Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, TN 37996, USA.
Oak Ridge National Laboratory, Bioenergy Science Center (BESC), Oak Ridge, TN 37830, USA.
Bioinformatics. 2018 Jan 1;34(1):16-23. doi: 10.1093/bioinformatics/btx564.
Genetic diversity of non-model organisms offers a repertoire of unique phenotypic features for exploration and cultivation for synthetic biology and metabolic engineering applications. To realize this enormous potential, it is critical to have an efficient genome editing tool for rapid strain engineering of these organisms to perform novel programmed functions.
To accommodate the use of CRISPR/Cas systems for genome editing across organisms, we have developed a novel method, named CRISPR Associated Software for Pathway Engineering and Research (CASPER), for identifying on- and off-targets with enhanced predictability coupled with an analysis of non-unique (repeated) targets to assist in editing any organism with various endonucleases. Utilizing CASPER, we demonstrated a modest 2.4% and significant 30.2% improvement (F-test, P < 0.05) over the conventional methods for predicting on- and off-target activities, respectively. Further we used CASPER to develop novel applications in genome editing: multitargeting analysis (i.e. simultaneous multiple-site modification on a target genome with a sole guide-RNA requirement) and multispecies population analysis (i.e. guide-RNA design for genome editing across a consortium of organisms). Our analysis on a selection of industrially relevant organisms revealed a number of non-unique target sites associated with genes and transposable elements that can be used as potential sites for multitargeting. The analysis also identified shared and unshared targets that enable genome editing of single or multiple genomes in a consortium of interest. We envision CASPER as a useful platform to enhance the precise CRISPR genome editing for metabolic engineering and synthetic biology applications.
https://github.com/TrinhLab/CASPER.
Supplementary data are available at Bioinformatics online.
非模式生物的遗传多样性为探索和培养合成生物学和代谢工程应用提供了独特的表型特征 repertoire。为了实现这一巨大潜力，对于这些生物体的快速菌株工程，快速菌株工程至关重要，以执行新的编程功能。
为了适应 CRISPR/Cas 系统在跨生物体基因组编辑中的使用，我们开发了一种名为 CRISPR 相关软件用于途径工程和研究（CASPER）的新方法，用于识别具有增强可预测性的 ON 和 OFF 靶点，以及分析非独特（重复）靶点，以协助用各种内切酶编辑任何生物体。利用 CASPER，我们分别证明了对 ON 和 OFF 靶点活性的预测分别提高了 2.4%和 30.2%（F 检验，P < 0.05），显著优于传统方法。此外，我们还使用 CASPER 在基因组编辑中开发了新的应用：多靶点分析（即使用单个向导 RNA 要求对目标基因组进行同时多点修饰）和多物种群体分析（即跨生物体联盟设计用于基因组编辑的向导 RNA）。我们对一组工业相关生物体的分析揭示了与基因和转座元件相关的许多非独特靶位点，这些靶位点可作为多靶点的潜在靶位点。该分析还确定了共享和非共享的靶标，这些靶标可用于在感兴趣的联盟中编辑单个或多个基因组。我们设想 CASPER 是一个有用的平台，可增强代谢工程和合成生物学应用中精确的 CRISPR 基因组编辑。
https://github.com/TrinhLab/CASPER。
补充数据可在生物信息学在线获得。
