工程化 CRISPR-Cas 介导系统用于特定位点 RNA 编辑的评估。
Evaluation of Engineered CRISPR-Cas-Mediated Systems for Site-Specific RNA Editing.
机构信息
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA; Stem Cell Program, University of California San Diego, La Jolla, CA 92093, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA 92093, USA.
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA; Stem Cell Program, University of California San Diego, La Jolla, CA 92093, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA 92093, USA.
出版信息
Cell Rep. 2020 Nov 3;33(5):108350. doi: 10.1016/j.celrep.2020.108350.
Site-directed RNA editing approaches offer great potential to correct genetic mutations in somatic cells while avoiding permanent off-target genomic edits. Nuclease-dead RNA-targeting CRISPR-Cas systems recruit functional effectors to RNA molecules in a programmable fashion. Here, we demonstrate a Streptococcus pyogenes Cas9-ADAR2 fusion system that uses a 3' modified guide RNA (gRNA) to enable adenosine-to-inosine (A-to-I) editing of specific bases on reporter and endogenously expressed mRNAs. Due to the sufficient nature of the 3' gRNA extension sequence, we observe that Cas9 gRNA spacer sequences are dispensable for directed RNA editing, revealing that Cas9 can act as an RNA-aptamer-binding protein. We demonstrate that Cas9-based A-to-I editing is comparable in on-target efficiency and off-target specificity with Cas13 RNA editing versions. This study provides a systematic benchmarking of RNA-targeting CRISPR-Cas designs for reversible nucleotide-level conversion at the transcriptome level.
靶向 RNA 编辑方法为纠正体细胞中的遗传突变提供了巨大的潜力，同时避免了永久性的脱靶基因组编辑。核酸酶失活的 RNA 靶向 CRISPR-Cas 系统以可编程的方式将功能性效应器募集到 RNA 分子上。在这里，我们展示了一种酿脓链球菌 Cas9-ADAR2 融合系统，该系统使用 3' 修饰的向导 RNA (gRNA) 实现了报告基因和内源性表达的 mRNA 上特定碱基的腺苷到肌苷 (A-to-I) 编辑。由于 3' gRNA 延伸序列的充分性，我们观察到 Cas9 gRNA 间隔序列对于定向 RNA 编辑是可有可无的，这表明 Cas9 可以作为 RNA 适体结合蛋白发挥作用。我们证明，基于 Cas9 的 A-to-I 编辑在靶效率和脱靶特异性方面与 Cas13 RNA 编辑版本相当。这项研究为基于 Cas9 的 RNA 靶向 CRISPR-Cas 设计在转录组水平上进行可逆核苷酸水平转换提供了系统的基准测试。
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