Harnessing Cpf1 for CRISPR-Based Antimicrobials

Beisel, C. L., North Carolina State University
Bacteria and archaea have a unique adaptive immune system called CRISPR-Cas whose RNA-guided nucleases have been used for applications ranging from genome editing and gene regulation to in vitro diagnostics and DNA imaging. The Type II CRISPR effector protein Cas9 has been the most thoroughly investigated RNA-guided DNA endonuclease and was the pioneer for genome editing and gene regulation applications. More recently, Cas9 has been applied as an antimicrobial, where it has been successfully used to target antibiotic-resistance genes encoded on plasmids or in the genome as well as non-essential chromosomal genes. Unfortunately, Cas9 exhibits a low level of off-target toxicity and cleavage by Cas9 is not always lethal which limits further development as a targeted antimicrobial. Recently, new types of CRISPR nucleases have been discovered that may better serve as discretionary antimicrobial agents. The Type V-A CRISPR effector protein Cas12a (Cpf1) functions similarly to Cas9 but has several advantages over Cas9. Cpf1 is smaller in size and requires only single RNA for targeting the bacterial genome. Cpf1 has been shown to be highly specific with lower off target killing than Cas9 antimicrobials. Here, we examine the potency of Cpf1 as a CRISPR-based antimicrobial. This antimicrobial functioned even in the presence of the RecA DNA repair pathway. Subsequent cleavage led to cell death and is not dependent on essential gene targets. We further demonstrate the utility of this antimicrobial against E. coli O9:HS, Salmonella enterica and Klebsiella pneumoniae. These results indicate that Cpf1 may be a highly desirable effector for future development of CRISPR antimicrobials.