A Speed Cloning Method for Editing Multiple Targets

• Published in: Journal of plant biology = Singmul Hakhoe chi Vol. 66; no. 4; pp. 311 - 316
• Main Authors: Moon, Sunok, Jung, Ki-Hong
• Format: Journal Article
• Language: English
• Published: Singapore Springer Nature Singapore 01-08-2023; Springer Nature B.V; 한국식물학회
• Subjects: Biomedical and Life Sciences; Cloning; Construction; CRISPR; Design; E coli; Editing; Enzymes; Genomes; gRNA; Life Sciences; Plant Breeding/Biotechnology; Plant Ecology; Plant Genetics and Genomics; Plant Sciences; Plant Systematics/Taxonomy/Biogeography; Redundancy; Transfer RNA; tRNA; 생물학; CRISPR/Cas9 vector; Editing multiple targets; Cloning; Rice; tRNA–gRNA structure
• ISSN: 1226-9239; 1867-0725
• DOI: 10.1007/s12374-023-09400-w

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.