Base editing is a promising technique, allowing precise single-base mutagenesis in genomes without double-strand DNA breaks or donor templates. Cytosine base editors (CBEs) convert cytosine to thymidine. In particular, CBEs can transform four codons, CAA, CAG, CGA, and TGG, into stop codons, providing a new means to rapidly inactivate a gene of interest and enabling loss-of-function study in recombination-deficient species and the construction of gene-inactivation libraries. However, designing single guide RNAs (sgRNAs) for gene inactivation is more complicated and more restricted in applicability than using the lustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (CRISPR/Cas9) system only, especially for researchers who do not specialize in the bioinformatics skills needed to design and evaluate sgRNAs. Here, we present a new user-friendly designing tool kit, namely, CRISPR-CBEI (cytosine base editor-mediated gene inactivation), including a Web tool and a command-line tool. The Web tool is dedicated to the design of sgRNAs for CBE-mediated gene inactivation and integrates various functions, including open reading frame (ORF) identification, CBE customization, sgRNA designing, summarizing, and front-end off-target searching against user-defined unlimited-file-size local genome files without the necessity of uploading to the server. The command-line version serves the same purpose but for a larger number of coding DNA sequences (CDSs), for instance, for designing a CBE-inactivation library in a target species which provides comprehensive evaluations of CBEs and target genomes. We envision that this tool would contribute to CBE-inactivation design.

中文翻译：

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CRISPR-CBEI：用于胞嘧啶碱基编辑器介导的基因失活的设计和分析工具包。

碱基编辑是一种很有前途的技术，它允许在基因组中进行精确的单碱基诱变，而不会出现双链 DNA 断裂或供体模板。胞嘧啶碱基编辑器 (CBE) 将胞嘧啶转化为胸苷。特别是，CBE 可以将四个密码子 CAA、CAG、CGA 和 TGG 转化为终止密码子，提供了一种新的方法来快速灭活感兴趣的基因，并能够在重组缺陷物种中进行功能丧失研究和构建基因失活文库。然而，与仅使用有规律间隔的短回文重复序列 (CRISPR)/CRISPR 相关 (CRISPR/Cas9) 系统相比，设计用于基因灭活的单向导 RNA (sgRNA) 更复杂，适用性也更受限制，尤其是对于不专注于设计和评估 sgRNA 所需的生物信息学技能。这里，Ç ytosine b ASE Ë ditor介导的基因我nactivation)，包括一个 Web 工具和一个命令行工具。该 Web 工具致力于设计用于 CBE 介导的基因失活的 sgRNA，并集成了各种功能，包括开放阅读框 (ORF) 识别、CBE 定制、sgRNA 设计、总结和针对用户定义的前端脱靶搜索无限文件大小的本地基因组文件，无需上传到服务器。命令行版本具有相同的目的，但适用于更多的编码 DNA 序列 (CDS)，例如，用于在目标物种中设计 CBE 灭活文库，提供 CBE 和目标基因组的综合评估。我们设想该工具将有助于 CBE 灭活设计。