acatcctgcc ggaattc gtt attcacaata acaattactc tgctagcgag 540 aaggaagaga aaacccaagt cataaagctc ttttcccggt tcgccacttc atttaaagat 600 tatttccgca accgcgcaaa ttgctttagc gccgacgata tcagttctag ctcctgtcat 660 cggattgtga acgacaatgc tgaaatcttc ttttcaaacg cccttgtata ccgccggatt 720 gtgaaaaatc tgagcaacga tgacataaat aagatcagtg gagatattaa agactctttg 780 aaggagatga gcctggaaga gatctattcc tacgaaaaat atggggagtt cattacccag 840 gaaggcatat cattttacaa cgatatctgc ggtaaggtta atagcttcat gaacctctat 900 tgtcagaaaa ataaggagaa caaaaatctt tacaagctgc gcaaattgca caagcaaatt 960 ctgtgcatcg cagacacaag ttatgaagtc ccttacaaat ttgagtctga tgaagaggtg 1020 tatcagagcg taaacggctt cctcgacaat atttcctcaa agcatatagt ggaacggctt 1080 cgcaaaatcg gagataacta caatgggtat aacctggaca agatttacat cgttagcaaa 1140 ttttatgaga gtgtctctca gaagacctac cgggattggg aaactattaa taccgccttg 1200 gagatacact ataacaatat cctgcccggc aacggtaaaa gcaaggctga caaagtgaag 1260 aaagccgtaa agaatgatct ccaaaaatcc attacagaaa tcaacgagct tgtgtcaaat 1320 tacaagctgt gtccggacga taacattaaa gcagaaacct atatacatga gatcagccac 1380 attttgaata acttcgaagc ccaggagctg aagtacaatc cagaaatcca tctcgttgag 1440 agtgaactta aagcttctga gctgaagaac gtcttggacg tgattatgaa tgcctttcac 1500 tggtgcagcg tattcatgac tgaagagctg gtggataaag acaacaattt ttatgcagaa 1560 ctcgaggaaa tatacgatga gatctatacc gttatttccc tttacaacct ggtccgcaat 1620 tatgtgacac agaagcccta ctcaaccaaa aagatcaaat tgaacttcgg cattccgact 1680 ctggcccgcg gatggagcaa gagtaaagaa tattctaata acgctataat cctcatgcgg 1740 gataatcttt actatctggg gatttttaac gccaagaata aacctgacaa gaaaatcatt 1800 gagggcaaca ccagcgaaaa taagggtgat tacaaaaaga tgatatataa cttgctgccc 1860 ggcccgaata aaatgatccc aaaggtattc ctctcctcaa aaacaggagt ggagacctac 1920 aagcccagcg catatattct tgaagggtac aaacaaaaca agcatctgaa aagttctaag 1980 gactttgata tcactttctg tcacgacttg attgattatt ttaaaaattg catagccatc 2040 catccggagt ggaagaactt cggctttgac ttcagcgata cctccacata cgaagacatt 2100 tcaggttttt atcgcgaggt tgaactgcag ggctacaaaa tcgattggac ctatattagc 2160 gagaaggaca tagatctcct tcaggaaaaa ggacaactgt acttg ttcca gatctataat 2220 aaggacttta gtaaaaagtc tactgggaac gataatctgc acaccatgta cctcaaaaac 2280 cttttcagcg aggaaaatct gaaggacatt gtcttgaaac tgaacggcga ggctgaaatc 2340 tttttccgga agtcctcaat taaaaatcct ataatccata agaaaggtag cattctcgtg 2400 aaccgcacat atgaggccga agagaaggat cagtttggca atatccaaat tgtacggaaa 2460 accatacccg aaaacatcta ccaggagctt tataagtact tcaatgacaa aagtgataag 2520 gaactgtctg acgaggcagc caaattgaag aacgtggttg gacaccatga agctgccact 2580 aatattgtca aagattatcg ctacacctat gacaagtact ttctgcacat gccgatcaca 2640 attaacttca aagcaaataa gaccagcttt ataaacgatc ggattctcca gtatattgcc 2700 aaagagaaga atcttcatgt gatcgggatt gaccgcggcg aacggaacct gatatacgta 2760 tccgtgatcg atacttgtgg taatattgtt gagcaaaaat cattcaacat cgtcaatggc 2820 tatgactacc agattaagtt gaaacagcaa gaaggagctc gccagatagc ccggaaggag 2880 tggaaggaaa tcgggaaaat taaggagatc aaagaaggct atctgagcct cgtgattcac 2940 gagataagta agatggtaat caaatacaac gcaattatcg ccatggaaga tctttcttat 3000 ggttttaaga aaggccgctt caaggtggag cggcaagttt accagaaatt tgaaaccatg 3060 ctgattaata agttgaacta tctggtcttc aaagacataa gcatcacaga gaatggaggg 3120 ctccttaagg gctaccagct gacctatatt ccagataaat tgaagaacgt gggtcatcaa 3180 tgcggctgta tcttttacgt acccgctgcc tatacttcca aaattgaccc gaccacagga 3240 ttcgtgaata tatttaagtt caaagatctg accgttgacg caaagcgcga atttatcaaa 3300 aagttcgatt caattcggta cgacagcgag aaaaagctct tttgcttcac ttttgattat 3360 aacaatttca tcacccagaa cacagtcatg agtaaatcta gctggtccgt gtacacctat 3420 ggggtacgca ttaagcggcg ctttgtgaat ggccggttct caaacgaaag cgacactata 3480 gatatcacca aagacatgga gaagacactt gaaatgaccg atattaattg gcgcgacggt 3540 cacgatctgc ggcaggacat cattgattac gagatagttc aacatatctt tgaaattttc 3600 cgcttgactg tccagatgcg gaacagtctg tctgagctcg aagaccgcga ttatgaccgg 3660 cttatcagcc ctgtgctgaa tgagaacaat attttttacg attccgccaa agctggcgac 3720 gccttgccca aggatgcaga cgccaacgga gcttattgta tagccctgaa agggctctac 3780 gaaatcaagc agattaccga gaattggaaa gaagatggca agttctcacg cgacaaactt 3840 aagatcagca acaaagattg gtttgacttc attcaaaata agcggtatct gggcgg ctcc 3900 cctgctgcta aacgtgttaa gcttgatggg ggtagcccgg cagccaagag agtcaaactc 3960 gacggcggct ccccagcggc aaaaagggtg aaactagacg ggggtagccc cgccgcgaag 4020 cgtgtaaagc tggatggagg atcgcctgcg gctaagcgag tcaaattaga cggtgggtca 4080 ccggctgcaa agcgcgtcaa attggatgct agcggcagcc accaccacca ccaccactga 4140 4140 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220 > <223> HPRT1<400> 21 ggttaaagat ggttaaatga t 21

Claims (11)

Cas12a 단백질에 있어서,
상기 단백질은 서열번호 4로 이루어진 myc-NLS(nuclear localization sequences)가 서열번호 1의 아미노산 서열로 이루어진 단백질의 C-말단에 연결된 것이고,
상기 myc-NLS는 6개가 반복연결된 것이고,
상기 서열번호 1의 아미노산 서열 중 169번째 위치의 라이신(Lys)이 아르기닌(Arg)으로 치환된 것이고,
상기 서열번호 1의 아미노산 서열 중 529번째 위치의 아스파르트산(Asp)이 아르기닌(Arg)으로 치환된 것인 Cas12a 단백질.
In the Cas12a protein,
In the protein, myc-NLS (nuclear localization sequences) consisting of SEQ ID NO: 4 is linked to the C-terminus of the protein consisting of the amino acid sequence of SEQ ID NO: 1,
The myc-NLS is a repeat connection of 6,
Lysine (Lys) at position 169 in the amino acid sequence of SEQ ID NO: 1 is substituted with arginine (Arg),
A Cas12a protein wherein aspartic acid (Asp) at position 529 in the amino acid sequence of SEQ ID NO: 1 is substituted with arginine (Arg).
 제 1 항에 있어서, 상기 연결은 Gly-Gly-Ser 아미노산 서열에 의한 것인 Cas12a 단백질.
The Cas12a protein according to claim 1, wherein the connection is by the Gly-Gly-Ser amino acid sequence.
 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 제 1 항에 있어서, 상기 Cas12a 단백질은 서열번호 19의 아미노산 서열로 이루어진 것인 Cas12a 단백질.
The Cas12a protein according to claim 1, wherein the Cas12a protein consists of the amino acid sequence of SEQ ID NO: 19.
 제 1 항의 Cas12a 단백질 또는 이를 암호화하는 DNA; 및
표적 뉴클레오티드(target nucleotide) 서열과 혼성화 가능한 뉴클레오티드 서열을 포함하는 가이드 RNA 또는 이를 암호화하는 DNA
를 포함하는 유전체 편집용 조성물.
The Cas12a protein of claim 1 or DNA encoding the same; and
Guide RNA containing a nucleotide sequence hybridizable with a target nucleotide sequence or DNA encoding the same
Genome editing composition comprising a.
 제 9 항의 유전체 편집용 조성물을 분리된 세포 또는 유기체에 도입하는 단계를 포함하는 유전체 편집 방법.
A genome editing method comprising introducing the genome editing composition of claim 9 into a separated cell or organism.
 제 9 항의 유전체 편집용 조성물을 분리된 세포 또는 인간을 제외한 유기체에 도입하는 단계를 포함하는 형질 전환체의 제조 방법.A method for producing a transformant comprising the step of introducing the genome editing composition of claim 9 into isolated cells or organisms other than humans.
KR1020200121989A 2020-09-22 2020-09-22 Novel CRISPR Associated Protein and Use thereof KR102497690B1 (en)

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