Table_2_Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.XLSX

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system that has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities. This system was first identified in Leptotrichia shahii. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a, and other CRISPR-Cas systems. Various types of CRISPR-Cas systems were found to be unevenly distributed among the Leptotrichia genomes, including types I-B (10/29, 34.4%), II-C (1/29, 2.6%), III-A (6/29, 15.4%), III-D (6/29, 15.4%), III-like (3/29, 7.7%), and VI-A (11/29, 37.9%), while 8 strains (20.5%) had no CRISPR-Cas system at all. The Cas13a effectors were found to be highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii, but their collateral ssRNA cleavage activities leading to impediment of bacterial growth were conserved. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 4.4% of spacers (40/889) were shared by two strains. The organization and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III, and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.

成簇规律间隔短回文重复序列（Clustered regularly interspaced short palindromic repeats, CRISPR）-Cas13a，此前又称CRISPR-C2c2，是目前最新发现的兼具靶向切割与旁切单链RNA（single-stranded RNA, ssRNA）活性的RNA引导型RNA靶向CRISPR-Cas系统。该系统最初于沙阿纤毛菌（Leptotrichia shahii）中被鉴定。本研究完成了11株纤毛菌的全基因组测序，并与18株公开可获取的纤毛菌基因组开展比对分析，以探究CRISPR-Cas13a及其他CRISPR-Cas系统的组成、存在情况与多样性。结果显示，各类CRISPR-Cas系统在纤毛菌基因组中分布不均：其中I-B型（10/29，34.4%）、II-C型（1/29，2.6%）、III-A型（6/29，15.4%）、III-D型（6/29，15.4%）、III样型（3/29，7.7%）与VI-A型（11/29，37.9%）均有检出，另有8株（20.5%）未携带任何CRISPR-Cas系统。Cas13a效应蛋白序列高度多样，与沙阿纤毛菌的Cas13a氨基酸序列相似性介于61%~90%之间，但其介导细菌生长受阻的旁切ssRNA活性仍保持保守。CRISPR间隔序列记录了菌株既往遭遇外源入侵物的经历，所保留的间隔序列可反映菌株的进化谱系或亲缘关系。对纤毛菌物种间间隔序列的组成与数量分析显示，其多样性显著，仅4.4%的间隔序列（40/889）可在两株菌中共享。对所有已注册纤毛菌所编码的I~VI型CRISPR-Cas系统的组织形式与分布情况分析表明，CRISPR-Cas系统的效应蛋白或间隔序列差异显著，I、III、VI型系统的检出率基本相当，仅1株菌携带II型系统，未发现携带IV型或V型系统的菌株。本研究结果为纤毛菌物种间CRISPR-Cas系统的特征与分化提供了新的认识。