A Multiplexed CRISPR/Cas9 Editing System Based on the Endogenous tRNA Processing

  • Kabin Xie
  • Yinong YangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1917)


The CRISPR-Cas9 system has become a powerful and popular tool for genome editing due to its efficiency and simplicity. Multiplex genome editing is an important feature of the CRISPR-Cas9 system and requires simultaneous expression of multiple guide RNAs (gRNAs). Here we describe a general method to efficiently produce many gRNAs from a single gene transcript based on the endogenous tRNA-processing system. A step-by-step protocol is provided for the design and construction of the polycistronic tRNA-gRNA (PTG) gene. The PTG method has been demonstrated to be highly efficient for multiplex genome editing in various plant, animal, and microbial species.

Key words

CRISPR-Cas9 tRNA gRNA Multiplex Genome editing 



This study was supported by the National Natural Science Foundation of China to KX (31571374 and 31622047) and National Science Foundation Plant Genome Research Project Grant (1740874) to YY. This work was also supported by the USDA National Institute of Food and Agriculture Hatch project PEN04659.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.National Key Laboratory for Crop Genetic Improvement and Plant Gene Research Center (Wuhan)Huazhong Agricultural UniversityWuhanChina
  2. 2.Department of Plant Pathology and Environmental Microbiology, The Huck Institutes of the Life SciencesThe Pennsylvania State UniversityPennsylvaniaUSA

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