A Simple and Low-Cost CRISPR/Cas9 Knockout System Widely Applicable to Insects

Simple Summary Editing (deletion, insertion, and substitution) of target genes is a commonly used method for studying gene function, which has significant applications in disease treatment, agricultural improvement, and environmental protection. The CRISPR/Cas9 system is currently the most widely used gene-editing technology due to its low cost and high efficiency. Continuous improvements in this system by researchers have resulted in a wide range of applications and greatly advanced gene function research. However, many CRISPR/Cas9 systems are complex and difficult to share, limiting their use to a few laboratories. In this study, we present a simple, low-cost, and universal CRISPR/Cas9 knockout system. We provide detailed information on the plasmid sequences, reagent codes, and methods. This study can help researchers establish gene knockout systems and facilitate better gene function research.Abstract The CRISPR/Cas9 gene-editing system is a standard technique in functional genomics, with widespread applications. However, the establishment of a CRISPR/Cas9 system is challenging. Previous studies have presented numerous methodologies for establishing a CRISPR/Cas9 system, yet detailed descriptions are limited. Additionally, the difficulties in obtaining the necessary plasmids have hindered the replication of CRISPR/Cas9 techniques in other laboratories. In this study, we share a detailed and simple CRISPR/Cas9 knockout system with optimized steps. The results of gene knockout experiments in vitro and in vivo show that this system successfully knocked out the target gene. By sharing detailed information on plasmid sequences, reagent codes, and methods, this study can assist researchers in establishing gene knockout systems.