Systematic interrogation of CRISPR-based antimicrobials in Klebsiella pneumoniae reveals nuclease-, guide-, and strain-specific factors influencing antimicrobial activity
CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform as antimicrobials across target sites and strains remains poorly explored. Here, we address this knowledge gap by systematically interrogating the use of CRISPR antimicrobials against multidrug-resistant and hypervirulent strains of Klebsiella pneumoniae. Comparing different Cas nucleases, we found that AsCas12a exhibited robust targeting across different strains. The elucidated modes of escape from this nuclease varied widely, restraining opportunities to enhance killing. We also encountered individual guide RNAs yielding different extents of clearance across strains. The differences were attributed to improper RNA folding, leading to inefficient DNA cleavage and subsequent repair via homologous recombination. To explore features that could improve targeting across strains, we performed a genome-wide screen in different K. pneumoniae strains that yielded guide design rules and trained an algorithm for predicting guide efficiency. Finally, we showed that Cas12a antimicrobials can be exploited to eliminate K. pneumoniae when encoded in phagemids delivered by T7-like phages. Altogether, our results highlight the importance of evaluating antimicrobial activity of CRISPR antimicrobials across relevant strains and define critical parameters for efficient CRISPR-based targeting. Overall design: A library (11,900 targets and 100 non-targeting guides) was designed to target a non-redundant collection of complete genomes of Klebsiella pneumoniae, by randomly choosing targets with a proper TTTV PAM shared by all the genomes in the collection. The library was prepared in E. coli MG1655, transfered to E. coli MFDpir, and delivered by conjugation to 3 K. pneumoniae strains (NTUH-K2044, KPPR1 and SB5442). Samples were harvested after induction with 1 nM anhydrotetracycline (aTc) at time zero, 3 hours and 16 hours. Finally, plasmids were extracted from 10 mL of these cultures and the donor strain culture. The samples were prepared for amplicon sequencing and sequencing was then performed with Illumina sequencing primers using a NextSeq 500 benchtop sequencer