Repositorium
Engineering Introns to Express RNA Guides for Cas9- and Cpf1-Mediated Multiplex Genome Editing
287
Journal Article / 2018
Ding, Dan; Chen, Kaiyuan; Chen, Yuedan; Li, Hong; Xie, Kabin
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.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
682 |
Xie, Kabin China |
Oryza sativa |
CRISPR/Cas9 PDS |
Albino phenotype SDN1 |
Basic research Basic research |
683 |
Xie, Kabin China |
Oryza sativa |
CRISPR/Cas9 MPK1 |
No information SDN1 |
Basic research Basic research |
684 |
Xie, Kabin China |
Oryza sativa |
CRISPR/Cas9 MPK2 |
No information SDN1 |
Basic research Basic research |
685 |
Xie, Kabin China |
Oryza sativa |
CRISPR/Cas9 MPK5 |
No information SDN1 |
Basic research Basic research |
686 |
Xie, Kabin China |
Oryza sativa |
CRISPR/Cpf1 PDS |
Albino phenotype SDN1 |
Basic research Basic research |
687 |
Xie, Kabin China |
Oryza sativa |
CRISPR/Cpf1 MPK2 |
No information SDN1 |
Basic research Basic research |