摘要
The clustered regularly interspaced short palindromic repeat （CRISPR） system has emerged as the revolu- tionary platform for DNA targeting. This system uses a site-specific RNA guide to direct a CRISPR effector （e.g., Cas9 and Cpfl） 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 Cpfl. This method utilizes the endogenous tRNA processing system or crRNA processing activity of Cpfl 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 Cpfl-mediated genome editing using intronic tRNA-crRNA and crRNA-crRNA arrays. Interestingly, hybrid genes containing Cpfl and intronic crRNA array exhibited remarkably increased efficiency compared with the conventional Cpfl 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.
The clustered regularly interspaced short palindromic repeat （CRISPR） system has emerged as the revolu- tionary platform for DNA targeting. This system uses a site-specific RNA guide to direct a CRISPR effector （e.g., Cas9 and Cpfl） 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 Cpfl. This method utilizes the endogenous tRNA processing system or crRNA processing activity of Cpfl 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 Cpfl-mediated genome editing using intronic tRNA-crRNA and crRNA-crRNA arrays. Interestingly, hybrid genes containing Cpfl and intronic crRNA array exhibited remarkably increased efficiency compared with the conventional Cpfl 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.
基金
This work was supported by the National Transgenic Science and Technology Program （2016ZX08010-002）, the National Natural Science Foundation of China （31571374 and 31622047）, and Fundamental Research Funds for the Central Universities （2662015PY212） to K.X.
