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《Plant Biotechnology Journal》:我科學家揭示CRISPR/Cas系統在水稻中産生突變的機制 
作者:中國水稻信息網
來源:中國水稻信息網
發布時間:2014-07-22

日前,國際學術期刊Plant Biotechnology Journal發表了我國科學家有關CRISPR/Cas研究新進展的論文。來自中科院的研究團隊揭示了CRISPR/Cas系統在水稻中産生突變的機制。

CRISPR (clustered regularly interspersed short palindromic repeats)/Cas是源于細菌及古細菌中的一種後天免疫系統,它可利用靶位點特異性的RNA指導Cas蛋白對靶位點序列進行修飾。自2013年以來,CRISPR/Cas系統已經成功應用于人類、小鼠、斑馬魚、家蠶、果蠅、酵母、擬南芥及水稻等多個物種中。也已經利用CRISPR/Cas系統成功實現了對模式植物擬南芥和農作物水稻中特定基因的定點突變,但對于CRISPR/Cas系統在水稻中産生突變的特點、效率、遺傳性及特異性等目前還不清楚。

在本項研究中,研究小組進一步測試了2個水稻亞種(粳稻日本晴和籼稻Kasalath)11個靶基因中CRISPR/Cas9誘導産生突變的效率、特點、遺傳性及特異性等。對T0代轉基因植株的檢測表明,CRISPR/Cas9系統在所有11個靶基因位點都誘導産生了突變,突變效率高達66.7%,且超過一半(6/11)的靶基因位點在T0代獲得純合突變體。同時研究表明,CRISPR/Cas9誘導産生的基因突變在後代的遺傳傳遞符合經典的孟德爾定律。對CRISPR/Cas9系統産生的突變類型分析表明,單堿基的突變類型超過70%,且大部分(53.9%)爲單堿基的插入;超過10個堿基的突變類型僅爲3.7%。通過全基因組重測序及檢測與靶序列高度同源的序列,研究小組僅在只有一個堿基不同的潛在脫靶位點檢測到突變,這表明CRISPR/Cas9系統在水稻中有很高的特異性。該研究表明CRISPR/Cas9系統可實現對水稻特定基因的高效、可穩定遺傳的及特異性的定點突變。這項研究成果爲CRISPR/Cas9系統在水稻中的穩定應用及進一步應用該技術提高水稻的産量、抗性及品質等提供了理論基礎。

這篇題爲“The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation”的論文于2014年5月23日在線發表于國際學術期刊Plant Biotechnology Journal 雜志上,7月17日被選爲該雜志第12卷第6期的封面文章。

根據2013年ISI發布的JCR(Journal Citation Reports)數據,2012年Plant Biotechnology Journal 的影響因子爲6.279,在總計197種期刊的植物科學(PLANT SCIENCES)學科中排名第9位;在總計160種期刊的生物工程和應用微生物(BIOTECHNOLOGY & APPLIED MICROBIOLOGY)學科中排名第12。

論文摘要:

The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9-induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9-induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large-scale off-targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off-target sites of a large number of T0 plants, low-frequency mutations were found in only one off-target site where the sequence had 1-bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering.

詳細信息:

http://www.cnrri.cn/zjww/Detail.aspx?id=20025786

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