Bioinformatics Identification of Anti-CRISPR Loci by Using Homology, Guilt-by-Association, and CRISPR Self-Targeting Spacer Approaches
Bioinformatics Identification of Anti-CRISPR Loci by Using Homology, Guilt-by-Association, and CRISPR Self-Targeting Spacer Approaches
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DOI：
10.1128/msystems.00455-19
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发表时间：
2019-09-01
期刊：
影响因子：
6.4
通讯作者：
Entwistle, Sarah
中科院分区：
文献类型：
--
作者：
Yin, Yanbin;Yang, Bowen;Entwistle, Sarah
抗CRISPR（Acr）基因座/操纵子编码Acr蛋白和Acr相关（Aca）蛋白。45个Acr家族已被实验表征为抑制CRISPR-Cas系统的7种亚型。我们已经开发了一个生物信息学管道，以确定基因组位点含有Acr同源物和/或Aca同源物相结合的三种计算方法：同源性，内疚的协会，和自我靶向间隔。同源性搜索在细菌和病毒基因组中发现了数千个Acr同源物，但大多数与AcrIIA 7和AcrIIA 9同源。研究这些Acr同源物的基因邻域发现，其中只有一小部分（细菌中为23.0%，病毒中为8.2%）具有相邻的Aca同源物，从而形成Acr-Aca操纵子。令人惊讶的是，尽管自靶向间隔区是基因组中Acr基因存在的强烈指示剂，但在没有自靶向间隔区或甚至没有完整的CRISPR-Cas系统的细菌基因组中发现了大百分比的Acr-Aca基因座。此外，对于来自具有自靶向间隔区的基因组的Acr同源物，基于同源性的Acr家族分配并不总是与自靶向CRISPR-Cas亚型一致。最后，通过研究Acr基因组中与自靶向间隔区共存的基因座，推断出Acr的5种已知亚型（I-C、I-E、I-F、II-A和II-C）和5种新亚型（I-B、III-A、III-B、IV-A和V-U4）。基于这些发现，我们得出结论，新的抗CRISPR的发现不应仅限于具有自靶向间隔区的基因组和具有Acr同源物的基因座。CRISPR-Cas系统和抗CRISPR系统的进化军备竞赛可能已经驱动了这些元件在密切相关的基因组中的适应性和快速的获得和丢失。重要信息作为一种天然存在的适应性免疫系统，CRISPR-Cas（clustered regularly interspersed short palindromic repeats-CRISPR-associated genes）系统广泛存在于细菌和古细菌中，以防御病毒。自2013年以来，各种细菌CRISPR-Cas系统的应用变得非常流行，因为它们发展成为具有编辑几乎任何基因组的能力的靶向和可编程基因组工程工具。作为CRISPR-Cas系统的天然关闭开关，抗CRISPR具有很大的潜力，可以作为CRISPR-Cas工具的调节剂，实现更安全、更可控的基因组编辑。这项研究将有助于了解三种生物信息学方法对新Acr发现的相对有用性，并指导未来开发新的生物信息学工具，以促进抗CRISPR研究。在这项研究中发现的数千个Acr同源物和数百个新的抗CRISPR基因座将成为基因组工程师寻找新的CRISPR-Cas调节剂的宝贵数据资源。
Anti-CRISPR (Acr) loci/operons encode Acr proteins and Acr-associated (Aca) proteins. Forty-five Acr families have been experimentally characterized inhibiting seven subtypes of CRISPR-Cas systems. We have developed a bioinformatics pipeline to identify genomic loci containing Acr homologs and/or Aca homologs by combining three computational approaches: homology, guilt-by-association, and self-targeting spacers. Homology search found thousands of Acr homologs in bacterial and viral genomes, but most are homologous to AcrIIA7 and AcrIIA9. Investigating the gene neighborhood of these Acr homologs revealed that only a small percentage (23.0% in bacteria and 8.2% in viruses) of them have neighboring Aca homologs and thus form Acr-Aca operons. Surprisingly, although a self-targeting spacer is a strong indicator of the presence of Acr genes in a genome, a large percentage of Acr-Aca loci are found in bacterial genomes without self-targeting spacers or even without complete CRISPR-Cas systems. Additionally, for Acr homologs from genomes with self-targeting spacers, homology-based Acr family assignments do not always agree with the self-targeting CRISPR-Cas subtypes. Last, by investigating Acr genomic loci coexisting with self-targeting spacers in the same genomes, five known subtypes (I-C, I-E, I-F, II-A, and II-C) and five new subtypes (I-B, Ill-A, Ill-B, IV-A, and V-U4) of Acrs were inferred. Based on these findings, we conclude that the discovery of new anti-CRISPRs should not be restricted to genomes with self-targeting spacers and loci with Acr homologs. The evolutionary arms race of CRISPR-Cas systems and anti-CRISPR systems may have driven the adaptive and rapid gain and loss of these elements in closely related genomes.IMPORTANCE As a naturally occurring adaptive immune system, CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated genes) systems are widely found in bacteria and archaea to defend against viruses. Since 2013, the application of various bacterial CRISPR-Cas systems has become very popular due to their development into targeted and programmable genome engineering tools with the ability to edit almost any genome. As the natural off-switch of CRISPR-Cas systems, anti-CRISPRs have a great potential to serve as regulators of CRISPR-Cas tools and enable safer and more controllable genome editing. This study will help understand the relative usefulness of the three bioinformatics approaches for new Acr discovery, as well as guide the future development of new bioinformatics tools to facilitate anti-CRISPR research. The thousands of Acr homologs and hundreds of new anti-CRISPR loci identified in this study will be a valuable data resource for genome engineers to search for new CRISPR-Cas regulators.