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Engineering introns to express RNA guides for Cas9- and Cpf1-mediated multiplex genome editing

信息来源:作物遗传改良国家重点实验室    发布时间:2018-02-28
Kaiyuan Chen,  Hong Li, 
 
 
DOI: https://doi.org/10.1016/j.molp.2018.02.005,Published by the Molecular Plant 
 
 

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system has emerged as the revolutionary platform for DNA targeting. This system uses a site-specific RNA guide to direct a CRISPR effector (e.g., Cas9 and Cpf1) to a DNA target. Here, we elaborate a general strategy to simultaneously express multiple guide RNAs (gRNA) and CRISPR RNAs (crRNA) from introns of Cas9 and Cpf1. This method utilizes the endogenous tRNA processing system or crRNA processing activity of Cpf1 to cleave the spliced intron which contains tRNA-gRNA polycistron or crRNA-crRNA array. We demonstrated that the tRNA-gRNA intron is able to fuse with Cas9 as one gene. Such hybrid gene could be expressed using one polymerase II promoter and exhibited high efficiency and robustness to simultaneously targeting multiple sites. We also implemented this strategy in Cpf1-mediated genome editing using intronic tRNA-crRNA and crRNA-crRNA array. Interestingly, hybrid genes containing Cpf1 and intronic crRNA array exhibited remarkably increased efficiency than the conventional Cpf1 vectors. Taken together, this study presents a method to express CRISPR reagents from one hybrid gene to increase genome editing efficiency and capacity. Owing to its simplicity and versatility, this method could be broadly used to develop sophisticated CRISPR tools in eukaryotes.

Key words:

CRISPR, intron, Cas9, Cpf1, multiplex
 
 

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