中国农业科学
中國農業科學
중국농업과학
SCIENTIA AGRICULTURA SINICA
2015年
9期
1669-1677
,共9页
CRISPR/Cas系统%基因组编辑%植物基因工程
CRISPR/Cas繫統%基因組編輯%植物基因工程
CRISPR/Cas계통%기인조편집%식물기인공정
CRISPR/Cas system%genome editing%plant genetic engineering
当外源DNA通过转基因技术导入植物细胞后，会以同源重组或非同源重组两种不同的方式整合到基因组中，进而获得相应的目标性状。外源DNA与受体细胞序列相同或相近的位点发生重新组合，从而整合到受体细胞的染色体上称之为同源重组；当发生了 DNA 双链断裂的细胞为了避免 DNA 或染色体断裂而造成DNA降解或对生命力的影响，而强行将2个DNA断端彼此连接在一起时则为非同源重组。发生非同源重组的细胞其基因组常出现核苷酸片段的插入和/或缺失以及其他突变等多种情况，使得研究者无法得到精确控制的突变结果；而发生同源重组的细胞基因组序列通常不变，通过加入同源重组的供体DNA，可以实现对基因组的精确修饰和改造。由于在植物中产生自发同源重组的概率很低，对植物基因组进行精确修饰和改造非常困难，位点特异性核酸酶的出现和应用，大大提升了同源重组的效率，使基因组编辑变得更加高效和精确，从而使得对包括植物在内的任何物种进行基因组编辑都将成为可能。锌指核酸酶（ZFN）和 TALE 核酸酶（TALENs）是能够使DNA的靶位点产生DNA双链断裂进而实现基因组定点编辑的常用系统，但在具体应用中发现这两种系统存在着许多缺陷和不足，如脱靶效应、与基因组进行特异结合与染色体位置及邻近序列有关等，另外技术难度大、构建组装时间长也限制了其应用。CRISPR/Cas系统广泛存在于细菌及古生菌中,是机体长期进化形成的RNA指导的降解入侵病毒或噬菌体DNA的适应性免疫系统。Ⅱ型CRISPR/Cas系统经过密码子优化等改造后已成为继锌指核酸酶ZFNs和TALENs后的新型高效定点编辑的新技术，具有突变效率高、制作简单、易操作及成本低的特点。目前，该技术成功应用于人类细胞、斑马鱼、小鼠以及细菌的基因组精确编辑，编辑的类型包括基因的定点插入、小片段的缺失、多个位点同时突变、基因定点的indel突变等。目前，CRISPR/Cas 系统在植物中的应用还比较有限，但该技术为植物基因工程的发展呈现了美好的前景。文中首先简要介绍了CRISPR/Cas系统的组成和基本原理，进而详细综述了该技术在植物内源基因和外源基因定点编辑中的应用，主要列举了自CRISPR/Cas系统改造成功以来利用该系统对单子叶和双子叶植物进行基因组定点编辑的案例，最后对基因组编辑技术在农业和植物基因工程上的应用进行了展望，希望能够为开展该领域研究的科研工作者提供参考。
噹外源DNA通過轉基因技術導入植物細胞後，會以同源重組或非同源重組兩種不同的方式整閤到基因組中，進而穫得相應的目標性狀。外源DNA與受體細胞序列相同或相近的位點髮生重新組閤，從而整閤到受體細胞的染色體上稱之為同源重組；噹髮生瞭 DNA 雙鏈斷裂的細胞為瞭避免 DNA 或染色體斷裂而造成DNA降解或對生命力的影響，而彊行將2箇DNA斷耑彼此連接在一起時則為非同源重組。髮生非同源重組的細胞其基因組常齣現覈苷痠片段的插入和/或缺失以及其他突變等多種情況，使得研究者無法得到精確控製的突變結果；而髮生同源重組的細胞基因組序列通常不變，通過加入同源重組的供體DNA，可以實現對基因組的精確脩飾和改造。由于在植物中產生自髮同源重組的概率很低，對植物基因組進行精確脩飾和改造非常睏難，位點特異性覈痠酶的齣現和應用，大大提升瞭同源重組的效率，使基因組編輯變得更加高效和精確，從而使得對包括植物在內的任何物種進行基因組編輯都將成為可能。鋅指覈痠酶（ZFN）和 TALE 覈痠酶（TALENs）是能夠使DNA的靶位點產生DNA雙鏈斷裂進而實現基因組定點編輯的常用繫統，但在具體應用中髮現這兩種繫統存在著許多缺陷和不足，如脫靶效應、與基因組進行特異結閤與染色體位置及鄰近序列有關等，另外技術難度大、構建組裝時間長也限製瞭其應用。CRISPR/Cas繫統廣汎存在于細菌及古生菌中,是機體長期進化形成的RNA指導的降解入侵病毒或噬菌體DNA的適應性免疫繫統。Ⅱ型CRISPR/Cas繫統經過密碼子優化等改造後已成為繼鋅指覈痠酶ZFNs和TALENs後的新型高效定點編輯的新技術，具有突變效率高、製作簡單、易操作及成本低的特點。目前，該技術成功應用于人類細胞、斑馬魚、小鼠以及細菌的基因組精確編輯，編輯的類型包括基因的定點插入、小片段的缺失、多箇位點同時突變、基因定點的indel突變等。目前，CRISPR/Cas 繫統在植物中的應用還比較有限，但該技術為植物基因工程的髮展呈現瞭美好的前景。文中首先簡要介紹瞭CRISPR/Cas繫統的組成和基本原理，進而詳細綜述瞭該技術在植物內源基因和外源基因定點編輯中的應用，主要列舉瞭自CRISPR/Cas繫統改造成功以來利用該繫統對單子葉和雙子葉植物進行基因組定點編輯的案例，最後對基因組編輯技術在農業和植物基因工程上的應用進行瞭展望，希望能夠為開展該領域研究的科研工作者提供參攷。
당외원DNA통과전기인기술도입식물세포후，회이동원중조혹비동원중조량충불동적방식정합도기인조중，진이획득상응적목표성상。외원DNA여수체세포서렬상동혹상근적위점발생중신조합，종이정합도수체세포적염색체상칭지위동원중조；당발생료 DNA 쌍련단렬적세포위료피면 DNA 혹염색체단렬이조성DNA강해혹대생명력적영향，이강행장2개DNA단단피차련접재일기시칙위비동원중조。발생비동원중조적세포기기인조상출현핵감산편단적삽입화/혹결실이급기타돌변등다충정황，사득연구자무법득도정학공제적돌변결과；이발생동원중조적세포기인조서렬통상불변，통과가입동원중조적공체DNA，가이실현대기인조적정학수식화개조。유우재식물중산생자발동원중조적개솔흔저，대식물기인조진행정학수식화개조비상곤난，위점특이성핵산매적출현화응용，대대제승료동원중조적효솔，사기인조편집변득경가고효화정학，종이사득대포괄식물재내적임하물충진행기인조편집도장성위가능。자지핵산매（ZFN）화 TALE 핵산매（TALENs）시능구사DNA적파위점산생DNA쌍련단렬진이실현기인조정점편집적상용계통，단재구체응용중발현저량충계통존재착허다결함화불족，여탈파효응、여기인조진행특이결합여염색체위치급린근서렬유관등，령외기술난도대、구건조장시간장야한제료기응용。CRISPR/Cas계통엄범존재우세균급고생균중,시궤체장기진화형성적RNA지도적강해입침병독혹서균체DNA적괄응성면역계통。Ⅱ형CRISPR/Cas계통경과밀마자우화등개조후이성위계자지핵산매ZFNs화TALENs후적신형고효정점편집적신기술，구유돌변효솔고、제작간단、역조작급성본저적특점。목전，해기술성공응용우인류세포、반마어、소서이급세균적기인조정학편집，편집적류형포괄기인적정점삽입、소편단적결실、다개위점동시돌변、기인정점적indel돌변등。목전，CRISPR/Cas 계통재식물중적응용환비교유한，단해기술위식물기인공정적발전정현료미호적전경。문중수선간요개소료CRISPR/Cas계통적조성화기본원리，진이상세종술료해기술재식물내원기인화외원기인정점편집중적응용，주요열거료자CRISPR/Cas계통개조성공이래이용해계통대단자협화쌍자협식물진행기인조정점편집적안례，최후대기인조편집기술재농업화식물기인공정상적응용진행료전망，희망능구위개전해영역연구적과연공작자제공삼고。
When exogenous DNA was imported into plant cell by transgenic technology, DNA fragment will integrate into the genome by homologous recombination or nonhomologous recombination. In addition, the plants seedling will achieve corresponding target traits. Homologous recombination occurred when the exogenous DNA and the same or similar sequences in receptor cells recombined and integrated to the receptor cell’s chromosomes, so the sequence will be possible to precisely modified and transformed. However, in some cases, to avoid the fracture caused by DNA or chromosomal DNA degradation or the impact on the vitality, the two double-stranded DNA break ends will be joined without considering the sequence similarity by error-prone nonhomologous end joining. As a result, precise mutations control is more difficult to achieve because insertion and/or deletion and other variety of mutations are high-frequently occurred in non-homologous recombination than in homologous recombination. Unfortunately, the frequency of homologous recombination is very low in plants which results the undesirable genome editing. Site-specific nucleases make genome editing more efficient and more precise by the great improvement of the efficiency in the homologous recombination. Such nucleases, zinc finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs), have been demonstrated to efficiently produce a DNA double-strand break at target site and to induce genome modification in a variety of organisms including plants. However, some defects found in the specific application, such as off-target effects, specific combined site with genome affected by chromosomal location and adjacent sequence, furthermore, technical complexity and time-consuming for assemble limit its application. The clustered, regularly interspaced, short palindromic repeats(CRISPR) system is a prokaryotic adaptive immune system which widely exists in bacteria and archaea. As the result of the long-term evolution, the system can defense against the degradation of RNA to guide the invasion of the virus or phage DNA. Recent advances in the study demonstrated CRISPR/Cas typeⅡ system was a promising system of genome editing strategy with high efficiency, affordability and easy to engineer compared with ZFNs and TALENs. Many precise genome editing cases by CRISPR/Cas system were found successfully in human cells, zebra fish, mice and bacterial, including gene insertion, deletion, mutation in multiple sites or in specific locus. Although the application in plants is still in a relatively limited range, there is an outstanding prospect of CRISPR/Cas system in plant genetic engineering. A brief summary of the composition and principles was presented firstly. Then, the authors emphasis on citing numerous cases involved its application of exogenous and endogenous genes editing in cotyledon and dicotyledonous plants which demonstrate that the CRISPR/Cas9 system has become a powerful tool in plant genome engineering. Finally, the future of the genome technology application in agriculture and plant genetic engineering was discussed which will provide a reference for researchers in genetic modification.
