遗传
遺傳
유전
Hereditas
2015年
10期
953-973
,共21页
基因组编辑%ZFN%TALEN%CRISPR/Cas9%同源重组%脱靶突变
基因組編輯%ZFN%TALEN%CRISPR/Cas9%同源重組%脫靶突變
기인조편집%ZFN%TALEN%CRISPR/Cas9%동원중조%탈파돌변
genome editing%ZFN%TALEN%CRISPR/Cas9%homologous recombination%off-target mutation
基因组编辑技术已经在多个模式植物、动物以及其他生物中得到成功应用。基因组编辑是利用序列特异核酸酶(Sequence-specific nucleases, SSNs)在基因组特定位点产生DNA双链断裂(Double-strand breaks, DSBs)，从而激活细胞自身修复机制——非同源末端连接(Non-homologous end joining, NHEJ)或同源重组(Homologous recombination, HR)，实现基因敲除、染色体重组以及基因定点插入或替换等。锌指核酸酶(Zinc finger nuclease, ZFN)、TALEN(Transcription activator-like effector nuclease)和CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9)系统是最主要的3类SSNs。ZFN和TALEN是利用蛋白与DNA结合方式靶向特定的基因组位点，而最新的 CIRISPR/Cas9系统则是利用更简单的核苷酸互补配对方式结合在基因组靶位点，其构建简单、效率更高效，因而促进了基因组编辑在植物中的广泛应用。利用基因组编辑技术除了实现植物基因定点突变外，还可以将 SSNs 的 DNA 结合域与其他功能蛋白融合，实现基因的靶向激活、抑制和表观调控等衍生技术。本文从基因组编辑技术的原理与优势、SSNs 组成及构建方法、基因组编辑及衍生技术在植物中应用、优化SSNs突变效率和减少脱靶突变方法等方面进行了系统介绍，并对未来需要迫切解决的一些问题进行了分析和展望。
基因組編輯技術已經在多箇模式植物、動物以及其他生物中得到成功應用。基因組編輯是利用序列特異覈痠酶(Sequence-specific nucleases, SSNs)在基因組特定位點產生DNA雙鏈斷裂(Double-strand breaks, DSBs)，從而激活細胞自身脩複機製——非同源末耑連接(Non-homologous end joining, NHEJ)或同源重組(Homologous recombination, HR)，實現基因敲除、染色體重組以及基因定點插入或替換等。鋅指覈痠酶(Zinc finger nuclease, ZFN)、TALEN(Transcription activator-like effector nuclease)和CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9)繫統是最主要的3類SSNs。ZFN和TALEN是利用蛋白與DNA結閤方式靶嚮特定的基因組位點，而最新的 CIRISPR/Cas9繫統則是利用更簡單的覈苷痠互補配對方式結閤在基因組靶位點，其構建簡單、效率更高效，因而促進瞭基因組編輯在植物中的廣汎應用。利用基因組編輯技術除瞭實現植物基因定點突變外，還可以將 SSNs 的 DNA 結閤域與其他功能蛋白融閤，實現基因的靶嚮激活、抑製和錶觀調控等衍生技術。本文從基因組編輯技術的原理與優勢、SSNs 組成及構建方法、基因組編輯及衍生技術在植物中應用、優化SSNs突變效率和減少脫靶突變方法等方麵進行瞭繫統介紹，併對未來需要迫切解決的一些問題進行瞭分析和展望。
기인조편집기술이경재다개모식식물、동물이급기타생물중득도성공응용。기인조편집시이용서렬특이핵산매(Sequence-specific nucleases, SSNs)재기인조특정위점산생DNA쌍련단렬(Double-strand breaks, DSBs)，종이격활세포자신수복궤제——비동원말단련접(Non-homologous end joining, NHEJ)혹동원중조(Homologous recombination, HR)，실현기인고제、염색체중조이급기인정점삽입혹체환등。자지핵산매(Zinc finger nuclease, ZFN)、TALEN(Transcription activator-like effector nuclease)화CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9)계통시최주요적3류SSNs。ZFN화TALEN시이용단백여DNA결합방식파향특정적기인조위점，이최신적 CIRISPR/Cas9계통칙시이용경간단적핵감산호보배대방식결합재기인조파위점，기구건간단、효솔경고효，인이촉진료기인조편집재식물중적엄범응용。이용기인조편집기술제료실현식물기인정점돌변외，환가이장 SSNs 적 DNA 결합역여기타공능단백융합，실현기인적파향격활、억제화표관조공등연생기술。본문종기인조편집기술적원리여우세、SSNs 조성급구건방법、기인조편집급연생기술재식물중응용、우화SSNs돌변효솔화감소탈파돌변방법등방면진행료계통개소，병대미래수요박절해결적일사문제진행료분석화전망。
Genome editing technologies using engineered nucleases have been widely used in many model or-ganisms. Genome editing with sequence-specific nuclease (SSN) creates DNA double-strand breaks (DSBs) in the genomic target sites that are primarily repaired by the non-homologous end joining (NHEJ) or homologous recombi-nation (HR) pathways, which can be employed to achieve targeted genome modifications such as gene mutations, insertions, replacements or chromosome rearrangements. There are three major SSNs—zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic re-peats/CRISPR-associated 9 (CRISPR/Cas9) system. In contrast to ZFN and TALEN, which require substantial protein engineering to each DNA target, the CRISPR/Cas9 system requires only a change in the guide RNA. For this reason, the CRISPR/Cas9 system is a simple, inexpensive and versatile tool for genome engineering. Furthermore, a modified version of the CRISPR/Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression, such as activation, depression and epigenetic regulation. In this review, we summarize the develop-ment and applications of genome editing technologies for basic research and biotechnology, as well as highlight chal-lenges and future directions, with particular emphasis on plants.
