CRISPR/Cpf1-Mediated Multiplex and Large-Fragment Gene Editing in <i>Staphylococcus aureus</i>

Abstract

<i>Staphylococcus aureus</i> is a major human pathogen that causes a variety of infections, including life-threatening diseases. Research on <i>S. aureus</i> is constrained by complex and limited genetic manipulation methods. Here, we report a CRISPR/Cpf1-mediated system, pCpfSA, for rapid and versatile genome editing in <i>S. aureus</i>. In direct comparison with the existing CRISPR/Cas9-mediated genome-editing system, the pCpfSA system exhibits enhanced colony-forming units (CFUs) after editing and an expanded targetable range with comparable editing efficiency. Given the precursor crRNA (pre-crRNA) processing activity of Cpf1, the pCpfSA system also allows multiplex gene editing and large-fragment DNA knockout simply by introducing two crRNAs and the corresponding donor templates, which is difficult to achieve using the CRISPR/Cas9 system, thereby greatly expanding the genome editor toolbox for <i>S. aureus</i>.