Doctorate Program of Biology, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia.
Microbiology Laboratory, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia.
J Mol Diagn. 2024 Dec;26(12):1045-1054. doi: 10.1016/j.jmoldx.2024.08.009.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) application for targeted sequencing has made a breakthrough in the genomic research era. High diversity in the capsular polysaccharide (cps) locus of Streptococcus pneumoniae has hampered identification of the serotype. This study developed a new serotyping method for S. pneumoniae using CRISPR/Cas9-targeted sequencing with the Oxford Nanopore Technologies platform. A probe was designed at the position of the cps locus using an excision approach on two sides flanking genes between the dexB and aliA genes with approximately 20 kb. A native barcoding method was used for multiplexing. The probe will attach to a specific side followed by attachment of CRISPR/Cas9 to cut the recognition area. The study used de novo assembly to reconstruct sequence reads, which were analyzed using PneumoCRISPR, a new serotyping pipeline for Oxford Nanopore Technologies sequencing data output. Four CRISPR/Cas9 probes have been designed and recognize the cps locus of S. pneumoniae. Serotyping results align precisely with serotyping data from whole-genome sequencing. This serotyping method also allows researchers to use multiple samples in a single run. The new serotyping method based on CRISPR/Cas9-targeted sequencing holds immense promise for serotype identification of S. pneumoniae.
CRISPR/Cas9 靶向测序在基因组研究时代取得了突破性进展。肺炎链球菌荚膜多糖（cps）基因座的高度多样性阻碍了血清型的鉴定。本研究利用 CRISPR/Cas9 靶向测序和牛津纳米孔技术平台，开发了一种新的肺炎链球菌血清分型方法。采用切除法在 dex B 和 aliA 基因之间的侧翼基因两侧设计 cps 基因座位置的探针，约 20 kb。采用原生条形码方法进行多重化。探针将附着在特定的一侧，然后 CRISPR/Cas9 附着在识别区域进行切割。该研究使用从头组装重建序列读取，使用 PneumoCRISPR 进行分析，这是一种针对牛津纳米孔技术测序数据输出的新的血清分型管道。已经设计了四个 CRISPR/Cas9 探针，可识别肺炎链球菌的 cps 基因座。血清分型结果与全基因组测序的血清分型数据完全吻合。这种血清分型方法还允许研究人员在单个运行中使用多个样本。基于 CRISPR/Cas9 靶向测序的新型血清分型方法为肺炎链球菌的血清型鉴定带来了巨大的希望。
