PNAS：提高CRISPR的多重编辑能力

规律成簇的间隔短回文重复CRISPR与内切酶Cas9，原本是细菌抵御病毒的重要武器。现在它们已经成为了基础研究、分子治疗和作物改良中的有力工具，帮助人们在多种生物中进行基因组编辑，包括人、小鼠、大鼠、斑马鱼、秀丽隐杆线虫、植物以及细菌。不过，CRISPR/Cas9的靶向能力和多重编辑效率，往往会受到引导RNA(gRNA)表达策略的限制。

为此，宾夕法尼亚州立大学的研究团队开发了从多顺反子基因生产大量gRNA的通用策略。这项研究发表在三月二日的美国国家科学院院刊PNAS杂志上，文章的通讯作者是宾夕法尼亚州立大学的副教授Yinong Yang。Yinong Yang博士早年毕业于浙江大学(前杭州大学)，现在他已经成为国际水稻分子病理领域的知名专家，近年来开始从事水稻基因组编辑的研究工作。

CRISPR/Cas体系包括一个称为Cas9的DNA剪切酶，和一段与目标dna片段匹配的引导性短RNA(gRNA)。Cas9内切酶在gRNA的引导下切割特定的DNA位点，引入多个gRNA可以同时修饰多个基因实现多重基因编辑。不过，CRISPR/Cas9的靶标能力很大程度上受到gRNA表达策略的影响。

tRNA加工系统能够精确切割tRNA前体的两端，研究人员决定利用这一点提高CRISPR/Cas9系统的靶向和多重编辑能力。他们将tRNA与gRNA结合起来，合成了一个多顺反子基因。研究显示，内源tRNA加工系统能够精确加工这个合成基因，有效生成大量携带正确靶向序列的gRNA，引导Cas9编辑多个染色体目标。这一策略能够在稳定的转基因水稻中，高效(可达到100%)实现多重基因组编辑和染色体片段删除。

tRNA及其加工系统在所有生物中是相当保守的，因此上述策略可以广泛用于小rna表达和基因组工程，显著提高CRISPR/Cas9的靶向能力和编辑效率。宾夕法尼亚州立大学已经为这一技术提出了专利申请。

原文链接：Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of thetRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplexediting capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy,multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability andediting efficiency of CRISPR/Cas9 toolkits.

作者：Yinong Yang