Culture-free DNA sequencing of patient samples has the potential to improve pathogen surveillance and antimicrobial treatment strategies compared to current laboratory-based approaches. Here, we demonstrate a novel CRISPR-Cas9 enrichment strategy combined with Oxford Nanopore sequencing for targeted characterisation of Klebsiella pneumoniae strains directly from complex patient samples. We developed 60 CRISPR-Cas9 guides targeting highly conserved transfer RNA (tRNA), MLST and AMR genes in K. pneumoniae via population genomics and sequence alignment against the Genome Taxonomy Database. We validated our guides against 21 K. pneumoniae isolates each with distinct STs and AMR profiles in pure culture. We then compared this approach to unenriched metagenomic sequencing in human faecal samples spiked with a known K. pneumoniae strain. CRISPR-Cas9 guides successfully enriched DNA yield of genomic regions surrounding tRNA genes in all isolates (median 52x vs untargeted regions, IQR 12-126x). Sequence conservation analysis suggested that tRNA guides could also be applied to many common pathogens across Enterobacterales, rather than being limited to K. pneumoniae. When performed on human faecal samples, enriched sequencing generated 11.3-28.8x higher abundance of reads aligning to MLST genes compared to unenriched metagenomics. Enriched sequencing detected target AMR genes in 6/6 faecal samples compared to 1/6 samples following unenriched sequencing. CRISPR-Cas9 enrichment shows promise for improved pathogen characterisation from clinical samples over traditional unenriched metagenomics, potentially at a lower financial cost due to reduced sequencing requirements. This approach could enable efficient culture-free surveillance screening of patient samples for problematic pathogens, including K. pneumoniae.