The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful-focusing on CRISPRi as a potential application-as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species.IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.

中文翻译：

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用于微生物基因组中 CRISPR/Cas9 靶向的通用全基因组 GuideFinder。

CRISPR/Cas 系统具有促进各种细菌物种基因编辑的巨大潜力。CRISPR 干扰 (CRISPRi) 和 CRISPR 激活 (CRISPRa) 代表了 CRISPR/Cas9 系统的修改，它们分别利用无催化活性的 Cas9 蛋白进行转录抑制和激活。尽管 CRISPRi 和 CRISPRa 具有系统研究细菌基因功能的巨大潜力，但很少有程序专门用于识别全基因组细菌基因组草图中的指南。此外，很少有程序提供具有用于细菌靶向的灵活设计参数的开源代码。为了解决这些限制，我们创建了 GuideFinder，这是一个可定制的、用户友好的程序，可以为任何带注释的细菌基因组设计指南。GuideFinder 通过使用用户输入的带注释的基因组和 FASTA 文件，为任意数量的基因设计来自 NGG protospacer-adjacentmotif (PAM) 位点的指南。指南根据用户定义的设计参数进行过滤，如果它们包含任何脱靶匹配，则将其删除。降低参数阈值的迭代允许程序为没有产生具有更严格参数的指南的基因设计指南，这是 GuideFinder 独有的几个功能之一。GuideFinder 还可以识别针对多重性的配对指南，我们通过实验测试了其有效性。GuideFinder 已经在各种不同的细菌基因组上进行了测试，平均为 95% 的基因找到了指南。而且，该程序设计的指南在功能上是有用的——专注于 CRISPRi 作为潜在的应用——正如两个葡萄球菌物种的基本基因敲除所证明的那样。通过大规模生成指南，这种开放获取软件将提高对各种细菌物种的 CRISPR/Cas 研究的可访问性。重要这些生物是非常需要的。CRISPR/Cas9 技术彻底改变了对多种模式生物基因功能的研究。系统的基因打靶需要高效的 CRISPR 引导设计。然而，现有工具不适用于微生物靶向的广泛需求，其中包括非凡的物种和亚种遗传多样性，其中绝大多数以基因组草图为特征。此外，指南设计参数的灵活性对于考虑可能影响指南效力的广泛因素很重要，其中许多可能是物种和菌株特异性的。我们设计了 GuideFinder，这是一个可定制的、用户友好的程序，它解决了现有软件的局限性，可以为任何带注释的细菌基因组设计指南，该指南具有众多功能，可促进各种微生物的指南设计。