Diverse CRISPRs Evolving in Human Microbiomes

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, together with cas (CRISPR–associated) genes, form the CRISPR/Cas adaptive immune system, a primary defense strategy that eubacteria and archaea mobilize against foreign nucleic acids, including phages and conjugative plasmids. Short spacer sequences separated by the repeats are derived from foreign DNA and direct interference to future infections. The availability of hundreds of shotgun metagenomic datasets from the Human Microbiome Project (HMP) enables us to explore the distribution and diversity of known CRISPRs in human-associated microbial communities and to discover new CRISPRs. We propose a targeted assembly strategy to reconstruct CRISPR arrays, which whole-metagenome assemblies fail to identify. For each known CRISPR type (identified from reference genomes), we use its direct repeat consensus sequence to recruit reads from each HMP dataset and then assemble the recruited reads into CRISPR loci; the unique spacer sequences can then be extracted for analysis. We also identified novel CRISPRs or new CRISPR variants in contigs from whole-metagenome assemblies and used targeted assembly to more comprehensively identify these CRISPRs across samples. We observed that the distributions of CRISPRs (including 64 known and 86 novel ones) are largely body-site specific. We provide detailed analysis of several CRISPR loci, including novel CRISPRs. For example, known streptococcal CRISPRs were identified in most oral microbiomes, totaling ∼8,000 unique spacers: samples resampled from the same individual and oral site shared the most spacers; different oral sites from the same individual shared significantly fewer, while different individuals had almost no common spacers, indicating the impact of subtle niche differences on the evolution of CRISPR defenses. We further demonstrate potential applications of CRISPRs to the tracing of rare species and the virus exposure of individuals. This work indicates the importance of effective identification and characterization of CRISPR loci to the study of the dynamic ecology of microbiomes.

CRISPR（成簇规律间隔短回文重复序列，Clustered Regularly Interspaced Short Palindromic Repeats）与cas（CRISPR相关，CRISPR–associated）基因共同构成CRISPR/Cas适应性免疫系统，这是真细菌与古菌抵御包括噬菌体、接合性质粒在内的外源核酸的主要防御策略。由重复序列分隔的短间隔序列源自外源DNA，可介导对后续感染的干扰作用。人类微生物组计划（Human Microbiome Project, HMP）已产出数百条鸟枪法宏基因组数据集，这使得我们得以探索与人类共生的微生物群落中已知CRISPR系统的分布与多样性，并挖掘全新的CRISPR系统。我们提出一种靶向组装策略以重建CRISPR阵列——这类阵列无法通过全宏基因组组装实现识别。针对每一种从参考基因组中鉴定得到的已知CRISPR类型，我们利用其直接重复序列的共有序列，从每份HMP数据集中招募测序读段，随后将招募得到的读段组装为CRISPR基因座；此后便可提取独特的间隔序列用于后续分析。我们还从全宏基因组组装得到的重叠群（contig）中鉴定出新型CRISPR系统或新的CRISPR变体，并通过靶向组装策略在多个样本中更全面地识别这类CRISPR系统。研究发现，CRISPR系统的分布（涵盖64种已知类型与86种新型类型）在很大程度上呈现身体部位特异性。我们对多种CRISPR基因座开展了详细解析，其中包括新型CRISPR系统。例如，我们在大多数口腔微生物组中鉴定得到链球菌属的已知CRISPR系统，总计包含约8000个独特的间隔序列：从同一受试者同一口腔部位采集的样本共享的间隔序列最多；同一受试者不同口腔部位共享的间隔序列则显著减少，而不同受试者之间几乎无共有间隔序列，这表明细微的生态位差异对CRISPR防御系统的演化具有关键影响。我们进一步证实了CRISPR系统在追踪稀有物种及个体病毒暴露史方面的潜在应用价值。本研究凸显了对CRISPR基因座进行高效鉴定与特征解析，对于微生物组动态生态学研究的重要意义。