Yin Xiaojia, Biswal Akshaya K, Dionora Jacqueline, Perdigon Kristel M, Balahadia Christian P, Mazumdar Shamik, Chater Caspar, Lin Hsiang-Chun, Coe Robert A, Kretzschmar Tobias, Gray Julie E, Quick Paul W, Bandyopadhyay Anindya
International Rice Research Institute, DAPO, 7777, Metro Manila, Philippines.
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Plant Cell Rep. 2017 May;36(5):745-757. doi: 10.1007/s00299-017-2118-z. Epub 2017 Mar 27.
CRISPR-Cas9/Cpf1 system with its unique gene targeting efficiency, could be an important tool for functional study of early developmental genes through the generation of successful knockout plants. The introduction and utilization of systems biology approaches have identified several genes that are involved in early development of a plant and with such knowledge a robust tool is required for the functional validation of putative candidate genes thus obtained. The development of the CRISPR-Cas9/Cpf1 genome editing system has provided a convenient tool for creating loss of function mutants for genes of interest. The present study utilized CRISPR/Cas9 and CRISPR-Cpf1 technology to knock out an early developmental gene EPFL9 (Epidermal Patterning Factor like-9, a positive regulator of stomatal development in Arabidopsis) orthologue in rice. Germ-line mutants that were generated showed edits that were carried forward into the T2 generation when Cas9-free homozygous mutants were obtained. The homozygous mutant plants showed more than an eightfold reduction in stomatal density on the abaxial leaf surface of the edited rice plants. Potential off-target analysis showed no significant off-target effects. This study also utilized the CRISPR-LbCpf1 (Lachnospiracae bacterium Cpf1) to target the same OsEPFL9 gene to test the activity of this class-2 CRISPR system in rice and found that Cpf1 is also capable of genome editing and edits get transmitted through generations with similar phenotypic changes seen with CRISPR-Cas9. This study demonstrates the application of CRISPR-Cas9/Cpf1 to precisely target genomic locations and develop transgene-free homozygous heritable gene edits and confirms that the loss of function analysis of the candidate genes emerging from different systems biology based approaches, could be performed, and therefore, this system adds value in the validation of gene function studies.
CRISPR-Cas9/Cpf1系统凭借其独特的基因靶向效率，通过培育成功的基因敲除植物，可能成为早期发育基因功能研究的重要工具。系统生物学方法的引入和应用已鉴定出多个参与植物早期发育的基因，基于这些知识，需要一种强大的工具来对由此获得的假定候选基因进行功能验证。CRISPR-Cas9/Cpf1基因组编辑系统的开发为创建感兴趣基因的功能缺失突变体提供了便利工具。本研究利用CRISPR/Cas9和CRISPR-Cpf1技术在水稻中敲除一个早期发育基因EPFL9（表皮模式因子样9，拟南芥气孔发育的正向调节因子）的同源基因。当获得无Cas9的纯合突变体时，所产生的生殖系突变体显示出的编辑能够传递到T2代。纯合突变体植株在编辑后的水稻植株叶背表面的气孔密度降低了八倍多。潜在的脱靶分析显示没有明显的脱靶效应。本研究还利用CRISPR-LbCpf1（毛螺菌科细菌Cpf1）靶向相同的OsEPFL9基因，以测试该II类CRISPR系统在水稻中的活性，发现Cpf1也能够进行基因组编辑，并且编辑能够世代传递，表现出与CRISPR-Cas9相似的表型变化。本研究证明了CRISPR-Cas9/Cpf1在精确靶向基因组位置以及开发无转基因的纯合可遗传基因编辑方面的应用，并证实可以对基于不同系统生物学方法产生的候选基因进行功能缺失分析，因此，该系统在基因功能研究的验证中具有重要价值。
