Engineering Introns to Express RNA Guides for Cas9-and Cpf1-Mediated Multiplex Genome Editing

被引:85
作者
Ding, Dan [1 ,2 ,3 ]
Chen, Kaiyuan [1 ,2 ,3 ]
Chen, Yuedan [1 ,2 ,3 ]
Li, Hong [1 ,2 ,3 ]
Xie, Kabin [1 ,2 ,3 ]
机构
[1] Huazhong Agr Univ, Natl Key Lab Crop Genet Improvement, Wuhan 430070, Peoples R China
[2] Huazhong Agr Univ, Natl Ctr Plant Gene Res Wuhan, Wuhan 430070, Peoples R China
[3] Huazhong Agr Univ, Hubei Key Lab Plant Pathol, Wuhan 430070, Peoples R China
基金
中国国家自然科学基金;
关键词
CRISPR; intron; Cas9; Cpf1; multiplex; HUMAN-CELLS; TARGETED MUTAGENESIS; POLYUBIQUITIN GENES; CRISPR-CAS9; SYSTEM; CRISPR/CAS SYSTEM; BINARY VECTORS; IN-VIVO; PLANTS; CPF1; RICE;
D O I
10.1016/j.molp.2018.02.005
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 that 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 a hybrid gene could be expressed using one polymerase II promoter, and exhibited high efficiency and robustness in simultaneously targeting multiple sites. We also implemented this strategy in Cpf1-mediated genome editing using intronic tRNA-crRNA and crRNA-crRNA arrays. Interestingly, hybrid genes containing Cpf1 and intronic crRNA array exhibited remarkably increased efficiency compared with 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.
引用
收藏
页码:542 / 552
页数:11
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