KR20190093171A - A method for producing avian leukosis virus(alv)-resistant avian line using crispr/cas9 system - Google Patents
A method for producing avian leukosis virus(alv)-resistant avian line using crispr/cas9 system Download PDFInfo
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Abstract
Description
본 발명은 CRISPR/Cas9 시스템을 이용하여 제작한 닭 백혈병 바이러스(Avian leukosis virus, ALV) 저항성 조류 및 이의 제조방법에 관한 것이다. 보다 구체적으로는 CRISPR/Cas9 시스템을 통한 유전자 편집을 실시하여 닭의 chNHE1 유전자 내 Trp38, Thr37 및 Gln40 중에서 선택된 하나 이상의 부위를 특이적으로 변형시켜 제조한 ALV 하위그룹 J(Avian Leukosis Virus subgroup J)에 대한 저항성을 가진 ALV-J 저항성 조류 및 이의 제조방법에 관한 것이다.The present invention relates to avian leukosis virus (ALV) resistant birds prepared using the CRISPR / Cas9 system and a method for preparing the same. More specifically, gene editing through the CRISPR / Cas9 system was performed to ALV subgroup J, which was prepared by specifically modifying one or more sites selected from Trp38, Thr37, and Gln40 in the chNHE1 gene of chicken. The present invention relates to an ALV-J resistant bird and a method of manufacturing the same.
최근 3세대 유전자 가위인 CRISPR/Cas9 시스템을 이용한 유전자 편집(genome editing)이 생명공학 분야에서 새로운 혁신을 가져오고 있다. CRISPR/Cas9 시스템은 Cas9(CRISPR associated protein 9) 단백질과 guide RNA의 결합을 통해 표적 유전자를 효과적으로 절단하여 DNA 이중가닥 절단(double strand breaks, DSBs)을 일으킨다. 이중가닥절단이 형성되면, 잘린 DNA는 크게 비상동성 말단 접합(non-homologous end joining, NHEJ) 또는 상동 재조합(homologous recombination, HR)의 두 가지 경로에 의해 수선되는데, 이 때 표적 특이적인 돌연변이 및 유전자 변형이 일어나게 된다. 이와 같은 CRISPR/Cas9 시스템에 기반하여 표적화된 유전자 조절이 가능하다.Recently, genome editing using the CRISPR / Cas9 system, a third-generation gene shear, is bringing new innovations in biotechnology. The CRISPR / Cas9 system efficiently cleaves target genes through the combination of Cas9 (CRISPR associated protein 9) protein and guide RNA, resulting in DNA double strand breaks (DSBs). When double stranded cuts are made, the cut DNA is repaired by two major pathways: non-homologous end joining (NHEJ) or homologous recombination (HR), with target-specific mutations and genes. Deformation will occur. Targeted gene regulation is possible based on this CRISPR / Cas9 system.
닭 백혈병 바이러스는 숙주 수용체와 특이적으로 결합하여 질병을 전파하여, 국내 가금분야는 물론 전세계적으로 꾸준히 막대한 경제적 피해를 야기하고 있다. 닭 백혈병 바이러스(Avian Leukosis Virus, ALV)는 표면 당단백질의 종류에 따라 하위 그룹이 나뉘고, 각각의 그룹은 특정 숙주 수용체와 결합한다는 것이 알려져 있다. 특히, 닭 백혈병 바이러스 중 하위 그룹 J(Avian Leukosis Virus subgroup J, ALV-J)는 Na+/H+ exchange 1(NHE1)이라는 수용체와 특이적으로 결합한다.Chicken leukemia virus is specifically bound to host receptors to spread the disease, causing a steady economic damage in the domestic poultry field as well as worldwide. Chicken Avian Leukosis Virus (ALV) is known to be divided into subgroups according to the type of surface glycoprotein, and each group binds to a specific host receptor. In particular, Avian Leukosis Virus subgroup J (ALV-J) of chicken leukemia virus specifically binds a receptor called Na + / H + exchange 1 (NHE1).
이처럼 닭 백혈병 바이러스가 특이적으로 결합하는 숙주 수용체에 대한 정보는 기존의 연구를 통해 잘 알려져 있지만, 기술적 한계에 의해 숙주의 수용체 유전자 편집을 통한 저항성 획득 연구에 관해서는 관련 연구가 진행된 바가 거의 없다. As such, information on host receptors specifically binding to chicken leukemia virus is well known through previous studies. However, due to technical limitations, few studies have been conducted on the acquisition of resistance through editing of host genes.
이에, 본 발명자들은 상기 숙주 수용체의 변이를 통해 조류에서 ALV-J 바이러스의 침투를 억제할 수 있을 것으로 판단하고, CRISPR/Cas9을 사용하여 특정 유전자 염기서열을 인지한 후 그 부위에 염기서열 이중나선 결손을 유도하여 유전자 편집을 실시하였다. 나아가, 정확한 유전자 편집을 위해 단일가닥 올리고데옥시뉴클레오티드(single-stranded oligodeoxynucleotide, ssODN)를 CRISPR/Cas9 벡터와 함께 도입하였다. 그 결과, 유전자 내 특정 부위 또는 특정 아미노산의 결손만을 유도하여 효율적으로 chNHE1 유전자를 변형시켜 ALV-J에 저항성을 가진 조류모델을 제조할 수 있음을 확인하고 본 발명을 완성하였다.Accordingly, the present inventors determined that the mutation of the host receptor can inhibit the invasion of ALV-J virus in birds, and after recognizing a specific gene sequence using CRISPR / Cas9, a nucleotide double helix at the site Deletion was induced to perform gene editing. Furthermore, single-stranded oligodeoxynucleotides (ssODNs) were introduced with the CRISPR / Cas9 vector for accurate gene editing. As a result, it was confirmed that the algae model having resistance to ALV-J can be prepared by efficiently modifying the chNHE1 gene by inducing only a deletion of a specific region or a specific amino acid in the gene and completing the present invention.
본 발명의 목적은 제1 프로모터 및 이와 작동가능하게 연결된 chNHE1(chicken Na+/H+ exchange 1)의 gRNA(guide RNA); 및 제2 프로모터 및 이와 작동가능하게 연결된 Cas9(CRISPR associated protein 9)을 암호화하는 유전자를 포함하는 ALV-J(Avian Leukosis Virus-J, 닭 백혈병 바이러스 하위그룹 J) 저항성 조류모델 제작용 재조합 벡터를 제공하는 데에 있다.An object of the present invention is a guide RNA (gRNA) of a first promoter and chNHE1 (chicken Na + / H + exchange 1) operably linked thereto; And ALV-J (Avian Leukosis Virus-J, sub-group J) resistant avian model, comprising a second promoter and a gene encoding Cas9 (CRISPR associated protein 9) operably linked thereto. It's there.
또한, 본 발명의 목적은 본 발명에 따른 재조합 벡터 및 ssODN(single-stranded oligodeoxynucleotide, 단일 가닥 올리고데옥시뉴클레오티드)을 포함하는 것을 특징으로 하는 유전체 교정용 조성물을 제공하는 데에 있다.It is also an object of the present invention to provide a composition for genome calibration, comprising a recombinant vector and a single-stranded oligodeoxynucleotide (single-stranded oligodeoxynucleotide) according to the present invention.
또한, 본 발명의 목적은 본 발명에 따른 재조합 벡터 또는 유전체 교정용 조성물이 도입된 ALV-J 저항성 조류모델 제작용 형질전환 세포주를 제공하는 데에 있다.It is also an object of the present invention to provide a transformed cell line for preparing ALV-J resistant avian model into which a recombinant vector or genome calibration composition according to the present invention is introduced.
또한, 본 발명의 목적은 본 발명에 따른 재조합 벡터 또는 유전체 교정용 조성물을 제조하는 단계; 및 상기 재조합 벡터 또는 유전체 교정용 조성물을 조류 체세포에 도입하는 단계;를 포함하는 ALV-J 저항성 조류모델 제작용 형질전환 세포주의 제조방법 및 상기 형질전환 세포주를 이용한 ALV-J 저항성 조류모델의 제조방법을 제공하는 데에 있다.In addition, an object of the present invention is to prepare a composition for recombinant vector or genome calibration according to the present invention; And introducing the recombinant vector or genome corrective composition into an avian somatic cell. A method of preparing a transformed cell line for preparing an ALV-J resistant bird model, and a method of preparing an ALV-J resistant bird model using the transformed cell line. To provide.
상기와 같은 목적을 달성하기 위하여, 본 발명은 제1 프로모터 및 이와 작동가능하게 연결된 chNHE1(chicken Na+/H+ exchange 1)의 gRNA(guide RNA); 및 제2 프로모터 및 이와 작동가능하게 연결된 Cas9(CRISPR associated protein 9)을 암호화하는 유전자를 포함하는 ALV-J(Avian Leukosis Virus-J, 닭 백혈병 바이러스 하위그룹 J) 저항성 조류모델 제작용 재조합 벡터를 제공한다.In order to achieve the above object, the present invention provides a first promoter and gRNA (guide RNA) of chNHE1 (chicken Na + / H + exchange 1) operably linked thereto; And ALV-J (Avian Leukosis Virus-J, sub-group J) resistant avian model, comprising a second promoter and a gene encoding Cas9 (CRISPR associated protein 9) operably linked thereto. do.
또한, 본 발명은 본 발명에 따른 재조합 벡터 및 ssODN(single-stranded oligodeoxynucleotide, 단일 가닥 올리고데옥시뉴클레오티드)을 포함하는 것을 특징으로 하는 유전체 교정용 조성물을 제공한다.In another aspect, the present invention provides a composition for genome correction, comprising a recombinant vector according to the invention and ssODN (single-stranded oligodeoxynucleotide, single stranded oligodeoxynucleotide).
또한, 본 발명은 본 발명에 따른 재조합 벡터 또는 유전체 교정용 조성물이 도입된 ALV-J 저항성 조류모델 제작용 형질전환 세포주를 제공한다.The present invention also provides a transformed cell line for preparing an ALV-J resistant avian model into which a recombinant vector or genome calibration composition according to the present invention is introduced.
또한, 본 발명은 본 발명에 따른 재조합 벡터 또는 유전체 교정용 조성물을 제조하는 단계; 및 상기 재조합 벡터 또는 유전체 교정용 조성물을 조류 체세포에 도입하는 단계;를 포함하는 ALV-J 저항성 조류모델 제작용 형질전환 세포주의 제조방법을 제공한다.In addition, the present invention comprises the steps of preparing a composition for recombinant vector or genome calibration according to the present invention; It provides a method for producing a transformed cell line for producing ALV-J resistant avian model comprising the step of introducing the recombinant vector or genomic correction composition to a somatic cell.
또한, 본 발명은 본 발명에 따른 형질전환 세포주를 수용체 조류의 배아에 이식하는 단계; 를 포함하는 ALV-J 저항성 조류모델의 제조방법을 제공한다.In addition, the present invention comprises the steps of transplanting the transformed cell line according to the invention into the embryo of the receptor bird; It provides a method of manufacturing an ALV-J resistant bird model comprising a.
본 발명에 따른 닭 백혈병 바이러스(Avian Leukosis Virus, ALV) 저항성 조류는 바이러스 감염의 위험이 없어 축산품으로서의 가치가 높다. 또한, 본 발명의 유전자 편집 방법은 바이러스와 수용체간의 결합을 억제할 수 있는 특정 유전자형의 발굴 및 세포주를 활용한 바이러스-수용체 결합 연구에 활용될 수 있고, 나아가 닭 백혈병 바이러스에 저항성을 가진 조류를 생산하는 시스템을 확립할 수 있을 것으로 기대된다.Chicken Avian Leukosis Virus (ALV) resistant birds according to the present invention have a high value as a livestock product because there is no risk of viral infection. In addition, the gene editing method of the present invention can be utilized for the discovery of a specific genotype capable of inhibiting the binding between the virus and the receptor, and the virus-receptor binding research utilizing the cell line, and furthermore, the production of algae resistant to chicken leukemia virus. It is expected to be able to establish a system.
도 1은 본 발명에 따른 CRISPR/Cas9 벡터의 개열지도 및 본 발명에 따른 ALV-J 저항성 세포주 확립 과정을 도식적으로 나타낸 것이다. CRISPR/Cas9 벡터는 Cas9 단백질 코딩 서열, chNHE1 타겟 gRNA(guide RNA), 네오마이신/퓨로마이신 저항성 유전자를 포함하며 DF-1 세포에 도입하여 형질전환시켰다. U6: 인간 U6 프로모터(human U6 promoter), CBh: 닭 베타-액틴 프로모터(chicken beta-actin short promoter), Amp: 엠피실린(ampicillin), Tk: 티미딘 키나아제 프로모터(thymidine kinase promoter), Neo: 네오마이신 저항성 유전자, Puro: 퓨로마이신 저항성 유전자, LTR: 긴 말단 반복(long terminal repeat), GFP: 녹색 형광 단백질(green fluorescent protein).
도 2는 DF-1 섬유아세포에서 CRISPR/Cas9을 이용한 chNHE1 유전자 내 변형을 나타내는 도이다. 도 2의 A는 chNHE1 유전자의 구조 및 NHE1#3, NHE1#4 CRISPR/Cas9 벡터의 인식 부위를 나타낸 도이며, 파란 선은 guide RNA 인식 부위를, 빨간 선은 PAM(protospacer-adjacent motif) 서열을 의미한다(Scale bar = 1 kb). 도 2의 B는 NHE1#3-neo (N3N), NHE1#4-neo (N4N), NHE1#3-puro (N3P) 및 NHE1#4-puro (N4P)로 감염시킨 DF-1 세포의 T7E1 분석 결과를 나타낸 도이다(숫자는 indel 효율을 의미). 도 2의 C는 감염된 DF-1 세포의 서열 분석 결과를 나타낸 도이며, 주황색 화살표는 chNHE1 유전자의 38번째 트립토판 잔기(Trp38)를, 빨간색 화살표는 CRISPR/Cas9 벡터의 타겟 부위를 의미한다.
도 3은 chNHE1 유전자 변형이 확인된 DF-1 클론에서 바이러스 감염 확인 및 유세포 분석(flow cytometry)을 실시한 결과를 나타낸 도이다.
도 4는 DF-1 섬유아세포에서 ssODN(single-stranded oligodeoxynucleotide)을 사용한 chNHE1 유전자 편집을 나타낸 도이다.
도 5는 chNHE1 유전자의 Trp38을 타겟팅한 DF-1 클론의 바이러스 감염 및 유세포 분석 결과를 나타낸 도이다. 도 5의 A는 NHE1#3-puro + ssODN (N3Pss)로 감염된 DF-1로부터 얻은 chNHE1 유전자의 Trp38을 타겟팅한 DF-1 클론의 유세포 분석 결과를 나타낸 도이며, 총 9개의 DF-1 클론이 관찰되었다(Scale bar = 200μm). 도 5의 B는 chNHE1 조기 종결 코돈을 생성하는 indel 유도된 DF-1 클론, C는 기타 변형이 유도된 DF-1 클론을 형광현미경으로 관찰한 결과 및 유세포 분석 결과를 나타낸 도이다(Scale bar = 200μm). 각 막대는 모든 실험을 3회 반복으로 행한 표준편차를 의미한다.
도 6은 chNHE1 단백질 서열의 생물정보학적 분석 결과를 나타낸 도이다. 도 6의 A는 chNHE1의 구조를 도식적으로 나타낸 도이며, 세포 외(extracellular) 및 세포질(cytoplasmic)에 존재하는 부위를 각각 노란색, 하늘색으로 나타내었다. N-말단 세포 외 서열은 신호 펩타이드 서열에 밑줄 표시로 나타내었다. PSIPRED에 의해 예측된 이차 구조는 해당 서열 위에 별표(*, α-나선) 및 점(, β-가닥)으로 나타내었다. DISOPRED3에 의해 예측된 disordered 단백질 결합 부위는 confidence score > 0.8에 대하여 빨간색 글자로 나타내었다. 도 6의 B는 DISOPRED3에 의해 예측된 chNHE1의 disorder 경향을 나타낸 도이며, confidence score > 0.8에 대하여 빨간 선으로 나타내었다.
도 7은 원시생식세포(Primordial germ cells, PGC)에서 CRISPR/Cas9을 이용한 chNHE1 유전자 내 Trp38 indel 유도를 확인한 결과를 나타낸 도이다. 도 7의 A는 NHE1#3 PGC로 감염시킨 PGC의 T7E1 분석 결과를 나타낸 도이다. 도 7의 B는 감염된 PGC의 서열 분석 결과를 나타낸 도이다.1 schematically shows a cleavage map of a CRISPR / Cas9 vector according to the present invention and an ALV-J resistant cell line establishment process according to the present invention. CRISPR / Cas9 vectors include Cas9 protein coding sequence, chNHE1 target gRNA (guide RNA), neomycin / puromycin resistance gene and were introduced and transformed into DF-1 cells. U6: human U6 promoter, CBh: chicken beta-actin short promoter, Amp: ampicillin, Tk: thymidine kinase promoter, Neo: neo Mycin resistance gene, Puro: puromycin resistance gene, LTR: long terminal repeat, GFP: green fluorescent protein.
Figure 2 is a diagram showing the modification in the chNHE1 gene using CRISPR / Cas9 in DF-1 fibroblasts. 2A is a diagram showing the structure of the chNHE1 gene and recognition sites of NHE1 # 3 and NHE1 # 4 CRISPR / Cas9 vectors, the blue line represents the guide RNA recognition site, and the red line represents the PAM (protospacer-adjacent motif) sequence. (Scale bar = 1 kb). 2B shows T7E1 analysis of DF-1 cells infected with NHE1 # 3-neo (N3N), NHE1 # 4-neo (N4N), NHE1 # 3-puro (N3P) and NHE1 # 4-puro (N4P) A diagram showing the results (numbers indicate indel efficiency). 2C is a diagram showing the results of sequencing of infected DF-1 cells, the orange arrow indicates the 38th tryptophan residue (Trp38) of the chNHE1 gene, and the red arrow indicates the target site of the CRISPR / Cas9 vector.
Figure 3 is a diagram showing the results of virus infection identification and flow cytometry (flow cytometry) in chNHE1 gene modification confirmed DF-1 clone.
Figure 4 is a diagram showing the editing of chNHE1 gene using single-stranded oligodeoxynucleotide (ssODN) in DF-1 fibroblasts.
5 is a diagram showing the results of virus infection and flow cytometry of the DF-1 clone targeting the Trp38 of the chNHE1 gene. FIG. 5A shows the flow cytometry of DF-1 clones targeting Trp38 of chNHE1 gene obtained from DF-1 infected with NHE1 # 3-puro + ssODN (N3Pss). A total of nine DF-1 clones Observed (Scale bar = 200 μm). 5B is an indel-induced DF-1 clone generating chNHE1 premature termination codon, C is a diagram showing the results of fluorescence microscopy and flow cytometry analysis of other modified DF-1 clone (Scale bar = 200 μm). Each bar represents the standard deviation of all experiments repeated three times.
Figure 6 shows the results of bioinformatics analysis of the chNHE1 protein sequence. FIG. 6A is a schematic diagram showing the structure of chNHE1, and the sites present in the extracellular and cytoplasmic cells are shown in yellow and light blue, respectively. N-terminal extracellular sequence is underlined in the signal peptide sequence. Secondary structures predicted by PSIPRED are indicated by asterisks (*, α-helix) and dots (, β-strand) on the sequence. The disordered protein binding site predicted by DISOPRED3 is shown in red letters for the confidence score> 0.8. FIG. 6B is a diagram showing the disorder tendency of chNHE1 predicted by DISOPRED3, and is indicated by a red line for confidence score> 0.8.
7 is a diagram showing the results of confirming the induction of Trp38 indel in chNHE1 gene using CRISPR / Cas9 in primitive germ cells (PGC). 7A is a diagram showing the results of T7E1 analysis of PGCs infected with NHE1 # 3 PGCs. Figure 7 B is a diagram showing the results of the sequence analysis of the infected PGC.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 명세서에서 용어 “CRISPR/Cas9” 또는 “CRISPR/Cas9 시스템”은, 크리스퍼(Clustered regularly interspaced short palindromic repeat, CRISPR) 유전자 가위라 불리는 게놈 편집 방법으로, 특정 염기서열에 특이적으로 결합하는 RNA(gRNA)와 특정한 염기서열을 자르는 가위 역할인 Cas9 단백질로 구성된다. 이러한 CRISPR/Cas9 시스템을 이용하면 세포나 동물에 플라스미드(Plasmid) DNA를 도입하여 특정 유전자의 기능을 억제할 수 있는 녹-아웃(knock-out)이 가능하다. As used herein, the term “CRISPR / Cas9” or “CRISPR / Cas9 system” is a genome editing method called a clustered regularly interspaced short palindromic repeat (CRISPR) gene scissors, which specifically binds to a specific nucleotide sequence (RNA). gRNA) and Cas9 protein, which is a scissors to cut a specific sequence. The CRISPR / Cas9 system enables knock-out that can inhibit the function of specific genes by introducing plasmid DNA into cells or animals.
본 명세서에서 용어 “Cas9(CRISPR associated protein 9) 단백질”은, CRISPR/Cas9 시스템에서 필수적인 단백질 요소를 의미하고, CRISPR RNA(crRNA) 및 트랜스-활성화 crRNA (trans-activating crRNA, tracrRNA)로 불리는 두 RNA와 복합체를 형성할 때, 활성 엔도뉴클레아제 또는 니카아제(nickase)를 형성한다.As used herein, the term “Cas9 (CRISPR associated protein 9) protein” refers to an essential protein element in the CRISPR / Cas9 system, and is referred to as two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). When complexed with, it forms an active endonuclease or nickase.
본 명세서에서 용어 "프로모터"는, RNA 중합효소에 대한 결합 부위를 포함하고 프로모터 다운스트림(downstream) 유전자의 mRNA로의 전사 개시 활성을 가지는, 암호화 영역의 상위(upstream)의 비해독된 핵산 서열을 말한다.As used herein, the term "promoter" refers to a non-translated nucleic acid sequence upstream of a coding region that contains a binding site for an RNA polymerase and has a transcription initiation activity to a mRNA of a promoter downstream gene. .
본 명세서에서 용어 "벡터"는, 목적하는 DNA 단편을 숙주균 등에 도입시켜 주고 증식할 수 있는 DNA로서, 클로닝 운반체(cloning vehicle)라고도 하는데, 벡터(vector) DNA는 제한효소 등으로 절단하여 개환하고 여기에 목적으로 하는 DNA 단편을 삽입하여 연결하고 숙주균에 도입시킨다. 목적 DNA 단편을 연결한 벡터 DNA는 숙주균이 증식됨에 따라 복제하여 균의 분열과 더불어 각 낭세포로 분배되어 목적 DNA 단편을 대대로 유지하여 이어져 나가며, 플라스미드 (plasmid), 파지 염색체를 주로 사용한다. As used herein, the term "vector" is a DNA capable of introducing and propagating a desired DNA fragment into a host bacterium and the like, and is also referred to as a cloning vehicle. The vector DNA is cut and opened by restriction enzymes and the like. The DNA fragment of interest is inserted, linked thereto, and introduced into a host bacterium. The vector DNA connecting the target DNA fragment replicates as the host bacteria proliferate, divides the bacteria, and distributes them to each follicular cell to maintain the target DNA fragment for generations, and mainly uses plasmids and phage chromosomes.
본 명세서에서 용어 "재조합 벡터"는, CRISPR/Cas9 시스템을 사용하여 특정 유전자 부위에 염기서열 이중나선 결손을 유도하여 유전자 편집을 실시하고, 적절한 숙주 세포에서 효율적으로 타겟 유전자를 변형시키는 벡터로서 사용될 수 있다. 숙주 세포는 바람직하게는 진핵세포일 수 있으며, 숙주세포의 종류에 따라 프로모터(promoter), 종결자(terminator), 인핸서(enhancer) 등과 같은 발현 조절 서열, 막 표적화 또는 분비를 위한 서열 등을 적절히 선택하고 목적에 따라 다양하게 조합할 수 있다.As used herein, the term "recombinant vector" can be used as a vector for inducing gene editing by inducing nucleotide double helix deletions at specific gene sites using the CRISPR / Cas9 system and efficiently modifying target genes in appropriate host cells. have. The host cell may preferably be a eukaryotic cell, and according to the type of host cell, appropriate selection of expression control sequences such as promoters, terminators, enhancers, and the like for membrane targeting or secretion, etc. And various combinations depending on the purpose.
본 명세서에서 용어 “gRNA(guide RNA)”는, RNA를 편집할 때 수식반응의 주형이 되는 염기순서정보를 갖는 45~70 뉴클레오티드가량의 작은 RNA를 지칭하는데, gRNA의 5′ 측 영역은 RNA 편집되는 mRNA의 일부와 상보적 순서로 되어 있고, 이 영역을 매개로 하여 mRNA에 결합되며, 3′ 측 영역에는 편집 후의 최종적 mRNA의 순서에 상보적 염기순서가 존재하고, 그 순서정보에 따라 우리딘(uridine) 염기의 삽입이나 결실이라는 RNA수식반응이 일어난다.As used herein, the term “gRNA (guide RNA)” refers to a small RNA of about 45 to 70 nucleotides having nucleotide sequence information which is a template of a modification reaction when editing RNA, and the 5 ′ side region of the gRNA is RNA editing. Complementary sequence with a part of mRNA, and is bound to mRNA through this region, Complementary base sequence exists in the sequence of final mRNA after editing in the 3 'side region, according to the order information (uridine) RNA modification reaction occurs by inserting or deleting a base.
본 명세서에서 용어 "세포주"는 세포를 분리해서 순수 배양하여 계대배양해 나갈때 세포계의 각 개체를 말하며, 이때 세포주는 유전적 형질에 의해 다른 세포주와 구별될 수 있으며, 계대배양에도 원 세포의 형질이 유지되는 것을 말한다.As used herein, the term "cell line" refers to each individual of the cell system when the cells are separated and purely cultured and passaged, wherein the cell line can be distinguished from other cell lines by genetic traits, and even when passaged, Say what is to be maintained.
본 명세서에서 용어 "형질전환"은 외부로부터 주어진 DNA에 의하여 생물의 유전적인 성질이 변하는 것으로, 즉 생물의 어떤 계통의 세포에서 추출된 핵산의 일종인 DNA를 다른 계통의 살아있는 세포의 주었을 때 DNA가 그 세포에 들어가서 유전형질이 변화하는 현상으로 형질변환, 형전환 또는 형변환 등이라고도 한다. 즉, "형질전환"이란 유전자를 숙주세포 내에 도입하여 숙주세포 내에서 발현시킬 수 있도록 하는 것을 의미한다.As used herein, the term "transformation" means that the genetic properties of an organism are changed by DNA given from the outside, that is, when DNA is a kind of nucleic acid extracted from a cell of a certain organism, the DNA is given to a living cell of another line. The phenomenon in which the genotype changes in the cell is also called transformation, transformation or transformation. In other words, "transformation" means that the gene can be introduced into the host cell to be expressed in the host cell.
본 발명은 제1 프로모터 및 이와 작동가능하게 연결된 chNHE1(chicken Na+/H+ exchange 1)의 gRNA(guide RNA); 및 제2 프로모터 및 이와 작동가능하게 연결된 Cas9(CRISPR associated protein 9)을 암호화하는 유전자를 포함하는 ALV-J(Avian Leukosis Virus-J, 닭 백혈병 바이러스 하위그룹 J) 저항성 조류모델 제작용 재조합 벡터를 제공한다.The present invention provides gRNA (guide RNA) of the first promoter and chNHE1 (chicken Na + / H + exchange 1) operably linked thereto; And ALV-J (Avian Leukosis Virus-J, sub-group J) resistant avian model, comprising a second promoter and a gene encoding Cas9 (CRISPR associated protein 9) operably linked thereto. do.
본 발명에 있어서, 상기 “chNHE1(chicken Na+/H+ exchange 1)”은 닭 백혈병 바이러스 ALV-J가 특이적으로 결합하는 수용체로, 본 발명자들은 CRISPR/Cas9 시스템을 이용한 숙주에서의 상기 수용체의 변이를 통해 조류에서 ALV-J 바이러스 저항성을 유도할 수 있음을 확인하였다. 본 발명의 일 실시예에서는, CRISPR/Cas9 시스템을 이용하여 chNHE1 유전자의 38번째 트립토판 잔기(Trp38, 또는 W38)에 변형을 유도하기 위하여 네오마이신 저항성 유전자를 포함하는 NHE1#3-Neo(N3N) 및 NHE1#4-Neo(N4N); 퓨로마이신 저항성 유전자를 포함하는 NHE1#3-Puro(N3P) 및 NHE1#4-Puro(N4P)의 CRISPR/Cas9 벡터를 제작하였다.In the present invention, the "chNHE1 (chicken Na + / H + exchange 1)" is a receptor specifically bound to chicken leukemia virus ALV-J, the inventors of the receptor in the host using the CRISPR / Cas9 system The mutation was confirmed to induce ALV-J virus resistance in birds. In one embodiment of the invention, NHE1 # 3-Neo (N3N) comprising a neomycin resistance gene to induce modifications to the 38th tryptophan residue (Trp38, or W38) of the chNHE1 gene using the CRISPR / Cas9 system and NHE1 # 4-Neo (N4N); CRISPR / Cas9 vectors of NHE1 # 3-Puro (N3P) and NHE1 # 4-Puro (N4P) containing a puromycin resistance gene were constructed.
한편, 야생형 chNHE1 유전자(wild-type chNHE1)의 서열은 다음과 같다: GCCCGCTGCTGCCCGGCCAGCGCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACCTGGGAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCGCCCCGCTGGCCACGGCCCAGGAGGTGCACCCGCTGAACAAACAGCACCACAACCACTC (서열번호 5)On the other hand, the sequence of the wild-type chNHE1 gene (wild-type chNHE1) is as follows: GCCCGCTGCTGCCCGGCCAGCGCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACCTGGGAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCGCCCCGCTGGCCACACACACACACACACAA
본 발명에 있어서, 상기 제1 프로모터는 gRNA의 발현을 개시할 수 있는 프로모터라면 제한 없이 사용할 수 있으며, 당업자에게 자명한 공지의 모든 프로모터를 사용할 수 있으나, 바람직하게는 CMV(cytomegalovirus), CAG(chicken beta-actin), CBh(hybrid form of the chicken beta-actin), U6 등의 유비쿼터스(ubiquitous) 프로모터 또는 조직 특이적 프로모터를 목적에 따라 사용할 수 있고, 보다 바람직하게는 인간 U6 프로모터(human U6 promoter)를 사용할 수 있다.In the present invention, the first promoter may be used without limitation as long as it is a promoter capable of initiating the expression of gRNA, all promoters known to those skilled in the art can be used, but preferably CMV (cytomegalovirus), CAG (chicken) Ubiquitous promoter or tissue specific promoters such as beta-actin), hybrid form of the chicken beta-actin (CBh), and U6 may be used depending on the purpose, and more preferably, human U6 promoter Can be used.
본 발명에 있어서, 상기 chNHE1(chicken Na+/H+ exchange 1)의 gRNA(guide RNA)는 서열번호 1 또는 서열번호 2로 표시되는 것일 수 있다.In the present invention, the gRNA (guide RNA) of the chNHE1 (chicken Na + / H + exchange 1) may be represented by SEQ ID NO: 1 or SEQ ID NO: 2.
상기 서열번호 00 또는 00으로 표시되는 염기서열의 변이체 또한 본 발명의 범위 내에 포함된다. 본 발명은 상기 염기서열의 등가물, 예를 들어, 일부 염기서열이 결실(deletion), 치환(substitution) 또는 삽입(insertion)에 의해 변형되었지만, chNHE1(chicken Na+/H+ exchange 1) gRNA(guide RNA)와 기능적으로 동일한 작용을 할 수 있는 변이체(variants)를 포함하는 개념이다. 구체적으로, chNHE1(chicken Na+/H+ exchange 1) gRNA(guide RNA)는 각 서열번호 00 또는 00의 염기 서열과 각각 70% 이상, 더욱 바람직하게는 80% 이상, 더더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.Variants of the nucleotide sequence represented by SEQ ID NO: 00 or 00 are also included within the scope of the present invention. In the present invention, an equivalent of the above nucleotide sequence, for example, some nucleotide sequences have been modified by deletion, substitution or insertion, but chNHE1 (chicken Na + / H + exchange 1) gRNA (guide) RNA is a concept that includes variants that can function functionally the same. Specifically, chNHE1 (chicken Na + / H + exchange 1) gRNA (guide RNA) is at least 70%, more preferably at least 80%, even more preferably at least 90%, each with the base sequence of each SEQ ID 00 or 00 And, most preferably, base sequences having 95% or more sequence homology. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).
본 발명에 있어서, 상기 제2 프로모터는 바람직하게는 CMV(cytomegalovirus), CAG(chicken beta-actin), CBh(hybrid form of the chicken beta-actin), U6 등의 유비쿼터스(ubiquitous) 프로모터 또는 조직 특이적 프로모터를 목적에 따라 사용할 수 있고, 보다 바람직하게는 닭 베타-액틴 프로모터(chicken beta-actin short promoter)일 수 있으나, 이에 제한되지 않는다.In the present invention, the second promoter is preferably a ubiquitous promoter or tissue specific such as CMV (cytomegalovirus), CAG (chicken beta-actin), CBh (hybrid form of the chicken beta-actin), U6, etc. The promoter may be used according to the purpose, and more preferably, it may be a chicken beta-actin short promoter, but is not limited thereto.
본 발명에 있어서, 상기 재조합 벡터는 도 1(좌측)에 기재된 개열 지도를 갖는 것으로, 본 발명의 chNHE1 유전자 변형을 달성할 수 있는 벡터의 구성이라면 이에 제한되지 않는다.In the present invention, the recombinant vector has the cleavage map described in FIG. 1 (left), and is not limited thereto as long as it is a constitution of a vector capable of achieving the chNHE1 gene modification of the present invention.
또한, 본 발명은 본 발명에 따른 재조합 벡터 및 ssODN(single-stranded oligodeoxynucleotide, 단일 가닥 올리고데옥시뉴클레오티드)을 포함하는 것을 특징으로 하는 유전체 교정용 조성물을 제공한다. 본 발명의 일 실시예에서는 CRISPR/Cas9 시스템을 사용함과 동시에, DNA를 보다 효율적으로 변형시키기 위해 단일가닥 올리고데옥시뉴클레오티드(single-stranded oligodeoxynucleotide, ssODN)를 CRISPR/Cas9 벡터와 함께 도입하여 보다 높은 효율로 정확하게 ALV-J에 저항성을 가진 조류모델을 제조할 수 있음을 확인하였다.In another aspect, the present invention provides a composition for genome correction, comprising a recombinant vector according to the invention and ssODN (single-stranded oligodeoxynucleotide, single stranded oligodeoxynucleotide). In one embodiment of the present invention, while using the CRISPR / Cas9 system, a single-stranded oligodeoxynucleotide (ssODN) is introduced together with the CRISPR / Cas9 vector for more efficient modification of DNA. As a result, it was confirmed that the algae model with resistance to ALV-J can be manufactured.
본 명세서에서 용어 “ssODN(single-stranded oligodeoxynucleotide, 단일 가닥 올리고데옥시뉴클레오티드)”는 한가닥의 DNA로 구성되어 있고, 비정상 염기(base)를 정상 염기로 교체하기 위하여 정상 유전자의 공여자(donor)로 사용된다.As used herein, the term “ssODN (single-stranded oligodeoxynucleotide)” consists of a single strand of DNA and is used as a donor of a normal gene to replace an abnormal base with a normal base. do.
본 발명에 있어서, 상기 ssODN은 chNHE1 유전자에 상보적으로 결합하며 서열번호 12의 염기서열로 표시되는 것일 수 있으나 이에 제한되는 것은 아니다. 또한, 상기 ssODN은 chNHE1 유전자에서 Trp38 결실 및 BsaI 제한효소 부위를 생성하기 위한 T96G 치환을 유도하는 것을 특징으로 할 수 있다. 본 발명의 일 실시예에서는 서열번호 00의 염기서열로 표시되며 chNHE1 유전자에 상보적으로 결합하여 chNHE1 유전자에서 Trp38 결실 및 BsaI 제한효소 부위를 생성하는 ssODN과 CRISPR/Cas9 재조합 벡터를 결합하여 세포를 형질전환하고, Trp38 결실 및 T96G 치환이 유도된 DF-1 세포의 단일 클론(single clone)을 확립하였다.In the present invention, the ssODN is complementary to the chNHE1 gene and may be represented by the nucleotide sequence of SEQ ID NO: 12, but is not limited thereto. In addition, the ssODN may be characterized by inducing T96G substitution to generate Trp38 deletion and BsaI restriction site in the chNHE1 gene. In one embodiment of the present invention represented by the nucleotide sequence of SEQ ID NO: 00 and binds to the chNHE1 gene complementary to ssODN and CRISPR / Cas9 recombinant vector to generate a Trp38 deletion and BsaI restriction enzyme site in the chNHE1 gene to transform the cells A single clone of DF-1 cells induced with Trp38 deletion and T96G substitution was established.
본 명세서에서 용어 “유전체 교정(genome editing)”은, 특별한 언급이 없는 한, 표적 유전자의 표적 부위에서의 절단에 의한 핵산 분자(하나 이상, 예컨대 1-100,000bp, 1-10,000bp, 1-1000bp, 1-100bp, 1-70bp, 1-50bp, 1-30bp, 1-20bp 또는 1-10bp)의 결실, 삽입, 치환 등에 의하여 유전자 기능을 상실, 변경, 및/또는 회복(수정) 시키는 것을 의미하기 위하여 사용될 수 있다.As used herein, the term “genome editing”, unless stated otherwise, refers to a nucleic acid molecule (eg, 1-100,000 bp, 1-10,000 bp, 1-1000 bp) by cleavage at a target site of a target gene. , 1-100bp, 1-70bp, 1-50bp, 1-30bp, 1-20bp or 1-10bp) means loss, alteration and / or recovery (modification) of gene function Can be used to
또한, 본 발명은 본 발명에 따른 재조합 벡터 또는 유전체 교정용 조성물이 도입된 ALV-J 저항성 조류모델 제작용 형질전환 세포주를 제공한다.The present invention also provides a transformed cell line for preparing an ALV-J resistant avian model into which a recombinant vector or genome calibration composition according to the present invention is introduced.
본 발명에 있어서 상기 세포주는 난모세포주, 섬유아세포주 또는 신장세포주일 수 있으며, 바람직하게는 섬유아세포주를 이용할 수 있으나, 이에 본 발명이 제한되는 것은 아니다.In the present invention, the cell line may be an oocyte cell line, a fibroblast cell line or a kidney cell line, and preferably a fibroblast cell line, but the present invention is not limited thereto.
본 발명의 재조합 벡터를 세포주에 도입하여 형질전환하는 방법은 본 발명의 재조합 벡터를 당업계에 공지된 방법, 예를 들어 이에 한정되지는 않으나, 일시적인 형질감염(transient transfection), 미세주사, 형질도입(transduction), 세포 융합, 칼슘 포스페이트 침전법, 리포좀 매개된 형질감염(liposem-mediated transfection), DEAE 덱스트란-매개된 형질감염(DEAE Dextran-mediated transfection), 폴리브렌-매개된 형질 감염(polybrene-mediated transfection), 전기 침공법(electroporation) 등의 공지 방법으로 진핵세포에 도입하여 형질전환시킬 수 있으며, 바람직하게는 미세주사 방법을 이용하여 형질전환시킬 수 있다.The method of transforming the recombinant vector of the present invention into a cell line may be performed by a method known in the art, such as, but not limited to, transient transfection, microinjection, and transduction. transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection It can be transformed by introducing into eukaryotic cells by known methods such as mediated transfection), electroporation, and the like, and preferably by microinjection.
상기 형질전환된 세포의 선별은 당업계에서 공지된 유전자 교정 여부를 확인하는 방법으로 선별할 수 있다. 본 발명의 일 실시예에서는 G418, 네오마이신 및 퓨로마이신과 같은 항생제 내성 유전자를 사용하여 형질전환된 DF-1 세포를 선별하고 있으나, 이에 제한되는 것은 아니다.Selection of the transformed cells may be selected by a method for confirming genetic correction known in the art. In one embodiment of the present invention, transformed DF-1 cells are selected using antibiotic resistance genes such as G418, neomycin and puromycin, but are not limited thereto.
본 발명에서는 상기 형질전환된 DF-1 세포의 바람직한 실시예의 하나로서, G418을 이용하여 chNHE1 유전자 변형이 확인된 DF-1 세포 선별 후, NHE1#3-Neo(N3N), NHE1#3-Puro(N3P) 또는 NHE1#4-Puro(N4P) 벡터로 형질감염된 DF-1 섬유아세포로부터 다양한 indel 변형을 갖는 클론을 확립하였다. 또한, Trp38 결실에 따른 보다 정확한 효과를 확인하기 위하여 NHE1#3-Puro(N3P) 또는 NHE1#4-Puro(N4P)와 함께 ssODN을 함께 처리한 후 퓨로마이신을 이용하여 형질전환이 이루어진 DF-1 세포 선별 후, Trp38 결실 및 T96G 치환이 유도된 단일 클론을 확립하였다.In the present invention, as one of the preferred embodiments of the transformed DF-1 cells, after screening for DF-1 cells in which chNHE1 gene modification was confirmed using G418, NHE1 # 3-Neo (N3N), NHE1 # 3-Puro ( Clones with various indel modifications were established from DF-1 fibroblasts transfected with N3P) or NHE1 # 4-Puro (N4P) vectors. In addition, DF-1 was transformed using puromycin after treatment with ssODN together with NHE1 # 3-Puro (N3P) or NHE1 # 4-Puro (N4P) to confirm the more accurate effect of Trp38 deletion. After cell selection, a single clone was induced that induced Trp38 deletion and T96G substitution.
특히, Trp38 결실과 함께 T96G 치환을 포함하는 N3Pss#12의 경우, CRISPR/Cas9 벡터 및 ssODN으로 형질전환됨에 따라 chNHE1 유전자에서 Trp38을 정확히 타겟팅한 유전자 편집이 이루어짐을 확인하였다. 상기의 특징을 가지는 형질전환 세포주를 2018년 4월 25일 한국세포주연구재단 (KCLRF, Korean Cell Line Research Foundation)에 수탁번호 KCLRF-BP-00433로 기탁하였다.In particular, in the case of
본 발명에 있어서, 본 발명에 따른 재조합 벡터 또는 유전체 교정용 조성물이 도입되는 상기 형질전환 세포주는 공여체 조류의 원시생식세포(primordial germ cells, PGCs)인 것을 특징으로 할 수 있다. In the present invention, the transformed cell line into which the recombinant vector or genome correction composition according to the present invention is introduced may be characterized as primordial germ cells (PGCs) of donor birds.
상기 재조합 벡터 또는 유전체 교정용 조성물은 당업계에 공지된 다양한 방법에 따라 조류 원시생식세포에 도입될 수 있다. 예를 들어, 원시생식세포로의 유전자 전달은 전기천공법(electroporation), 리포좀-매개 전달방법 및 레트로바이러스-매개 전달방법 등을 참고하여 실시할 수 있다.The recombinant vector or genome calibration composition may be introduced into avian primordial germ cells according to various methods known in the art. For example, gene transfer to primordial germ cells can be carried out with reference to electroporation, liposome-mediated delivery and retrovirus-mediated delivery.
본 발명에서 이용될 수 있는 원시생식세포는 다양한 소스로부터 얻을 수 있으나, 가장 바람직하게는 조류의 배아의 생식기(embryonic gonad)로부터 분리된 것이다. 원시생식세포의 소스로서 배아를 이용하는 경우, 본 발명에서 이용되는 공여체 배아는 다양한 단계에 진입한 것이 이용될 수 있으며, 1-10일령 배아가 이용될 수 있고, 바람직하게는 배 발달 스테이지 약 24-36 (약 4 일령-8 일령), 가장 바람직하게는 배 발달 스테이지 배 발달 스테이지 약 28 (약 5.5 일령)의 배아가 이용될 수 있다. 원시생식기로부터 원시생식세포를 수득하는 방법은 다양하게 실시될 수 있으며, 본 발명자들의 특허 제 0305715 호 및 특허 제 0267633 호에 개시된 것을 참고할 수 있다. 예컨대, 난황이 제거된 배아로부터 원시생식기를 회수하고, 원시생식기에 단백질분해효소 (예: 트립신)를 처리하여, 원시생식기 세포를 분리한다. 이러한 원시생식기 세포는 다양한 세포종의 군집으로서, 원시생식세포 이외에 기저세포 (stroma cells) 등이 포함되어 있다. 본 명세서에서 용어 “원시생식기 세포 (gonadal cells)” 는 원시생식기에 존재하는 모든 세포 종의 군집을 의미하며, 용어 “원시생식기-유래 원시생식세포 (gonadal primordial germ cells)” 는 후에 생식세포가 되는 원시생식기 세포의 일종을 나타내며, 약어 “gPGCs” 또는 “PGC"로 나타낼 수 있다.Primitive germ cells that can be used in the present invention can be obtained from a variety of sources, but most preferably are isolated from the embryonic gonad of an embryo of avian. When using an embryo as a source of primordial germ cells, donor embryos used in the present invention may be used which have entered various stages, embryos 1-10 days old may be used, preferably embryo development stage about 24- Embryos of 36 (about 4 days old-8 days old), most preferably embryonic development stage embryonic development stage about 28 (about 5.5 days old) can be used. The method for obtaining the primordial germ cells from the primordial genitalia may be carried out in various ways, and reference may be made to those disclosed by the inventors of Patent Nos. 0305715 and 0267633. For example, primitive genitalia are recovered from embryos from which yolk has been removed, and progenitor cells are treated with proteolytic enzymes (such as trypsin) to isolate primitive genital cells. Such primitive genital cells are a population of various cell types, and include basal cells in addition to primitive germ cells. As used herein, the term “gonadal cells” refers to a population of all cell species present in the primordial genitalia, and the term “gonadal primordial germ cells” later becomes germ cells. It represents a type of primitive genital cell and may be represented by the abbreviation “gPGCs” or “PGC”.
유전자가 편집된 원시생식세포는 수용체 배아에 주입된다. 원시생식세포를 수용체 배아에 주입하는 방법은 다양한 방법에 따라 실시될 수 있으며, 바람직하게는 배아의 대동맥에 주입하여 실시된다. 예를 들어, 적합한 개수의 원시생식세포를 포함하는 세포 현탁액을 배아의 등쪽 대동맥에 미세바늘로 주입하고, 배아를 포함하는 난을 밀봉한 다음 적절한 시간 동안 배양하여 실시된다. 그런 다음, 상기 수용체 배아를 포함하는 난을 배양 및 부화하고, 성성숙에 이른 수용체를 검정교배를 통해 유전자 편집된 조류를 생산한다. 유전자 편집 조류의 확인은 예를 들어, 유전자 편집 조류의 깃털수질(feather pulp) 또는 혈액으로부터 얻은 유전체 DNA를 주형으로 이용하여 PCR(polymerase-chain reaction), 실시간 PCR 방법 또는 염기서열분석법으로 서열이 편집되어 있는 지 여부를 검사하여 실시할 수 있다.Primitive germ cells whose genes have been edited are injected into receptor embryos. The method of injecting the primordial germ cells into the receptor embryo can be carried out according to various methods, preferably by injecting into the aorta of the embryo. For example, a cell suspension containing an appropriate number of primordial germ cells is injected by microneedle into the dorsal aorta of the embryo, the eggs containing the embryo are sealed and then incubated for an appropriate time. The eggs containing the receptor embryos are then cultured and incubated, and the maturated receptors are produced via genetically edited algae. Identification of genetically modified algae can be performed by sequence editing, for example, by polymerase-chain reaction (PCR), real-time PCR or sequencing using genomic DNA obtained from feather pulp or blood of genetically modified algae as a template. It can be carried out by checking whether it is.
본원에 따른 방법은 상기 방법에 의해 생산된 유전자 변형체를 검정교배하여, 동형접합 유전자 변형체 선별하는 단계를 추가로 포함할 수 있다.The method according to the present invention may further comprise the step of assaying the genetic variants produced by the method, selecting homozygous genetic variants.
또한, 본원에 따른 방법은 유전자 변형이 발생한 원시생식세포의 선별을 위해, 형광 단백질을 코딩하는 핵산을 조류 원시생식세포에 도입하는 단계를 추가로 포함할 수 있다. 형광 단백질은 조류의 세포에서 발현될 수 있는 한 특별히 제한되는 것은 아니며, 예를 들면 Green Fluorescent Protein (GFP)를 들 수 있다. 이러한 단백질은 조류에서 작용하는 벡터에 도입되어, PGC에 함께 도입될 수 있으며, 도입 후에 형광단백질을 발현하는 세포를 FACS와 같은 방법으로 선별한 후, 이러한 세포만을 수용체의 배아에 도입하여, 형질전환 조류의 생산 효율을 높일 수 있다.In addition, the method according to the present invention may further comprise the step of introducing a nucleic acid encoding a fluorescent protein into avian primordial germ cells, for the selection of primordial germ cells having genetic modification. The fluorescent protein is not particularly limited as long as it can be expressed in cells of algae, for example, Green Fluorescent Protein (GFP). These proteins can be introduced into a vector that acts on algae and co-introduced into PGCs. After introduction, cells expressing fluorescent proteins are selected by a method such as FACS, and then only these cells are introduced into the embryos of the receptor for transformation. It can increase the production efficiency of algae.
본 발명에 있어서, 상기 공여체 조류는 닭, 메추라기, 칠면조, 오리, 거위, 꿩 및 비둘기를 포함하며, 바람직하게는 닭인 것을 특징으로 할 수 있다.In the present invention, the donor bird includes chicken, quail, turkey, duck, goose, pheasant and pigeon, preferably may be characterized in that the chicken.
또한, 본 발명에 있어서, 상기 형질전환 세포주는 수탁번호가 KCLRF-BP-00432임을 특징으로 할 수 있다. In addition, in the present invention, the transgenic cell line may be characterized in that the accession number is KCLRF-BP-00432.
본 발명의 일 실시예에서는, 본 발명에 따른 CRISPR/Cas9 재조합 벡터로 형질감염시킨 PGC(NHE1#3 PGC)에서 chNHE1 유전자의 Trp38 위치를 정확히 타겟팅한 유전자 편집이 이루어짐을 확인하였다. 상기의 특징을 가지는 형질전환 세포주를 2018년 4월 25일 한국세포주연구재단 (KCLRF, Korean Cell Line Research Foundation)에 수탁번호 KCLRF-BP-00432로 기탁하였다.In an embodiment of the present invention, it was confirmed that the gene editing was precisely targeted to the Trp38 position of the chNHE1 gene in PGC (NHE1 # 3 PGC) transfected with the CRISPR / Cas9 recombinant vector according to the present invention. Transgenic cell lines having the above characteristics were deposited with Korean Cell Line Research Foundation (KCLRF) on April 25, 2018 under accession number KCLRF-BP-00432.
또한, 본 발명은 본 발명에 따른 재조합 벡터 또는 유전체 교정용 조성물을 제조하는 단계; 및 상기 재조합 벡터 또는 유전체 교정용 조성물을 조류 체세포에 도입하는 단계;를 포함하는 ALV-J 저항성 조류모델 제작용 형질전환 세포주의 제조방법을 제공한다.In addition, the present invention comprises the steps of preparing a composition for recombinant vector or genome calibration according to the present invention; It provides a method for producing a transformed cell line for producing ALV-J resistant avian model comprising the step of introducing the recombinant vector or genomic correction composition to a somatic cell.
또한, 본 발명은 본 발명에 따른 형질전환 세포주를 수용체 조류의 배아에 이식하는 단계; 를 포함하는 ALV-J 저항성 조류모델의 제조방법을 제공한다. 상기 형질전환 세포주는 공여체 조류의 원시생식세포(primordial germ cells, PGCs)인 것을 특징으로 한다.In addition, the present invention comprises the steps of transplanting the transformed cell line according to the invention into the embryo of the receptor bird; It provides a method of manufacturing an ALV-J resistant bird model comprising a. The transformed cell line is characterized in that the primordial germ cells (PGCs) of the donor bird.
본 발명에 따른 형질전환 세포주는 바이러스 감염 실험 결과 매우 낮은 GFP 발현을 나타내어 ALV-J에 대한 저항성을 획득할 수 있으므로, 닭 백혈병 바이러스(Avian Leukosis Virus-J, ALV-J)에 저항성을 가진 조류 생산에 유용하게 이용될 수 있다.The transgenic cell line according to the present invention exhibits very low GFP expression as a result of virus infection experiments and thus can acquire resistance to ALV-J, thus producing algae resistant to chicken leukosis virus (Avian Leukosis Virus-J, ALV-J). It can be usefully used.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다. Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the present invention is not limited to the contents of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
실시예Example 1. One. CRISPRCRISPR /Of Cas9Cas9 시스템을 통한 Through the system chNHE1chNHE1 타겟target 재조합 벡터 제조 Recombinant Vector Manufacturing
chNHE1(chicken Na+/H+ exchange 1) 유전자의 38번째 트립토판 잔기에 변형을 유도하기 위하여 네오마이신 저항성 유전자를 포함하는 2개의 CRISPR/Cas9 벡터 NHE1#3-Neo(N3N) 및 NHE1#4-Neo(N4N); 퓨로마이신 저항성 유전자를 포함하는 2개의 CRISPR/Cas9 벡터 NHE1#3-Puro(N3P) 및 NHE1#4-Puro(N4P)를 제작하였다. 상기 재조합 벡터의 개열지도를 도 1에 나타내었다.Two CRISPR / Cas9 vectors NHE1 # 3-Neo (N3N) and NHE1 # 4-Neo containing neomycin resistance genes to induce modifications at the 38th tryptophan residue of the chNHE1 (chicken Na + / H + exchange 1) gene (N4N); Two CRISPR / Cas9 vectors NHE1 # 3-Puro (N3P) and NHE1 # 4-Puro (N4P) were constructed containing the puromycin resistance gene. A cleavage map of the recombinant vector is shown in FIG. 1.
구체적으로, chNHE1 수용체를 타겟으로 하기 위한 재조합 벡터를 제조하기 위해 네오마이신 저항성 유전자 및 퓨로마이신 저항성 유전자를 NotI 제한효소(New England Biolabs, Ipswich, MA, USA)를 이용하여 CRISPR/Cas9 벡터에 도입하였다. 또한, guide RNA를 CRISPR/Cas9 벡터에 도입하기 위해 센스 및 안티센스 올리고뉴클레오티드(Bionics, Seoul, Korea)를 합성하여 95℃에서 30초, 72℃에서 2분, 37℃에서 2분 및 25℃에서 2분 조건으로 어닐링하였다. chNHE1 gRNA 2개 (각각 서열번호 1, 서열번호 2), 사용한 모든 프라이머 및 올리고뉴클레오티드의 서열을 표 1 및 표 2에 각각 나타내었다. CBh 프로모터는 Addgene(Cambridge, MA)에서 구매한 pX330A 플라스미드에 존재하는 염기서열을 사용하였고, 본 발명에 사용한 chNHE1 gRNA는 닭의 chNHE1 유전자 내 Trp38 부분(W38)을 효과적으로 타겟팅하기 위하여 임의로 제작하였다.Specifically, neomycin resistance genes and puromycin resistance genes were introduced into CRISPR / Cas9 vectors using NotI restriction enzymes (New England Biolabs, Ipswich, MA, USA) to prepare recombinant vectors for targeting the chNHE1 receptor. . In addition, a sense and antisense oligonucleotide (Bionics, Seoul, Korea) was synthesized to introduce guide RNA into the CRISPR / Cas9 vector, followed by 30 seconds at 95 ° C, 2 minutes at 72 ° C, 2 minutes at 37 ° C, and 2 at 25 ° C. Annealed under min conditions. The sequences of two chNHE1 gRNAs (SEQ ID NO: 1, SEQ ID NO: 2, respectively) and all primers and oligonucleotides used are shown in Tables 1 and 2, respectively. The CBh promoter used a nucleotide sequence present in the pX330A plasmid purchased from Addgene (Cambridge, Mass.), And the chNHE1 gRNA used in the present invention was arbitrarily constructed to effectively target the Trp38 portion (W38) in the chNHE1 gene of chicken.
이탤릭체로 표시된 염기는 gRNA 스캐폴드(scaffold) 부분을 의미한다.Underline refers to the human U6 promoter. Bases shown in bold represent NHE1 # 3 or
Bases indicated in italics refer to gRNA scaffold moieties.
GCG G GTCTCCGAGCCCACC(TGG)GAGCAGCCGTGGGGAGA
GCCCGGGGGTATCACCGCCGCCCCGCTGGCCACGGCCCAG
GAGGTGCACCCGCTGAACAAACAGCACCACAACCACTCGCCCGCTGCTGCCCGGCCAGCGCTTGCAGGCCGACGCCAC
GCG G GTCTC CGAGCCCACC (TGG) GAGCAGCCGTGGGGAGA
GCCCGGGGGTATCACCGCCGCCCCGCTGGCCACGGCCCAG
실시예Example 2. 재조합 벡터를 이용한 섬유아세포의 형질전환 2. Transformation of fibroblasts using recombinant vectors
상기 실시예 1에서 제조한 CRISPR/Cas9 재조합 벡터(3 ㎍)를 Opti-MEM(Thermo Fisher-Invitrogen)에서 Lipofectamine 2000 시약(Thermo Fisher-Invitrogen)과 혼합하고, 상기 혼합물을 5 x 105 개의 DF-1 세포에 도입하여 형질전환 시켰다. 이때 사용한 섬유아세포 DF-1은 60-70%의 상대습도, 5% 이산화탄소 및 37℃ 조건에서 10% 소태아혈청(FBS; Hyclone) 및 1x 안티바이오틱-안티마이코틱(ABAM; Thermo Fisher-Invitrogen, Carlsbad, CA, USA)이 첨가된 DMEM(Hyclone, Logan, UT, USA) 배지 내에서 유지하였다. 형질전환 6시간 후, 형질전환 혼합물을 DF-1 배양 배지로 교환하였다. 형질전환을 수행하고 1일 경과 후, G418(300 ㎍/ml) 및 퓨로마이신(1 ㎍/ml)을 배지에 첨가하였다. G418 및 퓨로마이신을 이용한 선별 과정은 7일까지 소요되었다.The CRISPR / Cas9 recombinant vector (3 μg) prepared in Example 1 was mixed with Lipofectamine 2000 reagent (Thermo Fisher-Invitrogen) in Opti-MEM (Thermo Fisher-Invitrogen), and the mixture was mixed with 5 × 10 5 DF- 1 cell was transformed. The fibroblast DF-1 used at this time was 60-70% relative humidity, 5% carbon dioxide and 10% fetal bovine serum (FBS; Hyclone) and 1x antibiotic-antimycotic (ABAM; Thermo Fisher-Invitrogen) at 37 ° C. , Carlsbad, CA, USA) was maintained in DMEM (Hyclone, Logan, UT, USA) medium added. After 6 hours of transformation, the transformation mixture was exchanged with DF-1 culture medium. One day after the transformation was performed, G418 (300 μg / ml) and puromycin (1 μg / ml) were added to the medium. The selection process with G418 and puromycin took up to 7 days.
실시예Example 3. 3. chNHE1chNHE1 유전자의 Gene Trp38Trp38 indelindel 유도 및 녹아웃 확인 Induction and knockout check
상기 실시예 2에서 생산한 형질전환 섬유아세포에서 chNHE1 유전자의 Trp38 위치에 indel이 정상적으로 유도되었는지 여부를 확인하기 위하여 T7 엔도뉴클라아제 1(T7E1) 분석을 수행하였다. 구체적으로 이형접합(heteroduplex) DNA 구조를 형성하기 위하여 상기 PCR 산물을 변성 이후 리어닐링하였고, 그 후 리어닐링한 산물을 37℃에서 20분 동안 T7E1(New England Biolabs) 효소로 분해하였다. T7E1으로 분해된 산물은 1% 아가로스 겔을 사용하여 전기영동시키고, 이후 이를 Gel Doc gel imaging system(Bio-Rad Laboratories, Hercules, CA, USA)를 사용하여 정량화하였다. chNHE1 유전자의 구조 및 NHE1#3, NHE1#4 CRISPR/Cas9 벡터의 인식 부위를 도 2의 A에 나타내었으며, 상기 T7E1 분석 결과를 도 2의 B에 나타내었다.T7 endonuclease 1 (T7E1) analysis was performed to confirm whether indel is normally induced at the Trp38 position of the chNHE1 gene in the transformed fibroblasts produced in Example 2. Specifically, in order to form a heteroduplex DNA structure, the PCR product was renealed after denaturation, and then the renealed product was digested with T7E1 (New England Biolabs) enzyme at 37 ° C. for 20 minutes. Products digested with T7E1 were electrophoresed using 1% agarose gel and then quantified using Gel Doc gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA). The structure of the chNHE1 gene and the recognition sites of the NHE1 # 3 and
도 2의 B에 나타낸 바와 같이, G418(왼쪽) 또는 퓨로마이신(오른쪽)으로 선별하기 전 세포에 비해 선별된 세포에서 타겟 위치의 indel 변형 비율이 높음을 확인하였다. 구체적으로, indel 유도 효율은 N3N, N4N, N3P 및 N4P에서 각각 18.0%, 19.1%, 23.9% 및 34.0%으로 나타남을 확인하였다.As shown in B of FIG. 2, it was confirmed that the ratio of indel modification at the target position was higher in the selected cells as compared to the cells before selection with G418 (left) or puromycin (right). Specifically, indel induction efficiency was found to be 18.0%, 19.1%, 23.9% and 34.0% in N3N, N4N, N3P and N4P, respectively.
추가적으로 chNHE1 유전자의 Trp38 위치에 녹아웃이 정상적으로 발생하였는지 여부를 확인하기 위하여 PCR을 수행하였다. 먼저, 형질전환 세포의 유전체 DNA는 G418을 이용한 세포 선별 후 DF-1에서 추출하였다. CRISPR/Cas9 타겟 부위를 포함하는 유전자 부위는 상기 표 2에 나타낸 프라이머 세트를 이용하여 증폭하였다. PCR은 총 20㎕의 100ng의 유전체 DNA에 대하여 10x PCR 버퍼(BioFACT, Daejeon, Korea), 0.4㎕ dNTPs(각각 10mM), 각 프라이머 10pmol 및 0.5 U Taq 폴리머레이즈(BioFACT)를 사용하여 다음과 같은 조건으로 수행하였다: 5분 동안 94℃에서 1차 변성을 1회 반복하였고, 후에 20초 동안 94℃에서 변성, 40초 동안 61℃에서 어닐링(annealing) 및 120초 동안 72℃에서 연장하는 단계를 35회 반복하였으며, 마지막으로 10분 동안 72℃에서 후-연장을 1회 반복하였다. 그 결과를 도 2의 C에 나타내었다.In addition, PCR was performed to confirm whether knockout occurred normally at the Trp38 position of the chNHE1 gene. First, the genomic DNA of the transformed cells was extracted from DF-1 after cell selection using G418. Gene regions comprising CRISPR / Cas9 target sites were amplified using the primer sets shown in Table 2 above. PCR was performed using 10 × PCR buffer (BioFACT, Daejeon, Korea), 0.4 μl dNTPs (10 mM each), 10 pmol of each primer and 0.5 U Taq polymerase (BioFACT) for a total of 20 μl of 100 ng of genomic DNA. The first denaturation was repeated once at 94 ° C. for 5 minutes, followed by denaturation at 94 ° C. for 20 seconds, annealing at 61 ° C. for 40 seconds and extending at 72 ° C. for 120 seconds. Repeated one time, the last post-extension was repeated once at 72 ° C. for 10 minutes. The results are shown in C of FIG.
도 2의 C에 나타낸 바와 같이, CRISPR/Cas9 재조합 벡터가 도입된 DF-1 섬유아세포의 chNHE1 유전자에서 삽입, 치환 또는 결실을 포함하는 변형이 발생하였음을 확인하였다.As shown in C of FIG. 2, it was confirmed that modifications including insertion, substitution or deletion occurred in the chNHE1 gene of DF-1 fibroblasts into which the CRISPR / Cas9 recombinant vector was introduced.
실시예Example 4. 4. chNHE1chNHE1 유전자 변형이 확인된 세포주의 클론 확립 Establish clones of cell lines with genetic modification
상기 실시예 3에서 확인한 CRISPR/Cas9 재조합 벡터가 도입된 DF-1 섬유아세포로부터 DF-1 클론을 확립하였다. 이 과정에서, 타겟 위치에서 다양한 indel 변형을 갖는 수개의 클론을 확립하였다.The DF-1 clone was established from DF-1 fibroblasts into which the CRISPR / Cas9 recombinant vector identified in Example 3 was introduced. In this process, several clones with various indel modifications at target locations were established.
구체적으로, G418를 이용하여 유전자 변형이 확인된 세포 선별 후, CRISPR 벡터가 도입된 DF-1 세포들을 100μL의 DF-1 배양 배지가 들어있는 96-웰 플레이트에 분주하였다. 분주된 세포들이 각 웰에 충분히 부착(confluency)되었을 때, 세포들을 48-웰 플레이트에 계대배양 하고 유전체 DNA 추출에 사용하였다. CRISPR/Cas9 타겟 부위를 포함하는 유전자 부위를 상기 표 2에 나타낸 프라이머 세트를 이용하여 증폭시키고, ABI Prism 3730 XL DNA Analyzer (Thermo Fisher-Applied Biosystems, Foster City, CA, USA)를 사용하여 PCR 산물의 염기서열을 분석하였다.Specifically, after selection of the cells in which genetic modification was confirmed using G418, DF-1 cells into which the CRISPR vector was introduced were divided into 96-well plates containing 100 µL of DF-1 culture medium. When the dispensed cells were sufficiently adherent to each well, the cells were passaged to 48-well plates and used for genomic DNA extraction. The gene region comprising the CRISPR / Cas9 target site was amplified using the primer set shown in Table 2 above, and the PCR product was prepared using an ABI Prism 3730 XL DNA Analyzer (Thermo Fisher-Applied Biosystems, Foster City, CA, USA). The sequence was analyzed.
그 결과, N3N으로 형질감염된 DF-1 섬유아세포에서 총 26개의 클론을 확립하였고, 시퀀싱 분석 결과 14개의 클론에서 indel 변형을 확인하였다. 동일한 방법으로 N3P로 형질감염된 실험군에서 총 18개의 클론을 확립하였고, 이 중 6개의 클론에서 indel 변형을 확인하였다. N4P로 형질감염된 DF-1 섬유아세포에서는 총 22개의 클론을 확립하였고, 이 중 16개의 클론에서 변형을 확인하였다. Indel 변형이 유도된 DF-1 클론의 서열분석 결과를 하기 표 3에 나타내었다. 하기 표 3에 나타낸 바와 같이, 닭 DF-1 섬유아세포에서 indel 변형을 유도함에 있어서 CRISPR/Cas9 시스템이 효과적인 도구임을 확인하였다.As a result, a total of 26 clones were established in DF-1 fibroblasts transfected with N3N, and sequencing analysis confirmed indel modifications in 14 clones. In the same way, a total of 18 clones were established in the experimental group transfected with N3P, and 6 of them identified indel modifications. A total of 22 clones were established in DF-1 fibroblasts transfected with N4P, of which 16 were identified for modification. The results of sequencing of the Indel modification-induced DF-1 clone are shown in Table 3 below. As shown in Table 3 below, it was confirmed that the CRISPR / Cas9 system is an effective tool for inducing indel modification in chicken DF-1 fibroblasts.
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC (서열번호 13)TTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC (SEQ ID NO: 13)
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GaAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
G a AGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GaAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
G a AGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TG G
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCA A C TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TG G
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TG G
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TG G
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TG G
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TG G
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCG A G ATA C A GAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCA a CC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCA a CC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
AGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGA A CCCACC TGG G
AGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
TGGGAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTC A CCGAGC tcggtg T C T CC
TGG GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCA a CC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
실시예Example 5. 5. ALVALV -J 바이러스에 감수성을 보유하는 클론 선별Clones susceptible to -J virus
상기 실시예 4에서 확립한 모든 chNHE1 유전자 변형된 DF-1 클론 중 바이러스 감염 확인 및 유세포 분석(flow cytometry)을 실시할 20개의 클론을 선별하였다. 그 후, 확립된 DF-1 클론의 ALV-J에 대한 감수성을 평가하기 위하여 RCAS-J-EGFP 벡터를 도입하여 chNHE1 유전자를 타겟으로 하는 DF-1 클론에 ALV-J 바이러스를 감염시켰다. 상기 벡터에 ALV-J gag, env 및 pol 유전자와 함께 녹색 형광 단백질(GFP)-발현 카세트를 포함시켜 야생형(wild-type, WT) 세포와 비교하여 GFP가 발현되었는지 여부에 따라 바이러스 감염 여부를 판단하였다. 이와 같은 벡터의 개열지도는 도 1의 우측에 나타내었다. Of all the chNHE1 genetically modified DF-1 clones established in Example 4, 20 clones were selected for viral infection identification and flow cytometry. Then, to assess the susceptibility of the established DF-1 clone to ALV-J, the RCAS-J-EGFP vector was introduced to infect the ALV-J virus to the DF-1 clone targeting the chNHE1 gene. Including the green fluorescent protein (GFP) -expressing cassette together with the ALV-J gag, env, and pol genes in the vector to determine whether the virus is infected according to whether GFP is expressed compared to wild-type (WT) cells. It was. The cleavage map of such a vector is shown on the right side of FIG.
구체적으로, RCAS-J-EGFP DNA 5 μg을 Opti-MEM(Thermo Fisher-Invitrogen)에서 Lipofectamine 2000 시약(Thermo Fisher-Invitrogen)과 혼합하고, 상기 혼합물을 5 x 105 개의 DF-1 세포에 도입하여 형질전환 시켰다. 형질전환 6시간 후, 형질전환 혼합물을 DF-1 배양 배지로 교환하였다. 형질전환을 수행하고 1일 경과 후, DF-1 세포에서 바이러스 감염을 의미하는 녹색 형광을 검출하였다. 세포를 계대배양하고, 계대 하루 경과시 배양 배지를 교체하였다. 1일 후, 바이러스를 포함하는 배지를 수확(harvest)한 후 사용 전까지 -70℃에서 동결 보관하였다. 이후, 바이러스 감염 수행 시에는 바이러스를 포함하는 배지를 37℃로 녹여 DF-1 클론에 첨가하였다. 4 dpi(days post-infection)에 DF-1 클론을 형광현미경(TU-80; Nikon, Tokyo, Japan) 하에서 관찰하고 FACSCalibur software (BD Biosciences, San Jose, CA, USA)를 사용하여 형광을 분석하였다. 그 결과를 도 3에 나타내었다.Specifically, 5 μg of RCAS-J-EGFP DNA was mixed with Lipofectamine 2000 reagent (Thermo Fisher-Invitrogen) in Opti-MEM (Thermo Fisher-Invitrogen), and the mixture was introduced into 5 x 10 5 DF-1 cells. Transformed. After 6 hours of transformation, the transformation mixture was exchanged with DF-1 culture medium. One day after the transformation, green fluorescence was detected in DF-1 cells, indicating viral infection. Cells were passaged and culture medium was replaced one day after passage. After 1 day, the medium containing the virus was harvested and stored frozen at −70 ° C. until use. Then, when performing viral infection, the medium containing the virus was dissolved at 37 ° C. and added to the DF-1 clone. At 4 dpi (days post-infection), the DF-1 clone was observed under a fluorescence microscope (TU-80; Nikon, Tokyo, Japan) and analyzed for fluorescence using FACSCalibur software (BD Biosciences, San Jose, CA, USA). . The results are shown in FIG.
도 3의 A는 DF-1 세포의 GFP 발현을 나타낸 것으로, 총 20개의 DF-1 클론을 형광현미경으로 관찰한 결과를 나타내었다(Scale bar = 200μm). 도 3의 A에 나타낸 바와 같이, N3P#12는 야생형(WT) DF-1과 유사한 정도의 강한 GFP 발현을 보였으며, N3N#13, N4P#11 및 N4P#23은 중등도의 GFP 발현을 나타냄을 확인하였다. 이를 제외한 나머지 16개의 클론은 매우 낮은 GFP 발현을 나타내었다. 3A shows GFP expression of DF-1 cells, and a total of 20 DF-1 clones were observed by fluorescence microscopy (Scale bar = 200 μm). As shown in A of FIG. 3,
도 3의 B에 chNHE1 조기 종결 코돈(premature stop codon)을 가진 바이러스 감염된 DF-1 클론, C에는 chNHE1 조기 종결 코돈이 없고, Trp38의 결실이 있거나/없는 바이러스 감염된 DF-1 클론의 유세포 분석 결과를 나타내었다(각 막대는 모든 실험을 3회 반복으로 행한 표준편차를 의미). 도 3의 B에 나타낸 바와 같이, chNHE1 수용체 상에 조기 종결 코돈을 가진 DF-1 클론들(N3N#1, N3N#8, N3N#10, N3N#18, N3N#19, N3N#20, N3N#21, N3N#22 및 N3N#27)은 GFP 발현이 거의 없었으며, 이를 통해 ALV-J에 대한 절대적인 저항성을 획득하였음을 확인하였다. 또한, 도 3의 C에 나타낸 바와 같이, 인공적으로 생성된 조기 종결 코돈이 없고, indel 변형이 유도된 DF-1 클론들은 다양한 ALV-J 바이러스 감수성을 나타냄을 확인하였다. 구체적으로, Trp38 결실이 있고, indel 변형이 유도된 DF-1 클론들(N3N#37, N4P#2, N4P#12, N4P#15, N4P#16 및 N4P#27)은 Trp38 결실이 없고, indel 변형이 유도된 DF-1 클론들(N3N#13, N3P#12, N4P#11 및 N4P#23)보다 GFP 발현이 현저히 낮게 나타남을 확인하였다. The flow cytometry results of the virus infected DF-1 clone with chNHE1 premature stop codon in FIG. 3B and the virus infected DF-1 clone without CNHE1 early stop codon and with / without deletion of Trp38 in C (Each bar represents the standard deviation of all experiments repeated three times). As shown in FIG. 3B, DF-1 clones with early termination codons on the chNHE1 receptor (
실시예Example 6. 6. 보다 정확한More accurate 유전자 편집을 위한 For gene editing ssODN의ssODN 도입 Introduction
Trp38 결실에 따른 보다 정확한 효과를 확인하기 위하여, CRISPR/Cas9 벡터와 함께 Trp38 결실 및 BsaI 제한효소 부위를 생성하기 위한 T96G 치환을 포함하는 ssODN을 도입하여 chNHE1 유전자의 상동 재조합을 수행하였다. 이를 통해 CRISPR/Cas9 벡터와 ssODN을 결합하여 세포를 형질전환시키고, Trp38 결실을 가지는 chNHE1 대립유전자를 얻을 수 있을 것으로 예상하였으며, ssODN을 주형으로 이용한 상동 재조합을 도 4의 A에 도식화하였다.To confirm the more accurate effect of Trp38 deletion, homologous recombination of the chNHE1 gene was performed by introducing ssODN containing a Trp38 deletion and a T96G substitution to generate a BsaI restriction site with the CRISPR / Cas9 vector. Through this, the CRISPR / Cas9 vector and the ssODN were combined to transform the cells, and the chNHE1 allele having the Trp38 deletion was expected to be obtained. Homologous recombination using ssODN as a template is illustrated in A of FIG. 4.
구체적으로, 상동 재조합(homologous recombination)을 위하여 단일가닥 올리고데옥시뉴클레오티드(single-stranded oligodeoxynucleotide, ssODN) 2nmol을 3μg의 CRISPR/Cas9 퓨로마이신 벡터와 함께 사용하였다. 사용한 ssODN의 염기서열은 상기 표 2에 나타내었다. 상기 실시예 2에서 수행한 방법과 동일하게, 형질전환 6시간 후 형질전환 혼합물을 DF-1 배양 배지로 교환하였다. 형질전환을 수행하고 1일 경과 후, 퓨로마이신(1 ㎍/ml)을 배지에 첨가하였다. 퓨로마이신을 이용한 선별이 완료된 후, N3Pss(NHE1#3-Puro + ssODN) 처리된 DF-1 세포들을 선택 및 증식시켰다.Specifically, 2 nmol of single-stranded oligodeoxynucleotide (ssODN) was used with 3 μg of CRISPR / Cas9 puromycin vector for homologous recombination. The base sequence of ssODN used is shown in Table 2 above. In the same manner as in Example 2 above, 6 hours after transformation, the transformation mixture was exchanged with DF-1 culture medium. One day after the transformation was performed, puromycin (1 μg / ml) was added to the medium. After selection with puromycin was completed, N3Pss (NHE1 # 3-Puro + ssODN) treated DF-1 cells were selected and expanded.
또한, chNHE1의 타겟 대립유전자(allele)를 확인하기 위하여, N3Pss(NHE1#3-Puro + SSODN) 또는 N4Pss(NHE1#4-Puro + SSODN) 처리한 DF-1 세포 및 N3Pss 단일 클론의 유전체 DNA를 분석하였다. CRISPR/Cas9 타겟 부위를 포함하는 유전자 부위는 상기 표 2에 나타낸 프라이머 세트를 이용하여 증폭하였다. PCR은 총 20㎕의 100ng의 유전체 DNA에 대하여 10x PCR 버퍼(BioFACT, Daejeon, Korea), 0.4㎕ dNTPs(각각 10mM), 각 프라이머 10pmol 및 0.5 U Taq 폴리머레이즈(BioFACT)를 사용하여 다음과 같은 조건으로 수행하였다: 5분 동안 94℃에서 1차 변성을 1회 반복하였고, 후에 20초 동안 94℃에서 변성, 40초 동안 61℃에서 어닐링(annealing) 및 120초 동안 72℃에서 연장하는 단계를 35회 반복하였으며, 마지막으로 10분 동안 72℃에서 후-연장을 1회 반복하였다. 앰플리콘(amplicon)들은 37℃에서 1시간 동안 BsaI 제한효소(New England Biolabs)로 처리한 후 1% 아가로즈 젤을 사용하여 전기영동하여 분석하였다. 이후 이를 Gel Doc gel imaging system(Bio-Rad Laboratories, Hercules, CA, USA)를 사용하여 정량화하였다. 그 결과를 도 4의 B 및 C에 나타내었다.In addition, in order to identify the target allele of chNHE1, genomic DNA of DF-1 cells and N3Pss monoclones treated with N3Pss (NHE1 # 3-Puro + SSODN) or N4Pss (NHE1 # 4-Puro + SSODN) was cloned. Analyzed. Gene regions comprising CRISPR / Cas9 target sites were amplified using the primer sets shown in Table 2 above. PCR was performed using 10 × PCR buffer (BioFACT, Daejeon, Korea), 0.4 μl dNTPs (10 mM each), 10 pmol of each primer and 0.5 U Taq polymerase (BioFACT) for a total of 20 μl of 100 ng of genomic DNA. The first denaturation was repeated once at 94 ° C. for 5 minutes, followed by denaturation at 94 ° C. for 20 seconds, annealing at 61 ° C. for 40 seconds and extending at 72 ° C. for 120 seconds. Repeated one time, the last post-extension was repeated once at 72 ° C. for 10 minutes. Amplicons were analyzed by electrophoresis using 1% agarose gel after treatment with BsaI restriction enzyme (New England Biolabs) at 37 ° C. for 1 hour. This was then quantified using a Gel Doc gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA). The results are shown in B and C of FIG. 4.
도 4의 B에 NHE1#3-puro + ssODN (N3Pss), NHE1#4-puro + ssODN (N4Pss) 처리된 DF-1 세포에 BsaI 제한효소를 처리한 결과를 나타내었다. 도 4의 B에 나타낸 바와 같이, 제한효소 절단 분석 결과 BsaI가 NHE1#3-puro + ssODN (N3Pss), NHE1#4-puro + ssODN (N4Pss)으로 형질감염된 DF-1 세포에서 각각 7.8% 및 6.7%의 효율로 절단 밴드를 생성하였음을 관찰하여, chNHE1 유전자에서 보다 정확한 유전자 편집이 이루어졌음을 확인하였다. 도 4의 C에는 CRISPR/Cas9 벡터 및 ssODN으로 형질전환된 DF-1 섬유아세포를 시퀀싱한 결과를 나타내었다. 도 4의 C에 나타낸 바와 같이, N3Pss로 형질감염된 DF-1 섬유아세포는 Trp38 결실과 함께 T96G 치환이 유도됨을 확인하였으며, N4Pss 실험군에서는 Trp38 결실 없이 indel 변형이 유도된 것을 확인하였다.4B shows the results of treatment of BsaI restriction enzymes in DF-1 cells treated with NHE1 # 3-puro + ssODN (N3Pss) and NHE1 # 4-puro + ssODN (N4Pss). As shown in FIG. 4B, restriction enzyme cleavage assay showed that BsaI was 7.8% and 6.7 in DF-1 cells transfected with NHE1 # 3-puro + ssODN (N3Pss) and NHE1 # 4-puro + ssODN (N4Pss), respectively. Observation of the generation of the cleavage band at the efficiency of% confirmed that more accurate gene editing was made in the chNHE1 gene. 4C shows the results of sequencing DF-1 fibroblasts transformed with CRISPR / Cas9 vector and ssODN. As shown in FIG. 4C, it was confirmed that DF-1 fibroblasts transfected with N3Pss induced T96G substitution along with Trp38 deletion, and that indel modification was induced without Trp38 deletion in the N4Pss experimental group.
한편, N3Pss 실험군의 염기서열 분석 결과 Trp38 결실 및 T96G 치환이 유도된 대립유전자의 존재가 예상되었기 때문에, 추가적으로 실험군에서 몇 개의 단일 클론(single clone)을 확립하였다. 총 15개의 클론을 확립하였으며, 하기 표 4에 Trp38 타겟 변형이 일어난 모든 클론의 염기서열 분석 결과를 나타내었다. 하기 표 4에 나타낸 바와 같이, 4개 클론들(N3Pss#4, N3P#12, N3P#13 및 N3P#18)은 Trp38 결실과 함께 T96G 치환을 포함하고 있었으며, 다른 4개 클론들(N3Pss#1, N3Pss#5, N3Pss#8 및 N3Pss#11)은 T96G 치환 없이 Trp38 결실만 포함하고 있음을 확인하였다. N3Pss#9는 또 다른 3-bp 결실과 함께 재조합 Trp38 대립 유전자를 가지고 있음을 확인하였다. 나아가, indel 변형이 유도된 그룹에서, 4개 클론들(N3Pss#6, N3Pss#7, N3Pss#10 및 N3Pss#17)은 chNHE1 수용체에서 조기 종결 코돈을 형성했으며, 다른 2개 클론들(N3Pss#14 및 N3Pss#16)은 조기 종결 코돈을 형성하지 않음을 확인하였다. N3Pss#18의 경우 변형 대립 유전자와 야생형(WT) 대립 유전자의 이형접합체(heterozygous)임을 확인하였다.On the other hand, since the presence of alleles induced by Trp38 deletion and T96G substitution as a result of sequencing of the N3Pss experimental group, several single clones were additionally established in the experimental group. A total of 15 clones were established, and Table 4 shows the results of sequencing of all clones in which the Trp38 target modification occurred. As shown in Table 4 below, four clones (
DF-1 클론의 이형접합성(heterozygosity)을 확인하기 위하여 PCR 산물에 BsaI 제한효소를 처리하고, 그 결과를 도 4의 D에 나타내었다. 도 4의 D에 나타낸 바와 같이, 상기 방법으로 ssODN 주형에서 유래한 BsaI 제한효소를 가진 DF-1 클론과 가지지 않은 DF-1 클론 간의 명확한 차이를 확인하였다(* 표시는 절단된 밴드를 의미). 상기 결과를 통해, N3Pss#18은 이형접합체(heterozygous)임을 확인하였다.In order to confirm the heterozygosity of the DF-1 clone, PCR products were treated with BsaI restriction enzymes, and the results are shown in FIG. 4D. As shown in FIG. 4D, the method identified a clear difference between the DF-1 clone with the BsaI restriction enzyme derived from the ssODN template and the DF-1 clone without it (* indicates cut band). Through the above results, it was confirmed that
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC (서열번호 13)TTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC (SEQ ID NO: 13)
GAGCAGCvCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCvCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCG G GTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAaGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GA a GCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCG G GTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCG G GTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TG G
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC /TTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACCTGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCCTTGCAGGCCGACGCCACGCG G GTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC / TTGCAGGCCGACGCCACGCGTGTCTCCGAGCCCACC TGG
GAGCAGCCGTGGGGAGAGCCCGGGGGTATCACCGCC
실시예Example 7. 바이러스 감염 실험 7. Virus Infection Experiment
상기 실시예 6에서 확립된 chNHE1 유전자의 Trp38을 정교하게 타겟팅한 DF-1 클론의 ALV-J에 대한 감수성을 평가하기 위하여 RCAS-J-EGFP 벡터를 도입하여 실시예 5와 동일한 방법으로 바이러스 감염 실험을 수행하였다. 그 결과를 도 5에 나타내었다.Viral infection experiment in the same manner as in Example 5 by introducing the RCAS-J-EGFP vector to evaluate the susceptibility to ALV-J of the DF-1 clone precisely targeting Trp38 of the chNHE1 gene established in Example 6 Was performed. The results are shown in FIG.
도 5의 A에 나타낸 바와 같이, chNHE1 유전자의 Trp38을 정교하게 타겟팅한 DF-1 클론들(N3Pss#1, N3Pss#4, N3Pss#5, N3Pss#8, N3Pss#9, N3Pss#11, N3Pss#12 및 N3Pss#13)은 매우 낮은 GFP 발현을 나타내어, ALV-J에 대한 저항성을 획득하였음을 확인하였다(이형접합성을 나타내는 N3Pss#18 제외). 또한, 도 5의 B에 나타낸 바와 같이, chNHE1 유전자 상에 조기 종결 코돈을 생성하는 indel 변형을 가진 DF-1 클론들(N3Pss#6, N3Pss#7, N3Pss#10 및 N3Pss#17) 역시 매우 낮은 GFP 발현을 나타냄을 확인하였다. 조기 종결 코돈이 없고, Trp38 결실을 포함하는 indel 변형이 유도된 DF-1 클론(N3Pss#16)의 경우 Trp38 결실을 포함하지 않는 indel 변형이 유도된 DF-1 클론(N3Pss#14)에 비하여 현저하게 낮은 GFP 발현을 나타내어, 이를 통해 Trp38이 ALV-J 감염에 중요한 역할을 함을 다시 한 번 확인하였다. 상기의 특징을 가지는 형질전환 세포주들 중 N3Pss#12 클론을 2018년 4월 25일 한국세포주연구재단 (KCLRF, Korean Cell Line Research Foundation)에 수탁번호 KCLRF-BP-00433로 기탁하였다.As shown in FIG. 5A, DF-1 clones precisely targeting Trp38 of the chNHE1 gene (
실시예Example 8. 8. chNHE1chNHE1 수용체의 단백질 서열 분석 Protein sequence analysis of the receptor
ALV-J 바이러스 감염에 영향을 미치는 chNHE1 수용체 잔기들을 확인하기 위하여, 하기 표 5와 같이 미스센스 돌연변이(missense mutation)를 가진 chNHE1 변형된 DF-1 클론의 추론된 아미노산 서열을 분석하였다. 하기 표 5에 나타낸 바와 같이, 돌연변이체와 바이러스 감염된 클론의 아미노산 서열을 비교한 결과, Trp38만큼은 아니지만 Thr37 및 Gln40에서의 단일 아미노산 결실 역시 바이러스 유입(entry)에 영향을 미침을 확인하였다. 또한, 돌연변이를 삽입시켜 아미노산 사슬이 연장되거나(N4P#26) 또는 큰 결실(N4P#12)이 유도된 DF-1 세포들이 낮은 GFP 발현을 나타냄을 통해, chNHE1 단백질의 이차 구조가 바이러스 감염에 중요한 역할을 함을 확인하였다.In order to identify chNHE1 receptor residues affecting ALV-J virus infection, the deduced amino acid sequences of chNHE1 modified DF-1 clones with missense mutations were analyzed as shown in Table 5 below. As shown in Table 5, when comparing the amino acid sequences of mutants and virus infected clones, it was confirmed that single amino acid deletions in Thr37 and Gln40, but not as much as Trp38, also affect viral entry. In addition, the secondary structure of the chNHE1 protein is important for viral infection through the insertion of mutations, resulting in low GFP expression in DF-1 cells with extended amino acid chains (N4P # 26) or large deletions (N4P # 12). It was confirmed to play a role.
[표 5]TABLE 5
다음으로, chNHE1 수용체의 구조적 특성을 확인하기 위하여 생물정보학적 분석을 수행하였다. TMHMM v2.0 software(Krogh et al., 2001)를 사용하여 chNHE1의 트랜스멤브레인 구조(transmembrane topology)를 분석하였다. 또한, chNHE1의 이차 구조를 PSIPRED(Jones, 1999) 및 웹서버 DISOPRED3(Jones and Cozzetto, 2015)로 분석하였다. 그 결과를 도 6에 나타내었다.Next, bioinformatics analysis was performed to confirm the structural characteristics of the chNHE1 receptor. The transmembrane topology of chNHE1 was analyzed using TMHMM v2.0 software (Krogh et al., 2001). In addition, the secondary structure of chNHE1 was analyzed by PSIPRED (Jones, 1999) and web server DISOPRED3 (Jones and Cozzetto, 2015). The results are shown in FIG.
chNHE1의 트랜스멤브레인 구조는 N-말단 부위를 제외하고는 이전의 연구결과(Chai and Bates, 2006; Kucerova et al., 2013)와 동일하게 나타남을 확인하였다. 도 6의 A에 나타낸 바와 같이, TMHMM을 사용한 분석 결과 chNHE1의 N-말단 부위(Met1-Ala28 잔기)는 막에 내재된 신호 펩타이드로서 α-나선 구조를 가짐을 예상할 수 있었다. 또한, 도 6의 B에 나타낸 바와 같이, DISOPRED3을 사용하여 단백질 결합 부위 및 disorder 경향(propensity)을 분석한 결과 잔기 1-10 및 잔기 21-83이 disordered 단백질 부위인 것으로 예상되었다(잔기 1-73에 대한 예측은 confidence score > 0.90에 따라 얻어졌다). 이를 통해, ALV-J 유입 저지에 중요한 역할을 수행하는 Ser34와 Gln40 사이 잔기들이 IDR(intrinsically disordered regions)에 속함을 확인하였다. 그러나, 상기 잔기들은 높은 confidence score(>0.8)로 disordered 단백질 결합 부위(잔기 1-12 및 잔기 53-72)에는 포함되지 않음을 확인하였다.The transmembrane structure of chNHE1 was confirmed to be the same as in previous studies (Chai and Bates, 2006; Kucerova et al., 2013) except for the N-terminal region. As shown in FIG. 6A, as a result of analysis using TMHMM, the N-terminal region (Met1-Ala28 residue) of chNHE1 can be expected to have α-helix structure as a signal peptide inherent in the membrane. In addition, as shown in FIG. 6B, analysis of protein binding site and disorder propensity using DISOPRED3 predicted that residues 1-10 and 21-83 were disordered protein sites (residues 1-73). Predictions were obtained according to a confidence score> 0.90). Through this, it was confirmed that residues between Ser34 and Gln40, which play an important role in inhibiting ALV-J influx, belong to intrinsically disordered regions (IDRs). However, the residues were confirmed to be not included in the disordered protein binding site (residues 1-12 and residues 53-72) with a high confidence score (> 0.8).
이를 통해, 단순히 Trp38뿐만 아니라 chNHE1 수용체의 ECL1(Extracellular loop 1) 영역(Met1-Val119)의 단백질 이차 구조도 ALV-J에 대한 민감도에 영향을 미침을 확인하였다.Through this, it was confirmed that not only Trp38 but also the protein secondary structure of the extracellular loop 1 (ECL1) region (Met1-Val119) of the chNHE1 receptor affects the sensitivity to ALV-J.
실시예Example 9. 재조합 벡터를 이용한 9. Recombinant Vectors 원시생식세포(PGC)의Of primitive germ cells (PGC) 형질감염 Transfection
상기 실시예 1에서 제조한 CRISPR/Cas9 재조합 벡터(3 ㎍)를 Opti-MEM(Thermo Fisher-Invitrogen)에서 Lipofectamine 2000 시약(Thermo Fisher-Invitrogen)과 혼합하고, 상기 혼합물을 5 x 105 개의 원시생식세포(Primordial germ cells, 이하 PGC)에 도입하여 형질전환 시켰다. 혼합물 도입 4시간 후, PGC 배양액으로 교체하였다. 유전자 도입 하루 또는 이틀 후, 퓨로마이신(1 ㎍/ml)을 배양액에 첨가하였다. 선별은 2일 동안 실시하였다.The CRISPR / Cas9 recombinant vector (3 μg) prepared in Example 1 was mixed with Lipofectamine 2000 reagent (Thermo Fisher-Invitrogen) in Opti-MEM (Thermo Fisher-Invitrogen), and the mixture was mixed with 5 × 10 5 primitive reproduction. The cells were transformed into primitive germ cells (hereinafter referred to as PGCs). Four hours after the introduction of the mixture, it was replaced with PGC culture. One or two days after transduction, puromycin (1 μg / ml) was added to the culture. Screening was carried out for 2 days.
이때 사용한 PGC는 20% FBS (Invitrogen), 2% chicken serum (Sigma-Aldrich, St. Louis, MO, USA), 1×nucleosides (Millipore, Temecula, CA, USA), 2 mM L-glutamine, 1×nonessential amino acids, β-mercaptoethanol, 10 mM sodium pyruvate, 1×antibiotic-antimycotic (Invitrogen), human basic fibroblast growth factor (10 ng/mL; Sigma-Aldrich)가 포함된 knockout DMEM(Invitrogen) 배양액을 사용하였으며, 37℃, 5% CO2, 60-70% 상대 습도 조건에서 배양되었다. 이 세포는 mitomycin-inactivated mouse embryonic fibroblasts (MEFs)위에서 배양되었으며, 5-6일 간격으로 계대 배양되었다.PGCs used were 20% FBS (Invitrogen), 2% chicken serum (Sigma-Aldrich, St. Louis, MO, USA), 1 × nucleosides (Millipore, Temecula, CA, USA), 2 mM L-glutamine, 1 × Knockout DMEM (Invitrogen) cultures containing nonessential amino acids, β-mercaptoethanol, 10 mM sodium pyruvate, 1 × antibiotic-antimycotic (Invitrogen), and human basic fibroblast growth factor (10 ng / mL; Sigma-Aldrich) were used. Incubated at 37 ° C., 5% CO 2 , 60-70% relative humidity conditions. The cells were cultured on mitomycin-inactivated mouse embryonic fibroblasts (MEFs) and passaged at 5-6 day intervals.
실시예Example 10. 10. chNHE1chNHE1 유전자의 Gene Trp38Trp38 indelindel 유도 확인 Induction confirmation
상기 실시예 9에서 생산한 형질전환 PGC에서 chNHE1 유전자의 Trp38 위치에 indel이 정상적으로 유도되었는지 여부를 확인하기 위하여 T7 엔도뉴클라아제 1(T7E1) 분석을 수행하였다. 구체적으로 이형접합(heteroduplex) DNA 구조를 형성하기 위하여 상기 PCR 산물을 변성 이후 리어닐링하였고, 그 후 리어닐링한 산물을 37℃에서 20분 동안 T7E1(New England Biolabs) 효소로 분해하였다. T7E1으로 분해된 산물은 1% 아가로스 겔을 사용하여 전기영동시켰다. PCR 산물은 pGEM-T easy 벡터 (promega)에 클로닝하여 염기서열 분석을 실시하였다. 그 결과를 도 7에 나타내었다.T7 endonuclease 1 (T7E1) analysis was performed to determine whether indel was normally induced at the Trp38 position of the chNHE1 gene in the transformed PGC produced in Example 9. Specifically, in order to form a heteroduplex DNA structure, the PCR product was renealed after denaturation, and then the renealed product was digested with T7E1 (New England Biolabs) enzyme at 37 ° C. for 20 minutes. The product digested with T7E1 was electrophoresed using 1% agarose gel. PCR products were cloned into pGEM-T easy vector (promega) for sequencing. The results are shown in FIG.
도 7에 나타낸 바와 같이, PGC(NHE1#3 PGC)에서 chNHE1 유전자의 Trp38 위치에 4 bp, 5 bp의 indel이 유도된 것을 T7E1, 염기서열 분석법을 통해 확인하였다. 상기의 특징을 가지는 형질전환 세포주를 2018년 4월 25일 한국세포주연구재단 (KCLRF, Korean Cell Line Research Foundation)에 수탁번호 KCLRF-BP-00432로 기탁하였다.As shown in FIG. 7, it was confirmed through T7E1, sequencing, that 4 bp and 5 bp of indel were induced at the Trp38 position of the chNHE1 gene in PGC (NHE1 # 3 PGC). Transgenic cell lines having the above characteristics were deposited with Korean Cell Line Research Foundation (KCLRF) on April 25, 2018 under accession number KCLRF-BP-00432.
상기 결과를 통해, 본 발명의 CRISPR/Cas9 시스템을 통한 유전자 편집은 닭 백혈병 바이러스 하위그룹 J(Avian leukosis virus-J, ALV-J)가 특이적으로 결합하는 chNHE1 수용체에 변이를 유도하여 저항성을 획득할 수 있음을 확인하였다. 특히, 정확한 유전자 편집을 위해 ssODN을 CRISPR/Cas9 벡터와 함께 도입하여 특정 아미노산의 결손만을 유도하여, 숙주 자체의 유전자를 성공적으로 편집함으로써 ALV-J 바이러스에 저항성을 획득할 수 있음을 확인하였다. 따라서, 본 발명에 따른 CRISPR/Cas9 시스템을 통한 유전자 편집은 질병 저항성 세포주, 질병 저항성 조류 생산에 널리 적용될 수 있다.Through the above results, gene editing through the CRISPR / Cas9 system of the present invention induces mutations in the chNHE1 receptor specifically bound to chicken leukemia virus subgroup J (Avian leukosis virus-J, ALV-J) to obtain resistance. It was confirmed that it can be done. In particular, ssODN was introduced together with the CRISPR / Cas9 vector for accurate gene editing to induce deletion of specific amino acids, and it was confirmed that resistance to ALV-J virus can be obtained by successfully editing the gene of the host itself. Therefore, gene editing via the CRISPR / Cas9 system according to the present invention can be widely applied to disease resistant cell lines, disease resistant algae production.
이상, 본 발명의 바람직한 실시예에 대하여 상세히 설명하였으나, 본 발명의 기술적 범위는 전술한 실시예에 한정되지 않고 특허청구범위에 의하여 해석되어야 할 것이다. 이때, 이 기술분야에서 통상의 지식을 습득한 자라면, 본 발명의 범위에서 벗어나지 않으면서도 많은 수정과 변형이 가능함을 고려해야 할 것이다.As mentioned above, although the preferred embodiment of this invention was described in detail, the technical scope of this invention is not limited to the above-mentioned embodiment, It should be interpreted by the claim. At this time, those of ordinary skill in the art should consider that many modifications and variations are possible without departing from the scope of the present invention.
<110> Seoul National University R&DB Foundation The Pirbright Institute <120> A METHOD FOR PRODUCING AVIAN LEUKOSIS VIRUS(ALV)-RESISTANT AVIAN LINE USING CRISPR/CAS9 SYSTEM <130> SNU1-140P-1 <150> KR 10-2018-0011871 <151> 2018-01-31 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NHE1#3 <400> 1 ctctccccac ggctgctccc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NHE1#4 <400> 2 gcgtgtctcc gagcccacct 20 <210> 3 <211> 346 <212> DNA <213> Artificial Sequence <220> <223> U6-NHE1#3-gRNA scaffold <400> 3 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240 cgaaacaccg ctctccccac ggctgctccc gttttagagc tagaaatagc aagttaaaat 300 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 346 <210> 4 <211> 346 <212> DNA <213> Artificial Sequence <220> <223> U6-NHE1#4-gRNA scaffold <400> 4 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240 cgaaacaccg gcgtgtctcc gagcccacct gttttagagc tagaaatagc aagttaaaat 300 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 346 <210> 5 <211> 157 <212> DNA <213> Artificial Sequence <220> <223> wild-type chNHE1 <400> 5 gcccgctgct gcccggccag cgcttgcagg ccgacgccac gcgtgtctcc gagcccacct 60 gggagcagcc gtggggagag cccgggggta tcaccgccgc cccgctggcc acggcccagg 120 aggtgcaccc gctgaacaaa cagcaccaca accactc 157 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> chNHE1 #3 F <400> 6 caccgctctc cccacggctg ctccc 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> chNHE1 #3 R <400> 7 aaacgggagc agccgtgggg agagc 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> chNHE1 #4 F <400> 8 caccggcgtg tctccgagcc cacct 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> chNHE1 #4 R <400> 9 aaacaggtgg gctcggagac acgcc 25 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> chNHE1 seq F <400> 10 gcacctcacg cctgtgcaac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> chNHE1 seq R <400> 11 gggatgcgga cgtgcgagta 20 <210> 12 <211> 157 <212> DNA <213> Artificial Sequence <220> <223> ssODN <220> <221> misc_feature <222> (60)..(62) <223> nucleotide sequence encoding Trp38 which is deleted from the ssODN <400> 12 gcccgctgct gcccggccag cgcttgcagg ccgacgccac gcgggtctcc gagcccacct 60 gggagcagcc gtggggagag cccgggggta tcaccgccgc cccgctggcc acggcccagg 120 aggtgcaccc gctgaacaaa cagcaccaca accactc 157 <210> 13 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> wild-type DF-1 fibroblast <400> 13 ttgcaggccg acgccacgcg tgtctccgag cccacctggg agcagccgtg gggagagccc 60 gggggtatca ccgcc 75 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> wild-type DF-1 fibroblast <400> 14 Arg Val Ser Glu Pro Thr Trp Glu Gln Pro Trp Gly Glu Pro Gly Gly 1 5 10 15 Ile <110> Seoul National University R & DB Foundation The Pirbright Institute <120> A METHOD FOR PRODUCING AVIAN LEUKOSIS VIRUS (ALV) -RESISTANT AVIAN LINE USING CRISPR / CAS9 SYSTEM <130> SNU1-140P-1 <150> KR 10-2018-0011871 <151> 2018-01-31 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NHE1 # 3 <400> 1 ctctccccac ggctgctccc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NHE1 # 4 <400> 2 gcgtgtctcc gagcccacct 20 <210> 3 <211> 346 <212> DNA <213> Artificial Sequence <220> <223> U6-NHE1 # 3-gRNA scaffold <400> 3 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240 cgaaacaccg ctctccccac ggctgctccc gttttagagc tagaaatagc aagttaaaat 300 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 346 <210> 4 <211> 346 <212> DNA <213> Artificial Sequence <220> <223> U6-NHE1 # 4-gRNA scaffold <400> 4 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240 cgaaacaccg gcgtgtctcc gagcccacct gttttagagc tagaaatagc aagttaaaat 300 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 346 <210> 5 <211> 157 <212> DNA <213> Artificial Sequence <220> <223> wild-type chNHE1 <400> 5 gcccgctgct gcccggccag cgcttgcagg ccgacgccac gcgtgtctcc gagcccacct 60 gggagcagcc gtggggagag cccgggggta tcaccgccgc cccgctggcc acggcccagg 120 aggtgcaccc gctgaacaaa cagcaccaca accactc 157 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> chNHE1 # 3 F <400> 6 caccgctctc cccacggctg ctccc 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> chNHE1 # 3 R <400> 7 aaacgggagc agccgtgggg agagc 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> chNHE1 # 4 F <400> 8 caccggcgtg tctccgagcc cacct 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> chNHE1 # 4 R <400> 9 aaacaggtgg gctcggagac acgcc 25 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> chNHE1 seq F <400> 10 gcacctcacg cctgtgcaac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> chNHE1 seq R <400> 11 gggatgcgga cgtgcgagta 20 <210> 12 <211> 157 <212> DNA <213> Artificial Sequence <220> <223> ssODN <220> <221> misc_feature (222) (60) .. (62) <223> nucleotide sequence encoding Trp38 which is deleted from the ssODN <400> 12 gcccgctgct gcccggccag cgcttgcagg ccgacgccac gcgggtctcc gagcccacct 60 gggagcagcc gtggggagag cccgggggta tcaccgccgc cccgctggcc acggcccagg 120 aggtgcaccc gctgaacaaa cagcaccaca accactc 157 <210> 13 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> wild-type DF-1 fibroblast <400> 13 ttgcaggccg acgccacgcg tgtctccgag cccacctggg agcagccgtg gggagagccc 60 gggggtatca ccgcc 75 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> wild-type DF-1 fibroblast <400> 14 Arg Val Ser Glu Pro Thr Trp Glu Gln Pro Trp Gly Glu Pro Gly Gly 1 5 10 15 Ile
Claims (16)
제2 프로모터 및 이와 작동가능하게 연결된 Cas9(CRISPR associated protein 9)을 암호화하는 유전자를 포함하는 ALV-J(Avian Leukosis Virus subgroup J, 닭 백혈병 바이러스 하위그룹 J) 저항성 조류모델 제작용 재조합 벡터.
GRNA (guide RNA) of chNHE1 (chicken Na + / H + exchange 1) operably linked to the first promoter; And
Recombinant vector for constructing ALV-J (Avian Leukosis Virus subgroup J) resistant avian model comprising a second promoter and a gene encoding Cas9 (CRISPR associated protein 9) operably linked thereto.
According to claim 1, wherein the first promoter is a U6 (human U6) promoter, ALV-J (Avian Leukosis Virus-J, chicken leukemia virus subgroup J) Recombinant vector for constructing a resistant avian model.
The method according to claim 1, wherein the gRNA (guide RNA) of chNHE1 (chicken Na + / H + exchange 1) is represented by SEQ ID NO: 1 or SEQ ID NO: 2, ALV-J (Avian Leukosis Virus-J, Chicken leukemia virus subgroup J) Recombinant vectors for constructing a resistant avian model.
According to claim 1, wherein the second promoter is CBh (chicken beta-actin) promoter, ALV-J (Avian Leukosis Virus-J, chicken leukemia virus subgroup J) Recombinant vector for the production of a resistant avian model.
The composition for genome calibration comprising the recombinant vector of claim 1 and ssODN (single-stranded oligodeoxynucleotide, single stranded oligodeoxynucleotide).
According to claim 5, The ssODN is complementary to the chNHE1 gene, characterized in that represented by the nucleotide sequence of SEQ ID NO: 12, genome calibration composition.
The composition of claim 6, wherein the ssODN induces Trp38 deletion and T96G substitution in the chNHE1 gene.
A transformed cell line for preparing an ALV-J resistant avian model, wherein the recombinant vector of any one of claims 1 to 4 or the genome correction composition of any one of claims 5 to 7 is introduced.
The transformed cell line of claim 8, wherein the transformed cell line is specifically modified at least one site selected from the group consisting of Trp38, Thr37, and Gln40 in the chNHE1 gene.
10. The method of claim 9, wherein the transformed cell line is characterized in that the accession number is KCLRF-BP-00433, ALV-J resistant avian model for the transformation cell line production.
The transformed cell line of claim 8, wherein the transformed cell line is primordial germ cells (PGCs) of a donor bird.
12. The transformed cell line of claim 11, wherein the primordial germ cells are derived from primordial genitalia of 1-10 day-old embryos of the donor alga.
13. The transformed cell line of claim 12, wherein the donor bird is a chicken.
The method of claim 11, wherein the transformed cell line is characterized in that the accession number KCLRF-BP-00432, ALV-J resistant avian model for the production of a transformed cell line.
상기 재조합 벡터 또는 유전체 교정용 조성물을 조류 체세포에 도입하는 단계;를 포함하는 ALV-J 저항성 조류모델 제작용 형질전환 세포주의 제조방법.
Preparing a recombinant vector according to any one of claims 1 to 4 or a composition for genome correction according to any one of claims 5 to 7; And
A method of producing a transformed cell line for preparing an ALV-J resistant avian model, comprising the step of introducing the recombinant vector or genomic correction composition into an avian somatic cell.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021141421A1 (en) * | 2020-01-07 | 2021-07-15 | 서울대학교산학협력단 | Method for producing genetically edited birds having resistance to avian influenza viruses |
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| KR20210126437A (en) | 2020-04-10 | 2021-10-20 | 차의과학대학교 산학협력단 | Vector system for light-inducible modulating of gene expression and uses thereof |
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| WO2022245110A1 (en) * | 2021-05-18 | 2022-11-24 | 서울대학교산학협력단 | Method for constructing bird that produces adiponectin and target protein |
| KR20230103866A (en) | 2021-12-29 | 2023-07-07 | 숙명여자대학교산학협력단 | CRISPR/Cas9 based irf3 specific sgRNA and use thereof |
| WO2024039236A1 (en) * | 2022-08-19 | 2024-02-22 | 서울대학교산학협력단 | System for substance delivery and gene editing within germ cells using exosomes |
| KR20240066516A (en) | 2022-11-03 | 2024-05-16 | 숙명여자대학교산학협력단 | CRISPR/Cas9 based on HepG2 cell irf3 specific sgRNA and Use thereof |
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