RESUMEN
Genome-wide screens have become powerful tools for elucidating genotype-to-phenotype relationships in bacteria. Of the varying techniques to achieve knockout and knockdown, CRISPR base editors are emerging as promising options. However, the limited number of available, efficient target sites hampers their use for high-throughput screening. Here, we make multiple advances to enable flexible base editing as part of high-throughput genetic screening in bacteria. We first co-opt the Streptococcus canis Cas9 that exhibits more flexible protospacer-adjacent motif recognition than the traditional Streptococcus pyogenes Cas9. We then expand beyond introducing premature stop codons by mutating start codons. Next, we derive guide design rules by applying machine learning to an essentiality screen conducted in Escherichia coli. Finally, we rescue poorly edited sites by combining base editing with Cas9-induced cleavage of unedited cells, thereby enriching for intended edits. The efficiency of this dual system was validated through a conditional essentiality screen based on growth in minimal media. Overall, expanding the scope of genome-wide knockout screens with base editors could further facilitate the investigation of new gene functions and interactions in bacteria.
Asunto(s)
Sistemas CRISPR-Cas , Escherichia coli , Edición Génica , Edición Génica/métodos , Escherichia coli/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Genoma Bacteriano/genética , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Streptococcus/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/enzimología , Aprendizaje Automático , ARN Guía de Sistemas CRISPR-Cas/genéticaRESUMEN
Protein adsorption after biomaterial implantation is the first stage of the foreign body response (FBR). However, the source(s) of the adsorbed proteins that lead to damaged associated molecular patterns (DAMPs) and induce inflammation have not been fully elucidated. This study examined the effects of different protein sources, cell-derived (from a NIH/3T3 fibroblast cell lysate) and serum-derived (from fetal bovine serum), which were compared to implant-derived proteins (after a 30 min subcutaneous implantation in mice) on activation of RAW 264.7 cells cultured in minimal (serum-free) medium. Both cell-derived and serum-derived protein sources when preadsorbed to either tissue culture polystyrene or medical-grade silicone induced RAW 264.7 cell activation. The combination led to an even higher expression of pro-inflammatory cytokine genes and proteins. Implant-derived proteins on silicone explants induced a rapid inflammatory response that then subsided more quickly and to a greater extent than the studies with in vitro cell-derived or serum-derived protein sources. Proteomic analysis of the implant-derived proteins identified proteins that included cell-derived and serum-derived, but also other proteinaceous sources (e.g., extracellular matrix), suggesting that the latter or nonproteinaceous sources may help to temper the inflammatory response in vivo. These findings indicate that both serum-derived and cell-derived proteins adsorbed to implants can act as DAMPs to drive inflammation in the FBR, but other protein sources may play an important role in controlling inflammation.