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科学网Plant Biotechnol J∣华南植物园黄宏文研究组关于对猕猴桃基...

2018-01-17 12:06栏目:通信

2018年1月13日,Plant Biotechnol J发表了题为“Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit”的文章。该文章为华南植物园黄宏文研究员课题组完成。


文章介绍了一种高效的多基因编辑系统,为猕猴桃分子育种和功能基因组研究提供了可靠的系统,也为其他植物基因编辑系统的开发提供给了参考。


摘要:

猕猴桃是一种重要的水果作物;然而,但仍受限于功能基因组和分子改进技术。CRISPR/Cas系统已成功应用于许多作物的遗传改良,但其编辑能力依赖于sgRNA和Cas9蛋白。因此,需要在特定物种中优化其使用条件,以实现高效的基因组编辑。在本研究中,我们开发了一种新的克隆策略,用于构建含有四个针对猕猴桃八氢番茄红素去饱和酶基因(AcPDS)的sgRNAs的paired-sgRNA/Cas9载体。与之前的paired-sgRNA克隆方法相比,我们的策略只需要合成两个含gRNA的引物,这大大降低了成本。我们进一步比较了含有不同sgRNA表达装置【包括多顺反子tRNA-sgRNA盒(PTG)和传统CRISPR表达盒】的paired-sgRNA/Cas9载体的效率。我们发现PTG/Cas9系统的诱变频率比CRISPR/Cas9系统高10倍,这与两种不同表达盒中sgRNA的相对表达一致。具体而言,我们鉴定了由PTG/Cas9系统的配对sgRNA诱导的大片段的染色体缺失。最后,正如预期的那样,我们发现两种系统都能成功地诱导由G418抗性愈伤组织系再生的猕猴桃幼苗的白化表型。我们得出的结论是,在猕猴桃基因组编辑方面,PTG/Cas9系统比传统的CRISPR/Cas9系统更加强大,为其他植物CRISPR/Cas9编辑系统的优化提供了有价值的线索。


Abstract:

Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In the present study, we developed a new cloning strategy for generating paired-sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired-sgRNA cloning, our strategy only requires the synthesis of two gRNA-containing primers which largely reduces the cost. We further compared efficiencies of paired-sgRNA/Cas9 vectors containing different sgRNA-expression devices, including both the polycistronic tRNA-sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system were 10-fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418-resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.


科学网Plant Biotechnol J∣华南植物园黄宏文研究组关于对猕猴桃基...



科学网Plant Biotechnol J∣华南植物园黄宏文研究组关于对猕猴桃基...