RECKLEEN is a lambda Red/CRISPR-Cas9 based single plasmid platform for enhanced genome editing in Klebsiella pneumoniae.

ABSTRACT: Klebsiella pneumoniae (Kp) has evolved as a major public health threat due to its multidrug-resistance (MDR) and hypervirulence. Current Kp genome-editing tools are constrained by cumbersome workflows, low flexibility, and limited scalability. Here, we present the RECKLEEN system -Recombineering/CRISPR-based KLebsiella Engineering for Efficient Nucleotide editing - as a single plasmid platform designed for precise genetic manipulation of Kp. RECKLEEN combines lambda Red recombineering with powerful CRISPR-Cas9-based targeted counterselection, achieving up to 99.998% killing efficiency. By implementing the near PAM-less SpG Cas9 variant in RECKLEEN, the compatible target sequence spectrum was significantly broadened. This approach enables deletions, point mutations, and DNA integrations, wi

SUBMITTER: Elsayed EM 

PROVIDER: S-EPMC12575629 | biostudies-literature | 2025 Oct

REPOSITORIES: biostudies-literature