A New Serotyping Method of Streptococcus pneumoniae Based on CRISPR/Cas9–Targeted Sequencing

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/Cas9etargeted 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/Cas9etargeted sequencing holds immense promise for serotype identification of S. pneumoniae. (J Mol Diagn 2024, 26: 1045e1054;