Genome Editing by CRISPR/Cas12 Recognizing AT-Rich PAMs in Shewanella oneidensis MR-1

Homologous recombination-mediated genomic editing is urgently needed to obtain high-performance chassis of electroactive microorganisms. However, the existing tools cannot meet the requirement of genome-wide editing in Shewanella oneidensis. Here, we develop different CRISPR-Cas systems that are ideal to be employed in AT-rich sequences as the supplements to Cas9. AsCpf1 and BhCas12b show low cell toxicity and superior ability to target sequences and are thus screened out in S. oneidensis MR-1. The PAMs of AsCpf1 and BhCas12b are 5'-TTTV-3' and 5'-ATTN-3'. For gene deletion, ∼1-kb gene is knocked out and the editing efficiency is 41.67% by BhCas12b-mediated system. For gene replacement, endogenous promoter of nagK was substituted to a constitutive promoter with the efficiency of 25% through BhCas12b system. For gene insertion, the integration efficiency was up to 94.4% and 83.9% via CRISPR-BhCas12b and AsCpf1 tools. This study implies a great potential of CRISPR-BhCas12b/AsCpf1 systems recognizing AT-rich PAMs for genomic editing in S. oneidensis to facilitate multifaceted gene manipulation.