KR20210126437A - 광 유도 유전자 발현 조절용 벡터 시스템 및 이의 용도 - Google Patents
광 유도 유전자 발현 조절용 벡터 시스템 및 이의 용도 Download PDFInfo
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- KR20210126437A KR20210126437A KR1020200044313A KR20200044313A KR20210126437A KR 20210126437 A KR20210126437 A KR 20210126437A KR 1020200044313 A KR1020200044313 A KR 1020200044313A KR 20200044313 A KR20200044313 A KR 20200044313A KR 20210126437 A KR20210126437 A KR 20210126437A
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Abstract
광 유도 유전자 발현 조절용 벡터 시스템 및 이의 용도에 관한 것으로, 일 양상에 따른 줄기세포의 형질전환용 키트는 다수의 표적 유전자에 대한 가이드 RNA(guide RNA: gRNA)를 하나의 벡터를 이용하여 동시에 줄기세포에 도입시켜 빛의 조사에 의해 다수의 표적 유전자를 동시에 안정적으로 발현하는 줄기세포를 제작할 수 있다. 또한, 일 양상에 따른 줄기세포의 표적 유전자 발현을 조절하는 방법에 의하면, 상기 키트를 이용하여 형질전환한 줄기세포에 특정 파장의 빛을 조사 또는 중단함으로써 간단한 조작에 의하여 줄기세포의 표적 유전자 발현을 증가시키거나 다시 감소시킬 수 있다. 따라서, 상기 줄기세포의 형질전환용 키트, 형질전환한 줄기세포 및 줄기세포의 표적 유전자 발현을 조절하는 방법은 표적 유전자와 관련된 기초과학 연구 및 세포 치료제 분야에 유용하게 활용될 수 있다.
Description
광 유도 유전자 발현 조절용 벡터 시스템 및 이의 용도에 관한 것이다.
CRISPR(Clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated protein) 시스템을 이용한 유전자 편집 기술은 유전자치료 및 새로운 형질전환 생물의 생산 등 기초과학과 의료산업 분야에서 다양하게 사용되고 있다. Cas 단백질은 가이드 RNA(guide RNA: gRNA)와 결합하여 효과적으로 표적 유전자를 절단하여 DNA 이중가닥 절단을 일으키며, 절단된 DNA은 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 일어나므로, 표적화된 유전자 편집이 가능하다.
최근에는, 기술적인 진보로 CRISPR/Cas 시스템을 기반으로 유전자를 조절하는 새로운 시스템들이 제작되고 있으며, 일 예로, DNA를 절단하는 효소활성을 결핍시킨 Cas9 돌연변이(dead Cas9: dCas9)와 gRNA를 이용하여 표적 유전자의 발현을 조절할 수 있는 기술이 개발되고 있다. 또한, CRISPR/Cas 시스템과 광유전학(Optogenetics) 기술을 결합하여 빛의 조사에 의해 유전자 발현을 조절하는 기술이 시도되고 있으나, 줄기세포 분야에서는 이러한 연구가 미미한 실정이다.
일 양상은 줄기세포의 형질전환용 키트를 제공하는 것이다.
다른 양상은 상기 형질전환용 키트를 이용하여 형질전환한 줄기세포를 제공하는 것이다.
다른 양상은 상기 형질전환한 줄기세포의 표적 유전자 발현을 조절하는 방법을 제공하는 것이다.
일 양상은 dCas (dead Cas) 단백질 및 제1 광특이적 결합 단백질을 각각 코딩하는 폴리뉴클레오티드를 포함하는 제1 벡터; 및 줄기세포에서의 발현을 조절하려는 표적 유전자의 가이드 RNA(guide RNA: gRNA) 및 제2 광특이적 결합 단백질을 각각 코딩하는 폴리뉴클레오티드를 포함하는 제2 벡터를 포함하는, 줄기세포의 형질전환용 키트를 제공한다.
본 명세서에서 사용된 용어, "dCas"는 Cas (CRISPR-associated protein) 단백질에서 DNA를 절단하는 효소활성이 결핍된 단백질로 "dead Cas", "nuclease-deficient Cas", 또는 "nuclease-deactivated Cas"등과 혼용될 수 있다.
상기 dCas 단백질은 dCas9 단백질을 포함하는 것일 수 있다. 상기 dCas9 단백질을 코딩하는 폴리뉴클레오티드는 서열번호 27의 염기서열을 포함하는 것일 수 있다.
본 명세서에서 사용된 용어, "광특이적 결합 단백질"은 광유도 이형이합체(heterodimer) 형성 단백질 또는 광유도 동형이합체(homodimer) 형성 단백질로, 특정 파장의 빛의 조사에 의하여 서로 상이하거나 동일한 종류의 단백질이 결합하여 이형이합체 또는 동형이합체를 형성할 수 있는 단백질을 의미할 수 있다. 예를 들어, 상기 광특이적 결합 단백질은 광유도 이형이합체 형성 단백질일 수 있고, 구체적으로, CIB1(cryptochrome-interacting basic helix-loop-helix protein), CIBN(N-terminal fragment of CIB1), CRY2 (cryptochrome 2), CRY2PHR(N-terminal photolyase homology domain of CRY2), PhyB(phytochrome B), PIF(phytochrome interacting factor), FKF1(Flavin-binding, Kelch repeat, F-box 1), 또는 GIGANTEA일 수 있으나, 이에 제한되지 않는다.
상기 제1 광특이적 결합 단백질은 CIB1 또는 이의 단편을 포함하는 것일 수 있고, 상기 CIB1의 단편은 예를 들어, CIBN을 포함하는 것일 수 있다. 상기 CIBN을 코딩하는 폴리뉴클레오티드는 서열번호 28의 염기서열을 포함하는 것일 수 있다.
상기 제2 광특이적 결합 단백질은 CRY2 또는 이의 단편을 포함하는 것일 수 있고, 상기 CRY2의 단편은 예를 들어, CRY2PHR을 포함하는 것일 수 있다. 상기 CRY2를 코딩하는 폴리뉴클레오티드는 서열번호 29의 염기서열을 포함하는 것일 수 있다.
상기 제1 광특이적 결합 단백질과 제2 광특이적 결합 단백질은 청색광, 예를 들어, 약 440 내지 490 nm의 파장을 갖는 빛이 조사되면 서로 결합하여 이형이합체를 형성하는 것일 수 있다.
상기 제1 벡터는 dCas 단백질 및 제1 광특이적 결합 단백질을 각각 코딩하는 폴리뉴클레오티드에 작동 가능하게 연결된 프로모터를 더 포함하는 것일 수 있다. 예를 들어, 상기 제1 벡터는 Ef1α 프로모터, UbC 프로모터 및 CAGG 프로모터로 이루어진 군으로부터 선택되는 프로모터를 더 포함하는 것일 수 있다. 일 구체예에서, 상기 프로모터는 dCas 단백질 및 제1 광특이적 결합 단백질의 융합 단백질에 대한 전사를 조절하는 것일 수 있다.
본 명세서에서 사용된 용어, "작동 가능하게 연결된"것이란 목적 단백질을 코딩하는 폴리뉴클레오티드의 전사를 개시 및 매개하도록 하는 프로모터 서열과 상기 유전자 서열이 기능적으로 연결되어 있는 것을 의미할 수 있다.
상기 제1 벡터는 서열번호 21의 염기서열을 포함하는 것일 수 있다.
상기 제2 벡터는 dCas 단백질을 코딩하는 폴리뉴클레오티드의 전사를 촉진하는 인자를 코딩하는 폴리뉴클레오티드를 더 포함하는 것일 수 있다. 상기 전사를 촉진하는 인자는 예를 들어, VP64 또는 VP16일 수 있으나, 이에 제한되지 않는다. 상기 VP64를 코딩하는 폴리뉴클레오티드는 서열번호 30의 염기서열을 포함하는 것일 수 있다.
상기 제2 벡터는 제2 광특이적 결합 단백질 및 dCas 단백질을 코딩하는 폴리뉴클레오티드의 전사를 촉진하는 인자를 각각 코딩하는 폴리뉴클레오티드에 작동 가능하게 연결된 프로모터를 더 포함하는 것일 수 있다. 예를 들어, 상기 제2 벡터는 Ef1α 프로모터, UbC 프로모터 및 CAGG 프로모터로 이루어진 군으로부터 선택되는 프로모터를 더 포함하는 것일 수 있다. 일 구체예에서, 상기 프로모터는 제2 광특이적 결합 단백질 및 전사 촉진 인자의 융합 단백질에 대한 전사를 조절하는 것일 수 있다.
상기 제2 벡터는 줄기세포에서의 발현을 조절하려는 표적 유전자의 gRNA를 코딩하는 폴리뉴클레오티드에 작동 가능하게 연결된 프로모터를 더 포함하는 것일 수 있다. 예를 들어, 상기 제2 벡터는 mU6 프로모터, hU6 프로모터, hH1 프로모터 및 h7SK 프로모터를 더 포함하는 것일 수 있으나, 이에 제한되지 않고, 표적 유전자의 gRNA를 코딩하는 폴리뉴클레오티드의 전사를 조절하는 프로모터라면 모두 포함될 수 있다.
상기 제2 벡터는 2종 이상의 표적 유전자의 gRNA를 코딩하는 폴리뉴클레오티드 및 각 표적 유전자의 gRNA에 대한 서로 다른 프로모터를 포함함으로써, 상기 형질전환용 키트를 이용하여 형질전환한 줄기세포에서 빛의 조사에 의해 동시에 2종 이상의 표적 유전자의 과발현을 유도하는 것일 수 있다.
상기 제2 벡터는 서열번호 22 내지 26의 염기서열 중 어느 하나의 염기서열을 포함하는 것일 수 있다.
상기 제1 벡터 및 제2 벡터는 바이러스 벡터 또는 비바이러스 벡터일 수 있고, 예를 들어, 바이러스 벡터로서, 렌티바이러스, 레트로바이러스(retrovirus), 아데노바이러스(adenovirus), 허피스바이러스(herpesvirus) 또는 아비폭스바이러스(avipoxvirus) 벡터 등일 수 있으나, 이에 제한되지 않는다.
상기 제1 벡터 및 제2 벡터는 적어도 하나의 선택 마커를 포함할 수 있고, 상기 선택 마커는 서로 다른 것일 수 있다. 일 구체예에서, 상기 제1 벡터는 선택 마커로서 퓨로마이신(puromycin: Puro)을 포함하고, 제2 벡터는 선택 마커로서 블라스티시딘(blasticidin: BSD)을 포함하는 것일 수 있다.
본 명세서에서 사용된 용어, "줄기세포(stem cell)"는 생물 조직을 구성하는 다양한 세포들로 분화할 수 있는 미분화 세포로, 만능성(pluripotent) 또는 다능성(multipotent)을 가지는 세포를 의미할 수 있다.
상기 줄기세포는 배아 줄기세포(embryonic stem cell: ESC), 성체 줄기세포(adult stem cell), 또는 유도 만능 줄기세포(induced pluripotent stem cell: iPSC)일 수 있다. 배아 줄기세포는 수정란이 모체의 자궁에 착상하기 직전인 포배기 배아에서 내세포괴(inner cell mass)를 추출하여 체외에서 배양한 것을 의미할 수 있다. 성체 줄기세포는 신체 각 조직에 극히 소량만이 존재하는 미분화된 세포로서 죽은 세포나 손상된 조직을 대체하는 세포를 의미할 수 있다. 유도 만능 줄기세포(induced pluripotent stem cell: iPSC)는 분화가 끝난 체세포에 세포 분화 관련 유전자를 주입하여 분화 이전의 세포 단계로 되돌려, 배아 줄기세포처럼 만능성을 유도한 세포를 의미할 수 있다. 상기 배아 줄기세포는 예를 들어 인간에서 유래한 인간 배아 줄기세포일 수 있다.
또한, 상기 배아 줄기세포는 체세포 복제된 배아 줄기세포일 수 있다. 본 명세서에서 사용된 용어, "체세포 복제된 배아 줄기세포"는 "체세포 핵 치환(Somatic cell nuclear transfer: SCNT) 기술에 의해 생성된 배아 줄기세포를 의미할 수 있다. 상기 "체세포 핵 치환"은 "체세포 핵 이식"과 혼용될 수 있다. 상기 체세포 핵 치환 기술은 난자의 핵을 제거한 후에, 체세포의 핵을 이식하여 복제하는 기술을 의미할 수 있다. 예를 들어, 체세포 복제된 배아 줄기세포는 체세포의 핵을 분리하고, 난자의 핵을 제거한 후, 핵이 제거된 난자에 상기 분리한 체세포 핵을 이식하고, 이를 배양하여 배아 줄기세포로 분화시키는 과정으로 수득되는 것일 수 있다. 상기 체세포 복제된 배아 줄기세포는 체세포 핵의 유전자를 가지므로 체세포의 유전적 특성을 가지는 것일 수 있다. 상기 체세포 복제된 배아 줄기세포는 HLA(human leukocyte antigen)에 대해 동형접합(homozygous)인 것일 수 있다. 예를 들어, HLA 동형접합인 체세포로 복제된 배아 줄기세포일 수 있다. 상기 HLA 동형접합인 체세포 복제된 배아 줄기세포는 면역 거부 반응이 적은 세포 치료제 개발에 활용될 수 있다.
상기 줄기세포에서의 발현을 조절하려는 표적 유전자는 2종 이상일 수 있다. 예를 들어, 상기 표적 유전자는 INS, GCK, PCSK1 및 PCSK2로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 것일 수 있으나, 이에 제한되지 않고, 줄기세포에서 빛의 조사에 의해 발현을 조절하고자 하는 모든 유전자를 포함할 수 있다.
상기 INS는 인슐린 단백질을 코딩하는 유전자를 의미하고, 상기 GCK는 글루코키나아제(glucokinase) 단백질을 코딩하는 유전자를 의미하고, 상기 PCSK1은 PCSK (Proprotein Convertase Subtilisin/Kexin) 타입 1 단백질을 코딩하는 유전자를 의미하고, 상기 PCSK2는 PCSK 타입 2 단백질을 코딩하는 유전자를 의미한다.
상기 INS의 gRNA를 코딩하는 폴리뉴클레오티드는 서열번호 1 내지 5의 염기서열 및 이들 각각의 상보적인 염기서열로 이루어진 군으로부터 선택되는 어느 하나의 염기서열을 포함하는 것일 수 있고, 상기 GCK의 gRNA를 코딩하는 폴리뉴클레오티드는 서열번호 6 내지 10의 염기서열 및 이들 각각의 상보적인 염기서열로 이루어진 군으로부터 선택되는 어느 하나의 염기서열을 포함하는 것일 수 있고, 상기 PCSK1의 gRNA를 코딩하는 폴리뉴클레오티드는 서열번호 11 내지 15의 염기서열 및 이들 각각의 상보적인 염기서열로 이루어진 군으로부터 선택되는 어느 하나의 염기서열을 포함하는 것일 수 있고, 상기 PCSK2의 gRNA를 코딩하는 폴리뉴클레오티드는 서열번호 16 내지 20의 염기서열 및 이들 각각의 상보적인 염기서열로 이루어진 군으로부터 선택되는 어느 하나의 염기서열을 포함하는 것일 수 있다.
다른 양상은 상기 줄기세포의 형질전환용 키트를 이용하여 형질전환한 줄기세포를 제공한다.
본 명세서에서 사용된 용어, "형질감염(transfection)"은 "형질주입"이라고도 하며, 숙주세포에 핵산을 주입하는 것을 의미할 수 있고, 일 구체예에서, 렌티바이러스 벡터를 이용하여 숙주세포에 핵산을 주입하는 것을 의미할 수 있다.
본 명세서에서 사용된 용어, "형질도입(transduction)"은 바이러스 벡터를 통하여 외부 DNA가 다른 세포에 도입되는 것을 의미할 수 있고, 일 구체예에서, 렌티바이러스 벡터에 의해 형질감염된 세포가 생산한 바이러스 입자를 다른 숙주세포에 처리하여 그 숙주세포에 표적 핵산이 도입되도록 하는 것을 의미할 수 있다.
본 명세서에서 사용된 용어, "형질전환"은 외부로부터 주어진 DNA에 의하여 생물의 유전적인 성질을 변화시키는 것을 의미할 수 있고, 예를 들어, 상기 "형질감염" 및 "형질도입"을 모두 포함하는 개념일 수 있다.
상기 줄기세포의 형질전환용 키트를 이용하여 형질전환하는 방법으로는 종래 알려진 다양한 방법, 예를 들면, 미세주입법(microinjection), 전기천공법(electroporation), 입자 분사법(particle bombardment), 정자를 이용하는 방법(sperm-mediated gene transfer), 바이러스 감염법(viral infection), 인슐레이터(insulator) 또는 트랜스포존(trnasposon)을 이용한 기법 등에서 적절하게 선택하여 적용할 수 있다. 일 구체예에서, 상기 형질전환은 렌티바이러스 벡터를 이용한 방법으로 수행될 수 있고, 구체적으로, 렌티바이러스 벡터를 1차 숙주세포에 형질감염시킨 후, 생산된 바이러스 입자를 2차 숙주세포인 줄기세포에 형질도입시킴으로써 수행되는 것일 수 있다.
상기 형질전환한 줄기세포는 청색광 조사에 의해 표적 유전자 발현이 증가하는 것일 수 있다. 예를 들어, 상기 형질전환한 줄기세포에 440 내지 490 nm 파장을 갖는 빛을 조사하면 상기 줄기세포의 표적 유전자 발현이 증가하는 것일 수 있다.
상기 표적 유전자는 INS, GCK, PCSK1 및 PCSK2로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 것일 수 있으나, 이에 제한되지 않고, 줄기세포에서 빛의 조사에 의해 발현을 조절하고자 하는 모든 유전자를 포함할 수 있다.
일 구체예에서, 상기 형질전환한 줄기세포는 440 내지 490 nm 파장을 갖는 빛의 조사에 의하여 INS, GCK, PCSK1 및 PCSK2로 이루어진 군으로부터 선택되는 2 이상의 표적 유전자의 발현이 동시에 증가하는 것일 수 있다.
상기 표적 유전자의 발현이 증가한 줄기세포에 상기 빛의 조사를 중단하면 증가한 표적 유전자의 발현이 다시 감소하는 것일 수 있다.
예를 들어, 상기 형질전환한 줄기세포는 상기 파장을 갖는 빛이 없는 상태에서는 gRNA의 서열을 인식한 dCas 단백질에 의해 dCas 단백질 및 제1 광특이적 결합 단백질의 융합 단백질이 표적 유전자 위치에 존재하고 있는데, 상기 파장을 갖는 빛이 조사되면, 제2 광특이적 결합 단백질 및 전사 촉진 인자의 융합 단백질이 상기 제1 광특이적 결합 단백질을 포함하는 융합 단백질에 결합함으로써 전사 촉진 인사의 작용에 의해 표적 유전자의 발현이 증가되는 것일 수 있다. 또한, 상기 파장을 갖는 빛의 조사가 중단되면, 다시 제1 광특이적 결합 단백질과 제2 광특이적 결합 단백질의 결합이 해제되면서 전사 촉진 인자가 표적 유전자에 대해 작용하지 못하게 되므로 표적 유전자의 발현이 다시 감소되는 것일 수 있다.
다른 양상은 상기 형질전환한 줄기세포에 440 내지 490 nm 파장을 갖는 빛을 조사하여 줄기세포의 표적 유전자 발현을 증가시키는 단계를 포함하는 줄기세포의 표적 유전자 발현을 조절하는 방법을 제공한다.
상기 빛은 청색을 나타내는 빛으로 440 내지 490 nm의 파장을 갖는 빛일 수 있으나, 상기 제1 광특이적 결합 단백질과 제2 광특이적 결합 단백질의 결합을 유도하는 빛이라면 상기 파장 범위에 제한되지 않는다.
상기 빛은 펄스(pulse)를 갖는 빛일 수 있고, 예를 들어, 10 내지 90%, 20 내지 80%, 30 내지 70%, 또는 40 내지 60%의 펄스를 갖는 빛일 수 있다.
상기 빛은 10 내지 2000 mA, 100 내지 2000 mA, 100 내지 1000 mA, 또는 300 내지 1000 mA, 또는 500 내지 800 mA의 전류로 조사하는 것일 수 있으나, 상기 제1 광특이적 결합 단백질과 제2 광특이적 결합 단백질의 결합을 유도하는 빛이라면 상기 전류 범위에 제한되지 않는다.
상기 빛은 형질전환한 줄기세포에 6 시간 이상, 예를 들어, 6 내지 120 시간, 10 내지 100 시간, 10 내지 80 시간, 10 내지 50 시간, 20 내지 100 시간, 20 내지 80 시간, 또는 20 내지 50 시간 동안 조사하는 것일 수 있으나, 상기 제1 광특이적 결합 단백질과 제2 광특이적 결합 단백질의 결합을 유도할 수 있는 시간 동안 조사하는 것이라면 상기 시간 범위에 제한되지 않는다.
상기 표적 유전자는 INS, GCK, PCSK1 및 PCSK2로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 것일 수 있으나, 이에 제한되지 않고, 줄기세포에서 빛의 조사에 의해 발현을 조절하고자 하는 모든 유전자를 포함할 수 있다.
상기 줄기세포의 표적 유전자 발현을 조절하는 방법은 상기 형질전환한 줄기세포에 440 내지 490 nm 파장을 갖는 빛을 조사하여 줄기세포의 표적 유전자 발현을 증가시키는 단계 이후에 상기 빛의 조사를 중단하여 증가된 줄기세포의 표적 유전자 발현을 다시 감소시키는 단계를 더 포함하는 것일 수 있다.
일 양상에 따른 줄기세포의 형질전환용 키트는 다수의 표적 유전자에 대한 gRNA를 하나의 벡터를 이용하여 동시에 줄기세포에 도입시켜 빛의 조사에 의해 다수의 표적 유전자를 동시에 안정적으로 발현하는 줄기세포를 제작할 수 있다. 또한, 일 양상에 따른 줄기세포의 표적 유전자 발현을 조절하는 방법에 의하면, 상기 키트를 이용하여 형질전환한 줄기세포에 특정 파장의 빛을 조사 또는 중단함으로써 간단한 조작에 의하여 줄기세포의 표적 유전자 발현을 증가시키거나 다시 감소시킬 수 있다. 따라서, 상기 줄기세포의 형질전환용 키트, 형질전환한 줄기세포 및 줄기세포의 표적 유전자 발현을 조절하는 방법은 표적 유전자와 관련된 기초과학 연구 및 세포 치료제 분야에 유용하게 활용될 수 있다.
도 1은 일 양상에 따른 pcDNA3.1-int_Ef1αP-CibN-dCas9-CibN 벡터의 제작 과정을 나타낸 도면이다.
도 2는 일 양상에 따른 Lenti-int_Ef1αP-CibN-dCas9-CibN-Puro 벡터(제1 벡터)의 제작 과정을 나타낸 도면이다.
도 3은 일 양상에 따른 pmU6-hINS gRNA (A), phU6-hGCK gRNA (B), phH1-hPCSK1 gRNA (C) 및 ph7SK-hPCSK2 (D) gRNA 벡터의 제작 과정을 나타낸 도면이다.
도 4는 일 양상에 따른 pcDNA3.1-BsmBI-int_Ef1αP-Cry2FL-VP64 벡터의 제작 과정을 나타낸 도면이다.
도 5는 일 양상에 따른 Lenti-BsmBI-int_Ef1αP-Cry2FL-VP64-BSD 벡터의 제작 과정을 나타낸 도면이다.
도 6은 일 양상에 따른 Lenti-4gRNA-int_Ef1αP-Cry2FL-VP64-BSD 벡터(제2 벡터)의 제작 과정을 나타낸 도면이다.
도 7은 일 양상에 따른 제1 벡터 및 제2 벡터를 제한효소 SpeI로 자른 후 전기영동한 결과를 나타낸 도면이다.
도 8은 인간 배아 줄기세포(human embryonic stem cells: hES)에서 청색광을 24시간 동안 조사하여 표적 유전자의 mRNA 발현량을 측정한 결과 그래프이다(A: hINS; B: hGCK; C: hPCSK1; 및 D: hPCSK2).
도 9는 hES에서 50%의 펄스(pulse)를 갖는 청색광을 16시간 또는 24시간 동안 조사하여 표적 유전자의 mRNA 발현량을 측정한 결과 그래프이다(A: hINS; B: hGCK; C: hPCSK1; 및 D: hPCSK2).
도 10은 hES에서 청색광을 24시간 또는 48시간 동안 조사하여 표적 유전자의 mRNA 발현량을 측정한 결과 그래프이다.
도 11은 일 양상에 따른 광 유도 유전자 발현 조절 형질전환 배아 줄기세포의 핵형 분석 결과를 나타낸 도면이다.
도 12는 일 양상에 따른 광 유도 유전자 발현 조절 형질전환 세포의 제작 과정을 나타낸 도면이다.
도 2는 일 양상에 따른 Lenti-int_Ef1αP-CibN-dCas9-CibN-Puro 벡터(제1 벡터)의 제작 과정을 나타낸 도면이다.
도 3은 일 양상에 따른 pmU6-hINS gRNA (A), phU6-hGCK gRNA (B), phH1-hPCSK1 gRNA (C) 및 ph7SK-hPCSK2 (D) gRNA 벡터의 제작 과정을 나타낸 도면이다.
도 4는 일 양상에 따른 pcDNA3.1-BsmBI-int_Ef1αP-Cry2FL-VP64 벡터의 제작 과정을 나타낸 도면이다.
도 5는 일 양상에 따른 Lenti-BsmBI-int_Ef1αP-Cry2FL-VP64-BSD 벡터의 제작 과정을 나타낸 도면이다.
도 6은 일 양상에 따른 Lenti-4gRNA-int_Ef1αP-Cry2FL-VP64-BSD 벡터(제2 벡터)의 제작 과정을 나타낸 도면이다.
도 7은 일 양상에 따른 제1 벡터 및 제2 벡터를 제한효소 SpeI로 자른 후 전기영동한 결과를 나타낸 도면이다.
도 8은 인간 배아 줄기세포(human embryonic stem cells: hES)에서 청색광을 24시간 동안 조사하여 표적 유전자의 mRNA 발현량을 측정한 결과 그래프이다(A: hINS; B: hGCK; C: hPCSK1; 및 D: hPCSK2).
도 9는 hES에서 50%의 펄스(pulse)를 갖는 청색광을 16시간 또는 24시간 동안 조사하여 표적 유전자의 mRNA 발현량을 측정한 결과 그래프이다(A: hINS; B: hGCK; C: hPCSK1; 및 D: hPCSK2).
도 10은 hES에서 청색광을 24시간 또는 48시간 동안 조사하여 표적 유전자의 mRNA 발현량을 측정한 결과 그래프이다.
도 11은 일 양상에 따른 광 유도 유전자 발현 조절 형질전환 배아 줄기세포의 핵형 분석 결과를 나타낸 도면이다.
도 12는 일 양상에 따른 광 유도 유전자 발현 조절 형질전환 세포의 제작 과정을 나타낸 도면이다.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
실시예 1. dCas9를 코딩하는 유전자 서열을 포함하는 제1 벡터의 제작
1-1. pcDNA3.1-int_Ef1α
P
-CibN-dCas9-CibN 벡터의 제작
1-1-1. int_Ef1α
P
의 증폭
lenti-EF1α-dCas9-KRAB-Puro (#99372, Addgene) 벡터에서 인트론 A 부위가 포함된 Ef1αP(이하, "int_Ef1αP")를 증폭시키기 위해 SpeI/SacI 링커를 연결한 프라이머를 제작하여(표 1), Herculase II PCR (agilent)을 수행한 후, 제한효소 SpeI(New England BioLabs: NEB) 및 SacI(NEB)로 부분 절단(partial digestion)하고, DNA gel elution 키트(FAVORGEN)를 이용하여 원하는 부위를 수득하였다.
| SpeI tagging oligo sequence (5'->3') | SacI tagging oligo sequence (5'->3') |
| ct ACTAGT CTAGGTCTTGAAAGGAGTGG | ac GAGCTC ACGTCACGACACCTGAAATG |
XL10-Gold®Ultracompetent Cells (STRATAGENE)을 이용하여 pJET1.2/blunt (Thermo)에 int_Ef1αP를 도입하고, Mini prep(GeneAll)한 후 제한효소 SpeI 및 SacI로 잘라 증폭한 int_Ef1αP를 수득하였다.
1-1-2. 벡터의 제작
pcDNA3.1-CMVP-CibN-dCas9-CibN (#60553, Addgene) 벡터의 CMVP를 제한효소 SpeI 및 SacI로 잘라 제거해주고, 여기에 T4 리가아제(Thermo)를 이용하여 상기 실시예 1-1-1에서 증폭한 int_Ef1αP를 연결하여 pcDNA3.1-int_Ef1αP-CibN-dCas9-CibN 벡터를 제작하였다(도 1).
1-2. Lenti-int_Ef1α
P
-CibN-dCas9-CibN-Puro 벡터의 제작
상기 실시예 1-1에서 제작한 pcDNA3.1-int_Ef1αP-CibN-dCas9-CibN 벡터에서 int_Ef1αP-CibN-dCas9-CibN 부위를 증폭시키기 위해 XbaI/BsrGI 링커를 연결한 프라이머를 제작하여(표 2), Herculase II PCR을 수행한 후, 제한효소 XbaI(NEB), BsrGI(NEB)로 잘라준 후, DNA gel elution 키트를 이용하여 원하는 부위를 수득하였다.
| XbaI tagging oligo sequence (5'->3') | BsrGI tagging oligo sequence (5'->3') |
| G TCTAGA AGATATACGCGTTGACATT | CG TGTACA AATATAATCCGTTTTCTCC |
수득한 int_Ef1αP-CibN-dCas9-CibN 부위를 제한효소 SpeI 및 SacI 대신 XbaI 및 BsrGI을 사용한 것을 제외하고는 상기 실시예 1-1-1에 기재된 것과 동일한 방법으로 증폭시켰다.
Lenti-Ef1α-dCas9-KRAB-Puro (#99372, Addgene) 렌티바이러스 벡터를 제한효소 NheI(NEB) 및 BsrGI로 잘라준 후, T4 리가아제를 이용하여 증폭시킨 int_Ef1αP-CibN-dCas9-CibN 부위를 연결하여 제1 벡터로서 Lenti-int_Ef1αP-CibN-dCas9-CibN-Puro 벡터를 제작하였다(도 2).
실시예 2. gRNA를 코딩하는 유전자 서열을 포함하는 제2 벡터의 제작
2-1. gRNA를 코딩하는 유전자 서열을 포함하는 벡터의 제작
pmU6-gRNA, phU6-gRNA, phH1-gRNA 및 ph7SK-gRNA 플라스미드의 각 키메릭 gRNA 부위에 표적 유전자인 hINS, hGCK, hPCSK1 및 hPCSK2 유전자에 대한 각 gRNA를 코딩하는 유전자 서열을 삽입하기 위하여, 하기와 같이 올리고뉴클레오티드를 제작하였다.
구체적으로, pmU6-gRNA (#53187, Addgene), phU6-gRNA (#53188, Addgene), phH1-gRNA (#53186, Addgene) 및 ph7SK-gRNA (#53189, Addgene) 플라스미드의 키메릭 gRNA 부위에서 BbsI 제한효소에 의해 절단되는 부위의 서열을 포함하고, gRNA를 코딩하는 각 5종씩의 유전자 서열을 포함하도록 표적 유전자 별로 각 5종씩 표 3의 올리고뉴클레오티드 쌍을 디자인 및 제작하였다. 각 올리고뉴클레오티드 서열의 디자인 및 제작은 http://crispr-era.stanford.edu/ 를 이용하였다.
| 표적 유전자 |
세트 번호 |
정방향 올리고뉴클레오티드 서열 (5’->3’) |
역방향 올리고뉴클레오티드 서열 (5’->3’) |
| hINS | 1 | TTGTTTG ACCCCAGGCCCTAATGGGCC | AAAC GGCCCATTAGGGCCTGGGGT CAA |
| 2 | TTGTTTG CTAATGACCCGCTGGTCCTG | AAAC CAGGACCAGCGGGTCATTAG CAA | |
| 3 | TTGTTTG TGAGACTATAAAGCCAGCGG | AAAC CCGCTGGCTTTATAGTCTCA CAA | |
| 4 | TTGTTTG CTCTGAGACTATAAAGCCAG | AAAC CTGGCTTTATAGTCTCAGAG CAA | |
| 5 | TTGTTTG GGAAGAGGTGCTGACGACCA | AAAC TGGTCGTCAGCACCTCTTCC CAA | |
| hGCK | 1 | CACCG AGAGTATTTGAGCAGCCGCC | AAAC GGCGGCTGCTCAAATACTCT C |
| 2 | CACCG GCCCGCACAAGGCTGAGGAA | AAAC TTCCTCAGCCTTGTGCGGGC C | |
| 3 | CACCG TAGCGGTCACTGTAGTGACA | AAAC TGTCACTACAGTGACCGCTA C | |
| 4 | CACCG AGTGGGGGATTCTCCTGCCA | AAAC TGGCAGGAGAATCCCCCACT C | |
| 5 | CACCG GATCCTCTGCTCATCAGAAC | AAAC GTTCTGATGAGCAGAGGATC C | |
| hPCSK1 | 1 | TCCCA AGCTGCTGGGACTCGCGACC | AAAC GGTCGCGAGTCCCAGCAGCT T |
| 2 | TCCCA ATCTATCCCCGCGACTCTCT | AAAC AGAGAGTCGCGGGGATAGAT T | |
| 3 | TCCCA CGCGACTCTCTAGGGAGCTC | AAAC GAGCTCCCTAGAGAGTCGCG T | |
| 4 | TCCCA CGCGGAGTAGCCTGGGAAGC | AAAC GCTTCCCAGGCTACTCCGCG T | |
| 5 | TCCCA CTTGGTAGTTATGTTCAAGT | AAAC ACTTGAACATAACTACCAAG T | |
| hPCSK2 | 1 | CCTCG GCCGCTGGCCTGATTACAGC | AAAC GCTGTAATCAGGCCAGCGGC C |
| 2 | CCTCG TGCGCTGCGCCCCCAGAAGG | AAAC CCTTCTGGGGGCGCAGCGCA C | |
| 3 | CCTCG GGCGGGCCCGGGGCGGCCCA | AAAC TGGGCCGCCCCGGGCCCGCC C | |
| 4 | CCTCG CCGCCCTCATTGGCCGCTGG | AAAC CCAGCGGCCAATGAGGGCGG C | |
| 5 | CCTCG GCCCGGGGCGGCCCAGGGCG | AAAC CGCCCTGGGCCGCCCCGGGC C |
상기 표 3의 서열에서 밑줄로 표시된 서열이 각 표적 유전자에 대한 gRNA를 코딩하는 서열에 해당하며, 나머지 부분의 서열은 각 플라스미드의 키메릭 gRNA 부위에서 BbsI 제한효소에 의해 절단되는 부위의 서열에 해당한다.
상기 표 3의 각 유전자에 대한 정방향 및 역방향의 올리고뉴클레오티드 각각을 동량(1:1)으로 실험용 1.5ml 튜브에 넣어 95℃에서 5분간 반응시킨 후, 상온이 될 때까지 점차적으로 온도를 낮춰 상보적 DNA (complementary DNA: cDNA)끼리 이중가닥이 형성되도록 하였다. pmU6-gRNA, phU6-gRNA, phH1-gRNA 및 ph7SK-gRNA 플라스미드를 제한효소 BbsI(Thermo scientific)로 잘라준 후, PCR로 증폭시킨 산물로부터 Gel elution 키트를 사용하여 잘라진 순수한 플라스미드를 얻었다. 도 3에 나타낸 바와 같이, 상기 각 플라스미드에 각 표적 유전자 gRNA의 DNA 이중가닥을 T4 리가아제 효소로 연결하여 pmU6-hINS gRNA, phU6-hGCK gRNA, phH1-hPCSK1 gRNA 및 ph7SK-hPCSK2 gRNA 플라스미드 각 세트 1 내지 5를 제작하였다.
2-2. pcDNA3.1-BsmBI-int_Ef1α
P
-Cry2FL-VP64 벡터의 제작
pLV hUbC-VP64 dCas9 VP64-T2A-GFP (#59791, Addgene) 벡터에서 BsmBI 부위를 증폭시키기 위해 NruI/MluI 링커를 연결한 프라이머를 제작하여(표 4), Herculase II PCR을 수행한 후, 제한효소 NruI(NEB), MluI(NEB)로 잘라준 뒤, DNA gel elution 키트를 이용하여 원하는 부위를 수득하였다.
| NruI tagging oligo sequence (5'->3') | MluI tagging oligo sequence (5'->3') |
| T TCGCGA TTACAGGGACAGCAGAGATCCAG | T ACGCGT GAGGCGCCAAAACCCGGCGCGGA |
수득한 BsmBI 부위를 제한효소 SpeI 및 SacI 대신 NruI 및 MluI을 사용한 것을 제외하고는 상기 실시예 1-1-1에 기재된 것과 동일한 방법으로 증폭시켰다.
상기 실시예 1-1-1 및 1-1-2에서, pcDNA3.1-CMVP-CibN-dCas9-CibN (#60553) 벡터 대신 pcDNA3.1-CMVP-Cry2FL-VP64 (#60554, Addgene) 벡터를 사용하여, pcDNA3.1-int_Ef1αP-Cry2FL-VP64 벡터를 제작하였다. 상기 벡터를 제한효소 NruI 및 MluI로 잘라준 후, T4 리가아제를 이용하여 증폭시킨 BsmBI 부위를 연결하여 pcDNA3.1-BsmBI-int_Ef1αP-Cry2FL-VP64 벡터를 제작하였다(도 4).
2-3. Lenti-BsmBI-int_Ef1α
P
-
Cry2FL-VP64-BSD 벡터의 제작
상기 실시예 2-2에서 제작한 pcDNA3.1-BsmBI-int_Ef1αP-Cry2FL-VP64 벡터에서 BsmBI-int_Ef1αP-Cry2FL-VP64 부위를 증폭시키기 위해 XbaI/BsrGI 링커를 연결한 프라이머로 하기 표 5의 프라이머를 사용한 것을 제외하고는 상기 실시예 1-2에 기재된 것과 동일한 방법으로 원하는 부위를 수득하였다.
| XbaI tagging oligo sequence (5'->3') | BsrGI tagging oligo sequence (5'->3') |
| G TCTAGA TTAGGGTTAGGCGTTTTGC | G TGTACA AGAAGCGTAGTCCGGAACG |
수득한 BsmBI-int_Ef1αP-Cry2FL-VP64 부위를 제한효소 SpeI 및 SacI 대신 XbaI 및 BsrGI을 사용한 것을 제외하고는 상기 실시예 1-1-1에 기재된 것과 동일한 방법으로 증폭시켰다.
Lenti-dCas9-KRAB-BSD (#89567, Addgene) 렌티바이러스 벡터를 제한효소 NheI 및 BsrGI로 잘라준 후, T4 리가아제를 이용하여 증폭시킨 BsmBI-int_Ef1αP-Cry2FL-VP64 부위를 연결하여 Lenti-BsmBI-int_Ef1αP-Cry2FL-VP64-BSD 벡터를 제작하였다(도 5).
2-4. Lenti-4gRNA-int_Ef1α
P
-
Cry2FL-VP64-BSD 벡터의 제작
상기 실시예 2-1에서 제작한 4개의 다른 프로모터를 가지고 있는 gRNA 플라스미드를 동일한 번호의 세트끼리 조합하고, 실시예 2-3에서 제작한 렌티바이러스 벡터와 함께 하기 표 6의 조성 및 용량으로 혼합하여 표 7의 반응 조건으로 골든 게이트 어셈블리(Golden Gate assembly)를 수행하였다.
| 시약 | 용량 |
| Lenti-BsmBI-int_Ef1αP-Cry2FL-VP64-BSD 벡터 | 200ng |
| pmU6-hINS gRNA | 200ng |
| phU6-hGCK gRNA | 200ng |
| phH1-hPCSK1 gRNA | 200ng |
| ph7SK-hPCSK2 gRNA | 200ng |
| T4 DNA 리가아제 | 1ul |
| 제한효소 BsmBI (Thermo scientific) | 1ul |
| T4 리가아제 버퍼 | 2ul |
| 총합 | 20ul |
| 반응 온도 | 반응 시간 |
| 37°C | 10분 |
| 16°C | 15분 |
| 37°C | 30분 |
| 80°C | 5분 |
| 4°C | ∞ |
상기 과정을 통해 최종적으로 하나의 벡터 내에서 4가지의 다른 프로모터로 4개의 gRNA가 발현되도록 하는 렌티바이러스 벡터(제2 벡터) 세트 1 내지 5를 제작하였다(도 6).
실시예 3. 제1 벡터 및 제2 벡터의 정상 구축 확인
상기 실시예 1 및 2에서 각각 제작한 제1 벡터 및 제2 벡터가 의도한 대로 구축되었는지 여부를 확인하기 위하여, 전기영동을 수행하였다.
구체적으로, 상기 Lenti-int_Ef1αP-CibN-dCas9-CibN-Puro 벡터(제1 벡터) 및 Lenti-4gRNA-int_Ef1αP- Cry2FL-VP64-BSD 벡터(제2 벡터) 내에 공통적으로 한 군데의 제한효소 SpeI의 인식서열이 존재하므로, 상기 각 벡터를 제한효소 SpeI로 잘라준 후 전기영동하였다.
그 결과, 도 7에 나타낸 바와 같이, 세트 1 내지 5의 제2 벡터에서는 9,533 bp 및 4,060 bp 위치의 밴드가 공통적으로 나타남을 확인하였고, 제1 벡터에서는 12,857 bp 및 2,382 bp 위치의 밴드가 나타남을 확인하였다. 상기 결과를 통하여, 각 벡터에서 두 조각으로 절단된 절편의 bp 크기의 합이 각 벡터 서열에 따른 이론적인 bp 크기에 해당하는 바, 각 벡터가 예상한 서열대로 구축되었음을 알 수 있었다.
실시예 4. 인간 체세포에서 청색광 유도에 의한 표적 유전자 발현 확인
4-1. 렌티바이러스의 제작
상기 실시예 1에서 제작한 제1 벡터(Lenti-int_Ef1αP-CibN-dCas9-CibN-Puro)와 상기 실시예 2에서 제작한 제2 벡터(Lenti-4gRNA-int_Ef1αP-Cry2FL-VP64-BSD) 세트 1 내지 5 각각을 렌티바이러스 패키징 플라스미드 psPAX2 (#12260, Addgene) 및 렌티바이러스 엔벨로프 플라스미드 pMD2.G (#12259, Addgene)와 함께 형질감염(transfection) 시약(Superfect transfection reagent, #301305, Qiagen)을 이용하여 293T 세포에 형질감염시켰다. 6 시간 후에 무혈청 발현배지(Serum-free Expression media)로 갈아준 뒤, 형질감염 26 시간, 38 시간, 50 시간 뒤 각각 바이러스가 들어있는 발현 배지를 모아서 얻은 샘플을 -80℃에 보관하였다. 초원심분리 필터(Amicon Ultra-15 Centrifugal Filter, UFC910024, Merck millipore)를 이용하여 많은 부피의 바이러스를 약 250ul 정도로 농축시켜 형질도입(transduction)을 진행하거나, 분주하여 -80℃에 보관하였다.
4-2. 청색광 매개의 hINS, hGCK, hPCSK1 및 hPCSK2 유전자 과발현 인간 포피 섬유아세포 및 인간 피부 섬유아세포의 제작
인간 포피 섬유아세포(Human Foreskin Fibroblast: hFF) 및 인간 피부 섬유아세포(Human Dermal Fibroblast: hDF)에 상기 실시예 4-1에서 제작한 렌티바이러스를 형질도입(transduction)하여 청색광 조사에 의해 hINS, hGCK, hPCSK1 및 hPCSK2 유전자 과발현이 유도되는 세포를 제작하였다.
구체적으로, hFF 및 hDF의 배지를 형질도입 효율을 높이기 위해 10 ug/mL의 폴리브렌(polybrene, TR-1003-G, Merck millipore)이 첨가된 배지로 갈아준 후, 실시예 4-1에서 제작한 int_Ef1αP-CibN-dCas9-CibN-Puro 렌티바이러스 농축액 약 20 내지 50 ul와 함께, 4gRNA-int_Ef1αP-Cry2FL-VP64-BSD 렌티바이러스 세트 1 내지 5의 농축액을 각각 약 20 내지 50 ul씩 첨가하였다. 이후, 선택 마커인 퓨로마이신(puromycin: Puro) 및 블라스티시딘(blasticidin: BSD)을 처리한 후, 상기 항생제 모두에 내성이 있는 세포를 상기 렌티바이러스가 모두 도입된 세포로 선별하였다.
4-3. 형질도입 hFF에서 청색광 조사에 의한 hINS, hGCK, hPCSK1 및 hPCSK2 유전자 발현 유도 확인
상기 실시예 4-2에서 제작한 형질도입 hFF에서 청색광 조사에 의해 표적 유전자인 hINS, hGCK, hPCSK1 및 hPCSK2 유전자 발현이 유도되는지 여부를 실시간 정량 역전사 PCR(Real-Time Quantitative Reverse Transcription PCR: 실시간 qRT-PCR)을 수행하여 확인하였다.
구체적으로, 상기 실시예 4-2에서 제작한 각 hFF에 청색광을 15 mA로 12 시간 동안 조사한 후, 세포를 회수하여, 키트(RNeasy Plus Mini Kit, Qiagen)를 이용하여 총 RNA를 추출하고, 이로부터 키트(TOPscriptTM RT DryMIX kit, Enzynomics)를 이용하여 cDNA를 합성하였다. 합성된 cDNA를 주형으로 하고, 하기 표 8의 프라이머들 및 SYBR 그린 마스터 믹스(SYBRTM Green Master Mix, ThermoFisher)를 이용하여 실시간 qRT-PCR을 수행하였다.
| 유전자 | Forward 5' → 3' | Reverse 5' → 3' |
| hGAPDH | GAC CCC TTC ATT GAC CTC AAC TAC | TTG ACG GTG CCA TGG AAT TT |
| hINS | TCA CTC TTG GTG CAA AGG AA | CGG CCC ATA TAA CTG ATG TTC |
| hGCK | TAT TCC CGA AGA GCA AAG GA | TGT CGT TTG TCT GAG CCT TTT |
| hPCSK1 | GCT TAA CAT CAT GAT GGA CCT | AGT TTA GGA GAC TCC ATA GCA |
| hPCSK2 | TTA GAC CCC TGA TTG CTG CT | CCT TGA AGG GGT TGT TGG TA |
각 유전자의 mRNA 발현량을 측정하여 GAPDH의 mRNA 발현량을 기준으로 표준화하고, ΔΔCt 방법을 이용하여 각 유전자의 상대적인 mRNA 발현량을 계산하고, 유전자를 조작하지 않은 야생형 hFF 대조군에 대한 표적 유전자의 mRNA 발현량 증가 배수를 계산하였다.
그 결과, 형질도입 hFF에서 청색광 조사에 의해 4종 표적 유전자의 과발현이 유도됨을 확인하였다.
실시예 5. 인간 배아 줄기세포에서 청색광 유도에 의한 표적 유전자 발현 확인
5-1. 렌티바이러스의 제작
293FT 세포에 상기 실시예 1에서 제작한 제1 벡터(Lenti-int_Ef1αP-CibN-dCas9-CibN-Puro)와 상기 실시예 2에서 제작한 제2 벡터(Lenti-4gRNA-int_Ef1αP-Cry2FL-VP64-BSD) 세트 1을 형질감염시켜 렌티바이러스를 제작하였다.
구체적으로, 형질감염 시약으로, 12 μg의 렌티바이러스 패키징 플라스미드 psPAX2, 10 μg의 렌티바이러스 엔벨로프 플라스미드 pMD2.G, 20 μg의 제1 벡터, 및 20 μg의 제2 벡터에 멸균 증류수를 450 μL 첨가하여 잘 섞어주었다. 여기에, 2X BBS 용액 500 μL 및 2.5 M CaCl2 용액 60 μL를 첨가하여 상온에서 20분 동안 정치하였다. 293FT 세포가 있는 10 cm 배양디쉬에 상기 시약 1 mL을 한 방울씩 떨어뜨려 첨가하고, 천천히 섞어준 후 16시간 동안 배양하였다. 이후, 상기 형질감염 시약이 포함되지 않은 기본 배양액으로 교체 후 48시간 동안 더 배양하였고, 세포 배양액을 회수하여 3,000 rpm에서 10분간 원심분리 후, 상층액을 0.45 μm 필터로 여과하였다. 여기에 바이러스 추출 시약(PEG-it Virus Precipitation Solution)을 전체 용액의 20 부피%로 첨가한 후, 혼합하여 24시간 동안 4℃에서 정치하였다. 이후, 상기 혼합액을 4℃ 및 1,500 g에서 30분간 원심분리한 후, 가라앉은 흰색 침전인 렌티바이러스 입자를 배지로 풀어서 이후 형질도입에 사용하였다.
5-2. 청색광 매개의 hINS, hGCK, hPCSK1 및 hPCSK2 유전자 과발현 인간 배아 줄기세포의 제작
인간 배아 줄기세포(human embryonic stem cells: hES)를 마트리겔 코팅된 4-웰 배양 디쉬에 mTeSR1 배양액으로 유지 배양 후, 60 내지 80% 정도 자랐을 때, 새로운 mTeSR1 배양액 300 μL/웰로 배양액 교체한 후, 상기 실시예 5-1에서 제작한 int_Ef1αP-CibN-dCas9-CibN-Puro 렌티바이러스 농축액, 4gRNA-int_Ef1αP-Cry2FL-VP64-BSD 렌티바이러스의 농축액, 및 8 μg/mL의 폴리브렌을 각 웰에 첨가하고 24시간 동안 배양하였다. 새로운 mTeSR1 배양액으로 교체하여 3일 동안 더 배양한 후, 이후, 선택 마커인 퓨로마이신 및 블라스티시딘을 처리한 후, 상기 항생제 모두에 내성이 있는 세포를 상기 렌티바이러스가 모두 도입된 세포로 선별하였다.
5-3. 형질도입 hES에서 청색광 조사에 의한 hINS, hGCK, hPCSK1 및 hPCSK2 유전자 발현 유도 확인
상기 실시예 5-2에서 제작한 형질도입 hES에서 청색광 조사에 의해 의해 표적 유전자인 hINS, hGCK, hPCSK1 및 hPCSK2 유전자 발현이 유도되는지 여부를 실시간 qRT-PCR을 수행하여 확인하였다.
구체적으로, 상기 실시예 5-2에서 제작한 hES에 455 nm의 청색광을 700 mA로 조사한 후, 세포를 회수하여, 하기 표 9의 프라이머들을 이용하고, 측정된 mRNA 발현량에 대하여 GAPDH의 발현량을 기준으로 표준화한 상대적인 표적 유전자의 mRNA 발현량을 계산하여 나타낸 것을 제외하고는 상기 실시예 4-3에 기재된 것과 동일한 방법으로 qRT-PCR을 수행하였다.
| 유전자 | Forward 5' → 3' | Reverse 5' → 3' |
| hGAPDH | GGT GGT CTC CTC TGA CTT CAA CA | GTG GTC GTT GAG GGC AAT G |
| hINS | GGA ACG AGG CTT CTT CTA CAC | ACA ATG CCA CGC TTC TGC |
| hGCK | CTT CCC TCA GTT TTT CGG TGG | TTG ATT CCA GCG AGA AAG GTG |
| hPCSK1 | AAG CAA ACC CAA ATC TCA CCT GGC | TCA CCA TCA AGC CTG CTC CAT TCT |
| hPCSK2 | AAG ATG GCT TTG CAG CAG GAA GGA | AGC CAC ATT CAA ATC AAG GCC AGG |
그 결과, 청색광을 24 시간 동안 조사한 경우, 도 8에 나타낸 바와 같이, 형질도입 hES에서 각 표적 유전자의 mRNA 발현량이 증가함을 확인하였다.
또한, 청색광을 16 시간 또는 24 시간 동안 조사하면서 빛에 50%의 펄스(pulse)를 준 경우, 도 9에 나타낸 바와 같이, 24 시간 동안 청색광을 조사한 경우에 형질도입 hES에서 각 표적 유전자의 mRNA 발현량이 크게 증가함을 확인하였다.
또한, 청색광을 24 시간 또는 48 시간 동안 조사하여, mRNA 발현량을 비교한 결과, 도 10에 나타낸 바와 같이, 형질도입 hES에 청색광을 조사한 결과 각 표적 유전자의 mRNA 발현량이 모두 증가하였으며, 24 시간에 비하여 48 시간 동안 조사한 경우에 모두 그 발현량이 더욱 증가함을 확인하였다.
실시예 6. 청색광 매개 표적 유전자 과발현 인간 배아 줄기세포의 핵형 분석
상기 실시예 5-2에서 제작한 형질도입 hES의 핵형을 분석하였다.
구체적으로, 상기 세포를 4-웰 플레이트에 배양한 후, 콜세미드(colcemid)를 웰 당 10 μL씩 첨가하여 30분 동안 더 배양하였다. 배양된 세포를 0.8% 소듐 시트레이트 및 0.075M KCl이 1:1의 부피비로 혼합된 저장액(hypotonic solution)으로 세척한 후, 상기 저장액을 웰 당 500 μL씩 첨가하여 23분 동안 더 배양하였다. 이후, 메탄올 및 아세트산을 3:1의 부피비로 혼합한 고정 시약을 첨가하여 1,800 rpm에서 10분간 원심분리 후 펠렛을 모아 상기 고정 시약으로 재현탁시키고, 동일한 조건으로 원심분리 후 재현탁시키는 과정을 3회 더 반복하였다. 현탁된 세포 용액을 슬라이드 글라스에 떨어뜨린 후, 80℃ 오븐에서 2시간 동안 방치한 후, 김자 염색(Giemsa staining)을 수행하여 염색체를 염색하여 시각화하였다(도 11).
<110> CHA University Industry-Academic Cooperation Foundation
Konkuk University Industrial Cooperation Corp
<120> Vector system for light-inducible modulating of gene expression
and uses thereof
<130> PN131429KR
<160> 30
<170> KoPatentIn 3.0
<210> 1
<211> 20
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<220>
<223> hINS gRNA 1
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accccaggcc ctaatgggcc 20
<210> 2
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<223> hINS gRNA 2
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ctaatgaccc gctggtcctg 20
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<223> hINS gRNA 3
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tgagactata aagccagcgg 20
<210> 4
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<223> hINS gRNA 4
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ctctgagact ataaagccag 20
<210> 5
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<223> hINS gRNA 5
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ggaagaggtg ctgacgacca 20
<210> 6
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<220>
<223> hGCK gRNA 1
<400> 6
agagtatttg agcagccgcc 20
<210> 7
<211> 20
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<223> hGCK gRNA 2
<400> 7
gcccgcacaa ggctgaggaa 20
<210> 8
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<223> hGCK gRNA 3
<400> 8
tagcggtcac tgtagtgaca 20
<210> 9
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<223> hGCK gRNA 4
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<223> hGCK gRNA 5
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gatcctctgc tcatcagaac 20
<210> 11
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<220>
<223> hPCSK1 gRNA 1
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agctgctggg actcgcgacc 20
<210> 12
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<213> Artificial Sequence
<220>
<223> hPCSK1 gRNA 2
<400> 12
atctatcccc gcgactctct 20
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<220>
<223> hPCSK1 gRNA 3
<400> 13
cgcgactctc tagggagctc 20
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<223> hPCSK1 gRNA 4
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cgcgactctc tagggagctc 20
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<223> hPCSK1 gRNA 5
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cttggtagtt atgttcaagt 20
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<223> hPCSK2 gRNA 1
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gccgctggcc tgattacagc 20
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<220>
<223> hPCSK2 gRNA 2
<400> 17
tgcgctgcgc ccccagaagg 20
<210> 18
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<223> hPCSK2 gRNA 3
<400> 18
ggcgggcccg gggcggccca 20
<210> 19
<211> 20
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<213> Artificial Sequence
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<223> hPCSK2 gRNA 4
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ccgccctcat tggccgctgg 20
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<223> hPCSK2 gRNA 5
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gcccggggcg gcccagggcg 20
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<212> DNA
<213> Artificial Sequence
<220>
<223> Lentivirus vector 1
<400> 21
aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga 60
gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag 120
tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag 180
gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga 240
cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc 300
tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc 360
tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt 420
ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta 480
ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta 540
acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga 600
atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa 660
caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa 720
gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat 780
cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag 840
aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg 900
cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt 960
ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa 1020
gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga 1080
gatccagttt ggttaattag ctagcagata tacgcgttga cattgattat tgactagtct 1140
aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc 1200
ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt 1260
ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg 1320
ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg 1380
ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt 1440
atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg 1500
agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc 1560
ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc 1620
accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac 1680
ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca 1740
ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat 1800
gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag 1860
ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg 1920
caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg 1980
ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca 2040
cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc 2100
gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt 2160
ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta 2220
ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt 2280
ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt 2340
gacgtgagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac 2400
gactcactat agggagaccc aagctggcta gcgccatgga ctacaaagac catgacggtg 2460
attataaaga tcatgacatc gatataaatg gagctatagg aggtgacctt ttgctcaatt 2520
ttcctgacat gtcggtccta gagcgccaaa gggctcacct caagtacctc aatcccacct 2580
ttgattctcc tctcgccggc ttctttgccg attcttcaat gattaccggc ggcgagatgg 2640
acagctatct ttcgactgcc ggtttgaatc ttccgatgat gtacggtgag acgacggtgg 2700
aaggtgattc aagactctca atttcgccgg aaacgacgct tgggactgga aatttcaaga 2760
aacggaagtt tgatacagag actaaggatt gtaatgagaa gaagaagaag atgacgatga 2820
acagagatga cctagtagaa gaaggagaag aagagaagtc gaaaataaca gagcaaaaca 2880
atgggagcac aaaaagcatc aagaagatga aacacaaagc caagaaagaa gagaacaatt 2940
tctctaatga ttcatctaaa gtgacgaagg aattggagaa aacggattat attcatcacg 3000
attacaagga tgacgatgac aagatggccc ccaagaagaa gaggaaggtg ggccgcggaa 3060
tggataagaa atactcaata ggcttagcta tcggcacaaa tagcgtcgga tgggcggtga 3120
tcactgatga atataaggtt ccgtctaaaa agttcaaggt tctgggaaat acagaccgcc 3180
acagtatcaa aaaaaatctt ataggggctc ttttatttga cagtggagag acagcggaag 3240
cgactcgtct caaacggaca gctcgtagaa ggtatacacg tcggaagaat cgtatttgtt 3300
atctacagga gattttttca aatgagatgg cgaaagtaga tgatagtttc tttcatcgac 3360
ttgaagagtc ttttttggtg gaagaagaca agaagcatga acgtcatcct atttttggaa 3420
atatagtaga tgaagttgct tatcatgaga aatatccaac tatctatcat ctgcgaaaaa 3480
aattggtaga ttctactgat aaagcggatt tgcgcttaat ctatttggcc ttagcgcata 3540
tgattaagtt tcgtggtcat tttttgattg agggagattt aaatcctgat aatagtgatg 3600
tggacaaact atttatccag ttggtacaaa cctacaatca attatttgaa gaaaacccta 3660
ttaacgcaag tggagtagat gctaaagcga ttctttctgc acgattgagt aaatcaagac 3720
gattagaaaa tctcattgct cagctccccg gtgagaagaa aaatggctta tttgggaatc 3780
tcattgcttt gtcattgggt ttgaccccta attttaaatc aaattttgat ttggcagaag 3840
atgctaaatt acagctttca aaagatactt acgatgatga tttagataat ttattggcgc 3900
aaattggaga tcaatatgct gatttgtttt tggcagctaa gaatttatca gatgctattt 3960
tactttcaga tatcctaaga gtaaatactg aaataactaa ggctccccta tcagcttcaa 4020
tgattaaacg ctacgatgaa catcatcaag acttgactct tttaaaagct ttagttcgac 4080
aacaacttcc agaaaagtat aaagaaatct tttttgatca atcaaaaaac ggatatgcag 4140
gttatattga tgggggagct agccaagaag aattttataa atttatcaaa ccaattttag 4200
aaaaaatgga tggtactgag gaattattgg tgaaactaaa tcgtgaagat ttgctgcgca 4260
agcaacggac ctttgacaac ggctctattc cccatcaaat tcacttgggt gagctgcatg 4320
ctattttgag aagacaagaa gacttttatc catttttaaa agacaatcgt gagaagattg 4380
aaaaaatctt gacttttcga attccttatt atgttggtcc attggcgcgt ggcaatagtc 4440
gttttgcatg gatgactcgg aagtctgaag aaacaattac cccatggaat tttgaagaag 4500
ttgtcgataa aggtgcttca gctcaatcat ttattgaacg catgacaaac tttgataaaa 4560
atcttccaaa tgaaaaagta ctaccaaaac atagtttgct ttatgagtat tttacggttt 4620
ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat gcgaaaacca gcatttcttt 4680
caggtgaaca gaagaaagcc attgttgatt tactcttcaa aacaaatcga aaagtaaccg 4740
ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg ttttgatagt gttgaaattt 4800
caggagttga agatagattt aatgcttcat taggtaccta ccatgatttg ctaaaaatta 4860
ttaaagataa agattttttg gataatgaag aaaatgaaga tatcttagag gatattgttt 4920
taacattgac cttatttgaa gatagggaga tgattgagga aagacttaaa acatatgctc 4980
acctctttga tgataaggtg atgaaacagc ttaaacgtcg ccgttatact ggttggggac 5040
gtttgtctcg aaaattgatt aatggtatta gggataagca atctggcaaa acaatattag 5100
attttttgaa atcagatggt tttgccaatc gcaattttat gcagctgatc catgatgata 5160
gtttgacatt taaagaagac attcaaaaag cacaagtgtc tggacaaggc gatagtttac 5220
atgaacatat tgcaaattta gctggtagcc ctgctattaa aaaaggtatt ttacagactg 5280
taaaagttgt tgatgaattg gtcaaagtaa tggggcggca taagccagaa aatatcgtta 5340
ttgaaatggc acgtgaaaat cagacaactc aaaagggcca gaaaaattcg cgagagcgta 5400
tgaaacgaat cgaagaaggt atcaaagaat taggaagtca gattcttaaa gagcatcctg 5460
ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta ttatctccaa aatggaagag 5520
acatgtatgt ggaccaagaa ttagatatta atcgtttaag tgattatgat gtcgatgcca 5580
ttgttccaca aagtttcctt aaagacgatt caatagacaa taaggtctta acgcgttctg 5640
ataaaaatcg tggtaaatcg gataacgttc caagtgaaga agtagtcaaa aagatgaaaa 5700
actattggag acaacttcta aacgccaagt taatcactca acgtaagttt gataatttaa 5760
cgaaagctga acgtggaggt ttgagtgaac ttgataaagc tggttttatc aaacgccaat 5820
tggttgaaac tcgccaaatc actaagcatg tggcacaaat tttggatagt cgcatgaata 5880
ctaaatacga tgaaaatgat aaacttattc gagaggttaa agtgattacc ttaaaatcta 5940
aattagtttc tgacttccga aaagatttcc aattctataa agtacgtgag attaacaatt 6000
accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg aactgctttg attaagaaat 6060
atccaaaact tgaatcggag tttgtctatg gtgattataa agtttatgat gttcgtaaaa 6120
tgattgctaa gtctgagcaa gaaataggca aagcaaccgc aaaatatttc ttttactcta 6180
atatcatgaa cttcttcaaa acagaaatta cacttgcaaa tggagagatt cgcaaacgcc 6240
ctctaatcga aactaatggg gaaactggag aaattgtctg ggataaaggg cgagattttg 6300
ccacagtgcg caaagtattg tccatgcccc aagtcaatat tgtcaagaaa acagaagtac 6360
agacaggcgg attctccaag gagtcaattt taccaaaaag aaattcggac aagcttattg 6420
ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt tgatagtcca acggtagctt 6480
attcagtcct agtggttgct aaggtggaaa aagggaaatc gaagaagtta aaatccgtta 6540
aagagttact agggatcaca attatggaaa gaagttcctt tgaaaaaaat ccgattgact 6600
ttttagaagc taaaggatat aaggaagtta aaaaagactt aatcattaaa ctacctaaat 6660
atagtctttt tgagttagaa aacggtcgta aacggatgct ggctagtgcc ggagaattac 6720
aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa ttttttatat ttagctagtc 6780
attatgaaaa gttgaagggt agtccagaag ataacgaaca aaaacaattg tttgtggagc 6840
agcataagca ttatttagat gagattattg agcaaatcag tgaattttct aagcgtgtta 6900
ttttagcaga tgccaattta gataaagttc ttagtgcata taacaaacat agagacaaac 6960
caatacgtga acaagcagaa aatattattc atttatttac gttgacgaat cttggagctc 7020
ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa acgatatacg tctacaaaag 7080
aagttttaga tgccactctt atccatcaat ccatcactgg tctttatgaa acacgcattg 7140
atttgagtca gctaggaggt gacccaattg ccggatccaa ggctagcccg aaaaagaaac 7200
gcaaagttgg gcgcgccacc atgaatggag ctataggagg tgaccttttg ctcaattttc 7260
ctgacatgtc ggtcctagag cgccaaaggg ctcacctcaa gtacctcaat cccacctttg 7320
attctcctct cgccggcttc tttgccgatt cttcaatgat taccggcggc gagatggaca 7380
gctatctttc gactgccggt ttgaatcttc cgatgatgta cggtgagacg acggtggaag 7440
gtgattcaag actctcaatt tcgccggaaa cgacgcttgg gactggaaat ttcaagaaac 7500
ggaagtttga tacagagact aaggattgta atgagaagaa gaagaagatg acgatgaaca 7560
gagatgacct agtagaagaa ggagaagaag agaagtcgaa aataacagag caaaacaatg 7620
ggagcacaaa aagcatcaag aagatgaaac acaaagccaa gaaagaagag aacaatttct 7680
ctaatgattc atctaaagtg acgaaggaat tggagaaaac ggattatatt tgtacaggtt 7740
caggggaggg aagaggttcc ctgctcacat gcggagacgt agaagagaat cccggaccca 7800
tgacctctag agagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg 7860
ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc 7920
cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc 7980
tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc 8040
cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga 8100
gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca 8160
aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc 8220
tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct 8280
tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca 8340
ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg 8400
cctgagaatt cgatatcaag cttatcggta atcaacctct ggattacaaa atttgtgaaa 8460
gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa 8520
tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat 8580
cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt 8640
gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc 8700
tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc 8760
ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg 8820
ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga 8880
cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc 8940
tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc 9000
tttgggccgc ctccccgcat cgataccgtc gacctcgaga cctagaaaaa catggagcaa 9060
tcacaagtag caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg 9120
aggaggaggt gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg 9180
cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact 9240
cccaacgaag acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg 9300
attggcagaa ctacacacca gggccaggga tcagatatcc actgaccttt ggatggtgct 9360
acaagctagt accagttgag caagagaagg tagaagaagc caatgaagga gagaacaccc 9420
gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtattagagt 9480
ggaggtttga cagccgccta gcatttcatc acatggcccg agagctgcat ccggactgta 9540
ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc 9600
cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt 9660
tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta 9720
gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 9780
gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 9840
tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 9900
ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 9960
gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat 10020
ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 10080
accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 10140
gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 10200
tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 10260
gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 10320
agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 10380
ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 10440
tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 10500
ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 10560
agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 10620
ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 10680
ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 10740
ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 10800
tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca attaatcatc 10860
ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 10920
gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 10980
cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 11040
cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 11100
ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 11160
gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 11220
ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 11280
ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 11340
aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt 11400
cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 11460
cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 11520
catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc 11580
atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 11640
agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 11700
tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 11760
aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 11820
cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 11880
ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 11940
ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 12000
cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 12060
actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 12120
cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 12180
tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 12240
gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 12300
caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 12360
agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 12420
tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 12480
tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 12540
gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 12600
gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 12660
aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 12720
atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 12780
gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 12840
acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 12900
ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 12960
tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 13020
ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 13080
ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 13140
atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 13200
taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 13260
catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 13320
atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 13380
acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 13440
aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 13500
ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 13560
cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 13620
atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 13680
ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 13740
cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat 13800
gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc 13860
gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg 13920
cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat 13980
tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat 14040
atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 14100
ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 14160
cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 14220
tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 14280
tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 14340
atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 14400
gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 14460
caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 14520
ggtaggcgtg tacggtggga ggtctatata agcagcgcgt tttgcctgta ctgggtctct 14580
ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 14640
gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 14700
tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc 14760
ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg 14820
cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt 14880
ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg 14940
agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa 15000
ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg 15060
ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca 15120
ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa 15180
aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaa 15239
<210> 22
<211> 13578
<212> DNA
<213> Artificial Sequence
<220>
<223> Lentivirus vector 2 (4gRNA set 1)
<400> 22
gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60
atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120
gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180
tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240
attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300
atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360
acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600
tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660
ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720
accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780
gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840
ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900
aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960
tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020
gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080
ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140
ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200
ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260
aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320
tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380
caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440
aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500
aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560
agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1620
gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680
ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740
acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800
ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860
ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920
tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980
aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2040
aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100
aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160
acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220
agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280
tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340
gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400
gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460
tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520
agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580
agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640
cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acattatacg 2700
aagttatgat agatccgacg ccgccatctc taggcccgcg ccggccccct cgcacagact 2760
tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820
actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880
attttacatg ataggcttgg atttctataa gagatacaaa tactaaatta ttattttaaa 2940
aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000
atatcccttg gagaaaagcc ttgtttgacc ccaggcccta atgggccgtt ttagagctag 3060
aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120
tgcttttttt gtacgctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180
tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaacgg cggctgctca 3240
aatactctcg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300
tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360
cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420
caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480
catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540
atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac ccagcgcgcg 3600
tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660
tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720
tataagttct gtatgagacc actctttccc aagctgctgg gactcgcgac cgttttagag 3780
ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840
tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900
aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa acgctgtaat 3960
caggccagcg gccgaggtac ccaagcggcg cacaagctat ataaacctga aggaagtctc 4020
aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080
aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140
aacggacttg atttccggct gttttgacac tatccagaat gccttgcaga tgggtggggc 4200
atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260
gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320
gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380
gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg 4440
ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500
aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560
aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620
ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680
tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740
ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800
cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt 4860
tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920
tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980
cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040
ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100
ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160
atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220
ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca 5280
cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340
tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400
gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc 5460
tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520
ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580
acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640
catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700
catgaagatg gacaaaaaga ctatagtttg gtttagaaga gacctaagga ttgaggataa 5760
tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820
tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880
tgctcactta tctcaatcct tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940
caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000
taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060
ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120
atactgcgaa aagggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180
tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240
ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300
accgagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360
actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420
tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480
cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540
agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600
atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660
ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720
ggtggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780
gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840
tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900
ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960
cgccaaatat gacccagaag gtgagtacat aaggcaatgg cttcccgagc ttgcgagatt 7020
gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080
tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140
actagctaaa gctatttcaa gaacccgtga agcacagatc atgatcggag cagcacctga 7200
tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260
ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320
aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380
tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcgagtgtgt ttttcgtttc 7440
gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500
tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560
cccgaaaaag aaacgcaaag ttgggcgcgc cgacgcgctg gacgatttcg atctcgacat 7620
gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680
tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740
aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800
aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860
gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920
ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980
tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc 8040
tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100
catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160
agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220
ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 8280
ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340
atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400
tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt 8460
caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520
tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580
ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640
caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700
cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760
ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820
tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgaga 8880
cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940
ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000
aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060
actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120
cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180
actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240
caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300
ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg 9360
agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420
ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480
agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540
gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc 9600
cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660
gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720
ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780
acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840
gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900
tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960
ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 10020
ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080
agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 10140
tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 10200
tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260
atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320
gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380
gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440
gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500
tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560
ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 10620
cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680
cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740
aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800
ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860
ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920
gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980
tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040
ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100
ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160
tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220
ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 11280
tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340
agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400
agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 11460
acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520
gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580
tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 11880
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240
tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540
tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 12600
agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660
gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720
ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780
gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840
cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900
acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960
cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 13020
cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080
ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140
tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200
atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 13260
tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320
actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380
aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440
ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500
ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560
cgaaaagtgc cacctgac 13578
<210> 23
<211> 13578
<212> DNA
<213> Artificial Sequence
<220>
<223> Lentivirus vector 2 (4gRNA set 2)
<400> 23
gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60
atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120
gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180
tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240
attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300
atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360
acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600
tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660
ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720
accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780
gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840
ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900
aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960
tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020
gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080
ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140
ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200
ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260
aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320
tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380
caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440
aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500
aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560
agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1620
gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680
ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740
acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800
ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860
ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920
tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980
aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2040
aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100
aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160
acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220
agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280
tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340
gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400
gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460
tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520
agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580
agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640
cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acattatacg 2700
aagttatgat agatccgacg ccgccatctc taggcccgcg ccggccccct cgcacagact 2760
tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820
actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880
attttacatg ataggcttgg atttctataa gagatacaaa tactaaatta ttattttaaa 2940
aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000
atatcccttg gagaaaagcc ttgtttgcta atgacccgct ggtcctggtt ttagagctag 3060
aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120
tgcttttttt gtacgctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180
tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaactt cctcagcctt 3240
gtgcgggccg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300
tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360
cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420
caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480
catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540
atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac ccagcgcgcg 3600
tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660
tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720
tataagttct gtatgagacc actctttccc aatctatccc cgcgactctc tgttttagag 3780
ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840
tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900
aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa acccttctgg 3960
gggcgcagcg cacgaggtac ccaagcggcg cacaagctat ataaacctga aggaagtctc 4020
aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080
aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140
aacggacttg atttccggct gttttgacac tatccagaat gccttgcaga tgggtggggc 4200
atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260
gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320
gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380
gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg 4440
ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500
aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560
aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620
ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680
tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740
ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800
cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt 4860
tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920
tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980
cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040
ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100
ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160
atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220
ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca 5280
cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340
tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400
gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc 5460
tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520
ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580
acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640
catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700
catgaagatg gacaaaaaga ctatagtttg gtttagaaga gacctaagga ttgaggataa 5760
tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820
tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880
tgctcactta tctcaatcct tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940
caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000
taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060
ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120
atactgcgaa aagggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180
tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240
ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300
accgagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360
actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420
tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480
cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540
agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600
atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660
ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720
ggtggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780
gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840
tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900
ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960
cgccaaatat gacccagaag gtgagtacat aaggcaatgg cttcccgagc ttgcgagatt 7020
gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080
tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140
actagctaaa gctatttcaa gaacccgtga agcacagatc atgatcggag cagcacctga 7200
tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260
ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320
aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380
tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcgagtgtgt ttttcgtttc 7440
gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500
tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560
cccgaaaaag aaacgcaaag ttgggcgcgc cgacgcgctg gacgatttcg atctcgacat 7620
gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680
tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740
aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800
aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860
gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920
ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980
tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc 8040
tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100
catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160
agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220
ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 8280
ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340
atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400
tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt 8460
caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520
tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580
ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640
caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700
cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760
ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820
tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgaga 8880
cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940
ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000
aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060
actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120
cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180
actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240
caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300
ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg 9360
agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420
ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480
agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540
gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc 9600
cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660
gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720
ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780
acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840
gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900
tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960
ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 10020
ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080
agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 10140
tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 10200
tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260
atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320
gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380
gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440
gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500
tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560
ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 10620
cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680
cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740
aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800
ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860
ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920
gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980
tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040
ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100
ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160
tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220
ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 11280
tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340
agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400
agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 11460
acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520
gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580
tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 11880
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240
tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540
tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 12600
agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660
gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720
ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780
gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840
cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900
acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960
cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 13020
cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080
ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140
tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200
atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 13260
tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320
actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380
aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440
ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500
ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560
cgaaaagtgc cacctgac 13578
<210> 24
<211> 13578
<212> DNA
<213> Artificial Sequence
<220>
<223> Lentivirus vector 2 (4gRNA set 3)
<400> 24
gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60
atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120
gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180
tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240
attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300
atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360
acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600
tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660
ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720
accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780
gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840
ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900
aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960
tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020
gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080
ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140
ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200
ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260
aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320
tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380
caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440
aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500
aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560
agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1620
gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680
ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740
acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800
ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860
ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920
tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980
aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2040
aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100
aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160
acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220
agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280
tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340
gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400
gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460
tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520
agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580
agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640
cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acattatacg 2700
aagttatgat agatccgacg ccgccatctc taggcccgcg ccggccccct cgcacagact 2760
tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820
actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880
attttacatg ataggcttgg atttctataa gagatacaaa tactaaatta ttattttaaa 2940
aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000
atatcccttg gagaaaagcc ttgtttgtga gactataaag ccagcgggtt ttagagctag 3060
aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120
tgcttttttt gtacgctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180
tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaactg tcactacagt 3240
gaccgctacg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300
tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360
cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420
caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480
catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540
atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac ccagcgcgcg 3600
tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660
tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720
tataagttct gtatgagacc actctttccc acgcgactct ctagggagct cgttttagag 3780
ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840
tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900
aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa actgggccgc 3960
cccgggcccg cccgaggtac ccaagcggcg cacaagctat ataaacctga aggaagtctc 4020
aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080
aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140
aacggacttg atttccggct gttttgacac tatccagaat gccttgcaga tgggtggggc 4200
atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260
gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320
gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380
gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg 4440
ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500
aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560
aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620
ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680
tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740
ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800
cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt 4860
tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920
tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980
cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040
ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100
ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160
atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220
ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca 5280
cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340
tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400
gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc 5460
tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520
ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580
acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640
catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700
catgaagatg gacaaaaaga ctatagtttg gtttagaaga gacctaagga ttgaggataa 5760
tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820
tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880
tgctcactta tctcaatcct tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940
caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000
taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060
ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120
atactgcgaa aagggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180
tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240
ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300
accgagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360
actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420
tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480
cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540
agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600
atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660
ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720
ggtggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780
gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840
tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900
ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960
cgccaaatat gacccagaag gtgagtacat aaggcaatgg cttcccgagc ttgcgagatt 7020
gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080
tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140
actagctaaa gctatttcaa gaacccgtga agcacagatc atgatcggag cagcacctga 7200
tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260
ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320
aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380
tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcgagtgtgt ttttcgtttc 7440
gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500
tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560
cccgaaaaag aaacgcaaag ttgggcgcgc cgacgcgctg gacgatttcg atctcgacat 7620
gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680
tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740
aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800
aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860
gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920
ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980
tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc 8040
tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100
catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160
agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220
ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 8280
ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340
atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400
tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt 8460
caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520
tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580
ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640
caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700
cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760
ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820
tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgaga 8880
cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940
ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000
aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060
actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120
cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180
actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240
caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300
ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg 9360
agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420
ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480
agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540
gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc 9600
cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660
gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720
ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780
acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840
gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900
tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960
ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 10020
ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080
agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 10140
tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 10200
tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260
atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320
gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380
gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440
gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500
tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560
ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 10620
cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680
cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740
aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800
ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860
ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920
gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980
tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040
ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100
ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160
tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220
ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 11280
tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340
agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400
agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 11460
acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520
gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580
tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 11880
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240
tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540
tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 12600
agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660
gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720
ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780
gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840
cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900
acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960
cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 13020
cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080
ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140
tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200
atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 13260
tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320
actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380
aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440
ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500
ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560
cgaaaagtgc cacctgac 13578
<210> 25
<211> 13578
<212> DNA
<213> Artificial Sequence
<220>
<223> Lentivirus vector 2 (4gRNA set 4)
<400> 25
gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60
atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120
gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180
tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240
attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300
atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360
acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600
tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660
ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720
accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780
gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840
ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900
aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960
tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020
gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080
ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140
ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200
ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260
aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320
tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380
caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440
aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500
aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560
agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1620
gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680
ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740
acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800
ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860
ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920
tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980
aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2040
aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100
aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160
acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220
agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280
tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340
gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400
gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460
tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520
agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580
agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640
cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acattatacg 2700
aagttatgat agatccgacg ccgccatctc taggcccgcg ccggccccct cgcacagact 2760
tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820
actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880
attttacatg ataggcttgg atttctataa gagatacaaa tactaaatta ttattttaaa 2940
aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000
atatcccttg gagaaaagcc ttgtttgctc tgagactata aagccaggtt ttagagctag 3060
aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120
tgcttttttt gtacgctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180
tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaactg gcaggagaat 3240
cccccactcg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300
tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360
cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420
caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480
catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540
atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac ccagcgcgcg 3600
tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660
tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720
tataagttct gtatgagacc actctttccc acgcggagta gcctgggaag cgttttagag 3780
ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840
tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900
aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa acccagcggc 3960
caatgagggc ggcgaggtac ccaagcggcg cacaagctat ataaacctga aggaagtctc 4020
aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080
aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140
aacggacttg atttccggct gttttgacac tatccagaat gccttgcaga tgggtggggc 4200
atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260
gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320
gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380
gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg 4440
ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500
aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560
aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620
ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680
tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740
ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800
cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt 4860
tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920
tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980
cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040
ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100
ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160
atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220
ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca 5280
cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340
tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400
gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc 5460
tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520
ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580
acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640
catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700
catgaagatg gacaaaaaga ctatagtttg gtttagaaga gacctaagga ttgaggataa 5760
tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820
tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880
tgctcactta tctcaatcct tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940
caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000
taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060
ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120
atactgcgaa aagggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180
tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240
ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300
accgagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360
actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420
tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480
cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540
agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600
atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660
ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720
ggtggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780
gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840
tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900
ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960
cgccaaatat gacccagaag gtgagtacat aaggcaatgg cttcccgagc ttgcgagatt 7020
gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080
tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140
actagctaaa gctatttcaa gaacccgtga agcacagatc atgatcggag cagcacctga 7200
tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260
ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320
aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380
tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcgagtgtgt ttttcgtttc 7440
gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500
tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560
cccgaaaaag aaacgcaaag ttgggcgcgc cgacgcgctg gacgatttcg atctcgacat 7620
gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680
tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740
aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800
aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860
gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920
ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980
tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc 8040
tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100
catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160
agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220
ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 8280
ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340
atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400
tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt 8460
caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520
tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580
ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640
caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700
cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760
ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820
tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgaga 8880
cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940
ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000
aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060
actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120
cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180
actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240
caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300
ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg 9360
agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420
ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480
agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540
gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc 9600
cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660
gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720
ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780
acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840
gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900
tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960
ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 10020
ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080
agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 10140
tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 10200
tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260
atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320
gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380
gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440
gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500
tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560
ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 10620
cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680
cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740
aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800
ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860
ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920
gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980
tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040
ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100
ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160
tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220
ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 11280
tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340
agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400
agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 11460
acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520
gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580
tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 11880
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240
tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540
tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 12600
agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660
gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720
ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780
gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840
cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900
acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960
cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 13020
cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080
ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140
tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200
atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 13260
tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320
actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380
aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440
ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500
ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560
cgaaaagtgc cacctgac 13578
<210> 26
<211> 13578
<212> DNA
<213> Artificial Sequence
<220>
<223> Lentivirus vector 2 (4gRNA set 5)
<400> 26
gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60
atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120
gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180
tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240
attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300
atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360
acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600
tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660
ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720
accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780
gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840
ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900
aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960
tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020
gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080
ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140
ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200
ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260
aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320
tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380
caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440
aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500
aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560
agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1620
gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680
ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740
acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800
ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860
ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920
tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980
aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2040
aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100
aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160
acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220
agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280
tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340
gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400
gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460
tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520
agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580
agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640
cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acattatacg 2700
aagttatgat agatccgacg ccgccatctc taggcccgcg ccggccccct cgcacagact 2760
tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820
actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880
attttacatg ataggcttgg atttctataa gagatacaaa tactaaatta ttattttaaa 2940
aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000
atatcccttg gagaaaagcc ttgtttggga agaggtgctg acgaccagtt ttagagctag 3060
aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120
tgcttttttt gtacgctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180
tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaacgt tctgatgagc 3240
agaggatccg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300
tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360
cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420
caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480
catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540
atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac ccagcgcgcg 3600
tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660
tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720
tataagttct gtatgagacc actctttccc acttggtagt tatgttcaag tgttttagag 3780
ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840
tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900
aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa accgccctgg 3960
gccgccccgg gccgaggtac ccaagcggcg cacaagctat ataaacctga aggaagtctc 4020
aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080
aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140
aacggacttg atttccggct gttttgacac tatccagaat gccttgcaga tgggtggggc 4200
atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260
gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320
gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380
gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg 4440
ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500
aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560
aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620
ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680
tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740
ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800
cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt 4860
tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920
tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980
cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040
ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100
ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160
atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220
ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca 5280
cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340
tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400
gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc 5460
tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520
ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580
acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640
catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700
catgaagatg gacaaaaaga ctatagtttg gtttagaaga gacctaagga ttgaggataa 5760
tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820
tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880
tgctcactta tctcaatcct tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940
caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000
taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060
ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120
atactgcgaa aagggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180
tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240
ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300
accgagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360
actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420
tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480
cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540
agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600
atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660
ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720
ggtggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780
gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840
tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900
ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960
cgccaaatat gacccagaag gtgagtacat aaggcaatgg cttcccgagc ttgcgagatt 7020
gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080
tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140
actagctaaa gctatttcaa gaacccgtga agcacagatc atgatcggag cagcacctga 7200
tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260
ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320
aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380
tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcgagtgtgt ttttcgtttc 7440
gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500
tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560
cccgaaaaag aaacgcaaag ttgggcgcgc cgacgcgctg gacgatttcg atctcgacat 7620
gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680
tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740
aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800
aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860
gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920
ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980
tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc 8040
tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100
catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160
agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220
ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 8280
ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340
atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400
tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt 8460
caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520
tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580
ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640
caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700
cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760
ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820
tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgaga 8880
cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940
ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000
aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060
actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120
cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180
actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240
caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300
ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg 9360
agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420
ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480
agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540
gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc 9600
cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660
gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720
ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780
acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840
gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900
tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960
ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 10020
ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080
agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 10140
tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 10200
tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260
atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320
gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380
gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440
gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500
tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560
ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 10620
cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680
cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740
aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800
ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860
ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920
gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980
tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040
ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100
ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160
tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220
ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 11280
tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340
agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400
agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 11460
acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520
gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580
tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 11880
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240
tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540
tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 12600
agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660
gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720
ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780
gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840
cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900
acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960
cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 13020
cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080
ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140
tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200
atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 13260
tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320
actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380
aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440
ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500
ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560
cgaaaagtgc cacctgac 13578
<210> 27
<211> 4104
<212> DNA
<213> Artificial Sequence
<220>
<223> dCas9
<400> 27
atggataaga aatactcaat aggcttagct atcggcacaa atagcgtcgg atgggcggtg 60
atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120
cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180
gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240
tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300
cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360
aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420
aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480
atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540
gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600
attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660
cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720
ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 780
gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840
caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900
ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 960
atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020
caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080
ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140
gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200
aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260
gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320
gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380
cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440
gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500
aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560
tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620
tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680
gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740
tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800
attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860
ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct 1920
cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980
cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040
gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100
agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160
catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220
gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280
attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2340
atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2400
gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460
gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatgcc 2520
attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct 2580
gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640
aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700
acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760
ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820
actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2880
aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940
taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3000
tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060
atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3120
aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180
cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240
gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300
cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3360
gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3420
tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3480
aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540
tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa 3600
tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660
caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720
cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780
cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840
attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900
ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960
cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020
gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080
gatttgagtc agctaggagg tgac 4104
<210> 28
<211> 510
<212> DNA
<213> Artificial Sequence
<220>
<223> CIBN
<400> 28
atgaatggag ctataggagg tgaccttttg ctcaattttc ctgacatgtc ggtcctagag 60
cgccaaaggg ctcacctcaa gtacctcaat cccacctttg attctcctct cgccggcttc 120
tttgccgatt cttcaatgat taccggcggc gagatggaca gctatctttc gactgccggt 180
ttgaatcttc cgatgatgta cggtgagacg acggtggaag gtgattcaag actctcaatt 240
tcgccggaaa cgacgcttgg gactggaaat ttcaagaaac ggaagtttga tacagagact 300
aaggattgta atgagaagaa gaagaagatg acgatgaaca gagatgacct agtagaagaa 360
ggagaagaag agaagtcgaa aataacagag caaaacaatg ggagcacaaa aagcatcaag 420
aagatgaaac acaaagccaa gaaagaagag aacaatttct ctaatgattc atctaaagtg 480
acgaaggaat tggagaaaac ggattatatt 510
<210> 29
<211> 1836
<212> DNA
<213> Artificial Sequence
<220>
<223> CRY2FL
<400> 29
atgaagatgg acaaaaagac tatagtttgg tttagaagag acctaaggat tgaggataat 60
cctgcattag cagcagctgc tcacgaagga tctgtttttc ctgtcttcat ttggtgtcct 120
gaagaagaag gacagtttta tcctggaaga gcttcaagat ggtggatgaa acaatcactt 180
gctcacttat ctcaatcctt gaaggctctt ggatctgacc tcactttaat caaaacccac 240
aacacgattt cagcgatctt ggattgtatc cgcgttaccg gtgctacaaa agtcgtcttt 300
aaccacctct atgatcctgt ttcgttagtt cgggaccata ccgtaaagga gaagctggtg 360
gaacgtggga tctctgtgca aagctacaat ggagatctat tgtatgaacc gtgggagata 420
tactgcgaaa agggcaaacc ttttacgagt ttcaattctt actggaagaa atgcttagat 480
atgtcgattg aatccgttat gcttcctcct ccttggcggt tgatgccaat aactgcagcg 540
gctgaagcga tttgggcgtg ttcgattgaa gaactagggc tggagaatga ggccgagaaa 600
ccgagcaatg cgttgttaac tagagcttgg tctccaggat ggagcaatgc tgataagtta 660
ctaaatgagt tcatcgagaa gcagttgata gattatgcaa agaacagcaa gaaagttgtt 720
gggaattcta cttcactact ttctccgtat ctccatttcg gggaaataag cgtcagacac 780
gttttccagt gtgcccggat gaaacaaatt atatgggcaa gagataagaa cagtgaagga 840
gaagaaagtg cagatctttt tcttagggga atcggtttaa gagagtattc tcggtatata 900
tgtttcaact tcccgtttac tcacgagcaa tcgttgttga gtcatcttcg gtttttccct 960
tgggatgctg atgttgataa gttcaaggcc tggagacaag gcaggaccgg ttatccgttg 1020
gtggatgccg gaatgagaga gctttgggct accggatgga tgcataacag aataagagtg 1080
attgtttcaa gctttgctgt gaagtttctt ctccttccat ggaaatgggg aatgaagtat 1140
ttctgggata cacttttgga tgctgatttg gaatgtgaca tccttggctg gcagtatatc 1200
tctgggagta tccccgatgg ccacgagctt gatcgcttgg acaatcccgc gttacaaggc 1260
gccaaatatg acccagaagg tgagtacata aggcaatggc ttcccgagct tgcgagattg 1320
ccaactgaat ggatccatca tccatgggac gctcctttaa ccgtactcaa agcttctggt 1380
gtggaactcg gaacaaacta tgcgaaaccc attgtagaca tcgacacagc tcgtgagcta 1440
ctagctaaag ctatttcaag aacccgtgaa gcacagatca tgatcggagc agcacctgat 1500
gagattgtag cagatagctt cgaggcctta ggggctaata ccattaaaga acctggtctt 1560
tgcccatctg tgtcttctaa tgaccaacaa gtaccttcgg ctgttcgtta caacgggtca 1620
aagagagtga aacctgagga agaagaagag agagacatga agaaatctag gggattcgat 1680
gaaagggagt tgttttcgac tgctgaatct tcttcttctt cgagtgtgtt tttcgtttcg 1740
cagtcttgct cgttggcatc agaagggaag aatctggaag gtattcaaga ttcatctgat 1800
cagattacta caagtttggg aaaaaatggt tgcaaa 1836
<210> 30
<211> 150
<212> DNA
<213> Artificial Sequence
<220>
<223> VP64
<400> 30
gacgcgctgg acgatttcga tctcgacatg ctgggttctg atgccctcga tgactttgac 60
ctggatatgt tgggaagcga cgcattggat gactttgatc tggacatgct cggctccgat 120
gctctggacg atttcgatct cgatatgtta 150
Claims (19)
- dCas (dead Cas) 단백질 및 제1 광특이적 결합 단백질을 각각 코딩하는 폴리뉴클레오티드를 포함하는 제1 벡터; 및
줄기세포에서의 발현을 조절하려는 표적 유전자의 가이드 RNA(guide RNA: gRNA) 및 제2 광특이적 결합 단백질을 각각 코딩하는 폴리뉴클레오티드를 포함하는 제2 벡터를 포함하는, 줄기세포의 형질전환용 키트. - 청구항 1에 있어서, 상기 dCas 단백질은 dCas9 단백질을 포함하는 것인, 줄기세포의 형질전환용 키트.
- 청구항 1에 있어서, 상기 제1 광특이적 결합 단백질은 CIB1 (cryptochrome-interacting basic helix-loop-helix protein) 또는 이의 단편을 포함하는 것인, 줄기세포의 형질전환용 키트.
- 청구항 1에 있어서, 상기 제2 광특이적 결합 단백질은 CRY2 (cryptochrome 2) 또는 이의 단편을 포함하는 것인, 줄기세포의 형질전환용 키트.
- 청구항 1에 있어서, 상기 제1 벡터는 Ef1α 프로모터, UbC 프로모터 및 CAGG 프로모터로 이루어진 군으로부터 선택되는 프로모터를 더 포함하는 것인, 줄기세포의 형질전환용 키트.
- 청구항 1에 있어서, 상기 제1 벡터는 서열번호 21의 염기서열을 포함하는 것인, 줄기세포의 형질전환용 키트.
- 청구항 1에 있어서, 상기 제2 벡터는 Ef1α 프로모터, UbC 프로모터 및 CAGG 프로모터로 이루어진 군으로부터 선택되는 프로모터를 더 포함하는 것인, 줄기세포의 형질전환용 키트.
- 청구항 1에 있어서, 상기 제2 벡터는 서열번호 22 내지 26의 염기서열 중 어느 하나의 염기서열을 포함하는 것인, 줄기세포의 형질전환용 키트.
- 청구항 1에 있어서, 상기 줄기세포는 배아 줄기세포(embryonic stem cell: ES), 성체 줄기세포(adult stem cell) 또는 유도 만능 줄기세포(induced pluripotent stem cell: iPSC)인 것인, 줄기세포의 형질전환용 키트.
- 청구항 9에 있어서, 상기 배아 줄기세포는 체세포 복제된 배아 줄기세포인 것인, 줄기세포의 형질전환용 키트.
- 청구항 1에 있어서, 상기 표적 유전자는 INS, GCK, PCSK1 및 PCSK2로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 것인, 줄기세포의 형질전환용 키트.
- 청구항 11에 있어서, 상기 INS의 gRNA를 코딩하는 폴리뉴클레오티드는 서열번호 1 내지 5의 염기서열 및 이들 각각의 상보적인 염기서열로 이루어진 군으로부터 선택되는 어느 하나의 염기서열을 포함하고;
상기 GCK의 gRNA를 코딩하는 폴리뉴클레오티드는 서열번호 6 내지 10의 염기서열 및 이들 각각의 상보적인 염기서열로 이루어진 군으로부터 선택되는 어느 하나의 염기서열을 포함하고;
상기 PCSK1의 gRNA를 코딩하는 폴리뉴클레오티드는 서열번호 11 내지 15의 염기서열 및 이들 각각의 상보적인 염기서열로 이루어진 군으로부터 선택되는 어느 하나의 염기서열을 포함하고; 및
상기 PCSK2의 gRNA를 코딩하는 폴리뉴클레오티드는 서열번호 16 내지 20의 염기서열 및 이들 각각의 상보적인 염기서열로 이루어진 군으로부터 선택되는 어느 하나의 염기서열을 포함하는 것인, 줄기세포의 형질전환용 키트. - 청구항 1 내지 12 중 어느 한 항의 줄기세포의 형질전환용 키트를 이용하여 형질전환한 줄기세포.
- 청구항 13에 있어서, 상기 형질전환한 줄기세포에 440 내지 490 nm 파장을 갖는 빛을 조사하면 상기 줄기세포의 표적 유전자 발현이 증가하는 것인, 줄기세포.
- 청구항 14에 있어서, 상기 표적 유전자는 INS, GCK, PCSK1 및 PCSK2로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 것인, 줄기세포.
- 청구항 14에 있어서, 상기 표적 유전자의 발현이 증가한 줄기세포에 상기 빛의 조사를 중단하면 증가한 표적 유전자의 발현이 다시 감소하는 것인, 줄기세포.
- 청구항 13의 형질전환한 줄기세포에 440 내지 490 nm 파장을 갖는 빛을 조사하여 줄기세포의 표적 유전자 발현을 증가시키는 단계를 포함하는 줄기세포의 표적 유전자 발현을 조절하는 방법.
- 청구항 17에 있어서, 상기 표적 유전자는 INS, GCK, PCSK1 및 PCSK2로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 것인, 줄기세포의 표적 유전자 발현을 조절하는 방법.
- 청구항 17에 있어서, 상기 빛은 30 내지 70%의 펄스(pulse)를 갖는 빛인 것인, 줄기세포의 표적 유전자 발현을 조절하는 방법.
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| KR20190093171A (ko) | 2018-01-31 | 2019-08-08 | 서울대학교산학협력단 | CRISPR/Cas9 시스템을 통한 닭 백혈병 바이러스(Avian Leukosis Virus, ALV) 저항성 조류의 제조방법 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20190093171A (ko) | 2018-01-31 | 2019-08-08 | 서울대학교산학협력단 | CRISPR/Cas9 시스템을 통한 닭 백혈병 바이러스(Avian Leukosis Virus, ALV) 저항성 조류의 제조방법 |
Non-Patent Citations (3)
| Title |
|---|
| Chem Biol. 2015 Feb 19, 22(2):169-74. * |
| Nat Chem Biol. 2015 Mar, 11(3):198-200. * |
| Nat Rev Mol Cell Biol. 2019 Aug, 20(8):490-507. * |
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