Designing and Assessing the Efficacy of Protein Inhibitors of IscB Endonucleases

Abstract

Many bacteria and archaea possess Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins, forming a CRISPR-Cas system that defends against viral infection. These microbes incorporate fragments of viral DNA as spacers between short DNA repeats, then transcribe these regions of alternating spacers and repeat units into guide RNA (gRNA) sequences that form complexes with Cas proteins. Upon subsequent viral attack, the gRNA sequences bind regions of complementary viral DNA, and the Cas proteins act as endonucleases, cleaving the DNA to curb the infection. The CRISPR-Cas system has been adapted into a gene editing technology, but viruses have evolved anti-CRISPR proteins to inhibit Cas proteins. There are many known anti-CRISPR proteins and they utilize different strategies to achieve the inhibition. However, no anti-CRISPR proteins have been described for the recently discovered group of transposon-encoded IS200/IS605 proteins known as IscB endonucleases, which are likely ancestors of the RNA-guided endonuclease Cas9. Our goal was to design artificial inhibitor proteins and assess their influence on the function of the IscB endonuclease.