T7 Polymerase Expression of Guide RNAs in vivo Allows Exportable CRISPR-Cas9 Editing in Multiple Yeast Hosts,ACS Synthetic Biology

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T7 Polymerase Expression of Guide RNAs in vivo Allows Exportable CRISPR-Cas9 Editing in Multiple Yeast Hosts
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-03-22 00:00:00 , DOI: 10.1021/acssynbio.7b00461
Nicholas J Morse ₁ , James M Wagner ₁ , Kevin B Reed ₁ , Madan R Gopal ₁ , Lars H Lauffer ₁ , Hal S Alper _{1,

2}

Affiliation

Efficient guide RNA expression often limits CRISPR-Cas9 implementation in new hosts. To address this limitation in fungal systems, we demonstrate the utility of a T7 polymerase system to effectively express sgRNAs. Initially, we developed a methodology in Saccharomyces cerevisiae using a modified version of the T7 P266L mutant polymerase with an SV40 nuclear localization signal to allow guide RNA expression immediately downstream of a T7 promoter. To improve targeting efficiency, guide RNA design was found to be tolerant to three mismatches or up to three additional bases appended to the 5′ end. The addition of three guanines to a T7-based guide RNA improved guide RNA expression 80-fold and achieved transcriptional output similar to the strong Pol III snr52 promoter. Resulting gene editing and dCas9-guided gene regulation with a T7-based guide RNA was on par with the commonly used snr52 system in S. cerevisiae. Finally, 96% and 60% genome editing efficiencies were achieved in Kluyveromyces lactis and Yarrowia lipolytica respectively with minimal optimization of this system. Thus, T7-based expression of sgRNAs offers an orthogonal method for implementing CRISPR systems in fungal systems.

中文翻译：

T7 聚合酶体内指导 RNA 表达允许在多个酵母宿主中进行可输出的 CRISPR-Cas9 编辑

高效的向导RNA表达通常限制CRISPR-Cas9在新宿主中的实施。为了解决真菌系统中的这一限制，我们证明了 T7 聚合酶系统有效表达 sgRNA 的效用。最初，我们在酿酒酵母中开发了一种方法，使用带有 SV40 核定位信号的 T7 P266L 突变聚合酶的改良版本，以允许紧邻 T7 启动子下游的指导 RNA 表达。为了提高靶向效率，指导 RNA 设计被发现可以容忍三个错配或在 5' 末端附加最多三个额外碱基。向基于 T7 的指导 RNA 添加三个鸟嘌呤将指导 RNA 表达提高了 80 倍，并实现了与强 Pol III snr52启动子类似的转录输出。由此产生的基因编辑和基于 T7 的向导 RNA 的 dCas9 引导基因调控与酿酒酵母中常用的snr52系统相当。最终，通过对该系统的最小优化，在乳酸克鲁维酵母和解脂耶氏酵母中分别实现了 96% 和 60% 的基因组编辑效率。因此，基于 T7 的 sgRNA 表达为在真菌系统中实施 CRISPR 系统提供了一种正交方法。

更新日期：2018-03-22

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