Center for Bio/Molecular Science & Engineering (Code 6900), US Naval Research Laboratory, Washington, DC, USA.
Centers for Disease Control and Prevention, Atlanta, GA, USA.
Sci Rep. 2022 Aug 17;12(1):13953. doi: 10.1038/s41598-022-17474-8.
All CRISPR/CAS systems utilize CRISPR guide RNAs (crRNAs), the design of which depend on the type of CAS protein, genetic target and the environment/matrix. While machine learning approaches have recently been developed to optimize some crRNA designs, candidate crRNAs must still be screened for efficacy under relevant conditions. Here, we demonstrate a high-throughput method to screen hundreds of candidate crRNAs for activation of Cas13a collateral RNA cleavage. Entire regions of a model gene transcript (Y. pestis lcrV gene) were tiled to produce overlapping crRNA sets. We tested for possible effects that included crRNA/target sequence, size and secondary structures, and the commercial source of DNA oligomers used to generate crRNAs. Detection of a 981 nt target RNA was initially successful with 271 out of 296 tested guide RNAs, and that was improved to 287 out of 296 (97%) after protocol optimizations. For this specific example, we determined that crRNA efficacy did not strongly depend on the target region or crRNA physical properties, but was dependent on the source of DNA oligomers used for RNA preparation. Our high-throughput methods for screening crRNAs has general applicability to the optimization of Cas12 and Cas13 guide RNA designs.
所有的 CRISPR/CAS 系统都利用 CRISPR 指导 RNA（crRNA），其设计取决于 CAS 蛋白的类型、遗传靶标和环境/基质。虽然最近已经开发出机器学习方法来优化一些 crRNA 的设计，但候选 crRNA 仍必须在相关条件下进行功效筛选。在这里，我们展示了一种高通量筛选数百种候选 crRNA 以激活 Cas13a 副产物 RNA 切割的方法。对模型基因转录本（Y. pestis lcrV 基因）的整个区域进行平铺，以产生重叠的 crRNA 集。我们测试了可能的影响因素，包括 crRNA/靶序列、大小和二级结构，以及用于生成 crRNA 的商业来源的 DNA 寡核苷酸。最初，在 296 个测试的向导 RNA 中有 271 个成功检测到 981 nt 的靶 RNA，经过方案优化后，这一比例提高到 296 个中的 287 个（97%）。对于这个特定的例子，我们确定 crRNA 的功效并不强烈依赖于靶区域或 crRNA 的物理性质，而是依赖于用于 RNA 制备的 DNA 寡核苷酸的来源。我们用于筛选 crRNA 的高通量方法具有普遍适用性，可以优化 Cas12 和 Cas13 向导 RNA 的设计。
