Scaffold RNA engineering in type V CRISPR-Cas systems: a potent way to enhance gene expression in the yeast Saccharomyces cerevisiae.

New, orthogonal transcription factors in eukaryotic cells have been realized by engineering nuclease-deficient CRISPR-associated proteins and/or their guide RNAs. In this work, we present a new kind of orthogonal activators, in Saccharomyces cerevisiae, made by turning type V CRISPR RNA into a scaffold RNA (ScRNA) able to recruit a variable number of VP64 activation domains. The activator arises from the complex between the synthetic ScRNA and DNase-deficient type V Cas proteins: dCas12e and denAsCas12a. The transcription activation achieved via the newly engineered dCas:ScRNA system is up to 4.7-fold higher than that obtained with the direct fusion of VP64 to Cas proteins. The new transcription factors have been proven to be functional in circuits such as Boolean gates, converters, multiplex-gene and metabolic-pathway activation. Our results extend the CRISPR-Cas-based technology with a new effective tool that only demands RNA engineering and improves the current design of transcription factors based on type V Cas proteins.通过工程化改造核酸酶缺陷型CRISPR相关蛋白（CRISPR-associated proteins, Cas）及其向导RNA（guide RNA, gRNA），现已在真核细胞中构建得到新型正交转录因子（orthogonal transcription factors）。本研究报道了一类全新的正交激活因子：在酿酒酵母（Saccharomyces cerevisiae）中，通过将V型CRISPR RNA改造为可招募任意数量VP64激活结构域（VP64 activation domains）的支架RNA（scaffold RNA, ScRNA），成功构建该类激活因子。该激活因子由人工合成的ScRNA与脱氧核糖核酸酶缺陷型V型Cas蛋白（dCas12e及denAsCas12a）结合形成的复合物构成。经该新型工程化改造的dCas:ScRNA系统实现的转录激活效率，最高可比将VP64直接融合至Cas蛋白的方案高出4.7倍。这类新型转录因子已被证实可在布尔逻辑门（Boolean gates）、转换器、多基因激活及代谢通路激活等基因回路中发挥功能。本研究成果为基于CRISPR-Cas的技术提供了一款仅需RNA工程化改造的高效新型工具，同时优化了当前基于V型Cas蛋白的转录因子设计方案。