CRISPR-Cas效应子是基因组和转录组靶向和编辑的强大工具。自然,这些蛋白质-RNA复合物是微生物先天免疫系统的一部分,它来自微生物和噬菌体之间的进化军备竞赛。这种协同进化也产生了所谓的抗CRISPR(Acr)蛋白,其抵消CRISPR-Cas适应性免疫。参与者组成性阻断同源CRISPR-Cas效应子,例如,通过干扰引导RNA结合,目标DNA/RNA识别,或目标切割。除了它们在微生物学和进化中的重要作用,Acrs最近因成为调节或微调CRISPR-Cas效应子活性的有用工具和开关而受到特别关注。由于它们通常尺寸小,高抑制效力,以及结构和机械的多功能性,Acrs为在异源系统中控制CRISPR效应子提供了广泛的潜在应用,包括哺乳动物细胞。这里,我们回顾了Acrs在哺乳动物细胞和生物体中的各种应用,并讨论了潜在的工程策略。这些应用包括(i)持续阻断CRISPR-Cas功能以创建写保护单元,(ii)减少CRISPR-Cas脱靶编辑,(iii)将CRISPR-Cas活性集中于特定的细胞类型和组织,(iv)基于工程的CRISPR效应子的时空控制,光电-,或化学遗传演员,和(v)Acrs用于选择性结合和检测复杂样品中的CRISPR-Cas效应子的用途。我们还将重点介绍Acrs在生物医学领域的潜在未来应用,并指出当前需要克服的挑战。
CRISPR-Cas effectors are powerful tools for genome and transcriptome targeting and editing. Naturally, these protein-RNA complexes are part of the microbial innate immune system, which emerged from the evolutionary arms race between microbes and phages. This coevolution has also given rise to so-called anti-CRISPR (Acr) proteins that counteract the CRISPR-Cas adaptive immunity. Acrs constitutively block cognate CRISPR-Cas effectors, e.g., by interfering with guide RNA binding, target DNA/RNA recognition, or target cleavage. In addition to their important role in microbiology and evolution, Acrs have recently gained particular attention for being useful tools and switches to regulate or fine-tune the activity of CRISPR-Cas effectors. Due to their commonly small size, high inhibition potency, and structural and mechanistic versatility, Acrs offer a wide range of potential applications for controlling CRISPR effectors in heterologous systems, including mammalian cells.Here, we review the diverse applications of Acrs in mammalian cells and organisms and discuss the underlying engineering strategies. These applications include (i) persistent blockage of CRISPR-Cas function to create write-protected cells, (ii) reduction of CRISPR-Cas off-target editing, (iii) focusing CRISPR-Cas activity to specific cell types and tissues, (iv) spatiotemporal control of CRISPR effectors based on engineered, opto-, or chemogenetic Acrs, and (v) the use of Acrs for selective binding and detection of CRISPR-Cas effectors in complex samples. We will also highlight potential future applications of Acrs in a biomedical context and point out present challenges that need to be overcome on the way.