Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated cas) systems constitute the adaptive immune system in prokaryotes, which provides resistance against bacteriophages and invasive genetic elements. The landscape of applications in bacteria and eukaryotes relies on a few Cas effector proteins that have been characterized in detail. However, there is a lack of comprehensive studies on naturally occurring CRISPR-Cas systems in beneficial bacteria, such as human gut commensal Bifidobacterium species. In this study, we mined 954 publicly available Bifidobacterium genomes and identified CRIPSR-Cas systems in 57% of these strains. A total of five CRISPR-Cas subtypes were identified as follows: Type I-E, I-C, I-G, II-A, and II-C. Among the subtypes, Type I-C was the most abundant (23%). We further characterized the CRISPR RNA (crRNA), tracrRNA, and PAM sequences to provide a molecular basis for the development of new genome editing tools for a variety of applications. Moreover, we investigated the evolutionary history of certain Bifidobacterium strains through visualization of acquired spacer sequences and demonstrated how these hypervariable CRISPR regions can be used as genotyping markers. This extensive characterization will enable the repurposing of endogenous CRISPR-Cas systems in Bifidobacteria for genome engineering, transcriptional regulation, genotyping, and screening of rare variants.
Highlights
Clustered regularly interspaced short palindromic repeats (CRISPR) and accompanying CRISPR-associated genes constitute the adaptive immune system in bacteria, which provides resistance against bacteriophage predation [1]
The dramatic advances in high-throughput sequencing technologies have revolutionized the study of genomics, expanding our knowledge of the genus Bifidobacterium through continued discovery of novel species
57% (548/954) of bifidobacterial genomes encoded CRISPR-Cas systems (Figure 1A), and displayed a slightly higher occurrence than the 46% prevalence observed across all bacteria [38]
Summary
Clustered regularly interspaced short palindromic repeats (CRISPR) and accompanying CRISPR-associated (cas) genes constitute the adaptive immune system in bacteria, which provides resistance against bacteriophage predation [1]. This immunity is orchestrated in three stages. With thousands of CRISPR-Cas systems occurring in nature across genera and species, only a handful have been characterized in detail and repurposed for various applications, notably genetic engineering and transcriptional regulation, among others. Many human commensal bacteria, probiotic strains, and other industrial workhorses harbor CRISPR-Cas systems in their genomes, allowing the repurposing of these systems for diverse applications without the need of heterologous expression [6]. The lack of a fundamental understanding by the scientific community of CRISPR-Cas biology in general, along with the repurposing of endogenous systems in particular, has represented a bottleneck which limits broad implementation
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