CRISPR/Cas9 유전자 편집 기술과 돼지에서 응용
CRISPR/Cas9 Gene Editing Technology and Its Applications in Pigs
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초록
새롭게 부상하는 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) 9 유전자 편집기술은 장기 이식(organ transplantation)과 같은 생의학 연구(biomedical research)와 동물 산업에 대한 전통적인 접근 방식을 빠르게 변화시키고있다. 돼지 생식 및 호흡기 증후군 바이러스(porcine reproductive and respiratory syndrome virus; PRRSV)와 전염성 위장염 바이러스(transmissible gastroenteritis coronavirus; TGEV)는 돼지 산업에 막대한 경제적 손실을 초래하는 치명적인 바이러스이다. 바이러스의 숙주 수용체단백질 CD163과 pAPN에 대한 이중 유전자 녹아웃(double knock-out; DKO) 돼지는 PRRSV와 TEGV에 내성을 나타내었으며, 정상(wild-type; WT) 돼지와 비교할 때 성장과 생식 특성의 차이가 없었다. 이러한 결과는 경제 동물 돼지에 CRISPR-Cas9 매개 유전자 편집 기술을 적용하여바이러스 저항성 유전자 변형에 의한 품종 개량이 달성될 수 있다는 것을 보여주며, 질병 저항성 돼지 생산을 위한 육종 시작점을 제공한다. 종간 배반포 보완(interspecies blastocyst complementation)은 이종 만능 줄기세포 유도체(xenogenic pluripotent stem cell derivatives)의 장기특이적 생산(organ-specific enrichment)을 가능하게 한다. CRISPR-Cas9 매개 접합자 유전자 편집(CRISPR-Cas9-mediated zygote gene editing) 을 이용하여 췌장 생성(pancreatogenesis), 신장 생성(nephrogenesis), 간 생성(hepatogenesis) 및 혈관 생성(vasculogenesis)이 불가능 생쥐 숙주를만들었으며, 이러한 숙주와 배반포 보완 플랫폼을 결합하여 키메라를 만들었다. 또한 돼지와 소 같은 유제류(ungulate)의 섬유아세포(fibroblasts)를이용하여 CRISPR-Cas9 매개 유전자 편집과 체세포 핵 치환(somatic cell nuclear transfer) 과정을 거쳐 복제 배아(genome-edited cloned embryos) 를 생산하였다. 복제 배아의 1차 배양 섬유아세포(primary cultured fibroblasts)를 재복제하여 배반포 보완을 위한 숙주 배아로 이용하였다. CRISPR-Cas9 유전자 편집 기술과 종간 배반포 보완 플랫폼 전략의 조합은 유전자 변형 돼지를 생산하는 데 유용하다. 본 논문에서는 CRISPR/Cas9 유전자 편집 기술과 배반포 보완 플랫폼, 질병 저항성(disease resistance) 돼지, 이종장기이식(xenotransplantation) 목적의 키메라 생산을 소개하고자한다.
Abstract
The emerging CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) 9 gene editing technology is rapidly changing the traditional approach to biomedical research and animal industry such as organ transplantation. Porcine reproductive and respiratory syndrome virus (PRRSV) and infectious gastroenteritis virus (TGEV) are lethal viruses that cause major economic losses to pig production. The double gene knockout (DKO) pigs against these viral receptor proteins CD163 and pAPN showed resistance to PRRSV and TEGV, and there were no differences in meat production and reproductive performance traits compared to wild-type (WT) pigs. These results show that breeding by viral resistance genetic modification can be achieved by applying CRISPR-Cas9 mediated gene editing technology to economic animal pigs, providing a breeding starting point for the production of disease-resistant pigs. Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell derivatives. CRISPR-Cas9-mediated zygote gene editing was used to create a mouse host incapable of pancreatogenesis, nephrogenesis, hepatogenesis and vasculogenesis. Chimera was created by combining this host with a blastocyst complementation platform. In addition, using fibroblasts from ungulates such as pigs and cows, genome-edited cloned embryos were produced. Primary cultured fibroblasts were used for generating cloned embryos as the host embryos for blastocyst complementation. The combination of CRISPR/Cas9 gene editing technology and an interspecies blastocyst complementation platform strategy is particularly useful for generating genetically modified pigs. Therefore, this review paper introduces CRISPR/Cas9 gene editing technology, interspecies blastocyst complementation platform, disease resistance pigs, and human-pig chimera production for xenotransplantation purposes.
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