UC San Diego

Widely distributed and highly conserved among all three domains of life, argonaute proteins generally function in binding and utilizing short nucleic acid guides, and some additionally perform guide-mediated cleavage of complementary DNA or RNA targets. Due to their functional analogy to CRISPR-Cas systems, catalytically active prokaryotic argonautes (pAgos) have been primarily highlighted in the field of biotechnology for applications in seamless genetic engineering, leading to the emergence of the first pAgo-based genetic modification technology. However, characterization of the in vivo role of pAgos is in its early stage and only a few catalytically active pAgos have been broadly analyzed such as the T. thermophilus TtAgo. Through bioinformatics, gene transfer assays, and RB-TnSeq, we identify and investigate a closely related homolog of TtAgo, SeAgo from the genetically tractable model cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus).

In this study, we explored the effect of SeAgo on natural transformation and conjugation in S. elongatus as well as the potential relationship between SeAgo and the predicted associated nuclease SeCas4. For the purpose of this study, we designed improved CRISPR-Cpf1 genetic engineering tools by constructing a CRISPR-CpfI module adapted for the CYANO-VECTOR platform in addition to five CRISPR-Cpf1 plasmids carrying an improved RSF1010 backbone which significantly reduced the difficulty of the subsequent cloning. Nevertheless, even with an improved RSF1010 backbone, the transfer of RSF1010 based plasmids in S. elongatus repeatedly fails or results in small unstable colonies. Here we show that loss of function of SeAgo drastically increases the efficiency of transfer of RSF1010-based plasmids. With new RSF1010-based CRISPR tools continuing to emerge, overcoming the challenges of utilizing these technologies in the model cyanobacterium S. elongatus could significantly advance our understanding of these photosynthetic prokaryotes and their defense mechanisms.