BE-dot:为单碱基编辑设计sgRNA及预测脱靶图谱的工具

BE-dot: a Tool for sgRNA Design and Off-target Profile Prediction of Base Editing

目的 单碱基编辑器作为修复基因组点突变的有力工具,在生物技术开发和临床应用中具有极大潜力。对于目标改造的单核苷酸变异(SNV),首先要选择可用于编辑的单碱基编辑器(base editors,BEs)和单向导RNA (single guide RNA,sgRNA)。目前,尽管有较多工具实现了设计sgRNA,但缺少将设计sgRNA与评估单碱基编辑器特异性完整结合起来的工具。方法 纳入主流的27种胞嘧啶碱基编辑器(CBEs)和12种腺嘌呤碱基编辑器(ABEs)对SNV设计编辑方案,并通过调用第三方工具BE-Hive对编辑方案进行效率预测。综合使用多个脱靶预测工具对编辑方案的脱靶图谱进行评估。最后结合BEs类型和脱靶位点,分析得到所有可能的脱靶编辑产物,再调用ANNOVAR这一变异注释工具进行脱靶产物的功能分析。结果 本文提出了一个综合性工具BE-dot,它可以实现对目标编辑的SNV从设计sgRNA到预测脱靶图谱的完整过程,并对脱靶产物进行功能注释。利用密码子的简并性,BE-dot除了提供DNA水平上的精确校正方案,还可以在蛋白质水平进行同义校正。在对单碱基编辑系统做脱靶图谱的预测时,BE-dot综合了Cas-OFFinder、CALITAS、CFD、u CRISPR、BEdeepoff等多个工具,可以更全面地评估单碱基编辑系统的特异性,为用户选择BEs和sgRNA提供参考。此外,BE-dot可以自动分析得到脱靶位点处所有可能的编辑产物,并转换为avinput格式供ANNOVAR进行功能注释,避免了以往手工注释的繁琐。结论 BE-dot能为单碱基编辑技术应用于纠正或引入SNV设计编辑方案,并且能够从编辑效率、脱靶图谱、脱靶带来的功能影响等方面对编辑方案进行全面的评估。

Objective As a powerful tool in correcting genomic point mutations, base editors(BEs) show a promising prospect for biotechnology development and therapeutic applications. While editing the target singlenucleotide variant(SNV), it’s primary to select competent BEs and design single guide RNA(sgRNA). Currently,although there are multiple sgRNA design tools, no tools are available for integrating the design of sgRNA with the assessment of the specificity of BEs. Methods 27 cytosine base editors(CBEs) and 12 adenine base editors(ABEs) were used to design base editing schemes. BE-Hive, a third-party tool, was provoked to predict the editing efficiency. The off-target profiles of editing schemes were evaluated by using a combination of multiple off-target prediction tools. Finally, all possible off-target editing products were calculated by considering both base editor types and off-target sites, and then ANNOVAR, a variant annotation tool, was called for functional analysis of off-target products. Results We propose a comprehensive tool, BE-dot, which enables the complete process from a given SNV to designing sgRNAs, predicting off-target profiles, and annotating off-target products’ functions. Besides providing precise correction schemes at DNA level, in order to expand the range of editable SNVs, BE-dot can perform synonymous corrections at protein level by degeneracy. When predicting offtarget profiles of single base editing systems, BE-dot integrates multiple tools such as Cas-OFFinder, CALITAS,CFD, uCRISPR and BEdeepoff, which allows BE-dot to evaluate the specificity of single base editing systems more comprehensively and provide users with consultations about BEs and sgRNA selection. In addition, BE-dot can automatically analyze all possible editing products at off-target sites, and convert them into avinput format for functional annotation by ANNOVAR, avoiding the tedious manual annotation. Conclusion BE-dot designs editing schemes for applying base editing to correct or introduce SNVs, and comprehensively evaluates the editing scheme in terms of editing efficiency, off-target profile, and off-target functional impact.