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

Area of Science:

• Genomics
• Microbiology
• Molecular Biology

Background:

• Streptococcus pneumoniae serotyping is crucial for public health.
• High diversity in the capsular polysaccharide (cps) locus complicates traditional identification methods.

Purpose of the Study:

• To develop a novel, efficient, and accurate serotyping method for Streptococcus pneumoniae.
• To leverage CRISPR/Cas9-targeted sequencing for improved serotype identification.

Main Methods:

• CRISPR/Cas9 system designed with specific probes targeting the cps locus (approx. 20 kb region between dexB and aliA genes).
• Utilized Oxford Nanopore Technologies platform for sequencing with native barcoding for multiplexing.
• Employed de novo assembly and a new analysis pipeline, PneumoCRISPR, for sequence read reconstruction and serotyping.

Main Results:

• Successfully designed four CRISPR/Cas9 probes that accurately recognize the S. pneumoniae cps locus.
• Serotyping results demonstrated high concordance with established whole-genome sequencing data.
• The method enables multiplexed sample analysis in a single sequencing run.

Conclusions:

• The developed CRISPR/Cas9-targeted sequencing method offers a promising advancement for S. pneumoniae serotype identification.
• This technique provides a robust and efficient alternative for epidemiological surveillance and diagnostics.
• The PneumoCRISPR pipeline facilitates rapid analysis of Nanopore sequencing data for serotyping.