Bioinformatics Discovery of Anti-CRISPR Operons in Human Gut Microbiome

项目摘要 各种细菌CRISPR-Cas系统及其变体已经被工程化并重新用于基因表达。 自2013年以来，在动物，植物和微生物中进行编辑。然而，目前的CRISPR-Cas基因组编辑工具 使用起来并不完全安全。这主要是由于缺乏有效的刹车系统和脱靶 这可能会在基因组中产生不必要的切割。正在制定不同的战略，以减少关闭- 使CRISPR-Cas更安全使用。 一个经常被基因组编辑的巨大成功所掩盖的事实是，在微生物世界中，CRISPR-Cas 是一种适应性抗病毒免疫机制，存在于约50%的细菌和约90%的古细菌基因组中。作为 作为一种对抗策略，抗CRISPR是由病毒和前病毒产生的，作为一种抗抗病毒机制， 以抑制其原核宿主的CRISPR-Cas系统。因此，作为天然存在的抑制剂， CRISPR-Cas、抗CRISPR（Acr）蛋白具有明显的优势，可用于开发更安全、更安全的 可控的CRISPR-Cas基因组编辑技术。 本项目的目标是开发一个基于基因组上下文的生物信息学数据分析工具（AOMiner 在人类肠道微生物组和病毒组中挖掘新的Acr操纵子。该项目的意义包括：（一） 它将加强新的抗CRISPR的实验表征，这是基础， 理解噬菌体-宿主相互作用，并进一步发展基于噬菌体的新技术 对抗病原菌和传染病;（ii）它将提供新的开源计算机程序 和在线数据库，以提供新的抗CRISPR候选药物，用于构建更安全，更安全的 可控的CRISPR-Cas基因组编辑工具。 我们最近开发了一个生物信息学软件包Acrfinder（http：//bcb.unl.edu/AcrFinder/），用于 Acr操纵子的自动发现，主要基于序列同源性搜索。然而，它的能力 鉴定新Acr是有限的，因为它主要基于同源性搜索。本项目的创新之处在于 AOMiner（http：//bcb.unl.edu/AOMiner）将实现一种基于基因组上下文的算法， 发现新的Acr操纵子而不是Acr蛋白。此外，该项目将是第一个大规模的 从人类肠道微生物组和病毒组中挖掘Acrs操纵子的基因组，这将发展成为一个 gutAO数据库（http：//bcb.unl.edu/gutAO）。 总之，该项目将以开源的形式提供一套生物信息学软件工具， 计算机程序和在线数据库，以帮助表征新的抗CRISPR。

PROJECT SUMMARY Various bacterial CRISPR-Cas systems and their variants have been engineered and repurposed for gene editing in animals, plants, and microbes since 2013. However, the current CRISPR-Cas genome editing tools are not perfectly safe to use. This is primarily due to the lack of an effective brake system and the off-target effect that may create unwanted cuts in the genome. Different strategies are being developed to reduce off- target effect and make CRISPR-Cas safer to use. A fact often overshadowed by the great success of genome editing is that, in the microbial world, CRISPR-Cas is an adaptive anti-viral immune mechanism present in ~50% of bacterial and ~90% of archaeal genomes. As a counterstrategy, anti-CRISPRs are produced by viruses and proviruses, as an anti-anti-viral mechanism, to inhibit the CRISPR-Cas systems of their prokaryote hosts. Thus, as the naturally occurring inhibitors of CRISPR-Cas, anti-CRISPR (Acr) proteins have obvious advantages to be used for developing safer and more controllable CRISPR-Cas genome editing technologies. The objective of this project is to develop a genomic context-based tool (AOMiner) for bioinformatics data mining of new Acr operons in human gut microbiome and virome. The significances of this project include: (i) it will enhance the experimental characterization of new anti-CRISPRs, which is fundamental to the understanding of phage-host interactions, and further to the development of novel phage-based technologies to combat pathogenic bacteria and infectious diseases; (ii) it will deliver new open source computer programs and online databases to provide novel anti-CRISPR candidates to be exploited for building safer and more controllable CRISPR-Cas genome editing tools. We recently developed AcrFinder (http://bcb.unl.edu/AcrFinder/), a bioinformatics software package for automated discovery of Acr operons, primarily based on sequence homology search. However, its ability to identify new Acrs is limited because it is largely based on homology search. The innovation of this project is that AOMiner (http://bcb.unl.edu/AOMiner) will implement a genomic context-based algorithm focusing on discovery of new Acr operons instead of Acr proteins. Additionally, this project will be the first large-scale genome mining for Acrs operons from human gut microbiome and virome, which will be developed into a gutAO database (http://bcb.unl.edu/gutAO). In summary, this project will deliver a suite of bioinformatics software tools in the form of open-source computer programs and online databases to assist the characterization of novel anti-CRISPRs.