CRISPR/Cas9 gene targeting technology has become the most widely used gene editing technology in both plants and animals. However, substantial off-target effect remains as a major imperfection hindering its further application. Here, Nicotiana benthamiana leaf cell-free system was used to simulate in vivo environment. And the effects of different CRISPR/Cas9 components on DNA stability in cell-free system were studied to explore possible mechanisms causing CRISPR off-target. The results showed that overexpressing Cas9, nCas9 and dCas9 significantly inhibited DNA cleavage in the cell extracts. While overexpressing RNPs accelerated the target DNA cleavage but inhibited non-target DNA digestion in cell extracts, overexpressing nRNP and dRNP blocked the cleavage of either target or non-target sequences. Meanwhile, analysis of whole-genome sequencing data from mice and rice edited by different CRISPR tools revealed that the main off-target mutations were SNVs (single nucleotide variants), rather than Indels (insertions and deletions) that were readily induced by DNA double-strand breaks. The off-target sites did not match the conventionally predicted places but were PAM-rich sites preferred. Our study suggests that PAM-dependent binding without cleavage of CRISPR/Cas9 to non-target sequences may increase off-target mutation risks through impeding the necessary cleavage process for repairing spontaneous or environmentally induced non-targeted DNA mutations.

Given the potential applications of gas vesicles (GVs) in multiple fields including antigen-displaying and imaging, heterologous reconstitution of synthetic GVs is an attractive and interesting study that has translational potential. Here, we attempted to express and assemble GV proteins (GVPs) into GVs using the model eukaryotic organism Saccharomyces cerevisiae. We first selected and expressed two core structural proteins, GvpA and GvpC from cyanobacteria Anabaena flos-aquae and Planktothrix rubescens, respectively. We then optimized the protein expression conditions and validated GV assembly in the context of cell flotation and GV shapes. We found that when two copies of AnaA were integrated into the genome, it resulted in cell floatation and GV production regardless of GvpC expression. Next, we co-expressed chaperone-RFP with the GFP-AnaA to aid the AnaA aggregation. The co-expression of individual chaperones (Hsp42, Sis1, Hsp104, and GvpN) with AnaA led to the formation of larger inclusions and enhanced the sequestration of AnaA into the perivacuolar site. To our knowledge, this represents the first study on reconstitution of GVs in S. cerevisiae. Our results could provide insights into optimizing conditions for heterologous protein expressions as well as the reconstitution of other synthetic microcompartments in yeast.