Interaction of Bare dSpCas9, Scaffold gRNA, and Type II Anti-CRISPR Proteins Highly Favors the Control of Gene Expression in the Yeast .

Type II CRISPR-(d)SpCas9 and anti-CRISPR proteins (AcrIIs) show evidence of coevolution and competition for survival between bacteria and phages. In biotechnology, CRISPR-(d)SpCas9 is utilized for gene editing and transcriptional regulation. Moreover, its activity is controlled by AcrIIs. However, studies of dSpCas9/AcrII-based transcription regulation in are rare. In this work, we used dSpCas9 as a template to engineer new transcription activators. We found that the most performant activation system requires the use of bare dSpCas9 in conjunction with scaffold gRNA (scRNA). This means that activation domains shall not be fused to dSpCas9 but rather interact with scRNA. We showed that a low amount of sgRNA is not a limiting factor in dSpCas9-driven transcription regulation. Moreover, a high quantity of sgRNA does not improve, generally, activation (and repression) efficiency. Importantly, we analyzed the performance of AcrIIA2, AcrIIA4, and AcrIIA5 in in depth. AcrIIA4 is the strongest of the three AcrIIs and also the only one able to induce high inhibition at low concentrations. However, the activation domains fused to dSpCas9 hindered interactions with the AcrIIs as well and limited their control of gene transcription regulation, confirming that bare dSpCas9 is the best solution for building synthetic genetic networks in yeast.