Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University, Keele, United Kingdom.
Department of Life Sciences, Imperial College London, London, United Kingdom.
CRISPR J. 2020 Oct;3(5):398-408. doi: 10.1089/crispr.2020.0029.
CRISPR guide RNAs (gRNAs) can be programmed with relative ease to allow the genetic editing of nearly any DNA or RNA sequence. Here, we propose novel molecular architectures to achieve RNA-dependent modulation of CRISPR activity in response to specific RNA molecules. We designed and tested, in both living cells and cell-free assays for rapid prototyping, -repressed RNA-interacting guide RNA (igRNA) that switch to their active state only upon interaction with small RNA fragments or long RNA transcripts, including pathogen-derived mRNAs of medical relevance such as the human immunodeficiency virus infectivity factor. The proposed CRISPR-igRNAs are fully customizable and easily adaptable to the majority if not all the available CRISPR-Cas variants to modulate a variety of genetic functions in response to specific cellular conditions, providing orthogonal activation and increased specificity. We thereby foresee a large scope of application for therapeutic, diagnostic, and biotech applications in both prokaryotic and eukaryotic systems.
CRISPR 指导 RNA（gRNA）可以相对轻松地编程，从而允许对几乎任何 DNA 或 RNA 序列进行基因编辑。在这里，我们提出了新的分子结构，以实现 RNA 依赖性的 CRISPR 活性调节，以响应特定的 RNA 分子。我们设计并测试了在活细胞和用于快速原型设计的无细胞测定中，-抑制性 RNA 相互作用指导 RNA（igRNA）仅在与小 RNA 片段或长 RNA 转录本相互作用时才会转换为其活性状态，包括具有医学相关性的病原体衍生 mRNA，如人类免疫缺陷病毒感染因子。所提出的 CRISPR-igRNA 是完全可定制的，并且易于适应大多数（如果不是全部）可用的 CRISPR-Cas 变体，以响应特定的细胞条件来调节各种遗传功能，从而提供正交激活和提高特异性。因此，我们预见到了在原核和真核系统中用于治疗、诊断和生物技术应用的广泛应用范围。
