Optimizing in vitro Transcribed CRISPR-Cas9 single-guide RNA Libraries for Improved Uniformity and Affordability

We describe a scalable and cost-effective sgRNA synthesis workflow that reduces costs by over 70% through the use of large pools of microarray-derived oligos encoding unique sgRNA spacers. These sub-pool oligos are assembled into full-length dsDNA templates via Golden Gate Assembly before in vitro transcription with T7 RNA polymerase. RNA-seq analysis reveals severe biases in spacer representation, with some spacers being highly overrepresented while others are completely absent. Consistent with previous studies, we identify guanine-rich sequences within the first four nucleotides of the spacer (immediately downstream of the T7 promoter) as the primary driver of this bias. To address this issue, we introduced a guanine tetramer upstream of all spacers, which reduced bias by an average of 19% in sgRNA libraries containing 389 spacers. However, this modification also increased the presence of high-molecular-weight RNA species after transcription. We also tested two alternative bias-reduction strategies: compartmentalizing spacers within emulsions and optimizing DNA input and reaction volumes. Both methods independently reduced bias in 2,626-plex sgRNA libraries, though to a lesser extent than the guanine tetramer approach. These advancements enhance both the affordability and uniformity of sgRNA libraries, with broad implications for improving CRISPR-Cas9 screens and optimizing guide RNA design for other CRISPR and nuclease systems.This is supplemental data file contains processed counts for all sequenced spacers. Each RNA-seq sample (and two DNA samples) has a corresponding csv file. Each file has three columns:seq - the sequence of the spacercount - the raw counts of the spacerclass - if this spacer is one of the ones designed (Target) or not (Mutated Spacer)An index.csv file describes the IVT parameters for each sample.我们报道了一种可扩展且高性价比的单引导RNA（single guide RNA, sgRNA）合成流程，通过使用编码独特sgRNA间隔序列的大型微阵列衍生寡核苷酸（microarray-derived oligos）池，将合成成本降低70%以上。该亚池寡核苷酸先通过Golden Gate组装（Golden Gate Assembly）构建为全长双链DNA（double-stranded DNA, dsDNA）模板，随后使用T7 RNA聚合酶（T7 RNA polymerase）进行体外转录。RNA测序（RNA-seq）分析显示，间隔序列的表征存在严重偏倚：部分间隔序列被高度富集，而另一些则完全缺失。与既往研究一致，我们发现位于间隔序列前四个核苷酸（紧邻T7启动子下游）的富鸟嘌呤序列是该偏倚的主要诱因。为解决该问题，我们在所有间隔序列的上游引入了一段四聚鸟嘌呤序列，在包含389个间隔序列的sgRNA文库中，该策略使偏倚平均降低了19%。但该修饰同时会导致转录后高分子量RNA产物的占比上升。我们还测试了两种替代的偏倚抑制策略：将间隔序列分隔于乳液微滴中，以及优化DNA投入量与反应体系体积。两种方法均可独立降低2626重sgRNA文库中的偏倚，不过其效果弱于四聚鸟嘌呤策略。这些技术进步同时提升了sgRNA文库的性价比与均一性，对优化CRISPR-Cas9筛选以及为其他CRISPR系统与核酸酶系统优化引导RNA设计具有广泛应用价值。 本补充数据文件包含所有已测序间隔序列的处理后计数。每个RNA测序样本（以及两个DNA样本）均对应一个csv文件。每个文件包含三列： seq：间隔序列的碱基序列 count：该间隔序列的原始计数 class：该间隔序列属于设计的靶标间隔序列（Target）还是突变间隔序列（Mutated Spacer）。另有一个index.csv文件，用于描述每个样本的体外转录参数。