Target Specificity of the CRISPR-Cas9 System in Arabidopsis thaliana , Oryza sativa , and Glycine max Genomes

作者：Pan Zou, Lijin Duan, Shasha Zhang, Xue Bai, Zhenghui Liu, Fengmei Jin, Haibo Sun, Wentao Xu, Rui Chen · 发表于：Journal of Computational Biology · 年份：2020 · DOI：10.1089/cmb.2019.0453 · 被引用次数：3 · 研究领域：CRISPR and Genetic Engineering、Plant Virus Research Studies、Insect symbiosis and bacterial influences

Clustered regularly interspaced short palindromic repeats (CRISPR), a class of immune-associated sequences in bacteria, have been developed as a powerful tool for editing eukaryotic genomes in diverse cells and organisms in recent years. The CRISPR-Cas9 system can recognize upstream 20 nucleotides (guide sequence) adjacent to the protospacer-adjacent motif site and trigger double-stranded DNA cleavage as well as DNA repair mechanisms, which eventually result in knockout, knockin, or site-specific mutagenesis. However, off-target effect caused by guide sequence misrecognition is the major drawback and restricts its widespread application. In this study, global analysis of specificities of all guide sequences in Arabidopsis thaliana , Oryza sativa (rice), and Glycine max (soybean) were performed. As a result, a simple pipeline and three genome-wide databases were established and shared for the scientific society. For each target site of CRISPR-Cas9, specificity score and off-target number were calculated and evaluated. The mean values of off-target numbers for A. thaliana , rice, and soybean were determined as 27.5, 57.3, and 174.7, respectively. Comparative analysis among these plants suggested that the frequency of off-target effects was correlated to genome size, chromosomal locus, gene density, and guanine-cytosine (GC) content. Our results contributed to the better understanding of CRISPR-Cas9 system in plants and would help to minimize the off-target effect during its app...