A Speed Cloning Method for Editing Multiple Targets.

Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) system has widespread usage in the process of gene knockout. Due to the phenomenon of gene redundancy in higher organisms, there has been an increasing need for CRISPR/Cas9 vectors capable of editing multiple sites in the genome. In rice, a CRISPR/Cas9 vector that utilized a tandem-arrayed tRNA–gRNA structure for editing multiple sites in the genome demonstrated a remarkably efficient multiplex editing capability. To construct a CRISPR/Cas9 vector that utilizes a tandem-arrayed tRNA–gRNA structure, the Golden Gate assembly method has been used, which needs multi-step manipulations. Here, we designed a simple method to construct CRISPR/Cas9 vector with tandem-arrayed tRNA–gRNA structure for knocking out multiple sites in rice genome. This method enables easy construction of tandem-arrayed tRNA–gRNA structure via simple PCR and ligation steps. [ABSTRACT FROM AUTHOR]